The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified . 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide  were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+ -evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3β4 nicotinic acetylcholine, α1β2γ2s GABAA or α1 glycine receptors at 30 μM. 1 displayed largely noncompetitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester ( The ZAC-HEK293, h5-HT3A-HEK293 and rα3β4-nAChR-HEK293, and hEAAT3-HEK293 cell lines were grown in the same culture medium supplemented with 1 mg/mL G-418. For the construction of the stable monoclonal ZAC-HEK293 cell line, 5 × 105 HEK293 cells were seeded in a 10 cm tissue culture dish, and 24 h later the cells were transfected with a total of 8 μg ZAC-pCIneo using PolyFect. 16 h after, the transfection medium were replaced with culture medium supplemented with 2 mg/mL G-418, and the cells were propagated in this medium for the following 3–4 weeks. Antibiotic-resistant individual cell clones were isolated and amplified in the continued presence of 1 mg/mL G-418. The individual clones were screened for functionality in the FMP assay (HEK293 cells were propagated in culture medium  at 18 °C for 2 days after injection.The cDNAs for the various WT and chimeric CLR subunits were linearized and subsequently transcribed and capped mMessage mMachine T7 RNA transcription kit . Different amounts of cRNA mixtures were injected for ZAC (9.2–36.8 nL in a concentration of 0.05 μg/μL), 1ια2, 0.1 mM MgCl2] and pH adjusted to 7.5 (if needed). Oocytes were placed in a recording chamber continuously perfused with this saline solution, and the compounds were applied in the perfusate. Both voltage and current electrodes were agar-plugged with 3 M KCl with a resistance of 0.2–2.0 MΩ. Oocytes were voltage-clamped at −50 mV (unless otherwise stated) by a Gene Clamp 500B amplifier , while current signals were digitized by a Digidata 1320. Currents were recorded using pCLAMP 10 . The recordings were performed at room temperature.On the day of experiment, all compound dilutions were prepared in a saline solution [115 mM NaCl, 2.5 mM KCl, 10 mM MOPS (pH 7.5), 1.8 mM CaCl2+ concentration giving rise to a maximal Zn2+ current (Zn2+ Imax) were applied to the perfusate, and it was verified that these consecutive applications elicited responses of comparable current amplitudes (±20%). The functional properties of the modulators at the CLRs were determined by pre-application of the modulator to the perfusate 30 sec and followed by co-application of the modulator and the agonist to the perfusate. In all recordings, washes of 1–5 min were executed between the applications to prevent receptor desensitization, the duration of these washes depending on the kinetics of the respective receptors and the “stickyness” of the respective compounds. For determination of the concentration-inhibtion relationships exhibited by the modulators at ZAC, agonist concentrations around EC50 (1 mM Zn2+ and 1 μM H+/pH 6.0) were used. For the selectivity and mode-of-action studies, the agonist concentrations used for different CLRs or chimeric CLRs were in the EC20–EC40 range (verified on the day of experiment), as this enabled us to investigate both for putative positive and negative receptor modulation exerted by TTFB -nicotine; m5-HT3AR, 2 μM 5-HT; hα1 GlyR, 100 μM glycine; hα1β2γ2S GABAAR, 30 μM GABA; m5-HT3A/ZAC, 0.3 μM 5-HT; ZAC/hɑ1-Gly, 2 μM Zn2+).In all recordings, the compounds or compound combinations were applied in the bath until the peak current decayed to a steady state (up to 30 sec at the most). At the beginning or end (wherever appropriate) of all recordings determining concentration-response relationships at ZAC, two consecutive applications of a Zn2.5.2+ and concentration-inhibition curves for compound 1 were constructed based on the difference in the relative fluorescence units (RFU) between the maximal fluorescence recorded before and after application of Cu2+. The data were fitted to sigmoidal curves with variable slopes using nonlinear regression, and EC50 (Cu2+) and IC50 (compound 1) values were derived from these equations.Data from the FMP assay was analysed using KaleidaGraph 3.08 . Concentration-response curves for Cu20–EC50-mediated response elicited by the agonist on each oocyte (specified in each case). Unless otherwise stated, data for agonist-induced responses in the presence of the antagonists were extracted from the plateau (or steady-state) of the agonist-evoked current. Concentration-response and concentration-inhibition curves were fitted in GraphPad Prism by nonlinear regression using the equation for sigmoidal dose-response with variable slope. I-V relationships were analyzed by fitting the curves with a third order polynomial model. Each data point represents the mean ± S.E.M. value of recordings performed on at least five oocytes from at least two different batches.Data analysis of the results from the TEVC recordings were performed using Clampfit software version 10.5  and GraphPad Prism version 7.0c . Data for the test compounds were normalized to either the maximal agonist-evoked current or to an EC3.3.1.2+ (200 μM) to elicit a significant increase in fluorescence levels (arising from the depolarization of the cells caused by ZAC activation). In the clone eventually used for the screening, Cu2+ consistently mediated concentration-dependent responses and displayed an average EC50 value of 26 μM  in the FMP assay , indicating that the Cu2+-induced response in the ZAC-HEK293 cells was indeed mediated through the receptor. On this basis we decided to perform the compound library screening.In search for novel ZAC modulators we developed a stable ZAC-HEK293 cell line to enable screening of a compound library at the receptor. ZAC-HEK293 clones were screened for functionality in the FMP assay by testing the ability of CuMP assay . Thus, Ccordings . Importa2+ (200 μM) as agonist -evoked response in the ZAC-HEK293 cells in the FMP assay, exhibiting an IC50 value of 6.1 μM  -nicotine-induced response in rα3β4-nAChR-HEK293 cells in the FMP assay significantly at concentrations up to 100 μM (data not shown). Thus, 1 seemed to exert its effects on the Cu2+-evoked response in ZAC-HEK293 cells in the FMP assay through the receptor.The 1,680 compounds in the compound library were screened for antagonist activity (at assay concentrations in the 10–30 μM range) at the ZAC-HEK293 cells in the FMP assay using Cuhe assay . Howeverassay 1,  and 1C. , n = 3) . Moreove3.2.1 at the ZAC-HEK293 cells in the FMP assay was subsequently verified at ZAC expressed in Xenopus oocytes in TEVC recordings. The signalling properties exhibited by ZAC in this assay has been characterized in detail in a previous study [2+ (~EC50) through ZAC-expressing oocytes in a concentration-dependent manner. Interestingly, 1 appeared to mediate partial inhibition of the receptor, since the fitted concentration-inhibition curve for the compound plateaued at a response level constituting ~31% of that evoked by Zn2+ (1 mM) on its own, with 1 displaying an IC50 of 4.1 μM  within this −69% inhibition range  at the receptor (2+ (1 mM)-induced response through ZAC (a fitted ~94% inhibition range), but with an IC50 of 3.2 μM  TC was equipotent with 1 as a ZAC antagonist   .1 at ZAC were next characterized in further detail by determining the concentration-response relationships for Zn2+ at the receptor in the absence and presence of various concentrations of the compound . However, much more pronounced than this effect on Zn2+ potency is the concentration-dependent 1-mediated suppression of the maximal current evoked by Zn2+ through ZAC, which is indicative of a non-competitive antagonism mode (left). In further support of this, extraction and depiction of the data (from left) in a different manner revealed that 1 displays very similar concentration-inhibition relationships at the currents evoked by 0.3 mM, 1 mM and 3 mM Zn2+ through ZAC, whereas the antagonist appears less potent at the Zn2+ (10 mM)-evoked current through the receptor (right). All in all, the antagonism exerted by 1 at ZAC thus appeared to be largely non-competitive in its nature.The antagonist properties of compound . Since ssomewhat , left. Hism mode , left. Iata from , left inreceptor , right. 3.3.1 and to search for analogs possessing higher antagonist potency at ZAC compared to this lead, we performed a SAR study in which the functional properties of 61 commercially available N-(thiazol-2-yl)-benzamide analogs were characterized at ZAC expressed in oocytes in TEVC recordings. In the following, the SAR results will be presented in the form of five series of analogs , which all were tested at ZAC at three concentrations   –5.3.3.1.Series 2 and 3, we probed the importance of the thiazole ring and different substitutions in the 4- and 5-positions of it for the ZAC activity in the N-(thiazol-2-yl)-benzamide analog  and 4-ethylacetyl (2c) analogs both displayed slightly more potent and efficacious ZAC inhibition than 1, whereas ZAC activity was reduced substantially by the introduction of a 4-(p-toIyl) substituent (2d) ((2e), a 5-cyclohexyl-1,3,4-thiadiazole (2f) or other bulky ring systems (2g-i) also resulted in substantial loss of activity  . Substitactivity .Series 3 all comprise a 3-bromobenzamido moiety, and thus differences in the functional properties exhibited by the analogs within this series are also directly attributable to their respective thiazole ring substituents. Since we were unable to obtain 2-(3-bromobenzamido)-4-methylthiazole-5-methyl ester from commercial sources, the functional consequences of elimination of the o-chloro substituent on the phenyl ring could not be assessed in a direct 1:1 comparison with 1. However, the fact that 2b and 3d, the corresponding 4-(tert-butyl)-thiazol-2-yl analogs from Series 2 and 3, exhibited comparable antagonist potencies suggested that the o-chloro substituent in the phenyl ring is not a key determinant of ZAC activity  and 3b  . Introduction of the bulky 5-bromothiophen-2-yl ring in this position eliminated ZAC activity completely (3e). The 5-nitro analog (3f) displayed an IC50 ~10 μM, whereas the 4,5-dimethyl analog 3g was a considerably weaker antagonist. Introduction of bulky aromatic/heteroaromatic substituents in the 4- and/or the 5-position of the thiazole ring essentially eliminated ZAC activity in the analogs (3h-j), and so did the substitution of thiazole for a 5-(tert-butyl)-l,3,4-thiadiazole ring (3k)  . The 4-ming (3k) .3.3.2.Series 4 and 5, we investigated the importance of the phenyl ring and the substitution pattern on it for the ZAC activity of the N-(thiazol-2-yl)-benzamide analog  and 5-ethyl ester (4i-o) groups on the thiazole ring ((4a-h), the o-tolyl (4a) and p-tolyl (4b) analogs were essentially inactive, whereas substitution of the 5-bromo-2-chlorophenyl ring in 1 with 3-iodophenyl (4c) or 3,5-dimethylphenyl (4d) rings did not alter its antagonist activity substantially  was completely devoid of antagonist activity at ZAC, and the fact that substitution of the 3-bromo substituent in 1 for a nitro group yielded an inactive analog (4f) further substantiated that a m-nitro group is unfavourable for ZAC activity. Finally, the introduction of a p-ethoxy group or substitution of the phenyl ring for a 4-pyridine resulted in inactive analogs . The 5-ethyl ester analogs (4i-o) were considerably less informative than the 5-methyl ester analogs, which in light of the inactivity of analog 3b was not particular surprising. Thus, the inactivity of six of these seven analogs could potentially be rooted in this 5-ethyl ester substituent on the thiazole ring. The o-chlorophenyl (4i) and 3,5-dichlorophenyl (4j) analogs and analog 4k comprising 5-chloro and 2-methoxy groups in the same positions of the phenyl ring as the 5-bromo and 2-chloro substituents in 1, respectively, were all inactive. The 3,4,5-trimethoxyphenyl analog (4l) displayed weak antagonist activity. Not surprisingly, the analog comprising a big N, N-diallylsulfamoyl group in the para-position of the phenyl ring (4m) was inactive, as were the 3-pyridin and 2-furan analogs   .2b compared to 1 suggested that introduction of a 4-tert-butyl group on the thiazole ring was beneficial for the antagonist activity thiazol-2-yl moiety (5a (N-(4-(tert-butyl)thiazol-2-yl)-3-flurobenzamide, TTFB) was roughly equipotent with 2b and mediated almost complete inhibition of ZAC at 10 μM, whereas the 2,3,4,5,6-pentafluoropentyl analog 5b was a considerably weaker ZAC antagonist (5c), ethoxy (5d) or acetyl (5e) groups in the 3-position of the phenyl ring, whereas the 3-dimethylamino analog (5f) was roughly equipotent with 2b  and o-chloro  substituents on the phenyl group. Finally, the fact that analogs 5i and 4l both were essentially inactive suggests that the 3,4,5-trimethoxy substitution pattern on the phenyl ring is detrimental to ZAC activity.The more potent and notable higher degree of ZAC inhibition mediated by activity . With Sel moiety . The 3-ftagonist . ZAC act with 2b . The 5-c3.3.3.Series 6 all comprised more than one modification to the N-(thiazol-2-yl)-benzamide scaffold compared to 1, and most of these analogs were completely inactive at ZAC -benzamide (6a and 6c). In light of the inactivity of analogs 2d and 3e the inactivity of analogs 6d-f that also comprise bulky substituents in the 4-position of the thiazole ring was also not particularly surprising. Since 6g comprises the same 4-ethylacetyl-substituted thiazol-2-yl ring as the potent 2c analog, the inactivity of this analog must be ascribed to its 3,5-dichlorophenyl moiety. The inactivity of analogs 6h-j could both arise from the 3-chloro or 2,4,6-tri-methyl substitutions at the phenyl ring or from the 4-acetate and the 5-carboxanride thiazole ring substients. Substitution of the 4-ferf-butyl group at the thiazole in 5f for a cyclopropyl ring (6l) resulted in decreased antagonist potency at ZAC, and introduction of a more bulky 3-substituent in the phenylring further reduced ZAC activity (6m) , the inactivity of analogs 6n-p was not surprising.The 17 analogs in e at ZAC . The inaity (6m) . Finally3.3.4.N-(thiazol-2-yl)-benzamide analogs were characterized in further detail at ZAC  were equipotent as ZAC antagonists , with 1 and 3f being weaker antagonists. Also notably, and in agreement with observations in the SAR study, 2b, 4c and 5a (TTFB) displayed substantially more efficacious ZAC antagonism than 1, inhibiting the Zn2+-induced responses through the receptor completely  as representative.The functional properties and mode of action of this novel class of ZAC antagonists were next characterized in further detail using TTFB (3.4.1.2+ and H+ as agonists and at the spontaneous ZAC activity in oocytes are given in 2+ and H+ in a concentration-dependent manner (left and left). Although this slow on-set of inhibition was observed both when using Zn2+ and H+ as agonists, the degree of it was particularly pronounced in the case of H+, which was reflected in the differences observed between the TTFB concentrations-inhibition relationships extracted from recorded peak and steady-state current amplitudes for the two agonists (right and right). Extraction of IC50 (PIC50 ± S.E.M.) values (from the steady-state currents) showed that TTFB displayed slightly higher antagonist potency at Zn2+-evoked than at H+-evoked ZAC signalling     and 7B. , n = 7) .+-evoked currents in ZAC-oocytes voltage-clamped at −60 mV and + 60 mV (left).We also investigated the current–voltage (I-V) relationship of TTFB-mediated inhibition of spontaneous ZAC activity and assessed whether its inhibition of agonist-induced ZAC currents was voltage-dependent . Analogo + 60 mV . Notably + 60 mV , left.3.4.2.3AR, the hα3β4 nAChR, the hα1β2γ2s GABAAR and the hα1 GlyR  did not display significant activity at any of these four representative classical CLRs . In this work, we took advantage of the fact that these two chimeric receptors comprise the ECD or the TMD-ICD of ZAC fused with the complementary domain from another CLR not modulated by TTFB . These black-and-white modulatory properties of TTFB (30 μM) exhibited at the two chimeric receptors strongly indicates that the binding site of the modulator resides within the TMD and/or ICD of ZAC.Functional receptors formed from chimeric subunits fusing the ECD of one CLR with the TMD and ICD of another have been used extensively to study the molecular basis for CLR signaling and to identify receptor domains involved in modulator binding –38. In a oocytes , left. I by TTFB  to delin4.In the present work we have discovered the first class of selective ZAC antagonists and delineated the functional properties and mode of action of these at the receptor.4.1.1 included in the study were informative with regard to the structural determinants for ZAC activity in the N-(thiazol-2-yl)-benzamide analog, and often the consequences of a modification in a certain position of this scaffold could not be assessed unambiguously due to differences in other parts of two analogs as well. Nevertheless, we propose that several interesting SAR observations about the N-(thiazol-2-yl)-benzamide analog as ZAC antagonist can be extracted from the study.When developing modulators based on a hit from a compound library screening, chances of optimizing the pharmacological properties of the hit as well as the ability to elucidate the SAR in a systematic manner are very dependent on the analogs accessible. These limitations are also evident in the present study, where the SAR was based exclusively on commercially available analogs. Not all 61 analogs of N-(thiazol-2-yl)-benzamide analog, as substitution of this ring for other ring systems  or fusion of it with phenyl or cycloalkyl rings  yielded inactive analogs. Introduction of small electron-withdrawing substituents  in the 5-position of the thiazole ring yielded some of the more potent analogs in the series , whereas analogs with electron-donating groups  in this position were inactive  , the inactivity displayed by all 5-ethoxy  and 5-amide  analogs was surprising  as well as both weak (3c) and potent ZAC antagonists , a range in potency primarily attributable to the identity of the 4-substituent in their respective thiazole rings. Introduction of bulky aliphatic and ester groups in the 4-position  increased ZAC activity substantially compared to analogs with a 4-methyl (3c), whereas ethyl or aromatic/heteroaromatic groups in this position was much less favourable for ZAC activity   B. The aprprising –5. This d, 3a,e) .N-(thiazol-2-yl)-benzamide analog, primarily with the 4a-h and 5a-i analogs. Introduction of a single ortho- or para-methyl group in the phenyl ring yielded inactive analogs . As evidenced by the potent ZAC antagonism exhibited by the 5-bromo-2-chlorobenzamido analogs 1 and 2b,c, an ortho-chloro substituent can be accommodated in the N-(thiazol-2-yl)-benzamide without loss of activity, albeit the similar antagonist potencies displayed by 2b and 3d also suggest that this substituent does not contribute significantly to binding affinity. The inactivity of 4g contrasts the potent ZAC activity displayed by 1 and suggests that para-substitution in the phenyl ring is unfavorable for modulator binding. Finally, analogs comprising a single methyl, fluoro, iodo or N,N-dimethylamide group in the meta-position possessed potent activity , whereas the m-aceto and m-ethoxy analogs 5d,e were inactive  and 6g  analogs. Moreover, the fact that most analogs comprising a disubstituted phenyl ring  were inactive raises another point. While no analogs comprising an unsubstituted phenyl group was included in the study, the m-fluoro analog TTFB (5a) was one of the most potent ZAC antagonists in the series, indicating that substitutions to the phenyl ring in the N-(thiazol-2-yl)-benzamide analog in general may not be beneficial for ZAC modulation.The benzamide moiety was retained in 59 of the 62 analogs in the SAR study, which thus mainly focused on the importance of the phenyl ring substitution pattern for the ZAC activity of the inactive B. Since 2b, 4c and 5a at ZAC still constitute improvement compared to those of 1 -benzamide analog , it would be interesting to probe whether combining these substituents in the N-(thiazol-2-yl)-benzamide analog would have additive or synergistic effects on ZAC antagonist potency.While we obviously could have hoped for analogs with even more increased antagonist potencies at ZAC to come out of the SAR study, the functional properties exhibited by analogs ose of 1 A. It is 4.2.N-(thiazol-2-yl)-benzamide analogs unequivocally demonstrate that the compounds act as NAMs at ZAC. In light of its pronounced selectivity for ZAC over m5-HT3AR and hα1 GlyR -benzamide analog binding site to reside within the TMD-ICD of ZAC -benzamide analog as a ZAC NAM could actually be argued to be premature, since it still remains a question whether Zn2+, Cu2+ and/or H+ or another yet unidentified transmitter is the “true” endogenous agonist for the receptor. However, given that the presently identified ZAC agonists act through the ECD and that any other putative “true” ZAC transmitter would have to be the first endogenous CLR agonist not acting through the ECD in order for the N-(thiazol-2-yl)-benzamide analog not to be a NAM, we nevertheless consider this a reasonable claim.Our investigations into the underlying mechanism for the ZAC inhibition exerted by the hα1 GlyR , the robD of ZAC . Since bD of ZAC , this algnalling . The claN-(thiazol-2-yl)-benzamide binding to ZAC cannot be completely ruled out based on the black-and-white modulation displayed by TTFB at the m5-HT3A/ZAC and ZAC/hα1-Gly receptors, it seems unlikely (N-(thiazol-2-yl)-benzamide binding site in the ZAC TMD in future studies.While a possible involvement of the ICD in unlikely . ClassicN-(thiazol-2-yl)-benzamide binding site in the ZAC TMD. However, the substantially larger difference between peak and steady-state currents observed for TTFB-mediated inhibition of H+-evoked currents than for its inhibition of Zn2+-evoked currents is interesting, as it could have both molecular and kinetic origins. In the molecular scenario, the state-dependency of the TTFB-mediated inhibition is agonist-specific, meaning that the structural composition of the TTFB binding site, and with that the ability of the antagonist to bind to it, differs in the Zn2+- and H+-stabilized active ZAC conformations. While TTFB thus may exhibit little preference for the Zn2+-bound active conformation over the resting ZAC conformation, resulting in a modest difference between peak and steady-state currents  will induce a higher maximal number of open channels during the initial phase of the agonist/TTFB co-application than the slower agonist (Zn+), and the change in the current arising from the subsequent establishment of the kinetic equilibrium between the agonist and TTFB (steady-state) will inevitably be bigger for the agonist having the bigger kinetic edge as there will be a greater accumulation of agonist-bound, blocked channels.The interesting slow on-set of the TTFB-mediated ZAC inhibition can not be ascribed to overall slow on-binding of the antagonist to the receptor. Whereas TTFB seems unable to establish binding equilibrium with ZAC during the 30 s-preincubation step, the subsequent co-application of it with agonist clearly facilitates its receptor binding  and 8B, currents , TTFB bicurrents  and 8B.  by Zn2+ , and giv4.3.While TTFB and the other active N-(thiazol-2-yl)-benzamide analogs in this series represent the first selective ZAC ligands published to date, some considerations should be given to their applicability as pharmacological tools.3AR, hα3β4 nAChR, hα1β2γ2S GABAAR and hα1 GlyR is likely to extend to the other members of the CLR superfamily (N-(thiazol-2-yl)-benzamide scaffold has been used extensively in medicinal chemistry development of ligands for a diverse range of targets (exemplified in N-(thiazol-2-yl)-benzamide scaffold that most likely would render them inactive at ZAC. Conversely, it can not be ruled out that the ZAC antagonists identified in this study could hold activity at some of these other targets, and a ligand displaying a functional potency of ~1 μM at its target can not be automatically assumed not to possess off-target activities in relevant concentration ranges.Although we only have probed the putative off-target effects of TTFB by testing it at one representative from each of the four classical CLR subfamilies, we propose that the pronounced selectivity exhibited by the antagonist for ZAC over m5-HTerfamily . ConsideN-(thiazol-2-yl)-benzamide analogs presented here potentially could be applied as a pharmacological tool for studies of ZAC in vivo. We have not investigated the pharmacokinetic properties of TTFB, but given its moderate target potency and its limited solubility it seems unlikely that systemic administration of the drug would produce sufficiently high concentrations of the free (unbound) compound to achieve significant ZAC inhibition in vivo. Moreover, the absence of an orthologous ZACN gene in rat and mouse genomes also rules out that such studies can be performed in the species most often used for in vivo investigations. Instead, TTFB and its analogs could be useful tools in focused electrophysiological or other functional in vitro/ex vivo studies of native ZAC signalling by use of tissue or cell lines derived from human or other mammalian species.Another question is whether TTFB and the other 5.N-(thiazol-2-yl)-benzamide analogs constitute the first class of selective antagonists of ZAC, and we propose that the antagonists represent a valuable addition to the limited pharmacological tool box presently available for ZAC and could aid future investigations into this enigmatic member of the CLR superfamily. The N-(thiazol-2-yl)-benzamide analogs were found to act as NAMs through a binding site in its TMD and to mediate state-dependent inhibition of ZAC, which further substantiates the complexity of CLR signalling and of the ligand-mediated modulation of them through allosteric sites.In conclusion, TTFB and the other active


These results have important consequences for the astrochemistry of interstellar ices and outer Solar System bodies, as they imply that the chemical products arising from the irradiation of amorphous ices (which may include prebiotic molecules relevant to biology) should be more abundant than those arising from similar irradiations of crystalline phases. In this present study, we have extended our work on this subject by performing comparative energetic electron irradiations of the amorphous and crystalline phases of the sulphur-bearing molecules H2S and SO2 at 20 K. We have found evidence for phase-dependent chemistry in both these species, with the radiation-induced exponential decay of amorphous H2S being more rapid than that of the crystalline phase, similar to the effect that has been previously observed for CH3OH. For SO2, two fluence regimes are apparent: a low-fluence regime in which the crystalline ice exhibits a rapid exponential decay while the amorphous ice possibly resists decay, and a high-fluence regime in which both phases undergo slow exponential-like decays. We have discussed our results in the contexts of interstellar and Solar System ice astrochemistry and the formation of sulphur allotropes and residues in these settings.Laboratory experiments have confirmed that the radiolytic decay rate of astrochemical ice analogues is dependent upon the solid phase of the target ice, with some crystalline molecular ices being more radio-resistant than their amorphous counterparts. The degree of radio-resistance exhibited by crystalline ice phases is dependent upon the nature, strength, and extent of the intermolecular interactions that characterise their solid structure. For example, it has been shown that crystalline CH It has been established for some time now that the laboratory irradiation of astrochemical ice analogues using energetic charged particles  or ultraviolet photons may lead to the production of prebiotic molecules relevant to biology, such as amino acids or nucleobases e.g., . Motivat3OH was found to be significantly less rapid than that of the amorphous phase. This was attributed to the existence of an extensive network of strong hydrogen bonds that exists in the α-crystalline phase. This network requires an additional energy input from the projectile electrons to be overcome, thus leaving less energy overall to drive radiolytic chemistry. Conversely, the amorphous CH3OH ice is characterised only by localised clusters of hydrogen bonded molecules. Such a structure does not benefit from the same stabilising effect supplied by the network of hydrogen bonds in the α-crystalline phase, particularly as hydrogen bonding in CH3OH is known to be a cooperative phenomenon in which the presence of one hydrogen bond in the network strengthens successive hydrogen bonds through electrostatic effects  which is of importance under conditions relevant to astrochemistry. This phase is orthorhombic, having eight molecules per unit cell and adopting the Pbcm space group. SO2 may also exist as an orthorhombic crystalline solid under astrochemical conditions, but in this case the Aba2 space group is adopted and there are only two molecules per unit cell  in dense interstellar clouds by producing large quantities of atomic sulphur or molecular sulphur chains and rings which are difficult to detect using current observation techniques  transmission absorption spectroscopy.The purpose of this study is thus two-fold: (i) to determine whether the phase of irradiated sulphur-bearing molecular ices influences the radiation-induced rate of decay as was previously demonstrated for non-sulphur-bearing ices; and (ii) to contribute further to our (comparatively poor) understanding of the extra-terrestrial chemistry of sulphur. To achieve these goals, the amorphous and crystalline phases of pure H−9 mbar which is achieved by the combined action of a dry rough vacuum pump and a turbomolecular pump. Within the centre of the chamber is a gold-coated oxygen-free copper sample holder which supports up to four ZnSe deposition substrates, onto which astrochemical ice analogues may be prepared. The temperature of the sample holder and the substrates may be cooled to 20 K using a closed-cycle helium cryostat, although an operational temperature range of 20–300 K is available.The irradiation experiments were performed using the Ice Chamber for Astrophysics-Astrochemistry (ICA); a custom-built experimental apparatus located at the Institute for Nuclear Research (Atomki) in Debrecen, Hungary. This apparatus  has been2S and SO2 astrochemical ice analogue phases was achieved via background deposition by allowing the relevant gas into a pre-mixing line before dosing it into the main chamber at a pressure of a few 10−6 mbar. Amorphous ice phases were prepared by deposition at 20 K, while crystalline H2S and SO2 ices were prepared by deposition at 60 and 90 K, respectively, before being cooled to 20 K. Once deposited, FT-IR spectra  of the ices were acquired, from which quantitative measurements of their molecular column densities N (molecules cm−2) and thicknesses d (μm) could be performed by measuring the peak area P (cm−1) of a characteristic absorption band (Z is the molar mass (g mol−1) of the molecular ice, NA is the Avogadro constant (6.02×1023 molecules mol−1), ρ is the density of the ice (g cm−3), and Aν is the band strength constant of the characteristic absorption band whose area is being measured (cm molecule−1). Information on the molecular column densities and thicknesses of the ices investigated in this study, as well as the physical parameters used to calculate these values, is given in The preparation of Hion band :d=10,002S and SO2 astrochemical ices were respectively irradiated using 2 and 1.5 keV electron beams (average flux = 4×1013 electrons cm−2 s−1) to a total fluence of about 8.3×1016 electrons cm−2, with projectile electrons impacting the target ices at an angle of 36° to the normal. Prior to commencing the irradiations, the beam current, spot size, and profile homogeneity were determined using the method described by 2S and SO2 ices were 155 and 70 nm, respectively. FT-IR spectra were collected at several intervals throughout the irradiation process so as to monitor the radiation chemistry occurring. All irradiations were carried out at 20 K so as to preclude any temperature-dependent effects on the mobility of radiolytically derived radicals. Moreover, the irradiation of each ice phase was performed three times so as to ensure good repeatability of the experiment.The deposited pure H2S and SO2 ice phases investigated in this study, both before and after irradiation by electrons at different fluences, are depicted in 2S presents a very broad absorption band which peaks at 2,550 cm−1 attributable to both the symmetric (ν1) and asymmetric (ν3) stretching modes. In the crystalline phase, this band is better resolved and the individual contributors may be observed. The asymmetric stretching mode is observed to peak at 2,546 cm−1, while the symmetric stretching mode is split into two components peaking at 2,534 and 2,522 cm−1. The FT-IR spectra of the pure H2 presents two distinct albeit broad absorption asymmetric and symmetric stretching mode bands which respectively peak at 1,320 and 1,148 cm−1. In the crystalline phase, these bands are observed to be better resolved, with three and two individual structures being observed in the asymmetric and symmetric stretching mode bands, respectively. These structures have been attributed to the various isotopologues of SO2 (−1 are ascribed to the transverse B1(TO) and B2(TO) modes of 32S16O2, and to 34S16O2 and 32S16O18O; while those peaking at 1,143 and 1,140 cm−1 are respectively attributed to the transverse A1(TO) mode of 32S16O2 and to 34S16O2. It is interesting to note that the naturally low abundances of 34S and 18O do not result in weaker band intensities for the SO2 isotopologues containing these isotopes due to intermolecular coupling between these isotopologues in the condensed phase  are still observable in the crystalline ices at the end of irradiation. As such, these irradiated ices are likely largely amorphous but with some small degree of remnant structural order.The onset of electron irradiation brings about noticeable changes in the appearances of the spectra of the pristine ices. Perhaps the most prominent of these is the significant broadening of the crystalline ice absorption bands, which also lose their resolved individual structures. This is due to radiation-induced amorphisation, which has been well documented in several ice species irradiated by ions, electrons, and ultraviolet photons; including HH3 e.g., . It is i2S ices efficiently yield H2S2 as well as higher order polysulphanes  in addition to allotropic forms of elemental sulphur  is thought to be largely mediated by HS radicals formed via the dissociation of the parent H2S molecules:The irradiation of molecular ices is known to initiate a rich chemistry leading to the formation of new species. Previous studies have established that irradiated H sulphur . In our 500 cm−1 . The che2S may pick up an electron to form HS−ions. These HS−ions may possibly participate in chemistry leading to the formation of other HS radicals via proton abstraction reactions with H2S, after which the resultant HS−ion may undergo electron auto-detachment to yield HS. A similar process has recently been demonstrated to occur in H2O ice with respect to radiolytically derived OH radicals and OH−ions  that is qualitatively similar to that of CH3OH. As such, it is likely that another factor should be invoked to account for the relative radio-resistance of the crystalline phases of these ices; that of lattice energies. The energetic advantage induced by the ordering of the molecular components of the solid ice must also be overcome and thus a proportion of the incident electrons’ kinetic energy must be expended upon overcoming both the lattice energy as well as the more extensive hydrogen bonding network in the crystalline phase, leaving less energy to induce the molecular dissociation that drives radiolytic chemistry.Holecules , althougectively . Despite2O, which demonstrated only a moderately more rapid decay rate of the former compared to the latter  does not really vary at low electron fluences, having an average normalised column density of 0.97 after a fluence of 8.2×1015 electrons cm−2 had been delivered. For comparison, by the point this fluence had been delivered to the crystalline SO2 ice, its average normalised column density had decreased to 0.83. However, similarly to the case of the crystalline SO2 ice, once a fluence of about 1.4×1016 electrons cm−2 had been delivered, the normalised column density was observed to undergo a slow exponential-like decay. Interestingly, beyond a delivered fluence of 1.4×1016 electrons cm−2, the rate of decay of the amorphous SO2 is greater than that of the crystalline SO2, and indeed the average decay trends cross one another at a fluence of about 5.3×1016 electrons cm−2.The radiation-induced decay trends of amorphous and crystalline SO2 , however2 decay trends is a challenging task. Measurements of the photo-desorption of SO2 molecules from an amorphous ice induced by soft x-rays allowed 2 to yield electronically excited SO2 may be a favourable process, thus largely precluding net SO2 dissociation within the ice. It is also possible that the irradiation of the amorphous SO2 ice results in its compaction, which may cause an increase in Aν of the measured band , due to the amorphous SO2 ice possibly resisting radiolytic decay. However, the greater abundance of SO3 in the irradiated crystalline ices persists even beyond this fluence, despite the more rapid decay of amorphous SO2 after this point. It should be noted, however, that after peaking at a fluence of about 5.5×1015 electrons cm−2, the SO3 column density within the irradiated amorphous SO2 ice also declines slightly  or is being desorbed or sputtered from the bulk ice. In either case, however, there is a fraction of the initially deposited sulphur that remains unobserved in the ice.As expected, the yield of Hslightly . The con14 electrons cm−2 in all of the ices apart from the amorphous SO2 ice, and that the quantity of unobserved sulphur as a fraction of that initially deposited continually grows during irradiation. In the case of the amorphous SO2 ice, unaccounted for sulphur is only registered after a fluence of 2.7×1016 electrons cm−2 has been supplied, possibly due to the resistance of SO2 to radiolytic dissociation as discussed earlier  should therefore be tested in future experiments.Given that Io orbits within the giant Jovian magnetosphere, its surface is continually exposed to ionising radiation in the form of energetic ions and electrons. The flux of 0.1–52 keV electrons at the surface of Io was given by fficient . However sulphur . Such re sulphur . It shoues >70 K . The effRosetta mission to comet 67P/Churyumov-Gerasimenko revealed the presence of a number of sulphur-bearing molecules within its icy nucleus, including H2S, SO2, SO, OCS, CS2, and S2  and a high-fluence regime in which a slow exponential-like decay trend is observed. This contrasts greatly with the crystalline ice, for which a rapid exponential decay is first observed in the low-fluence regime followed by a slower decay (which is slower than that of the amorphous phase) in the high-fluence regime. Interestingly, the formation of SO3 as a result of the irradiation of the crystalline ice was always greater than during the irradiation of the amorphous ice, possibly due to the initial resistance of the amorphous ice to radiolytic decay and its subsequent preferential formation of infrared inactive sulphur allotropes and residues.The irradiation of the SOWe suggest that our results are important not only in the context of further investigating the phase-dependent radiation chemistry of astrochemical ices, which has thus far been overlooked in the literature, but also in further understanding the chemistry of sulphur in extra-terrestrial environments. Our characterisation of this phase-dependent chemistry is directly applicable to understanding the sulphur chemistry on the surface of Io, as well as in the icy nuclei of comets. Moreover, our calculated sulphur budgets for each of the irradiation processes considered in this study  may aid in further constraining the exact molecular forms of sulphur in interstellar icy grain mantles and cometary ices. Finally, we conclude by noting that our experimental results further demonstrate the importance of incorporating ice phase as a factor when designing more complete ‘systems astrochemistry’ investigations .


Correction to: Journal of Interventional Cardiac Electrophysiologyhttps://doi.org/10.1007/s10840-022-01173-5Correction to original version of Figure 6B caption which now attributes source image provided in courtesy by Dr. Mansour Razminia. The authors regret this error.Figure 6 caption should have appeared as shown below.A ICE imaging of coronary sinus (CS) and cannulation of the catheter. Left panel, ICE image from within right atrium (RA) shows CS os. Right panel, the orientation of ICE catheter from within RA (from St. Jude brochure of ViewFlex Xtra ICE Catheter). B An electrophysiologic wire enters CS in a long-axis view. Image shown is courtesy of Mansour Razminia.The original article has been corrected.


Exposure to the Mus m 1 aeroallergen is a significant risk factor for laboratory animal allergy. This allergen, primarily expressed in mouse urine where it is characterized by a marked and dynamic polymorphism, is also present in epithelium and dander. Considering the relevance of sequence/structure assessment in protein antigenic reactivity, we compared the sequence of the variant Mus m 1.0102 to other members of the Mus m 1 allergen, and used Discotope 2.0 to predict conformational epitopes based on its 3D-structure. Conventional diagnosis of mouse allergy is based on serum IgE testing, using an epithelial extract as the antigen source. Given the heterogeneous and variable composition of extracts, we developed an indirect ELISA assay based on the recombinant component Mus m 1.0102. The assay performed with adequate precision and reasonable diagnostic accuracy (AUC = 0.87) compared to a routine clinical diagnostic test that exploits the native allergen. Recombinant Mus m 1.0102 turned out to be a valuable tool to study the fine epitope mapping of specific IgE reactivity to the major allergen responsible for mouse allergy. We believe that advancing in its functional characterization will lead to the standardization of murine lipocalins and to the development of allergen-specific immunotherapy. Laboratory animal allergy (LAA) is a relevant occupational health hazard for workers attending veterinary or research animal facilities. It was estimated that as many as 44% of personnel operating in contact with laboratory animals report symptoms related to occupational asthma or allergic rhinoconjunctivitis ,2.Since animal allergens can associate with dust particles and remain airborne for a period of time, aeroallergen exposure is considered the most significant risk factor for LAA ; among tMus m 1 is present in mouse epithelium, urine, serum, saliva, hair, and dander . Urine iThe three-dimensional structure of an allergen defines the epitopes that may interact with antibodies. In the sensitization phase of allergic diseases, B-cells are stimulated to produce IgE molecules against specific conformational epitopes exposed on the allergen surface. During a subsequent exposure to the same allergen, the recognition of those epitopes by IgE antibodies bound to specific sensitized effector cells results in clinically relevant outcomes of variable severity, even including anaphylactic reactions .In vitro diagnosis of mouse allergy is based on the detection of allergen-specific serum IgE (sIgE) using an extract of epithelial source as allergen ,12. WhilRecombinant Mus m 1.0102 protein (rMus m 1.0102) is a member of the MUPs family and has been included with the Mus m 1 allergen as an allergen component ,19,20. IWhile various studies have investigated the role of allergen molecules in the diagnosis of occupational allergens ,23,24, sOur approach, by determining the features of the allergen-antibody interactions, paves the way to the introduction of this allergen in molecular allergology.Recognizing the relevance of sequence/structure assessments in protein antigenic reactivity, (i) we searched for sequence similarities among mouse MUPs and Mus m 1.0102; and (ii) we performed a prediction of rMus m 1.0102 conformational epitopes based on its three-dimensional structure.We performed a BLAST sequence similarity search on the UniProt resource platform  and obtaHowever, when aligning the Mus m 1.0102 sequence with the peripheral MUPs pinned down by the BLAST search , we obseThe Discotope 2.0 server was used to predict discontinuous B-cell epitopes from the Mus m 1.0102 three-dimensional structure  . The outEpitope prediction, based on the Mus m 1.0102 three-dimensional structure , was also generated by peer methods. The SEPPA 3.0 prediction includes 26 epitope residues , coverinB-cell epitope prediction by Discotope 2.0 was performed also on the 3D-structure of Rat n 1 and Equ c 1 lipocalin allergens , mammalian lipocalins characterized by IgE cross-reactivity ,31. ResiWe conclude that: (i) few residues differentiate Mus m 1.0102 from central MUPs, mainly corresponding to Mus m 1.0102 solvent-exposed residues; (ii) major differences in surface composition and core interactions are observed when comparing Mus m 1.0102 with peripheral MUPs; (iii) rMus m 1.0102-predicted epitopes involve mainly polar and charged loop residues, exposed on the protein surface and presumably affected by fewer structural constraints compared to secondary structure regions; and (iv) rMus m 1.0102 structure shares predicted conformational epitopes with cross-reactive Rat n 1 and Equ c 1 allergens.Pichia pastoris, is secreted in the yeast supernatant , deg cmUrinary nMus m 1 allergen was also purchased from Indoor Biotechnologies . As reported by the company, it was purified from concentrated male mouse urine by gel filtration and ion-exchange HPLC. Protein purity was estimation by SDS-PAGE to be 95%, as declared by the manufacturer. This urinary nMus m 1 sample is referred to as “commercial preparation” throughout the manuscript.Eighteen serum samples from Mus m 1-sensitized individuals  and ten healthy nonatopic individuals (control sera) were provided by IRCCS Ospedale San Raffaele . Informed consent for the use of sera was obtained from all the subjects involved in this study (see ethics clearance below).Allergic sera (N = 18) were obtained from subjects that had access to a standard pathogen-free animal facility. Following exposition to mouse allergens, they had developed respiratory symptoms which allowed them to receive a clinical diagnosis of allergic rhinitis with or without allergic asthma, according to EAACI guidelines . Sensitig for 15 min at room temperature and used for IgE detection. The IgE repertoire of each serum was analyzed using the multiplex ImmunoCAP ISAC test . IgE quantification, as a measure of IgE allergen-specific reactivity, is expressed in arbitrary ISAC Standardized Units (ISU). According to the method, ISU values are classified into four categories: values < 0.3 ISU are defined as negative; values between 0.3 and 1 ISU as low-level positive; values between 1 and 15 ISU as moderately high; values > 15.0 ISU as very high positive. No IgE reactivity (ISU < 0.3) was detected in all the control sera.Serum samples were separated by centrifugation at 500× Serum IgE binding to recombinant and native proteins was detected by indirect IgE-ELISA. According to this technique, the protein antigen is immobilized to the surface of a multi-well plate, sIgE primary antibodies bind to the antigen, and a labelled secondary antibody against human IgE molecules binds to the primary antibodies, allowing for detection.2SO4. The reaction was quantified by measuring the absorbance at 450 nm.For this assay, 96-well plates  were coated overnight at 4 °C with the native or recombinant antigen . After 3 washes in phosphate-buffered saline solution containing 0.05% Tween 20 (PBST), the plates were blocked with 200 μL of blocking buffer , for 2 h at room temperature. After a washing step with PBST, diluted sera (1:20 with PBST containing 0.5% BSA) were loaded on the plates (100 μL) and incubated overnight at 4 °C. Then, plates were thoroughly washed with PBST (5 times). A polyclonal anti-human IgE secondary antibody conjugated to horseradish peroxidase  was diluted 1:4000 in PBST containing 0.5% BSA, applied to each well (100 μL) and incubated for 2.5 h at room temperature in the dark. Following washing with PBST (5 times), bound antibodies were detected by incubation with 100 μL of TMB Substrate Solution  for 30 min at room temperature and successive acidification with 100 μL of 0.16 M HAll samples were run in duplicate, and the mean optical density (O.D.) value was used for analysis. The cut-off for positive ELISA reactivity was determined using the mean O.D. values obtained with 10 sera of subjects without history of any allergy.To monitor intra-plate or plate-to-plate variation, intra- and inter-assay Coefficients of Variability (CV) were calculated. Samples were run in quintuplicate on two different plates. CV is calculated by dividing the standard deviation of a set of measurements by the mean of the set and expressed as a percentage. p < 0.05 or < 0.01 were considered to indicate a statistically significant difference.Statistical data analysis and graphing were performed using OriginPro, Version 2021 . The Mann–Whitney test was used for evaluating differences between the ELISA data sets obtained with Mus m 1 allergic and control sera. For correlation analysis, the Spearman correlation coefficient, specific for non-normally distributed data, was calculated. Values of The diagnostic performance of each ELISA assay was determined from receiver-operating characteristic (ROC) curves, computed in OriginPro, Version 2021. The threshold value of each ELISA assay was obtained by balancing optimal sensitivity and specificity.Based on our data, mouse sensitization was confirmed in a percentage of sera from mouse allergic patients ranging between 76.4% and 88.9%. ROC analysis attributed a good to very good accuracy level to the ELISA assays, with AUC values in the 0.77–0.89 interval. A remarkable diagnostic performance was demonstrated by rMus m 1.0102 (AUC = 0.87) which, together with its extensive structural characterization, makes it an important reagent for epitope mapping and mouse allergy diagnostics. It is worth mentioning that (i) further characterization and standardization of the recombinant allergen  might co


In vivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones in vivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation in vivo. Lastly, immunoblot analyses of rod grk1a−/− and cone grk1b−/− zebrafish and Nrl−/− mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation in vivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein–coupled receptor kinases (GRKs) is essential for sustained visual function. Previous  In vertebrates, visual interpretation of the surrounding environment relies on two types of specialized neurons in the retina: rod and cone photoreceptors. Rods are highly sensitive, requiring capture of only a single photon to initiate hyperpolarization of the photoreceptor , 2. Howein vitro at Ser21 and Ser36, respectively , as well as the Nrl−/− mouse , and found that GRK1 natively expressed in cones fails to undergo cAMP-dependent phosphorylation in both vertebrate species unlike GRK1 expressed in rods.In this report, we evaluate the effects of elevated cAMP on phosphorylation of GRK1 and GRK7 in cones as well as recovery of the cone photoresponse. We incubated WT and Grk-KO zebrafish larvae with forskolin (FSK), a potent activator of adenylyl cyclase that has been observed to increase intracellular cAMP in vertebrate photoreceptors , 32, andin vitro at Ser21 and Ser36, respectively . This antibody also recognizes phosphorylated Grk1 in retinal homogenates from dark-adapted adult zebrafish (B). Additionally, the anti-phosGrk1 antibody generated against zebrafish peptides was capable of detecting endogenous phosphorylated GRK1 in dark-adapted WT mouse retina but not in GRK1-S21A mice, in which the cAMP-dependent phosphorylation site is mutated from a serine to an alanine .The antibody against Grk7 was reported to recognize both paralogs of Grk7 in zebrafish, while the antibody against phosphorylated Grk7 showed cAMP-dependent phosphorylation of GRK7 at Ser36 (Ser33 in zebrafish) analysis , 29. Sinanalysis C.ex vivo display an increase in Grk7 phosphorylation  (A). We observed a 6-fold increase in Grk1 phosphorylation in light-adapted larvae treated with forskolin compared to those treated with vehicle as well as a phosphorylation-dependent electrophoretic mobility shift in Grk1 immunostaining in these larvae under these conditions. Immunoblot analysis and quantification also show a significant increase in Grk7 phosphorylation in larvae exposed to forskolin compared to vehicle regardless of background illumination (B). A significant increase in Grk7 phosphorylation was observed in forskolin-treated larvae under dark-adapted conditions compared to light adapted, and Grk7 phosphorylation was elevated 10-fold in vehicle-treated larvae under dark-adapted conditions compared to light adapted. These data suggest that under conditions where intracellular cAMP is elevated in vivo, both retinal Grks undergo phosphorylation.Recombinant human GRK1 and GRK7 undergo phosphorylation in HEK-293 cells treated with forskolin . Similarrylation . To detelfoxide) A. We obsmination B. A sign2 . To compare cone sensitivity in WT larvae treated with either vehicle or forskolin, responses were normalized (μV/μVMax) and fit with the Naka–Rushton equation . No significant differences in peak amplitudes or normalized sensitivity were observed between vehicle- or forskolin-treated WT larvae.To understand the effects of increased intracellular cAMP on the cone photoresponse, electroretinogram (ERG) recordings were performed on larvae incubated with forskolin. In larval zebrafish at 5 dpf, cones are electrophysiologically functional while nascent rods are not. The native photopic response at 5 dpf consists of a small a-wave elicited by the cone photoreceptors that is occluded by the larger b-wave response of the inner retina. Isolation of the cone mass receptor potential for recording was achieved through coincubation of larvae with 2-amino-4-phosphonobutyric acid (APB), an mGluR6 agonist, that blocks neurotransmission to ON bipolar cells and eliminates the b-wave , 34. Res2, 20 ms), followed by a probe flash of the same intensity at varying ISIs. Recovery was measured as the ratio of the cone mass receptor potential of the probe flash to the initial conditioning flash. Representative recovery waveforms using an ISI of 3 s are shown in Figure 3B (top panels). A time course of the cone mass receptor potential recovery shows a statistically significant delay in recovery for forskolin-treated larvae compared to vehicle-treated larvae  = 115.6; p < 0.0001) . This indicates that increasing levels of intracellular cAMP via incubation of larvae with forskolin leads to impaired recovery of the cone photoresponse.To determine if exposure to forskolin affects the cone photoresponse recovery, ERG responses to successive stimuli with variable interstimulus intervals (ISI) were analyzed. Vehicle- or forskolin-treated WT larvae were subjected to a conditioning flash of saturating white light  = 17.82; p <0.0001) (A). While the extent of the delayed recovery associated with forskolin treatment appears smaller in grk1b−/− larvae compared to WT (B), it should be noted that deletion of either Grk1b or Grk7a resulted in a significant delay in recovery of the cone photoresponse absent any pharmaceutical intervention  = 2.363; p = 0.1252) (B). These data suggest that the delay in cone recovery brought about by increased levels of cAMP is mediated by Grk7a rather than Grk1b in zebrafish larvae.We previously reported that both Grk1b and Grk7a contribute to the recovery of the cone photoresponse based on observations made using zebrafish lines in which either of the cone-specific Grk paralogs, Grk1b or Grk7a, was deleted through the use of transcription activator-like effector nucleases . To dete<0.0001) A. While ed to WT B, it shorvention . However 0.1252) B. These in vivo in response to cAMP led us to re-examine the nature of Grk1 phosphorylation in rods and cones. In order to determine whether cone-specific Grk1 is phosphorylated in response to changes in cAMP in vivo, the immunoblot analysis was modified to distinguish the Grk1 paralogs. Using the previously characterized grk1b−/− fish along with a longer SDS-PAGE running period (A and B) or a lower percentage acrylamide gel (C) allows for specific identification of immunoblot bands corresponding to each zebrafish Grk1 paralog (A). The lower band corresponding to Grk1b is undetectable in grk1b−/− fish and the ratio of Grk1a to Grk1b expression is higher in adult zebrafish (B) than in larval zebrafish (A), most likely because the rod to cone ratio is higher in zebrafish adult retinas  and adults (B), it was unclear if both Grk1 paralogs are phosphorylated in a cAMP-dependent manner or just rod-expressed Grk1a. To determine if the phosphorylated Grk1 band detected in fish retinal homogenates is Grk1a, a grk1a−/− zebrafish (17bpdel95aa) was generated using CRISPR as described in Experimental procedures. Immunoblot analysis demonstrates that phosphorylated Grk1 is undetectable in dark-adapted grk1a−/− fish compared to WT and grk1b−/− zebrafish larvae (C). When grk1b−/− and grk1a−/− larvae were treated with forskolin, grk1b−/− larvae exhibited a significant increase in Grk1 phosphorylation under both light-adapted and dark-adapted conditions (A), whereas no significant Grk1 phosphorylation is detected in forskolin-treated grk1a−/− larvae (B). Together, these data suggest that only rod-expressed Grk1a undergoes cAMP-dependent phosphorylation in zebrafish.Since the antibody against phosphorylated GRK1 does not discriminate between the Grk1a and Grk1b paralogs and phosphorylated Grk1 (white arrowheads) is detectable in dark-adapted − larvae A and adud adults B, it wash larvae C. When gnditions A, wherea− larvae B. TogethNrl−/− mice. Mice deficient for the Nrl transcription factor possess retinas lacking rods and instead consist mostly of modified S-cones  zebrafish expressing a dominant negative RI⍺ regulatory subunit (RI⍺B) that inhibits PKA activation and is driven by the cone transducin promotor (T⍺CP) were generated via tol2 transgenesis (A). To examine if the expression of RI⍺B in zebrafish cones can block the induction of Grk7a phosphorylation by forskolin under light-adapted conditions, WT and Tg(gnat2:prkar1aG323D) larvae were incubated in vehicle or forskolin for 30 min under light-adapted conditions. Compared to WT larvae, Tg(gnat2:prkar1aG323D) larvae treated with forskolin exhibited significantly lower levels of Grk7 phosphorylation (B). These data suggest that PKA is a critical effector in the phosphorylation of Grk7 in response to increased levels of cAMP in cones.While both Grk1 and Grk7 readily undergo phosphorylation by PKA⍺ channels , 39. To sgenesis . After lsgenesis A. To exarylation B. These in vivo phosphorylation of the photoreceptor GRKs in vertebrates, as well as the roles they may play in modulating cone phototransduction in response to dynamic intracellular cAMP levels. We chose zebrafish as our principal model, not only because they are an established model for the study of cone photoreceptor kinetics but also because they have a similar distribution profile of photoreceptor Grks as humans. In human retinas, Grk1 is expressed in both rods and cones while Grk7 expression is limited to cones  expression is deleted and that ex vivo treatment of frog retinas with forskolin is associated with an increase in Grk7 phosphorylation independent of light conditions  and which acts preferentially toward Grk1 rather than Grk7 (to account for our observations in zebrafish). Currently, the identity of any phosphatase that targets retinal GRKs is unknown. Previous work by our group showed that inactivation of the autophosphorylation sites important for kinase activity (K219 in GRK1 and K220 in GRK7) did not negatively affect phosphorylation at Ser21 by PKA in vitro or in cultured cells incubated with forskolin, suggesting that there are no obvious inhibitory phosphorylation sites on the retinal GRKs  against GRK1 for detection in zebrafish tissue  and for detection in mouse tissue , as well as secondary antibodies for immunoblot analysis  and immunocytochemistry  were purchased from ThermoFisher Scientific. IRDye800 goat antimouse IgG (#926-32210) secondary antibody and Intercept (PBS) blocking buffer (#927-70001) were purchased from Licor Biosciences. A mouse mAb against FLAG-tag #F1804) was purchased from MilliporeSigma. A rabbit polyclonal against phosphorylated GRK7 was generated by Bethyl Laboratories Inc . Rabbit 04 was pugrk1a were designed using CHOPCHOP (https://chopchop.cbu.uib.no/) and generated based on the methods in Hwang et al. (in vitro with T4 polynucleotide kinase (#M0201S) from New England Biolabs and ligated into pDR274 vector (#42250) from Addgene, which was previously linearized with BsaI . Following in vitro dephosphorylation using calf intestinal alkaline phosphatase , the construct was purified with a Qiaquick PCR purification Kit (#28104) from Qiagen. Following transformation into Dh5⍺ cells, colony selection, and screening by Sanger sequencing (Genewiz), plasmid clones containing the correct insert were linearized with DraI  and sgRNA was synthesized in vitro using a MAXIscript T7 Transcription Kit . Following cleanup of the sgRNAs using Micro Bio-Spin 6 columns  and quantification, 80 pg of sgRNA was coinjected with 200 pg of Cas9 protein  into 1-cell zebrafish embryos. Genomic DNA was prepared from uninjected and injected larvae at 5 dpf and the induction of indels was measured by high resolution melt analysis (HRMA). The sgRNA with the highest efficiency was 5′-GGACGTAGAGGAATACGACACGG-3’. Fish injected with this sgRNA were raised to sexual maturity (F0) and outcrossed to WT fish to create the F1 generation. F1 heterozygote larvae at 5 dpf were analyzed by HRMA to find an F0 parent with germline transmission and by Sanger sequencing to determine the sequence of the indel mutations. An F0 founder was identified that contained a 17 bp deletion in exon 1 of grk1a giving rise to an early termination codon at Tyr96 (pTyr96∗). Additional F1 progeny from this fish were raised to sexual maturity and fin-clipped to find individuals carrying the pTyr96∗ allele. Once identified, heterozygous pTyr96∗ F1 fish were in-crossed to produce F2 offspring. These F2 fish were raised to adulthood and fin-clipped for HRMA and Sanger sequencing to identify homozygous pTyr96∗ fish, which were used to continue the line, hereafter designated grk1a−/−.CRISPR single-guide RNAs (sgRNAs) targeting exon 1 of zebrafish g et al. . Sense aPrkar1a gene encoding the RI⍺ regulatory subunit of PKA in mice  from Promega, site-directed mutagenesis was performed as described in Liu & Naismith . Following confirmation of the RI⍺B G323D mutation by Sanger sequencing, the zebrafish RI⍺B fragment was cloned into the pME-MCS (#237) middle entry vector of the tol2kit (gnat2)  with NotI-linearized pCS2FA-transposase (#396) plasmid as a template. The T⍺CP-RI⍺B-polyA-pDestTol2CG2 plasmid (30 pg) and Tol2 transposase mRNA (25 pg) were coinjected into zebrafish embryos at the 1-cell stage. Live injected embryos were screened using fluorescence microscopy at 4 dpf to identify EGFP mosaic expression in cardiac muscle, as the pDestTol2CG2 destination vector also carries cmlc2:EGFP as a transgenesis marker. EGFP-positive larvae were raised to sexual maturity (F0) and outcrossed to WT fish to produce F1 zebrafish, which were similarly screened for heart EGFP expression to identify germline transmitting F0 founders. F1 zebrafish and subsequent generations that were EGFP-positive were used in these studies. Animals from this line are designated Tg(gnat2:prkar1aG323D).The dominant negative PKA RI⍺B mutant was originally generated by a G324D mutation in the  in mice , 72. ThiNaismith  using pr tol2kit . Simulta (gnat2)  was clong for 7 min. Protein concentration was determined using a DC Protein Assay (#5000112) from Bio-Rad. Following addition of bromphenol blue and β-mercaptoethanol , homogenates were analyzed by either 8% or 10% SDS-PAGE, followed by immunoblot analysis. Spectra Multicolor Broad Range Protein Ladder  was loaded in a lane of all gels/blots as a molecular weight marker. Nitrocellulose membranes were blocked in Intercept (PBS) Blocking Buffer  and incubated overnight at 4 °C in Intercept buffer with 0.1% Tween 20 (v/v) and primary antibodies. Blots were washed four times for 5 min each in 1× PBS containing 0.1% Tween 20, then incubated in Intercept buffer containing 0.1% Tween 20 and secondary antibodies for 1 h at room temperature. Following four additional washes in 1× PBS containing 0.1% Tween 20 for 5 min each and one wash in 1× PBS for 5 min, blots were analyzed using the Odyssey Infrared Imaging System (Licor Biosciences). All primary antibodies were used at a dilution of 1:10,000 except for anti-phosGrk7 (1:2000), and all secondary antibodies were used at a dilution of 1:15,000. For experiments requiring the use of two antibodies derived from the same host species, primary antibodies were directly conjugated to fluorophores using Mix-n-Stain CF Dye Antibody Labeling Kits (Biotium) according to the manufacturer’s instructions.Unless noted, all tissue samples for immunoblot analysis were collected in Hepes-Ringer buffer containing 10 mM Hepes, pH 7.5, 120 mM sodium chloride, 0.5 mM potassium chloride, 0.2 mM calcium chloride, 0.2 mM magnesium chloride, 0.1 mM EDTA, 10 mM glucose, and 1 mM DTT . Hot 95. SamplesTo determine levels of phosphorylation of retinal GRKs, tissue preparations varied by species and age. For zebrafish adults, both retinas were harvested and placed in 100 μl of Hepes-Ringer buffer to which 150 μl of hot (95 °C) 2× Laemmli buffer was added, then processed as described above. Following protein quantification, 25 μg of total protein per adult zebrafish retina sample was run in each lane. For 5 dpf zebrafish larvae, whole intact eyes from 20 larvae were harvested and placed in 16 μl of ice-cold Hepes-Ringer buffer to which 24 μl of hot (95 °C) 2× Laemmli buffer was added, then processed as described above. An entire zebrafish larval eye sample (40 eyes) was loaded in each lane.g for 30 min at 4 °C. FLAG-tagged zebrafish Grk1a or Grk1b was isolated using anti-FLAG M2 affinity gel (#A2220) from Millipore-Sigma as described previously  or λ phosphatase (P0753S) from New England Biolabs. Reactions were stopped by the addition of 2× Laemmli buffer to a final concentration of 1.2×, then subjected to immunoblot analysis.To determine the immunoreactivity of the novel anti-phosphoGrk1 antibody, recombinant FLAG-tagged zebrafish Grk1a and Grk1b were each inserted into the pFLAG-CMV2 vector . HEK-293 cells cultured in DME/F-12 with 10% fetal bovine serum were transfected with either of these constructs using FuGENE 6 transfection reagent (#E2691) from Promega. Approximately 72 h after transfection, cells were harvested, frozen once, then homogenized in Tris-buffered saline containing 0.5% n-dodecylmaltoside and cOmplete Protease Inhibitor Cocktail , followed by centrifugation at 40,000eviously . In vitr2. Recovery to successive stimuli was determined using a dual flash paradigm that utilized two 20-ms flashes of white LED light with an intensity of 1000 cd/m2 separated by variable ISIs of darkness.ERG analysis was performed as described previously , 78, 79.Tg(gnat2:prkar1aG323D) transgenic larvae, animals were dark adapted overnight, light adapted under white light (1200 lux) for at least 2 h, then dark adapted for 15 min prior to whole eye collection under oblique dim red or infrared light. The samples were then processed for immunoblot analysis as described.Animals indicated as ‘dark-adapted’ were dark adapted overnight for a minimum of 16 h. All dissections and sample preparations were performed under oblique dim red or infrared light until after the sample was homogenized and heated as described. Animals indicated as ‘light-adapted’ were dark adapted overnight, then placed under white light of 1200 lux for at least 2 h. In the experiment involving the For immunoblot analysis, larvae at 5 dpf were incubated in system water for 30 min with either forskolin (50 μM) or vehicle dimethyl sulfoxide (DMSO) . For ERG analysis, larvae were incubated in forskolin or vehicle for 25 min, followed by a 5 min coincubation with APB (500 μM).t test. For all analyses, a p-value of 0.05 was considered significant.For single flash ERGs, normalized peak amplitude responses were fit using the Naka Rushton function with GraphPad Prism (GraphPad Software Inc) . For duaAll data are contained within the article.This article contains supporting information.The authors declare that they have no conflicts of interest with the contents of this article.


There were six (2.4%) unaccepted screws, which were immediately revised during the same operation. There was no neurological injury noted in our patients. Conclusion: With the use of iCT-navigation, the rate of accepted screws at the revision site was found to be comparable to that at the virgin site. We concluded that iCT-navigation could achieve high accuracy and safety for transpedicular screw placement in revision spinal surgery and allow for the immediate revision of unaccepted screws.Background: Intraoperative CT-guided navigation (iCT-navigation) has been reported to improve the accuracy and safety of transpedicular screw placement in primary spinal surgery. However, due to a disrupted bony anatomy and scarring tissue, revision spinal surgery can be challenging. The purpose of this study was to evaluate the accuracy and safety of iCT-navigation for screw placement at the virgin site versus the revision site in revision thoracolumbar spinal surgery. Method: In total, 254 screws were inserted in 27 revision surgeries, in which 114 (44.9%) screws were inserted at the site with previous laminectomy or posterolateral fusion (the revision site), 64 (25.2%) were inserted at the virgin site, and 76 (29.9%) were inserted to replace the pre-existing screws. CT scans were conducted for each patient after all screws were inserted to intraoperatively confirm the screw accuracy. Results: In total, 248 (97.6%) screws were considered accepted. The rate of accepted screws at the virgin site was 98.4% (63/64) versus 95.6% (109/114) at the revision site ( The transpedicular screw (TPS) system is one of the most widely used fixation devices in spinal surgery. It provides a three-column fixation  of the sSeveral advanced image-guided techniques have emerged in the past two decades, including three-dimensional (3D) fluoroscopy ,6, O-armWe enrolled 27 consecutive patients who underwent TPS placement assisted with intraoperative CT  integrated with navigation  for revision thoracolumbar spinal surgeries. The study was approved by the Institutional Review Board of the Chang Gung Memorial Hospital.There were 27 patients (13 women and 14 men) in the present study, with an average age of 67 years (range 47–92 years). A total of 254 TPSs was inserted during the revision spinal surgeries, which were categorized into 3 groups: “revision site” screws (no. = 114) inserted over a previous laminectomy or posterolateral fusion field; “virgin site” screws (64) inserted at a site away from previous laminectomy or posterolateral fusion for the extension of posterior instrumentation; and “exchanged” screws (76), inserted to replace pre-existing screws with screws of a different style or brand through the previous trajectories.After general anesthesia, the patient was positioned prone on a Wilson radiolucent frame  on a Jackson table. A midline incision with paraspinal muscle stripping was performed to expose the previous operation site and extended to the virgin level if necessary. The pre-existing screws were exchanged for screws of a different brand, if necessary, which were inserted manually through previous trajectories without using iCT-navigation. Thereafter, the superior articular processes of the facets through which we planned to insert new TPSs were exposed. The reference array was then securely fixed at a vertebra distal to the expected lowest instrumented vertebra  by tightly clamping its spinal process.The operative field was scanned with the intraoperative CT, and the image data were transferred to the navigation system. A straight drilling guide was then registered to guide a 2.7 mm in diameter drill bit to create the optimal track for the TPS under iCT-navigation by checking the axial, sagittal and coronal views . A TPS wIntraoperatively, with the aid of the confirmatory CT scan, we evaluated the TPS accuracy and divided the results into 2 conditions: “accepted” and “unaccepted”. An accepted screw was defined as having a medial pedicle breach of ≤3 mm or a lateral pedicle or body wall breach of ≤3 mm. The position of the TPS was considered biomechanically optimal and safe from neurologic injury. An unaccepted screw was defined as having a medial pedicle breach of >3 mm or a lateral pedicle or body wall breach of >3 mm ; the TPSThe accuracy of the TPS placement was assessed with the first confirmatory CT. The rates of accepted screws and unaccepted screws were calculated for comparison between the virgin site and the revision site. Perioperative medical records were collected retrospectively, and postoperative neurological complications were evaluated.p < 0.05.Descriptive statistics were reported as percentages to describe the rates of accepted screws and unaccepted screws. Fisher’s exact test was used to compare the rates of accepted and unaccepted screws between the virgin site and the revision site. The test was considered significant if In total, 27 patients were categorized using the etiology for revision spine surgery: 12 patients (44.4%) were diagnosed with postlaminectomy instability with or without posterolateral fusion; 9 (33.3%) patients had adjacent segmental disease with spinal stenosis or instability; 4 (14.8%) patients had malpositions of a previous TPS with clinical neurologic symptoms; and the remaining 2 (7.4%) patients had a refractory postoperative infection .p = 0.422).Of the total 254 TPSs, 248 screw placements were considered accepted (97.6%) by the first confirmatory CT, including all of the “exchanged screws” (N = 76). The rates of accepted screws were comparable both at the virgin site  and the revision site . There was no significant difference between both groups (There were six screws (2.4%) considered unaccepted: one (1.6%) at the virgin site with a medial pedicle breach; and five (4.4%) at the revision site, including two with a medial pedicle breach and three with a lateral pedicle or body wall breach. The rates of unaccepted screws trended higher at the revision site than at the virgin site, but were not significantly different. All the unaccepted screws were revised immediately with the assistance of iCT-navigation, and, then, each proved to be accepted with a secondary confirmatory CT. No postoperative neurologic deterioration was observed related to the screw placement.p = 0.422). There was one (1.6%) unaccepted screw noted at the virgin site, and five (4.4%) at the revision site. The rates of unaccepted screws trended higher at the revision site than at the virgin site, but were not significantly different. We assumed that some of the TPSs in revision spinal surgery could be inserted at the virgin site for the extension of posterior instrumentation instead of right over the revision site. Thus, the accuracy of TPS placement at the revision site could be different from or inferior to that at the virgin site across the whole revision spinal surgery.With the use of iCT-navigation for revision spinal surgery, the rate of accepted screws was 97.6%, which was comparable to other studies ,11 for rIn the current study, most of the unaccepted screws were noted at the revision site. We considered dense postoperative fibrotic tissue or scarring to be the principal causes resulting in screw malpositioning at the revision site. Once the navigation device bent around the dense scar during the process of TPS placement, the navigation had the potential to fail. The scar tissue caused excessive pressure on the instrument and usually resulted in the medial deviation of the screw trajectory. Furthermore, if the surgeon exerted excessive bending force on the instrument (the drill or awl) to apply soft tissue pressure, the instrument could skive off the pedicle and result in the lateral skidding of the trajectory. The skiving effect might explain why there were more unaccepted screws presenting with a lateral wall breach  at the revision site. We suggest that the skiving effect could be diminished with the adequate release of soft tissue tension, and by using a high-speed drill with a shard bit for drilling .There have been few studies focused on the feasibility or accuracy of TPS placement in revision spinal surgery. Kim et al.  reportedSince unaccepted screws may not always result in neurologic deficits, it might not be necessary to revise all of them intraoperatively. Laine et al.  reportedIn spinal surgery with posterior instrumentation, the free-hand technique combined with intraoperative fluoroscopy is commonly used to confirm the entry point and trajectory of the TPS. The reported incidence of misplaced screws ranged from 10.5% to 21% ,16,17,18In the last two decades, several advanced imaging techniques have emerged that are able to provide 3D images. Usually, 3D fluoroscopy is limited by a small “field-of-view” scan area and an inferior image quality, with a screw accuracy inferior to iCT . The O-aCertain navigation techniques enable the diminution of CT scans for guiding TPS placement, while providing virtual but real-time images to guide TPS placement. The iCT-navigation technique is an effective method for TPS placement in complex spinal surgery for patients with spinal deformities  and unstThere were several limitations in our study. It might be relatively expensive to set up the iCT-navigation system; however, the installation charge should be considered against the cost of reoperation for patients with misplaced screws. We should have measured the dose of radiation to which patients were exposed, which might have been relatively high, since the entire workflow of iCT-navigation surgery contained at least two CT scans, including the registration scan and confirmatory scan. According to our previous studies  for primThe use of iCT-navigation for TPS placement in revision thoracolumbar spinal surgery was a useful technique for improving the accuracy and safety of TPS placement and allowed for the immediate intraoperative revision of unaccepted screws. The rate of accepted screws at the revision site was comparable to that at the virgin site in revision spinal surgery, although the rate of unaccepted screws at the revision site trended higher than at the virgin site. No postoperative neurological injury was noted in our patients.


The purpose of this research was to explore the impacts of different homogenization pressures, pasteurization conditions, and process sequence on the physical and chemical properties of soybean oil body (SOB)-substituted low-fat ice cream as well as the storage stability of SOB-substituted ice cream under these process parameters. With the increase of homogenization pressure (10–30 MPa), the increase of pasteurization temperature (65 °C for 30 min–85 °C for 15 min), and the addition of SOB before homogenization, the overrun and apparent viscosity of ice cream increased significantly, and the particle size, hardness, and melting rate decreased significantly. Thus, frozen dairy products of desired quality and condition could be obtained by optimizing process parameters. In addition, the SOB ice cream showed better storage stability, which was reflected in lower melting rate and hardness and more stable microstructure compared with the full-milk-fat ice cream. This study opened up new ideas for the application of SOB and the development of nutritious and healthy ice cream. Meanwhile, this research supplied a conceptual basis for the processing and quality optimization of SOB ice cream. Ice cream is frozen foam formed by infusing air during the freezing process . MeanwhiSoybean oil body (SOB) is an important organelle for storing lipids in soybean. It is a natural oil-in-water emulsion and remains stable under environmental stress, so SOB can be used in a variety of foods  preparation . SOB is In a previous study, the low-fat ice cream with SOB substitute for 40% cream had better melting properties, texture, sensory scores, and digestive properties compared to full-cream ice cream . Among tIn this research, the SOB substitute for cream to obtain the low-fat ice cream. The effects of different homogenization pressures, pasteurization conditions, and process sequences on the particle size and rheological properties of ice cream mixes as well as on the overrun, melting properties, and texture of ice cream were investigated. In addition, the changes of melting properties, hardness, color properties, and microstructure of SOB ice cream prepared by the optimal process during frozen storage were investigated. This study provides new ideas for the application of SOB and the development of nutritious and healthy ice cream. Meanwhile, this research also provides a theoretical foundation for the processing and quality optimization of SOB ice cream.w/w fat was obtained from Qingdao Nestle Co., Ltd. . Soybeans were provided by Soybean Institute of Northeast Agricultural University . Skimmed dried milk was obtained from Inner Mongolia Yili Industrial Group Co., Ltd. . All other reagents in this study were of analytical grade.Milk cream with 35% (w/v) for 12 h at a constant temperature of 4 °C. The swollen soybeans were ground in pre-cooled deionized water (4 °C) for 2 min by the tissue grinder. The homogenate was filtered through 4 layers of degreased gauze to collect raw soybean milk. The sucrose was added to the raw soybean milk to a final concentration of 20% , and mixed rapidly in the ice-water bath. The mixture was centrifuged  to collect SOB cream. The SOB cream was then resuspended in sucrose solution  and centrifuged . The above washing step was repeated three times, and the final wash medium did not contain sucrose to collect the SOB. The fat, protein, and moisture content in SOB were determined in previous studies as 25.39 ± 0.28%, 9.27 ± 0.19% and 56.41 ± 0.72%, respectively.The SOB was extracted referring to the report of Zhou et al. . SoybeanBased on the results of previous studies, low-fat ice cream had better physicochemical properties when SOB was substituted for 40% of milk fat . Thus, tWe first mixed 14 g of skim milk powder, 10 g of white sugar, 5 g of egg yolk, 7.2 g of cream, 4.8 g of SOB, and 59 g of water and filter. Among them, the ice cream prepared by replacing the SOB with cream was used as the control group . The mixed raw materials were homogenized, pasteurized, and aged (constant temperature of 4 °C for 12 h) to obtain the ice cream mixes. Then, the above mixes were frozen (constant temperature of −5 °C for 30 min) by an ice cream machine  and hardened  in the containers to obtain the ice cream samples by a low-temperature refrigerator . The fat, protein, and carbohydrate contents in SOB-substituted ice cream samples were determined in previous studies as 5.63 ± 0.02%, 5.92 ± 0.04%, and 18.48 ± 0.02%, respectively.When exploring the impact of homogenization pressure on SOB ice cream, the pasteurization condition was determined at 75 °C for 20 min, and the homogenization pressures were selected as 10 MPa, 15 MPa, 20 MPa, 25 MPa, and 30 MPa, respectively, by a high-pressure homogenizer .When exploring the effect of pasteurization conditions on SOB ice cream, the homogenization pressure was determined to be 15 MPa, and the pasteurization conditions were selected as 65 °C for 30 min, 75 °C for 20 min, and 85 °C for 15 min, respectively, by the Cherry-Burrell pasteurizer .When exploring the effect of process sequence on SOB ice cream, the homogenization pressure was determined to be 15 MPa, and the pasteurization condition was 75 °C for 20 min. The SOB added before homogenization and the SOB added after homogenization were named as process 1 and process 2, respectively.−1 and 4 °C, respectively.The determination of rheological properties was based on the method reported by Jiang et al. . The iceThe effect of different homogenization pressures, pasteurization conditions, and process sequences on the particle size distribution of ice cream mixes (4 °C) was determined based on the previously reported method ,19.1 and M2, respectively.The determination of overrun was based on the description of Wang et al. . The sam1. The mass of the melted ice cream was registered as W2.The determination of melting rate and the first dripping time was based on the method described by Kurt et al. . The iceThe texture profile analysis of SOB-substituted low-fat ice cream was determined by the back extrusion method . Low-fatTo assess consumer acceptance of SOB ice cream with different processing parameters, the appearance, ice crystals, smoothness, chewiness, and acceptability were evaluated. Ice cream samples were prepared before sensory testing, divided into individual test containers, and stored at −20 °C. Sensory scoring of ice cream samples was performed by 10 professionally trained sensory evaluation members, based on the nine-scale hedonic test.According to the results of the single-factor test, the SOB ice cream with the SOB added before homogenization, with a homogenization pressure of 20 MPa and a pasteurization condition of 85 °C for 15 min, was selected as the research object to further explore the effect of frozen storage on the SOB ice cream.SOB ice cream and full-milk-fat ice cream prepared according to the above parameters were stored at a constant temperature of −18 °C for 28 days. The ice creams were sampled weekly to determine melting properties, hardness, color properties, and microstructure during frozen storage.L*), red-green value (a*), and yellow-blue value (b*), by a colorimeter at the temperature of 4 °C .The determination of color properties of the ice cream samples (−18 °C) were according to the previously reported method , includiw/v) and 40 µL of Nile red  and reacted in the dark for 0.5 h. After that, 1.5 µL of the reaction solution was blocked on the glass slide. The microstructure of ice cream samples was observed by ultra-high distraction microscopy .All the samples were stained according to the previous method . A dilutn = 3) was used to express all date. One-way analysis of variance (ANOVA) was implemented through SPSS software (SPSS 25.0). p < 0.05 indicated significant difference with Duncan’s test.The mean ± standard deviation (p < 0.05) increase trend with increasing homogenization pressure (p < 0.05), thereby decreasing the speed of droplet motion, which was probably another reason for the increase in apparent viscosity  of the SOB ice cream mixes was no longer decreased, which was not significantly different from the D of the ice cream mixes at 20 and 25 MPa (p > 0.05). This might be because even if the homogenization pressure continued to increase, the fat globules that were too small would re-aggregate under the action of the emulsifier so that the average particle size of the ice cream mixes was no longer reduced. In addition, the polydispersity index (PDI) of the ice cream mixes also decreased significantly with increasing homogenization pressure, which indicated that particle distribution of the droplets was more uniform , and D of the ice cream mixes and a wider particle size distribution [The effect of process sequence on particle distribution of SOB ice cream mixes is presented in ribution .The smaller particle size and PDI meant that the ice cream mixes had better stability . Meanwhip < 0.05) (The overrun of SOB ice cream increased significantly with increasing homogenization pressure ( < 0.05) A. The ovp < 0.05), from (28.89 ± 0.37)% to (35.63 ± 0.49)% % B.p < 0.05), in comparison to ice cream with SOB added before homogenization   D. The mep < 0.05)  E. When tp < 0.05)  F. The fi2 = −0.963) [The reason for this might be that the homogenized fat globules were more likely to partially coalesce and form a continuous three-dimensional network during the freezing and whipping process of the ice cream mixes. It had been reported that partial aggregation and fat destabilization of fat globules during freezing are the ideal phenomena for maintaining the structural stability of ice cream and are negatively correlated with the melting rate (r −0.963) .Melting was defined by Kalicka et al. as the degree to which an ice cream sample resisted flow when force was applied in the mouth . Therefop < 0.05) (p < 0.05). Among them, the adhesiveness increased from (281.08 ± 7.04) g·s to (476.97 ± 3.79) g·s, and the chewiness increased from (279.94 ± 8.33) to (415.73 ± 2.19).The hardness of ice cream decreased significantly with increasing homogenization pressure ( < 0.05) . The harp < 0.05). The hardness of SOB ice cream decreased from (4218.87 ± 45.90) g to (3516.93 ± 75.84) g, the adhesiveness increased from (308.33 ± 4.09) g·s to (458.75 ± 7.43) g·s, and the chewiness increased from (337.84 ± 5.22) to (439.31 ± 9.18).In addition, with the increase of pasteurization temperature, the hardness of SOB ice cream significantly reduced, and the adhesiveness and chewiness of SOB ice cream significantly increased (p < 0.05), while the adhesiveness significantly decreased (p < 0.05), and conversely, the springiness of ice cream changed with no significant difference (p > 0.05).Furthermore, compared with ice cream with SOB added before homogenization, the ice cream with SOB added after homogenization significantly increased the hardness and chewiness  with the prolongation of storage time. The melting rates of SOB ice cream and full-milk-fat ice cream were (24.21 ± 0.65)% and (34.59 ± 0.94)% in the initial storage period and increased to (31.85 ± 0.88)% and (40.24 ± 0.53)%, respectively, after 28 days of frozen storage. However, there was no significant difference (p > 0.05) in the first drop time between SOB ice cream and full-milk-fat ice cream during storage  (The hardness of both ice creams increased significantly with prolonged storage time ( < 0.05) C. The haL* of ice cream was not significantly different (p > 0.05), but the a* and b* values increased significantly (p < 0.05). On the 28th day of storage, the a* values of SOB ice cream and full-milk-fat ice cream increased from 0.46 ± 0.03 and 0.61 ± 0.03 to 0.56 ± 0.02 and 0.68 ± 0.02, respectively, and the b* values increased from 20.82 ± 0.09 and 21.46 ± 0.15 to 22.36 ± 0.11 and 22.72 ± 0.11, respectively. In this study, the differences in color properties might be owing to changes in the air cells structure, protein, and fat globule states of ice cream during storage, which affected the light-reflecting properties of ice cream [The color properties of SOB ice cream with different storage times are shown in ce cream .The microstructure of fat and protein was collected using ultra high-resolution microscope (fat in green and protein in red). Compared with the initial stage of storage, the droplet size in both the SOB ice cream and the full-milk fat ice cream after 28 days of frozen storage increased significantly . Due to This study provides a new perspective on the effects of homogenization pressure, pasteurization conditions, and process sequence on the physicochemical properties of SOB ice cream. The study found that the homogenization pressure of 25 or 30 MPa, the pasteurization conditions of 85 °C for 15 min and SOB added before homogenization were the best conditions for SOB ice cream with the best acceptability. Meanwhile, based on the above processing parameters, the apparent viscosity, overrun, and melting properties of ice cream could be significantly improved, and the particle size and hardness of ice cream could be significantly decreased, which were crucial for improving the taste and quality of ice cream. In addition, contrasted with full-cream ice cream, SOB ice cream had better storage stability, which had positive significance for the transportation and sales of frozen dairy products. This research provides a theoretical foundation for the selection of process parameters for frozen dairy products. Meanwhile, this research provides new ideas for the utilization of SOB in food and the development of nutritious and healthy ice cream.