Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami  leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami. Spread of antibiotic resistance in bacteria is one of the most urgent issues in medicine, well-known and long used antibiotics are losing their efficacy over time. The most promising ways to overcome this problem are creating analogs based on existing antibiotics, searching for novel natural substances with antibacterial activity, as well as thorough study of antibacterials and the resistance mechanisms to them . AmicoumBacillus pumilus  lead to Ami resistance by the same mechanism. However, the antibiotic resistance due to amino acid substitutions in the domain IV of EF-G has not been described earlier. All found alterations G542V, G581A or ins544V in EF-G from E. coli, as well as G542V or G543S in EF-G from S. aureus (E. coli numbering) are located in the part of the factor that interacts with tRNA-mRNA complex on the ribosome , tRNAfMet(Prf20) from E. coli , fMet-tRNAfMet , Phe-tRNl, EF-Tu , yeast t E. coli , BPY-Metli  strain and purified using affinity chromatography according to the previously published procedure (N-terminus.The gene coding for an intact form (wt) or mutant variants  of EF-G from promoter . Intact rocedure . All pro4Cl, 30 mM KCl, and X mM MgCl2 (7 mM ≤ X ≤ 21 mM).All experiments were carried out in buffer TAKMx containing 50 mM Tris-HCl pH 7.5, 70 mM NHfMet, 2 μM IF1, 1 μM IF2, 1.5 μM IF3, 0.5 mM GTP, and the corresponding mRNA in buffer TAKM7 for 30 min at 37°C. Before 30S IC formation, 30S subunits were reactivated by incubation in buffer TAKM20 for 30 min at 37°C.30S initiation complex (IC) was formed by incubation of 1 μM reactivated 30S subunits with 0.5 μM BPY-Met-tRNA7 for 1 h at 37°C. Where necessary, ICs were purified by gel-filtration chromatography on a BioSuite 450 HR SEC column, 7.8 × 300 mm  in buffer TAKM7.70S IC was formed by incubation of 2 μM 70S ribosomes, 4 μM each of the initiation factors , 12 μM mRNA coding Met-Phe-…, 4 μM initiator tRNA, 1 mM GTP, and 1 mM DTT in buffer TAKMPhe was formed by incubation of 24 μM EF-Tu, 1 mM GTP, 1 mM DTT, 0.5 mg/ml pyruvate kinase , 3 mM phosphoenolpyruvate in buffer TAKM7 for 15 min at 37°C, then 12 μM Phe-tRNAPhe was added and mixture was incubated for another 5 min at 37°C. Phe-tRNAPhe was purified by HPLC and stored at −80°C, whereas Phe-tRNAPhe(Prf16/17) was prepared immediately before ternary complex formation. For this, 8 μM tRNAPhe(Prf16/17) was incubated with 0.2 mM Phe, 3 mM ATP, 6 μM 2-mercaptoethanol, 40 nM phenylalanine-tRNA synthetase, 40 nM tRNA nucleotidyltransferase in buffer TAKM7 for 30 min at 37°C.Ternary complex EF-Tu⋅GTP⋅Phe-tRNA2+ ions was increased to 21 mM, and the mixture of pretranslocation complexes was layered on 500 μl of the 1.1 M sucrose cushion (prepared in buffer TAKM21), followed by centrifugation in a SW55 rotor  at 55,000 rpm for 3 h at 4°C. The formed precipitate was dissolved in buffer TAKM21, shock frozen in liquid N2 and stored at −80°C.Pretranslocation complexes were formed by mixing 70S initiation and ternary complexes with subsequent incubation for 1 min at 25°C. For purification, concentration of MgfMet (initiator tRNA labelled with Bodipy FL at methionine moiety) and increasing concentrations of corresponding mRNA in buffer TAKM7 for 30 min at 37°C in the presence of 60 μM amicoumacin A, 30 μM edeine, 60 μM kasugamycin or without antibiotic. Detection of fluorescence changes was carried out on a Monolith NT.115 instrument , using standard capillaries . A green filter was applied, the power of monochromatic LED was 30%, and the intensity of IR laser was 40%. Each experiment was done in three replicates.To determine the affinity of mRNA to 30S IC microscale thermophoresis method (MST) was used . 30S IC 7, with 1 mM GTP and 1 mM DTT at 20°C (binding of aminoacyl-tRNA to the A site) (kapp), amplitude (A), and final signal amplitude (F0) according to equation F = F0 + A × exp(−kapp × t) where F is the fluorescence at time t. Where necessary, two exponential terms were used with two characteristic time constants , amplitudes , according to equation F = F0 + A1 × exp(−kapp1 × t) + A2 × exp(−kapp2 × t). A hyperbolic function was used to analyze the concentration dependence of the translocation apparent rate constants from the concentration of elongation factor EF-G. Calculations were performed using Prism 6.02 software . Standard deviations were calculated using the same software.Rapid kinetics were measured using a SX-20 stopped-flow apparatus . Proflavin fluorescence was excited at 460 nm, Bodipy FL fluorescence was excited at 470 nm and measured after passing a cut-off filter KV490 nm  in both cases. Light scattering experiments were carried out at 430 nm, followed by detection at 90° angle, without cut-off filter. Experiments were performed in buffer TAKM A site) , 25°C (f A site)  or 37°C  A site) . Where nfMet and 50S subunit joining at the 70S IC formation, 0.1 μM 30S IC programmed with an appropriate mRNA were rapidly mixed with 0.3 μM 50S subunits .To study accommodation of initiator BPY-Met-tRNAfMet (Prf20) (initiator tRNA labelled with proflavine at position 20 in the elbow region of tRNA) was rapidly mixed with ternary complex. fMet-tRNAfMet(Prf20) was obtained immediately before the IC formation, as described previously ] (Phe labelled with Bodipy FL at methionine moiety (BPY-Met-Phe-tRNAPhe) ] . To monitRNAPhe) . PretranfMet at the P site were mixed with equal volume of 1 μM ternary complex EF-Tu⋅GTP⋅[14C]Phe-tRNAPhe and incubated in buffer TAKM7, for 1 min at 25°C. Then samples were quenched with 1/10 volume of 5 M KOH and hydrolyzed for 30 min at 37°C. Samples were neutralized with 1/10 volume of glacial acetic acid and analyzed by reversed-phase HPLC. Percentage of synthesized dipeptide was determined by incorporation of the radioactive label, as described earlier  with EF-G wt or EF-G G542V (5 μM) was added to pretranslocation complexes (0.4 μM) in a quench-flow instrument KintTek RQF-3 . The experiments were performed in buffer TAKM7 with addition of 1 mM DTT and 1 mM GTP at 20°C. The reaction was stopped at certain time points by addition of 0.8 M KOH, and subsequent procedures for measurement of synthesized tripeptide fMet-[14C]Val-Phe were performed as described above for dipeptide formation analysis. The reaction rate constants were calculated using one or two exponential equations.To analyze formation of tripeptide fMet-Val-Phe, pretranslocation complexes, containing deacylated tRNAFor the analysis of dipeptide formation, ICs were formed in the presence of 30 μM amicoumacin A or 2 μM madumycin II; for the analysis of tripeptide formation, purified pretranslocation complexes were preliminarily incubated with 30 μM amicoumacin A for 10 min at 25°C, and then mixed with other reaction components.fMet at the P site and fMet-[14C]Val-tRNAVal or fMet-[14C]Phe-tRNAPhe at the A site, were purified by gel-filtration chromatography, and 3 nM wt or mutant form of EF-G was added. The reaction was carried out in buffer TAKM14 at 25°C. Where necessary, 30 μM amicoumacin A was added. For fMet-[14C]Val-Pmn or fMet-[14C]Phe-Pmn formation, posttranslocation complexes were mixed with 1 mM Pmn for 10 s at 37°C. The reaction was quenched by 1.5 M sodium acetate saturated with MgSO4. fMet-[14C]Val-Pmn or fMet-[14C]Phe-Pmn was extracted with ethyl acetate and quantified by radioactivity counting.The reaction was performed according to previously published method . For expfMet at the P site and fMet-[14C]Val-tRNAVal at the A site were purified by centrifugation through a sucrose cushion. To induce the dissociation of fMet-[14C]Val-tRNAVal from the A site, the Mg2+ concentration in buffer TAKM was adjusted to 7, 8.5, 10, 15, or 20 mM, and the amount of pept-tRNA bound to the A site at different incubation times at 37°C (with or without 30 μM amicoumacin A) was determined by nitrocellulose filtration. Dissociation and association elemental rate constants, koff and kon, were calculated from time courses of dissociation by numerical integration  of binding was calculated from the Kd values according to the equation ΔG0 = -RTln(Kd).Experiments and calculations were carried out according to previously published method , namely:egration . The folTm, °C), temperature at which half of a protein was unfolded, was determined by the fluorescence intensity measurements of aromatic amino acid residues at 330, 350, and 350/330 nm ratio. Proteins were diluted to a final concentration of 5 μM in buffer TAKM7 in the presence of 1 mM GTP. For the reaction, 10 μl of the sample was taken using standard capillaries . The melting experiment was performed with a step of 0.1°C/min in the temperature range 15–95°C, the laser intensity was 30%.To detect conformational stability and identity of intact and mutant forms of EF-G nanoDSF method of Prometheus NT.48  was used. The melting point  assembly of initiation factors, mRNA, and initiator fMet-tRNAfMet on the 30S subunit into a pre-IC complex (30S pre-IC), 2) conversion of the 30S pre-IC into a stable 30S IC upon recognition of the mRNA start codon by the fMet-tRNAfMet, 3) joining of the 50S subunit to the 30S IC resulting in the 70S IC formation  = 18 ± 9 nM; Kd(with Ami) = 169 ± 39 nM], whereas the amplitude was reduced only by 20% (fMet replaced by elongator Phe-tRNAPhe was shown to be impaired (Kd(UUC + Ami) = 584 ± 165 nM; Kd(AUG/Phe + Ami) = 940 ± 166 nM]  and kasugamycin (Ksg). Ede inhibits tRNA binding to the P site by preventing codon–anticodon interaction (Kd (Ede) = 568 ± 108 nM], implying that Ede not only violates codon-anticodon base pairing but also interferes with mRNA binding. Ksg distorts the mRNA path in the ribosome leading to hypothesis of preventing recognition of the start codon by the initiator tRNA (Kd (Ksg) = 59 ± 10 nM] were comparable with those obtained for canonical 30S IC   = 2.26 ± 0.02 s–1; kapp(Ami) = 0.148 ± 0.002 s–1]. As it was expected from the previous initiation experiments, in the presence of Ede no tRNA accommodation was detected, whereas Ksg demonstrated only moderate inhibiting effect with two-fold decrease of the signal amplitude without significant influence on the rate of the reaction [kapp(Ksg) = 1.70 ± 0.02 s–1]. The ICs containing mRNA with UUC start codon, regardless of the presence of Ami, demonstrated very low amplitude of fluorescence change  . Furtherormation . The decormation . Similare change . The sigTo summarize, three initiation inhibitors sharing overlapping binding sites on the ribosome perform differently. Ede significantly impairs formation of 30S IC, most probably, due to displacement of mRNA preventing codon–anticodon interaction in the P site. Ksg, in contrast, does not affect 30S initiation and only modestly violates 70S IC formation. As Ksg is a context-specific antibiotic, its mild inhibiting effect could be associated with mRNA sequence used in the study. In the presence of Ami, 30S IC programmed with the mRNA with the classical AUG start codon forms 70S IC, albeit at slower rates. At the same time, our data show that Ami enhances formation of erroneous 30S ICs with the UUC start codon; however, these complexes are not able to build 70S ICs, likely because of IF3 kinetically checking the transition to elongation .fMet and correct start codon (Kd(AUG) = 1,282 ± 58 nM, Kd(UUC) = 1,271 ± 210 nM], the titration curves amplitudes were rather large indicating that the complexes were formed (Kd(AUG + Ami) = 77 ± 18 nM, Kd(UUC + Ami) = 587 ± 42 nM], making the values comparable with those obtained for the 30S IC formed with all components of the translation initiation. Thus, Ami increases the mRNA affinity for 30S complexes lacking IF3 regardless of the initiation codon at the P site.As IF3 plays a particularly important role in the fidelity of selection of initiator tRNArt codon , we decie formed . The addkapp(AUG) = 25.6 ± 0.3 s–1; kapp(UUC) = 22.9 ± 0.8 s–1] with 30% drop of signal amplitude for UUC codon at the P site. The presence of the antibiotic did not substantially contribute to the rate or amplitude of the reaction . Thus, the mechanism of Ami inhibition during 70S IC formation appears to be IF3-dependent. The 30-fold rate reduction provoked by Ami (kapp(AUG) = 3.97 ± 0.02 s–1; kapp(UUC) = 3.68 ± 0.04 s–1; kapp(AUG + Ami) = 3.43 ± 0.02 s–1; kapp(UUC + Ami) = 3.63 ± 0.02 s–1]. As in the case of subunits joining, during tRNA accommodation complex with UUC start codon demonstrated lower signal amplitude that could be restored almost to maximum value (AUG case) by the addition of Ami  the speed of 70S IC formation, thereby significantly suppressing canonical initiation. At the same time, Ami promotes formation of incorrect 30S IC due to increase in the binding affinity of mRNA with an incorrect start codon. Although erroneous complexes are unable to progress along the path of translation initiation due to controlling action of IF3, formation of such complexes requires cellular resources that otherwise could be used for canonical initiation. Analysis of the system lacking IF3 supports a model where the mechanism of translation initiation inhibition is IF3-dependent. The absence of IF3 dramatically reduces the binding affinity of mRNA, however, the resulting complexes rapidly and effectively turn into the 70S IC. Erroneous complexes with an UUC start codon result in moderate decrease in amount of 70S IC formed, which could be readily rescued by addition of Ami, most probably due to an mRNA affinity gain. Thus, Ami increases the amount of incorrect 30S IC converted to the 70S IC that becomes possible in the absence of IF3. Our results highlight controlling function of IF3 as the complete translation initiation system does not allow formation of incorrect 70S IC regardless of Ami presence.Phe at the A site of the 70S IC was monitored by the fluorescence change of a fluorescent reporter group proflavine attached to dihydrouridine at position 20 located in the elbow region of initiator tRNA [fMet-tRNAfMet(Prf20)] (Phe in the process of its accommodation (kapp(without Ami) = 4.96 ± 0.05 s–1; kapp(Ami) = 4.31 ± 0.06 s–1]. On the contrary, there was essentially no fluorescence signal change upon application of well-known inhibitors of A-site reactions ] . The flumodation . The preeactions . Tetracyeactions . Since teactions , we canneactions . Kirromyeactions . Thus, p14C]Phe formed compared to free amino acid [14C]Phe. Ami-containing ribosomal complexes were as effective in the reaction of peptide bond formation as intact complexes with the amount of dipeptide fMet-[14C]Phe formed equal to 72 ± 7% and 73 ± 8%, respectively. Addition of specific inhibitor of peptide bond formation madumycin II  and dissociation constant (Kd) of peptidyl-tRNA binding at the A site on Mg2+ ions concentration (2+ (7 mM), the presence of the antibiotic did not change koff and kon values and had only moderate impact on the affinity of peptidyl-tRNA to the A site . At elevated Mg2+ concentration (up to 20 mM), the rate of the peptidyl-tRNA dissociation decreased three times, the rate of association decreased 17 times, and the affinity difference increased five-fold. The result indicates that Ami reduces the amount of Mg2+ ions required for dissociation and association, as well as for stabilization of peptidyl-tRNA at the A site. Nevertheless, Ami does not change the stability of peptidyl-tRNA binding at the A site at physiological conditions and does not affect the free energy (ΔG0) of binding calculated directly from the Kd values according to the equation ΔG0 = -RTln(Kd)   (Phe [fMet-Phe-tRNAPhe(Prf16/17)]  . To monif16/17)] .k = 42 s–1 for complexes containing reporter in the tRNA elbow region (Prf) and k = 22 s–1 for complexes containing reporter at the acceptor end of tRNA  and earlier .The kinetics of translocation was monitored by the fluorescence change upon addition of EF-G to fluorescently labelled pretranslocation complexes. Upon addition of saturating concentration of EF-G (4 μM) the displacement of peptidyl-tRNA occurred with the rate constant of  of tRNA . Intereseporters , that cok(without Ami) = 43.6 ± 0.4 s–1; k(Ami) = 22.3 ± 0.2 s–1; BPY: k(without Ami) = 21.7 ± 0.2 s–1; k(Ami) = 12.6 ± 0.1 s–1; in both cases amplitude of the fast phase A > 85%] . Even in this case the presence of the antibiotic diminished the rate of the peptidyl-tRNA displacement by three-fold both with labels at the elbow and acceptor end region (k (Vio) = 47.2 ± 0.6 s–1]  = 0.103 ± 0.010 min−1; k(Ami) = 0.020 ± 0.005 min−1], and reduced the amount of peptidyl-tRNA translocated from the A to the P site of the ribosome three times  that binds at the A site of the post-translocated ribosome, intercepts a nascent polypeptide from the peptidyl-tRNA, and dissociates . In fMet of EF-G . The preee times .Phe were added to the pretranslocation complexes containing deacylated tRNAfMet at the P site and fMet-[14C]Val-tRNAVal at the A site, followed by the sampling at certain time points. Tripeptide formation in the presence of the antibiotic revealed a two-phase curve in contrast to the one-phase curve obtained for intact complexes (k(without Ami) = 0.9 ± 0.1 s–1; k(Ami) = 0.9 ± 0.1 s–1. However, the fast phase reached only a half (35% of 75%) of the formed tripeptide amplitude curve, and the final level of the curve was lower even after extended incubation (the maximum amplitude was 61%). This indicates that a large portion of 70S complexes were impaired for translation.This effect could be associated with the change of EF-G engagement in the translocation or decrease of the ribosomal complex activity after a single round of translocation in the presence of Ami. To test this assumption, we performed the analysis of the tripeptide fMet-Val-Phe formation. EF-G and ternary complex EF-Tu⋅GTP⋅Phe-tRNAomplexes . Ami didAmi slows down movement of the peptidyl-tRNA from the A to the P site of the ribosome without influencing the conformational dynamics of tRNA displacement, as the rate of translocation monitored at different parts of the tRNA decreased by the same factor. However, the results of multiple turnover translocation and the tripeptide formation designate that Ami significantly decreases the portion of the ribosomes involved into the next elongation cycle.B (584–590 aa)] , providing Ami resistance, are located more than 16 Å away from the binding site of the antibiotic , being, 590 aa)] . These l590 aa)] . Thus, twt EF-G while variants of EF-G deviated less than 2°C  = 0.09 ± 0.02 min−1, k (EF-G G542V) = 0.10 ± 0.02 min−1, k (EF-G ins544V) = 0.09 ± 0.01 min−1, k (EF-G G581A) = 0.07 ± 0.01 min−1 at 3 nM EF-G (k = 4.1 ± 0.1 s–1) or ins544V (k = 5.2 ± 0.2 s–1) and 25 times with G542V (k = 2.1 ± 0.1 s–1). The ratio of translocation phases also changed with the amplitude of the fast phase decreased to 60%, increasing the contribution of the slow phase for all mutant forms of EF-G. Interestingly, modified variants of EF-G did not affect the displacement rate of the peptidyl-tRNA labelled at the acceptor end region , as well as the ratio of the translocation phases  = 1.65 ± 0.07 s–1; k(Ami) = 0.92 ± 0.02 s–1; BPY: k(without Ami) = 24.5 ± 0.2 s–1; k(Ami) = 8.9 ± 0.2 s–1]  = 0.099 ± 0.009 s–1; k(Ami) = 0.024 ± 0.009 s–1]. The same extent of inhibition by Ami was observed for intact EF-G. Interestingly, despite of equal rate reduction the amount of translocated peptidyl-tRNA was different. Modified variant of EF-G ensured maximal amplitude of the reaction, whereas the amount of translocated tRNA in the presence of native EF-G was reduced by a factor of three  = 0.73 ± 0.08 s–1; k(EF-G G542V) = 0.55 ± 0.06 s–1] , also known as loops I and II, which contact mRNA and the anticodon region of the peptidyl-tRNA at the P site of the posttranslocation complex and supposedly mRNA-tRNA duplex at the A site of the pretranslocation complex, accompanying it during translocation from the A to the P site of the ribosome  slows down the rate of peptidyl-tRNA movement at the elbow region without affecting translocation at the acceptor end of the tRNA  similarlAmi shares its binding site on the ribosome with several antibiotics, however, their mechanisms of protein synthesis inhibition are quite distinct. Edeine severely suppresses the translation initiation by interfering with the initiator tRNA binding to the P site . InhibitThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.EM, DV, and IO carried out the experiments. IO and PK provided materials. EM, DV, ON, and AP analyzed the data and created the pictures. EM, DV, and AK designed the experiments. OD, IO, PS, PM, AP, and AK conceived and curated the research and interpreted the results. EM, DV, AP, PM, and AK wrote the manuscript. All authors contributed to the article and approved the submitted version.AK is a founder of the company NanoTemper Technologies Rus , which provides services and devices based on MST and nanoDSF and represents NanoTemper Technologies GmbH (Germany). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using either bulk or fiber optics the profile of laser beams can be altered from Gaussian to top-hat or hollow beams allowing enhanced performance in applications like laser cooling, optical trapping, and fiber sensing. Here, we report a method based on multimode Plastic Optical Fibers (POF) long-tapers, to tweak the beam profile from near Gaussian to a hollow beam, by generating surface irregularities on the conical sections of the taper with a heat-and-pull technique. Furthermore, a cutback technique applied on long tapers expanded the output beam profile by more than twice the numerical aperture (NA) of the fiber. The enhanced sensitivity and detection efficiency of the extended profile was tested on a fiber optical ice sensor related to aviation safety. Shaping beam profiles from Gaussian to top-hat or hollow-beams has attracted a lot of attention due to the numerous applications in lithography, laser processing, fiber optic coupling, optical trapping, and medical research ,2. Beam We report the development and characterization of a multimode POF fiber taper, fabricated in a large diameter fiber with thin cladding using a heat-and-pull technique to form long bi-conical tapers, which are cut near the waste to produce two single tapers. Specifically, the adopted heating and pulling protocols alters the surface quality of the conical section transforming the Gaussian modal intensity to a hollow-beam with a much wider beam profile. In contrast with previous work reported in the literature ,10, whicg) near 25 °C to 135 °C. The heater was made of brass having dimension off 25 mm long and a diameter of 2.5 mm with a slot of 0.5 mm to allow free access of the fiber. The heating and elongation assembly were mounted vertically on a motorized linear translation stage, which executed linear or oscillatory motion to increase the heated volume. Two vertically mounted, computer controlled, linear stages pulled the fiber as shown schematically in We used PMMA-based POF fibers purchased from different manufacturers having OD of 250 μm and very thin cladding of 10 to 12 μm (core diameter of about 240 μm). Their refractive indices ranged from 1.45 to 1.47 with a NA of 0.35 to 0.47, transmitting in the visible at peak wavelengths ranging from 400 to 650 nm. The tapers were made by heating and pulling a small section of fiber, in a cylindrical temperature-stabilized electrical oven, to its transition glass temperature . A typical taper side-profile measurement is shown in With some elongation protocols the bi-conical tapers became progressively lossy, radiating light from the conical down-taper and the thin section of the fiber taper. The optical losses increased as the tapers became thinner and longer. Thus, we monitored the fabrication process, by measuring the light exiting from the side of the biconcave taper as shown in Bi-conical tapers were subsequently cleaved at the taper waste as shown in The fiber tapers were cleaved at the tip and clamped in the center of the goniometer as shown in g. The taper lengths ranged from 6 mm to 17 mm and formation of asymmetrical conical sections were observed when only one stage was allowed to move. As mentioned above, tapers drawn near Tg and at about 350 to 400 μm/min at temperatures of 125 to 135 °C, became lossy, radiating from the conical section  the initial tendency of the fiber to contract, due to memory processes in the PMMA which set the lower pulling limit and (ii) braking of the fiber when the pull rate exceeded the rate at which the fiber-core dropped below T section a. The inThe physical dimensions of the tapers were measured from the shadowgraphs using Image-J. The length of the conical sections was defined as the points were the diameters at the thin and thick sections were respectively, 10% and 90% of the final values, and ranged from 6 to 17 mm. The corresponding conical angles were from 1.0 ° to 2.5° and waists diameters from 30 to 90 μm with a typical profile shown in Under an optical microscope, the bi-conical fiber tapers exposed surface imperfections . In The output modal intensity profile from the tapers were measured using the goniometric set up, as described earlier in The “effective” diameter of hollow beams was measured at the full width half maximum (FWHM), with the maximum being defined relative to the average intensities of the twin peaks, as indicated in α, undergoing successive total internal reflections (TIR), which gradually reduce the reflected angle at the interface. Eventually the rays will exceed the critical angle and will be converted to cladding modes refracting out of the fiber taper as radiation modes [1, R2, ..., Rm, change as shown schematically in 1 as:To better understand the origin of the side losses it is necessary to model the expected exit angles from a long smooth MMF taper using the meridian ray optics model. In this first approximation model, the paraxial modes are not included (a) due to the large diameter of the fibers and (b) because the Gaussian modal intensity profile implies the light energy is concentrated in the center of the beam b. Therefon modes ,17,18. Iα ranging from 1° to 3°. This theoretical model ignores the thin cladding which eventually becomes even thinner by the drawing process shortening the optical paths. Therefore, the model predicts that when shaping a hollow beam, most of the light escapes from the sides of the smooth taper in angles 5° to 17° to the fiber axis while no light exits from the tip of the fiber , thus altering the ratio of light exiting the side and the cleaved end-facet of the taper, as shown in Using the experimental setup outlined in The normalized intensity variations as a function of angle for three distinct cutback lengths are depicted in One important application of the discussed beam expansion technique is in the development of a fiber optics ice detector. Ice accumulates on the leading edge of aerodynamic surfaces disrupting the normal airflow and occurs when aircrafts fly through cold moist air. These conditions are particularly dangerous when an aircraft flies at low altitudes near congested airports because lift decreases and drag increases ,20. The Previously, we have reported an alternative method for direct ice detection based on optical diffusion which measures, in real time, the thickness and type of ice accreted on the wings of aircraft using a fused-silica fiber optical sensor array . A schemIn principle, the method relies on illuminating and detecting the light from the accreting ice on the leading edge of the airfoil. Depending on the freezing rate of water, gases dissolved in the super-cooled droplets may or may not escape, forming micro-cracks and micro-bubbles in the ice volume. The density of these discontinuities varies, giving ice its optical characteristics, attributed primarily to Mie scattering .3. The optical intensity was recorded as a function ice thickness, measured by a shadowgraph technique using a perpendicular laser beam, which traced the front edge on the ice , defined as the ratio of ice that freezes directly on impact to that which remains liquid and freezes behind the impact area ,22. The In glazed ice (−5 to −10 °C), reflected light from the surface of the ice dominates and signals from the central fibers 3, 4 increase rapidly up to an ice thickness of 3 mm, and exhibit acute intensity spikes. The origin of these intensity spikes is primarily due to transient ice micro-crystals, formed on the surface of the thin transparent ice which can be detected by the inner fibers, being closest to the illumination fiber a. ConverAnother interesting point is that for a low NA fibers-array sensor in glazed ice, the backscattered light is near constant for a particular ice thickness, while the strong transient reflections, near the fiber facet will momentarily contribute a much higher percentage of the detectable power, thus distorting detectible signals. Conversely, if the acceptance angel is increased, light from a much wider “field of view” will be detected, so transient events will represent a lower percentage of the detectable signal, thus averaging out their influence on the signals. Similarly, if the accreting ice is illuminated by a wider beam profile, the overall near field intensity is reduced, thus also reducing the transient reflections. Therefore, this effect can be achieved by using high-NA, MM-POF tapers, both for illumination and detection, utilizing the cutback method as described above. ° to 140°. Typical results for large NA array sensor are shown in By retaining the same fiber-array sensor architecture shown in Similar intensity patens were obtained with the low NA, polished fiber optic array-sensor a,b and tWe have demonstrated the ability a POF taper to tweak the near Gaussian modal intensity profile of a cleaved fiber to a deep hollow beam profile with very low intensity dark spot in the central ID. Our experiments showed that the underlined mechanism of beam shaping was the generation of AS on the side of the taper which disrupted the TIR process on the guided modes, exiting the conical section by diffuse transmission from the side of the taper. Furthermore, by a suitable cutback technique the modal beam profile could be altered from a hollow-beam to near top-hat, and finally to an expanded Gaussian, by cutting back the tapers from 35% to 40% of their original length. This technique redistributes the ratio of the radiation to ending modes, which exit the sides of the taper and the flat polished fiber tip respectively, allowing the beam profile to expand almost 2.5 times the FWHM of the original fiber. Furthermore, we demonstrated the use of the modified POF tapers in ice sensing applications with improved signal-to-noise ratio in all aspects of the detection envelope and in particular in detecting glazed ice.


With the hypothesis that equine dorsal lamellar tissue can be desensitized by anesthesia injection into distal interphalangeal joint (DIPJ), the objective was to assess the mechanical nociceptive threshold of hoof dorsal lamellae following intra-articular (IA) administration of lidocaine into this joint.P < 0.05 considered statistically significant.The DIPJ of the forelimbs of six adult healthy horses were injected with either 5 mL of lidocaine, or 5 mL of lactated Ringer’s solution. Treatments were randomly distributed, with each forelimb undergoing a single treatment. The hooves were evaluated pre- and post-injection at pre-selected times over 4 h, using a pressure algometry model. Mechanical nociceptive thresholds (MNTs) were recorded for the sole , coronary band , heel bulbs , and dorsal lamellar region . The MNT means were compared over time using the Friedman test and between treatments using the Wilcoxon signed-rank test, with values of There were no differences between treatments for any region of the hoof during the evaluation period. However, MNT values indicating analgesia were recorded in the dorsal lamellar region in 50% of hooves following adminstration of lidocaine into the DIPJ.The administration of 5 mL of lidocaine into the DIPJ does not significantly increase the mechanical nociceptive threshold of the equine hoof. To date, diagnostic anesthesia remains the best method for localization of lameness pain in horses, although misinterpretation due unspecific analgesia may occur . The lacThe experiment was approved by the Ethics Committee on Animal Use of Universidade Federal de Minas Gerais (UFMG) . Six mixed-breed horses of varying sex and neuter status  were used in randomized blocks and repeated measurements over time design. The horses were 7–13 years of age , 142–164 cm tall , and weighed 350–460 kg . All horses were considered clinically healthy, determined via physical and orthopedic examination. A complete lameness examination was also performed on all horses to assure that no lameness was present prior to beginning the study .−1 intravenous (IV); Detomidin, Sintec) followed by intramuscular (IM) administration of tetanus antitoxin  and aseptic preparation of the hoof capsule. Two hoof wall defects were made in the center of the dorsal hoof wall of each fore foot, using a 10 mm drill bit mounted onto a drill . The first defect was located 2 cm distal to the coronary band and the second 4 cm distal to the coronary band. Hoof wall defects penetrated only the insensitive horn, stopping immediately before reaching the sensitive lamellae. Using a hoof knife, the sole of each hoof was prepared by removing the keratinized layer until reaching the sensitive tissue near the solar corium at three different points close to the palmarolateral, palmaromedial, and dorsal borders of the sole  and the other DIPJ receiving 5 mL of lactated Ringer’s solution (Samtec Biotechnology). The horses were restrained in stocks, and a nose twitch was applied. Complete aseptic preparation of the the injection site was performed prior to administering the treatment into each forelimb. A dorsolateral DIPJ injection technique was used, as previously described . The prePressure algometry was utilized to evaluate the MNT for each hoof. A portable dynamometer (Instrutemp 20kgf ITFG-5020), calibrated by the manufacturer for compression, was used to determined the force (kg) necessary to incite a response. Using a previously described technique , the dynTo avoid environmental distractions and visual perception of the algometer being applied, a blindfold was applied over the eyes of each horse during each evaluation. The basal MNT for each foot was determined prior to IA administration of each treatment. The minimum force required to stimulate withdrawal of the limb was recorded . Sites w−1; Ourofino) IV daily for 5 days.Preparation of the injectate was performed by an individual not blinded to the treatments. Joint injections and dynamometer measurements were performed by a single investigator blinded to the randomized treatments. Two additional evaluators, also blinded to the treatments administered, evaluated the horses response to the applied instrument pressure. The response was determined twice at each time in intervals of 2–4 s, and the average of the two readings was obtained. When divergence in a reading occurred between the evaluators, the value was disregarded and the procedure repeated. After the experimental period, all horses received phenylbutazone  over time or between treatment groups (2% lidocaine and lactated ringer’s solution) for any of the variables analyzed  in MNT within the dorsal lamellae, sole, coronary band, or heel bulbs. However, 3/6 horses in this study demonstrated partial desensitization in the dorsal lamellar region between 15 and 60 min post-injection. These results suggest that diffusion of lidocaine from the DIPJ to the innervation of the dorsal lamellae should be considered when interpreting the effects of a DIPJ block. For diagnostic purposes, it is recommended to use the minimum anesthetic volume necessary for effective desensitization (This study demonstrates that IA injection of the DIPJ with 5 mL of lidocaine does not result in statistical increases (tization . Moyer, tization . ConsequWhile some horses demonstrated anesthesia of the dorsal lamellae after administration of lidocaine into the DIPJ, there was no statistical difference between treatment groups. This could be explained by the variability in MNT responses in addition to a small sample size. Variability in MNT responses between individuals has been observed when evaluating back pain in horses . HoweverThe solar region of the hoof can become desensitized following administration of an anesthetic agent into the DIPJ . Using aWe also need to highlight that lidocaine was used in the present study and that most of the previous assays studying DIPJ anesthesia effects used mepivacaine, which could promote a different effect under the current set up/design. Another limitation of this study is the small number of horses and the lack of normal distribuition of the data. Under such circuntances, data was analysed by nonparametric tests that has lower power than do standard tests. By line of reason, a larger sample could indicate statistical difference between groups considering the MNT values of dorsal lamellae.Our findings suggest that the dorsal lamellae should be considered as a possible source of lameness pain following 2% lidocaine anesthetic block of the DIPJ in some horses. This information is potential relevant in face of increasing number of horses with subclinical signs of endocrinopathic laminitis reported nowadays . Further10.7717/peerj.9469/supp-1Data S1Click here for additional data file.


We study two-dimensional triangular-network models, which have degenerate ground states composed of straight or randomly-zigzagging stripes and thus sub-extensive residual entropy. We show that attraction is responsible for the inversion of the stable phase by changing the entropy of fluctuations around the ground-state configurations. By using a real-space shell-expansion method, we compute the exact expression of the entropy for harmonic interactions, while for repulsive harmonic interactions we obtain the entropy arising from a limited subset of the system by numerical integration. We compare these results with a three-dimensional triangular-network model, which shows the same attraction-mediated selection mechanism of the stable phase, and conclude that this effect is general with respect to the dimensionality of the system. Geometrically-constrained systems may show peculiar features compared to their unconstrained counterparts. In particular, geometric constraints can lead to frustration because the system cannot simultaneously minimize all local interaction energies, or free energies. Frustrated systems usually show a degenerate ground state and then they may posses a residual entropy. Frustration is relevant in physical and biological systems that range from water  and spinA prototypical geometrically-confined system is composed of short-range repulsive colloids confined in a slit pore of two plates. Varying density and plate separation, discontinuous phase transitions between layered, buckled, rhombic and adaptive prism crystal structures occur ,16,17. Iractions ,18, and ractions . Multipl spheres a or by a spheres b.N the number of particles in the system) [This ground-state degeneracy implies a subextensive residual entropy at  system)  so that  in Ref. . The ter in Ref. . In this in Ref. . The thrThis geometric mechanism underlying the ground state of the buckled colloidal system composed of straight or zigzagging stripes, has been realised, experimentally also by packing a granular system in a container under the effect of gravity ,23, and nitrides ). In theservoirs .For temperature slightly larger than zero, the degeneracy is removed by the order-by-disorder effect ,35,36,37tentials . By tunitentials . We estarepulsive harmonic potential, described in detail in the following section). This result suggests that the inversion of the stable phase by adding attraction to repulsively-interacting particles in triangular networks is a general mechanism, irrespective of the dimensionality of the system.In this paper we study a 2D isosceles triangular network which shows the same ground-state degeneracy as the Ising elastic antiferromagnet at c, and thus has a height K is the spring constant, which is assumed to be identical for all springs, l runs over three of the six neighbors each particle has to avoid double counting of bonds. The positions of the particles are described by the coordinates i and j, and its square is thus given byi and j, which can take the values c or The model we consider is composed of particles in a 2D triangular network linked with springs of two different lengths at rest such that every plaquette or triangle is formed by a longer edge α/2) see e. We wilr 2a see . Since wThe Hamiltonian of the straight-stripes configuration, with particle positions specified for the unit-cell in Using the relations m, and particle 1, which represents the particles with even m. Therefore, we set for particle 0, The Hamiltonian of the bent-stripes configuration, with particle positions specified for the unit-cell in cles see d: partics refers to straight and b to bent, and n belonging to the shells up to n particles are free to move, while the other n, For a harmonic interparticle potential, the Hamiltonian for straight and bent stripes configurations we expanded around mechanical equilibrium takes the quadratic form: onian is : Δs= see . The HamThe difference between the calculation of the harmonic and the repulsive harmonic partition function is that in the former case the functions Due to the symmetry of the straight-stripes configuration, thanks to which the reflection about the origin of each neighbor transforms it in another neighboring particle see , we can For the repulsive harmonic potential in the bent configuration we need to consider the azimuthal ranges coming from each one of the neighboring particles: I for that specific angle I for bent stripes (corresponding in In Equation . From Filues see b, that rWe studied a 2D triangular-network model composed of particles interacting through harmonic or repulsive harmonic springs. At olloidal  and Isinolloidal  antiferrBoth the Ising antiferromagnet on a deformable triangular lattice and the 2D isosceles triangular network model at  theorem . From th theorem , for whi theorem , for whi theorem . In ther


The aim of the present study was to evaluate the possible gut inhibitory role of the phosphodiesterase (PDE) inhibitor roflumilast. Increasing doses of roflumilast were tested against castor oil-induced diarrhea in mice, whereas the pharmacodynamics of the same effect was determined in isolated rabbit jejunum tissues. For in silico analysis, the identified PDE protein was docked with roflumilast and papaverine using the Autodock vina program from the PyRx virtual screening tool. Roflumilast protected against diarrhea significantly at 0.5 and 1.5 mg/kg doses, with 40% and 80% protection. Ex vivo findings from jejunum tissues show that roflumilast possesses an antispasmodic effect by inhibiting spontaneous contractions in a concentration-dependent manner. Roflumilast reversed carbachol -mediated and potassium -mediated contractile responses with comparable efficacies but different potencies. The observed potency against K+ was significantly higher in comparison to CCh, similar to verapamil. Experiments were extended to further confirm the inhibitory effect on Ca++ channels. Interestingly, roflumilast deflected Ca++ concentration–response curves (CRCs) to the right with suppression of the maximum peak at both tested doses (0.001-0.003 mg/mL), similar to verapamil. The PDE-inhibitory effect was authenticated when pre-incubation of jejunum tissues with roflumilast (0.03-0.1 mg/mL) produced a leftward deflection of isoprenaline-mediated inhibitory CRCs and increased the tissue level of cAMP, similar to papaverine. This idea was further strengthened by molecular docking studies, where roflumilast exhibited a better binding affinity  with the PDE protein than the standard papaverine . In conclusion, inhibition of Ca++ channels and the PDE-4 enzyme explains the pharmacodynamics of the gut inhibitory effect of roflumilast. Gastrointestinal (GI) motility plays an important role in the digestive and absorptive processes of the gut, essential for pushing intestinal material, mixing it with digestive juices, and preparing undigested foods for excretion. Irritable bowel syndrome (IBS) is a gastrointestinal disease related to intestinal motility problems and can be associated with pain, constipation, or diarrhea . DiarrheCurrently available drug treatment for diarrhea is non-specific, and generally, drugs are used to reduce the uneasiness and discomfort of recurrent bowel movements . AvailabThe cyclic nucleotides (cAMP and cGMP) act as intercellular second messengers by stimulating the role of many hormones, neurotransmitters, and inflammatory mediators ,12. ThesCurrently, the main focus of researchers is to target one specific PDE enzyme and find out various specific PDE enzyme inhibitors . The selRoflumilast, a known PDE4 inhibitor, has been known to reduce inflammatory reactions by activating cAMP/PKA pathways . In our Both orally administered doses of roflumilast showed dose-dependent protection of mice, whereas the saline group did not show any effect. At the lower tested dose of roflumilast (0.5 mg/kg), two out of five mice showed protection, thus showing 40% protection, while the higher dose of 1.5 mg/kg showed 80% protection. No diarrheal spot was observed in any cage of mice treated with loperamide (100% protection), as detailed in When tested against spontaneously contracting rabbit jejunum preparations, roflumilast caused dose-dependent (0.001–0.1 mg/mL) inhibition with a resultant EC50 value of 0.06 mg/mL , as shown in When tested for possible interaction with Ca++ channels, roflumilast was tested against high K+-induced contractions where it produced complete inhibition, similar to verapamil, with respective EC50 values of 0.002 mg/mL  and 0.1 µM , as shown in When tested against CCh-induced contractions, roflumilast produced dose-dependent (0.001-0.1 mg/mL) inhibition with a resultant EC50 value of 0.07 mg/mL , as shown in p < 0.01) and 142.71 ± 10.4 pmol/mL (p < 0.01) (p < 0.01) and 165.93 ± 6.80 pmol/mL (p < 0.01), at respective doses of 0.3 and 1µM  A. Papave and 1µM B. Furthermore, to understand the PDE-4 inhibition, roflumilast and papaverine were docked into the active pockets of PDE-4B and PDE-4D proteins having PDB ID 5WH5 and 5LAQ, respectively. Roflumilast exhibited better binding affinity  in comparison to papaverine  in both the isoforms, as shown in In view of the wide therapeutic potential of roflumilast, we previously reported studies showing its effectiveness in the treatment of inflammatory conditions and related diseases such as cardio and renal toxicities ,21. The After we observed the antidiarrheal response, our second objective was to determine the possible pharmacodynamics involved in this effect. For this purpose, the method of Bashir et al.  was follThe PDE inhibition concept was further proved concretely by the in silico molecular docking results. The molecular docking study helped us to gain an insight into the protein–ligand interaction in the active domain of PDE-4. PDE-4 is reported to have four subtypes  each with the potential to degrade cAMP, which is the second messenger to react to the inflammatory effect. Roflumilast is a selective and non-specific PDE4 inhibitor that is able inhibit all PDE-4 isoforms. Despite its pharmacological effects, roflumilast can cause side-effects, such as diarrhea, nausea and dizziness, due to a lack of specificity. As PDE-4B and PFDE-4D have been found to be more expressed and extensively studied, hence were selected for the present docking study. The standard drug papaverine exhibited two hydrogen bonding affinities, while roflumilast exhibited better binding affinity with two hydrogen bond and two hydrogen–halogen bonds. Halogen and hydrogen–halogen bonding has been considered more favorable, and this is why halogen-containing compounds are being further explored and believed to be dual agonists .The following analytical-grade chemicals, obtained from Sigma (USA), were used: carbamylcholine (CCh), loperamide, acetylcholine perchlorate (ACh), isoprenaline, verapamil, and papaverine. Castor oil was purchased from a local pharmacy. Rabbits (1-1.5 kg) procured from a local farm in Al-Kharj and Swiss albino mice (20-25 g) from the Animal Care Unit, College of Pharmacy, Prince Sattam bin Abdulaziz University, Saudi Arabia, were kept at optimum temperature (22 ± 1 °C), relative humidity 55% ± 5%, and equal exposure to light/dark cycle. All animals were provided with a standard pellet diet and water ad libitum. Before sacrifice, rabbits fasted for 24 h. Cervical dislocation was performed by a blow on the neck, and death was confirmed by the disappearance of ear reflexes. Due care was taken, and all experiments (in vivo and ex vivo) were performed by following the instructions detailed in NRC .Twenty mice were randomly allocated into four groups with equal numbers in each group. Following twenty-four-hour fasting, mice in the 1st group were exposed to oral gavage of saline (10 mL/kg) and were labeled as a negative control. After pilot screening for dose selection, the 2nd and 3rd groups (test groups) were exposed to two increasing dosages of 0.5 and 1.5 mg/kg of roflumilast, respectively. The last group (4th) of mice were given loperamide (10 mg/kg) and labeled as a positive control. Separate cages were assigned to each animal, with a blotting sheet on the floor of each cage for observation of the absence or presence of diarrhea by a blind observer. After an hour, all mice were orally exposed to castor oil (10 mL/kg) using a 1 mL syringe. After 4 h, all individual cages’ blotting sheets were inspected for typical diarrheal droppings. Protection was noted if no diarrheal drops were observed, as previously reported by Jebunnessa et al. (2009) . The method of Shah et al. (2011)  was follAfter observing the inhibitory effect of roflumilast on spontaneous contractions, experiments were further extended to determine the mechanism(s) of its antispasmodic effect. The inhibitory effect of roflumilast was tested against sustained spasms mediated by K+ (80 mM) and CCh (1 µM) for multiple pharmacodynamics including voltage-gated Ca++ channel blockade and inhibition of PDE. According to Godfraind et al. (1986) , K+  for Ca++ chelation. To further deplete the intracellular stores of Ca++, the method of Blattner et al. (1978) was followed . BrieflyAs roflumilast showed efficacy against CCh, which is an indication of PDE inhibition, indirect confirmation was carried out by dose-mediated inhibitory curves of isoprenaline against CCh in the absence and presence of roflumilast. Potentiation of curves to the left is indicative of a blockade of PDE, while papaverine, a standard PDE inhibitor, was used for comparison .Further confirmation of PDE inhibitory activity was done by biochemical method using cAMP enzyme immunoassay kit . Jejunal tissues, after contracting with CCh (1 µM), were exposed to relaxant doses of roflumilast and papaverine. Soon after the tissues reached to baseline, the tissues were snapped frozen in liquid nitrogen and were homogenized with 1 mL of sample diluent for every 100 mg of tissue, on ice, which was kept like this for 10 min. After centrifugation at ≥6000 g (4 °C for 15 min), the supernatant was collected and either analyzed immediately. Similar steps were repeated for tissues without administration of any drug and only contracted with CCh, which served as negative control. The cAMP content was expressed as picomole per mg protein.To gain an insight into a PDE protein and its interaction with roflumilast, co-crystallized ligand and papaverine, molecular docking studies were carried out on two isoforms, namely PDE-4B and PDE-4D, using the Autodock vina program from the PyRx virtual screening tool ,47. The 2) test. Statistical significance was considered only when p < 0.05. Graph Pad Prism (version 4) was used for the analysis of concentration–response curves (CRCs).Data are expressed as the mean ± standard error of the mean (SEM), where n is the number of experiments and EC50 represents the median effective concentration with 95% confidence interval (CI). Protection from diarrhea was statistically evaluated by comparing all groups with the saline control group using Chi-square (xThese findings show that roflumilast possesses antidiarrheal and antispasmodic effects, possibly mediated by blockade of voltage-gated Ca++ channels and PDE inhibition with resultant increase in cAMP levels, although the possibility of β2-adrenergic receptor and/or adenylyl cyclase activation mechanisms cannot be ignored.


In chronic kidney disease (CKD), the secretion of uremic toxins is compromised leading to their accumulation in blood, which contributes to uremic complications, in particular cardiovascular disease. Organic anion transporters (OATs) are involved in the tubular secretion of protein-bound uremic toxins (PBUTs). However, OATs also handle a wide range of drugs, including those used for treatment of cardiovascular complications and their interaction with PBUTs is unknown. The aim of this study was to investigate the interaction between commonly prescribed drugs in CKD and endogenous PBUTs with respect to OAT1-mediated uptake. We exposed a unique conditionally immortalized proximal tubule cell line (ciPTEC) equipped with OAT1 to a panel of selected drugs, including angiotensin-converting enzyme inhibitors , angiotensin receptor blockers , furosemide and statins (pravastatin and simvastatin), and evaluated the drug-interactions using an OAT1-mediated fluorescein assay. We show that selected ARBs and furosemide significantly reduced fluorescein uptake, with the highest potency for ARBs. This was exaggerated in presence of some PBUTs. Selected ACEIs and statins had either no or a slight effect at supratherapeutic concentrations on OAT1-mediated fluorescein uptake. In conclusion, we demonstrate that PBUTs may compete with co-administrated drugs commonly used in CKD management for renal OAT1 mediated secretion, thus potentially compromising the residual renal function. Chronic kidney disease (CKD) is a worldwide public health problem associated with considerable prevalence of comorbidities, impaired quality of life and premature mortality . In patiCKD patients are routinely treated with many drugs that require transporters and DMEs for their disposition ,16. The SLC22A6) and OAT3 (SLC22A8) are abundantly expressed at the basolateral membrane of proximal tubule cells, and appear to be the most important transporters involved in the renal uptake of endogenous metabolites, including PBUTs , with OAT1 dominating over OAT3 with respect to PBUT uptake [In renal tissue, the key mediators of transepithelial transport of many endogenous metabolites are the organic anion transporters (OATs), which play a central role in the cellular uptake of uremic retention solutes as a first step in their excretion ,29,30. AT uptake ,29,31,32T uptake . The intThe aim of this study was to investigate the pharmacokinetic interactions between commonly prescribed drugs in CKD management  and PBUTs with respect to OAT1-mediated uptake in an in vitro setting. First, the inhibitory potency of the selected PBUTs and drugs on OAT1 activity was evaluated. For this, we exposed a human conditionally immortalized proximal tubule cells line (ciPTEC) expressing OAT1 (ciPTEC-OAT1) previously developed and characterized ,35,36,37p < 0.0001 when compared to control).First, the effect of PBUTs on OAT1-mediated uptake of fluorescein by ciPTEC-OAT1 was evaluated. Fluorescein, an OAT1 model substrate, was used to evaluate the transporter activity as described earlier . To confThe reduction of fluorescein uptake in the presence of probenecid (500 µM) was in agreement with our previous findings . AdditioSLC22A2) was used to reflect the non-inhibition of OAT1 [To evaluate the role of OAT1 in the disposition of commonly prescribed drugs in CKD management, a panel of nine drugs selected based on their use within our hospital  and PBUTs  at uremic concentrations, together with fluorescein.50 values were affected by the presence of PBUTs as shown in In the presence of PBUTs, the inhibitory effect of the drugs on fluorescein uptake was maintained with a clear dose-response relationship. The ICWith KA alone, only a slight reduction in fluorescein uptake was noticed. In co-incubation with drugs, the inhibitory potencies of the drugs were not aggravated , Table 2With PCS, fluorescein uptake was strongly inhibited in the absence of drugs confirming the role of OAT1 in handling PCS . During ).The exposure to the mixture of PBUTs (UTox) resulted in a strong reduction of fluorescein uptake (34.4 ± 8.3%). With the addition of drugs, a further decrease was observed at the lowest concentration of each drug , with a more pronounced effect for losartan and valsartan. This drop remained stable with the increase of the drug concentration .Our major findings are that PBUTs may directly interact with drugs commonly prescribed in CKD management for OAT1-mediated renal transport at concentrations found in uremic serum. These interactions could exert widespread and unpredictable effects in CKD patients that already have a high burden of co-existing diseases, poor health-related quality of life and are prescribed many medications ,17. CKD The potential interaction of drugs and metabolites with the renal organic ion secretion system , and multidrug and toxin extrusion (MATE)) has been acknowledged by the US Food and Drug Administration (FDA). They released regulatory guidelines to study the contribution of  transporters in disposition of new pharmaceutical entities to identify and understand potential DDIs and (drug-induced) nephrotoxicity including evaluation of interaction with the transporters in vitro . Notewor50 values for losartan and valsartan , based on the uptake of uric acid by OAT1-expressing Flp-HEK293 cells. For statins, exposure to our in vitro cell model revealed higher affinities towards OAT1 than those reported in the literature  reported for the ciPTEC-OAT1 system [50 (IC50 = 6 ± 1 µM) [The simultaneous incubation of individual toxins at uremic concentrations  with the selected drugs resulted in a significant alteration of the inhibition potential of drugs on OAT1-mediated fluorescein uptake, when compared to the toxins alone. This indicates that the transporter is susceptible to modifications of the microenvironmental composition. In the case of IS- and PCS-drug co-incubation, at the lowest concentration of the drugs tested, the inhibitory potential was similar to that of toxin alone, suggesting that the competitive inhibition was mainly toxin-dominated. With increasing drug concentrations, which in case of furosemide, losartan and valsartan was still within their therapeutic window, a significant inhibition occurred, which is further evidence of OAT1-mediated handling of the drugs, concomitant with the PBUTs . Of note1 system . KA on t ± 1 µM) , which eα7 nicotinic receptors [For KA-ARBs co-incubation, the inhibitory potential was lower than when the cells were exposed to the drug alone, despite the increase in drug concentration, an indication that KA compromises the uptake of ARBs in favor of fluorescein, suggesting that the toxin acts as a negative allosteric modulator, an effect previously documented for eceptors . Such efThis study has some limitations that should be addressed in future research. First, PBUTs in plasma are highly bound to plasma proteins  and tested drugs  is available in A brief overview of the uremic toxins used within the study  CO2 to allow expansion and prior to the experiments seeded at a density of 63,000 cells/cm2. Subsequently, cells were grown 24 h at 33 °C, 5% (v/v) CO2 to allow adhesion and proliferation, then cultured for 7 days at 37 °C, 5% (v/v) CO2 for differentiation and maturation, refreshing the medium every other day. The temperature shift ensured the maturation of cells into fully differentiated epithelial cells able to form confluent monolayers.Conditionally immortalized proximal tubule epithelial cells, obtained from urine samples of healthy volunteers and overexpressing the organic anion transporter 1, (ciPTEC-OAT1) were cultured as described by Nieskens et al. . BrieflyThe potency of a panel of several commonly prescribed drugs in the CKD management to inhibit OAT1-mediated fluorescein uptake was investigated in ciPTEC-OAT1 mature monolayers using an inhibition assay, as previously described by Nieskens et al. . CiPTEC-To confirm the ability of the ciPTEC-OAT1 to handle uremic toxins, cells were co-incubated with fluorescein and PBUTs , following the procedure described above. In order to replicate the uremic condition present in CKD patients, a specific mixture of eight known anionic PBUTs , predomiAll data are expressed as mean ± standard deviation (SD) of at least three separate experiments. Inhibition data were fitted according to one-site total binding saturation curve using non-linear regression analysis ((inhibitor) vs. response-variable slope) and Vmax values were calculated according to Michaelis-Menten kinetics using non-linear regression analysis. Statistical analysis was performed using one-way ANOVA analysis followed by Tukey’s multiple comparison test  or two-way ANOVA analysis followed by Dunnett’s Multiple Comparison test  with GraphPad Prism version 8.4 . All experiments were performed four times.


Affected by the sea ice, the shear force and bending moment of the tower showed significant increases, and more attention needs to be paid to the tower bottom and action position of the sea ice. According to the dynamic similarity principle, finally paraffin was used to simulate sea ice, and shaking-table tests were performed for simulating dynamic ice-structure-water interactions. Results of shaking-table tests verified the rationality of our proposed simplified model.Ice-structure interaction threatens the safety of the offshore structure; however, dynamic seismic action even renders this process more sophisticated. This research constructed a simplified calculation model for the wind turbine tower, ice, and water under seismic loading, which could avoid solving the complex non-linear equations. Then, the seismic behaviour of the structure,  Ice load induced structural failure is one of the main disasters for the offshore structures; therefore, finding a reasonable computing method is urgent for seismic design of offshore structures.North China belongs to the earthquake-prone area, and there is a four-month freeze every year in the sea. For example, the freezing range could reach thirty nautical miles, and its thickness could reach the 0.6 m. Sea ice affects dynamic responses of wind turbine towers to seismic actions; however, influences of earthquakes and sea ice upon the offshore structures are taken into account separately in most design specifications. For example, the codes including Canadian Standards Association code (CSA)4, Yu et al.5, Sun et al.6, Paavilainen et al.7 and Brown et al.8 investigated the ice-structure interaction, and discussed on the structure-ice impact process; however, they did not mention ice-structure dynamic interplay under influences of seismic loading. It is considered that ice loading results from a complex process of the structure-ice interaction with lots of unknown factors9. Particularly, due to dynamic seismic actions, this process becomes more sophisticated. In addition, earthquakes can also induce hydrodynamic pressure. Jia and Han10 found that floating ice influences hydrodynamic pressure on structures. According to Kobayashi11, influences of sea ice are able to magnify hydrodynamic pressure, with the interaction effect non-ignorable. Yamauchi et al.12 investigated the effects of surrounding ice sheets on the design of foundations for gravity-based structures while an earthquake occurs, and found that the load combination of ice and earthquake depends on the indentation velocity and an interaction between structure and non-moving ice may mitigate the seismic load; Feng et al.13 found that the ice generated around the structure not only changes the constraints and boundary conditions of the structure, but also induces a greater force on the structure under seismic load; however, the effect of earthquake-induced hydrodynamic pressure is ignored in this study, and seismic dynamic behaviour of structures varied greatly if water-structure-ice dynamic interaction was considered simultaneously under earthquake action.Ice-structure dynamic interplay under seismic loading is not studied as extensively as the ice-structure interplay. For examples, Staroszczyk15, earthquake actions still bring difficulties when designing ice-structure dynamic interplays16. Kato et al.17 proposed a method using finite element modelling and the Monte Carlo simulation and offers a general procedure with which to evaluate the simultaneous ice loading in the design; Kouichi et al.18 simplified the structure and ice system to a three-particle system and investigated the effect of structural form on the internal force response of the interactions therein. Jia19 assessed the pier response to ice and earthquake actions through numerical analysis, and pointed out indicate that the analytical method proposed in this paper can provide references for the development of ice mechanics, and the method is feasible for the study of the response of deep-water piers. Using a new physical mechanism that links self-excited and forced vibrations, Xu et al.20 developed a model for dynamic ice-structure interplays of single degree-of-freedom. As mentioned above, plenty of works have been done for investigation of variations of methods for calculating the ice load. Among these researches, few studies could provide a simple calculation method for ice-structure-water dynamic interaction under seismic loading. In addition, few studies have been conducted into the seismic behaviour of wind turbines affected by sea ice; because such wind turbines are new structural forms, there is a lack of codified seismic design provision governing their analysis, especially for wind turbines located at sea.Although there are simplified approaches for calculating ice load available in design standardsIn seismic design of offshore structures built in ice zones, it is necessary to deal with interplays of sea ice, water, and wind turbines. For such purpose, this paper explores an approach on simulating dynamic ice-structure-water interplays in response to seismic loading, and established a simplified model for a wind turbine tower subjected to ice load, hydrodynamic pressure, and seismic load; then we analysed the seismic behaviour of the structure in the presence and absence of influences from sea ice. Finally, shaking-table tests were conducted for verifying the rationality of the simplified model and the accuracy of the calculated results.18, water can be considered as added mass, while the hydrodynamic pressure shows equivalence to damping force and inertia force generated by relative motions of structures and added mass under earthquake actions. Dampers and springs with the same damping and inertia force are used to simulate damping and inertia force. Due to inspiration of the above ideas, the sea ice is also considered as the added mass, however, it is different from water. The sea ice is as an independent added mass, and the connection between the structure and the sea ice through the springs and dampers. The dynamic behaviour of the springs and dampers are simulated by the dynamic ice force model. The added mass used to simulate the sea ice is free at the direction for inputting seismic excitations while has constraints at other directions   3) into Based on the above ideas, mass points and springs are used to take effects of free sea ice upon the structure, as shown in Fig. 19. Persistent ice-breaking force The research considers sea ice load on the tower by using added mass and springs, with stiffness of springs following the ice force model suggested by Croteau Fig. , in whic20, see Eq.  where water is 15 m deep. The tower is 60 m high, and length of the pile is 60 m as well, with a 20-m-high above-ground part and a 40-m-high part underground. The wind turbine tower has a cylindrical structure with varying cross section and varying wall thickness; the tower and above-ground pile are manufactured with Q345C steel. The structure has an outer diameter ranging from 3.1 to 4.5 m and wall thickness from 18 to 50 mm. Poisson ratio, yield strength, and elasticity modulus of the material are γ = 0.3σy = 345 MPa, and E = 2.06 × 1011 Pa, respectively. To adopt the proposed simplified calculation model, the mass of the structure was ascertained in Fig. A monopole wind turbine in the Yellow Sea was used in this study, where it is close to the coast of Jiangsu province in China and at 8-degree seismic fortification intensity according to the new seismic ground motion parameters zonation map of ChinaStandard Specification for Highway Bridges is used as the stress–strain relationship of steel23, simplifying the plasticity stage and enhancing the material stage into a diagonal line  were employed, separately termed as T1 and T2. The seismic waves and response spectrum with a damping coefficient of 0.05 were calculated  was adopted as an index for seismic intensity, and the index for structural damage was curvature at tower bottom. Then we could obtain the vulnerability curve of the structure. First, change laws of curvature with PGA are shown in Fig. 2; the structure is damaged in the presence of ice influences when the PGA is about 10 m/s2 under T2-II-1 earthquake action. Therefore, the wind turbine tower may be damaged due to the different types of seismic waves even under the same magnitude earthquake in the seismic design.As Fig. 26, we conducted the shanking table tests for verifying rationality of the simplified model.Considering that shaking-table tests could simulate dynamic behaviours of wind turbines under seismic actions22. To simulate the ice and water, a water tank was made. The test model is fixed inside the tank. The length of the tank is 2.0 m, the width is 1.3 m, and the height is 1.5 m. Considering that the shaking table measures 1.5 m × 1.5 m, the similarity coefficient is The parameter information of the shaking-table equipment can be seen in referenceWater, ice, and structure following a same similarity relation.Ensuring undistorted gravity of the structure, and adding mass blocks to the model top as mass of the upper structure on the basis of no influence on structural stiffness.Controlling parameters of applied dynamic load for satisfying performance requirements of the loading device.Before derivation of similar formulas, we make the following assumptions:27.In the shaking-table tests on interplays among ice, structure, and water, it is ideal when the model and prototype structures satisfy similarity relationships in terms of gravity, inertial force, and resilience; whereas, there are strict requirements on density and elasticity modulus of structural materials. Hence, researchers generally use a similarity law of elastic force-gravity when performing the tests, to ensure consistency of the Cauchy constant and Froude constant in prototype and tested structures28. Because this model is designed to be at the elastic response stage, materials are supposed to be consistent with fundamental assumptions in the general elasticity theory as much as possible. So, we can use steel for similar model design. Equation 10) are aBy comparing Eq.  with Eq.Because of limited equipment size in the tests, boundary conditions of water are restricted. Under the condition, we set a similarity constant entclass1pt{minimaParaffin is used to simulate the sea ice, and the compression test is conducted to determine the physical and mechanics parameters of the paraffin. After being cut into little cylindrical blocks, the paraffin is crushed under compression. We determined the stress and strain of each sample Fig. , and calAs shown in Table 22, and the sensor placement is shown in Fig. In the shaking-table test, five acceleration sensors and two displacement sensors are used to measure the displacement and acceleration for the test model. The sensor model can be seen in reference2. We then compared time histories and Fourier spectra for displacement and acceleration at the model top, as shown in Figs. To testify that the calculation results are accurate, T1-II-1 seismic waves were taken as examples, with peak acceleration is set as 1 m/sAs shown in Figs. Finally, calculated peak acceleration and displacement along height direction of the model were compared in the presence and absence of influences of sea ice Fig. .Figure 1As shown in Fig. The added mass and springs of certain damping and stiffness were used to simulate the effect of the ice on wind power tower, and a simplified calculation model for the wind turbine tower, ice, and water dynamic interaction under seismic load was established. This obviated the need to solve complicated non-linear equations and reduced the computational burden to a significant extent.When the ice mass is within a certain range, it exerted a significant influence upon curvature at the bottom of the tower, which could reflect the plastic deformation of the structure. Under effects of sea ice, the structure exhibited decreased deformation and energy dissipation capacity; hence, it needs to enlarge thickness of tower walls or stiffening ribs for improving ductility and seismic performance of the structure. The wind turbine tower may be damaged due to different seismic waves even under same magnitude earthquakes in the seismic design.The tower bottom and action point of sea ice showed significantly increased maximum bending moment and shear force; thus, it was deemed necessary to conduct anti-seismic design check-calculations on the tower bottom and action point under influences of unfavourable sea ice mass in seismic design of the structure. Based on the design principle of the similarity ratio, paraffin was used to simulate sea ice and shaking-table tests were conducted for verifying accuracy of the simplified model proposed, and calculated data were within accepted tolerances.The simplified model for dynamic interactions among ice, structure, and water was proposed for studying effects of sea ice upon seismic behaviours of the wind turbine tower. To verify rationality of our proposed simplified model, we conducted shaking-table tests. The following conclusions were drawn:Supplementary Information 1.


ABM300 was characterized in vitro  and in vivo  to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including β-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB However, antipsychotics are associated with extrapyramidal side effects, sedation, metabolic syndrome, and weight gain  CB1R agonist binding but functionally acts as an inhibitor of CB1R agonist-mediated signaling CP55,940 . Non-specific binding was defined in the presence of 10 µM WIN55,212-2.Binding assays in hCBhttps://www.eurofinsdiscoveryservices.com).The PathHunter® β-Arrestin assay was conducted by Eurofins Pharma Discovery Services  according to the manufacturer’s protocol, in hCB1R CHO cells plated at a density of 40,000 cells/well (96-well). ABM300 IC50 values were determined in the presence of increasing concentrations of ABM300 at the EC80 for CP55,940 (40 nM). Results are presented as the percent stimulation of ERK1/2 phosphorylation by CP55,940 alone.CB1R CHO cells were seeded at a density of 40,000 cells/well (white 96-well), and after 24 h were serum-starved in the presence of 0.1% Bovine Serum Albumin for 60 min prior to pretreatment for 30 min with increasing concentrations of ABM300 (10−10–10−5 M) and a final 30 min incubation with 40 nM CP55,940 and 5 μM forskolin. The cAMP BRET experiments were performed as previously described CP55,940 to hCB1R CHO  and an α value of 4.33 ± 0.79 (logα 0.622 ± 0.08) (Supplementary Table In line with our previous studies using a related compound (Org275) , we findCHO Fig.  with an 1R assay, CP55,940-stimulated β-arrestin recruitment with an EC50 value of 5.37 nM (pIC50 8.28 ± 0.06) and an Emax of 104.3 ± 0.90%. In the presence of the EC80 of CP55,940 (10 nM), ABM300 produced a concentration-related reduction in β-arrestin recruitment with an IC50 value of 49.7 nM (pIC50 7.31 ± 0.02)  Fig. .1R CHO cells. In the presence of vehicle, CP55,940 induced ERK1/2 phosphorylation with an EC50 of 12.1 nM (pEC50 8.14 ± 0.23) , to 40.3 ± 5.57% and 14.7 ± 5.22%, respectively  of AEA from 100.3 ± 0.20% to 80.23 ± 14.4% and 31.88 ± 6.09%, respectively  of CP55,940, ABM300 produced a concentration-related reduction in ERK1/2 phosphorylation with an IC50 value of 47.0 nM (pIC50 7.38 ± 0.12) . At CP55,940 EC80 (40 nM), ABM300 blocked this inhibition with an IC50 value of 379 nM (pIC50 6.45 ± 0.08)  Fig. . At concely Fig. . ABM300 ely Fig. . Additioely Fig. . In the 12) Fig. . CP55,9408) Fig. .We further characterized the real-time kinetic effect of ABM300 using a cAMP BRET sensor assay in HEK293 cells. Similar to previous observations with Org275 , ABM300 2, Dopamine D1 and D2, NMDA, 5HT1A, 5HT1B, 5HT2A, 5HT2B, and 5HT3 . Previous reports demonstrated adverse effects observed with CB1R orthosteric agonists/inverse agonists [p = 0.3335), whereas rimonabant significantly reduced open arm time compared to vehicle (p = 0.0059).We confirmed that ABM300 (10 mg/kg) did not have agonist activity via the cannabinoid-induced tetrad, compared to THC (10 mg/kg). In all four tetrad measures , and GluN1KD mice responded to ABM300 (p < 0.0001) when compared to vehicle. Significant effects of genotype were observed for stereotypy and vertical exploration . ABM300 significantly reduced exaggerated stereotypic activity . Sensorimotor gating deficits, along with acoustic startle response, did not respond to ABM300 or the atypical antipsychotic olanzapine  , indicating that ABM300 has genotype-specific effects on the exacerbated hyperactivity endophenotype in DATKO. There was an effect of genotype on exaggerated stereotypic movements and mania-like rearing behavior , which ABM300 had a beneficial effect in decreasing  . Neither genotype, nor treatment with ABM300, had an effect on the acoustic startle response in the DATKO model .The DATKO mouse, another model of hyperdopaminergia, displays hyperactivity, increased stereotypy and vertical exploration, with an impairment in sensorimotor gating , 31. Simly) Fig. . Furtherly) Fig. , with a p = 0.007). Furthermore, olanzapine, but not ABM300, enhanced escape responding during CAR  and an interaction between drug treatment and testing day (p = 0.034). Lastly, neither ABM300 nor olanzapine affected escape failures  [1R agonist-mediated β-arrestin recruitment , ERK phosphorylation , and cAMP inhibition .Our molecular docking data indicate that ABM300 binds to the recently elucidated binding site for the original allosteric modulator Org275 . The Orgund CB1R , which mo Org275 , 15, whe ± 0.79) , but act1R allosteric modulators such as Org275 and PSNCBAM-1 [1Rs can significantly differ in various neuronal cell types; CB1R on GABAergic interneurons have significantly higher agonist affinity than those found on glutamatergic terminals, but the coupling efficacy of glutamatergic CB1R is significantly higher [1R signaling in vivo and, potentially, selectively modulates the endocannabinoid system in specific neurotransmitter system pathways to ameliorate the dysregulated behaviors observed in these mice.As previously described , the uniSNCBAM-1 . This may higher , 46. Thi1R to the soma, while cholesterol, which binds to the same site as ABM300 and acts as a positive modulator, allows for the enrichment of CB1R at the axon [1R membrane localization by CB1R allosterics. The consequences of this in a complex neuronal network, and pathological states, are yet to be fully elucidated.Studies have demonstrated that administration of compounds that increase endocannabinoid levels modulate a number of schizophrenia-like responses in mice . Studiesthe axon . Thus, i2 receptor [To assess psychosis-like behaviors, and therapeutic efficacy of ABM300, we focused on two main behavioral categories: exploratory behavior (hyperactivity) and sensorimotor gating (disruption in PPI). The literature related to psychosis-like behaviors in genetically modified mouse models has classically focused on the same two categories . Althougreceptor . Thereforeceptor . Meanwhireceptor .1R antagonism and loss of function may enhance some forms of learning and memory, and it is possible that CB1R-negative allosteric modulators may be pro-cognitive in preclinical schizophrenic models [Here, we show that ABM300 restores dysregulated dopamine-mediated exploratory activity in both genetic models: decreasing exaggerated hyperactivity, stereotypy and rearing. Furthermore, ABM300 rescues PPI deficits in the DATKO model. Since psychosis symptomology is never present alone in a disease state such as schizophrenia, it would be intriguing to investigate the effects of ABM300 in other symptomatic domains, such as cognition. CBc models , 53.We saw no effect of the compound in CAR in rat even though olanzapine produced significant suppression of CAR as has been shown in previous studies . CAR is 1R in the in vivo effect of ABM300 in genetic models. Directly implicating CB1R is challenging. One approach would be to create a double knockout of CB1R-/- and DAT-/- or GluN1-/-. However, given the crucial role of the CB1R in dopaminergic and glutamatergic signaling, there is a strong likelihood that the double knockouts could have a novel phenotype (such as seizures) or that previous phenotypes would be exacerbated [1R has been shown to have effects on dopamine signaling [1R orthosteric antagonist would block the effects of ABM300, thereby implicating CB1R. However, there is also doubt as to whether an orthosteric CB1R antagonist would block the effects of a negative allosteric inhibitor (antagonist blocking inhibitor); it is conceivable that a synergistic effect might be observed. Taken together, these limitations highlight the complexity of directly implicating CB1R in the mechanism of action of ABM300 in vivo in these genetic models. We plan to make this the subject of future investigations involving a variety of in vivo and ex vivo approaches. The pharmacological profiling and assessment of the potential for off-target interactions of ABM300 in binding screens suggest that the compound is CB1R-selective. Taken together, the potent inhibitory effect of the compound on CB1R signaling at nM concentrations in vitro and the favorable PK and brain penetration data, there is substantial support for the hypothesis that the CB1R mediates the effects of ABM300 in vivo in the genetic mouse models of hyperdopaminergia. It is also notable that the present study only included acute administration of one dose of ABM300. Future experiments will focus on various dose regimens, chronic dosing, development of tolerance and the effects of ABM300 in combination with an antipsychotic.It is important to note that we do not have direct evidence for the involvement of the CBcerbated , 57. Furignaling , 59. Ano1R allosteric modulator, with a unique pharmacological profile, effectively ameliorates certain behavioral deficits in two distinct models of increased dopamine. These data highlight that the allosteric-binding pocket on the CB1R warrants further investigation as a potentially important therapeutic target in psychiatry.The data presented here offer the first evidence that acute administration of a CBThe authors declare the following financial and biomedical conflict of interests: Ruth A. Ross, Catharine A. Mielnik, Amy J. Ramsey, Iain R. Greig, Laurent A. Trembleau, Mostafa H. Abdelrahman are co-inventors on a patent application related to ABM300 and structural analogs. Kim S. Sugamori, David B. Finlay, Hayley H.A. Thorpe, Matthieu Schapira, Nirunthan Sivananthan, Chun Kit Li, Vincent M. Lam, Sean Harrington, W. McIntyre Burnham, Jibran Y. Khokhar, Ali Salahpour, Michelle Glass reported no biomedical financial interests or potential conflicts of interest. W. McIntyre Burnham received Δ9- (THC) as a gift from MedReleaf. The authors would like to gratefully acknowledge Wendy Horsfall for mouse colony maintenance. The work was funded by grants to RAR from CIHR , CIHR funding to AJR (MOP119298) and CIHR funding to AS (PJT-15619).Mielniketal2020_Supplemental Material