Background: There is a dearth of studies to assess the effectiveness of cryotherapy as a possible adjuvant in anesthesia. Therefore, this study aimed to compare the impact of pretreatment intraoral ice packs, application of Endo-ice, and a combination of Endo-ice and intrapulpal ice sticks use on the effectiveness of pulpal anesthesia as an adjunct in the management of pain and dental anxiety in mandibular second molars with symptomatic irreversible pulpitis (SIP) against traditional inferior alveolar nerve block (IANB) technique.Materials and methods: The randomized controlled double-blind clinical study comprised of 200 subjects within the age group of 20-45 years with SIP involving mandibular second molars and divided into four groups of 50 each. Conventional IANB was administered in group 1. Intraoral compact ice packs, Endo-ice, and combined use of Endo-ice and intrapulpal ice sticks were employed following conventional IANB in groups 2, 3, and 4, respectively. The intensity of pain and anxiety before and after the intervention was documented using Heft-Parker visual analog scale (HP-VAS) and Corah's Dental Anxiety Scale-Revised (DAS-R).Results: The average age of the overall study sample was 30.8±2.08 years, and the differences in age and gender distribution were found to be statistically insignificant. The mean HP-VAS scores on access opening and pulpectomy, using ANOVA paired with Tukey's post hoc multiple comparison tests were evaluated to be statistically highly significant (p<0.001). The greatest reduction in HP-VAS score was observed in group 4 receiving combined use of both Endo-ice and intrapulpal ice sticks. While the pretreatment DAS-R score was determined to be statistically insignificant between groups, group 1 subjects had the highest DAS-R score postoperatively (p<0.001). The effectiveness of pulpal anesthesia was found to be 84%, 96%, 92%, and 98% for groups 1 through 4, respectively, and demonstrated a statistically significant difference.Conclusion: The combined use of Endo-ice and intrapulpal ice sticks used as an adjuvant to local anesthesia (LA) was found to be significantly effective in lowering pain compared to the control groups in molars with SIP. Cold administration before and during the procedure was more effective than conventional LA in lowering intraoperative anxiety. Furthermore, the use of Endo-ice and intrapulpal cold after IANB significantly improves the effectiveness of pulpal anesthesia in mandibular second molars with SIP. The inflamed pulp in symptomatic irreversible pulpitis (SIP) causes acute pain that may either be spontaneous, or induced by heat stimuli and can last for 30 seconds or even further following the removal of stimulus, establishing the necessity for endodontic therapy. It is critical to accomplish effective pulpal anesthesia during root canal therapy of molars with SIP for pain control and anxiety management. It is particularly challenging for individuals with SIP in mandibular molars as the inferior alveolar nerve block (IANB) typically fails to deliver substantial anesthesia . ClinicaSeveral clinical investigations were conducted to address the challenges of IANB in patients with SIP. These researches evaluated various LA solutions to lidocaine, which presently constitutes the most often administered anesthetic in dentistry and can be regarded as the benchmark . PreoperNot many investigations have been conducted to assess the effectiveness of cryotherapy as a possible adjuvant in anesthesia . It has Therefore, the study aimed to compare the impact of preoperative intraoral small ice packs, application of Endo-ice, and a combination of the use of Endo-ice and intrapulpal ice sticks on the effectiveness of pulpal anesthesia as an adjunct in the management of pain and dental anxiety in mandibular second molars with SIP against conventional IANB technique.This double-blind randomized controlled clinical study was designed following the Declaration of Helsinki and in compliance with the consolidated standards of reporting trials statement . The insBefore administering LA, each patient was provided with an extensive overview of the HP-VAS, which was categorized into four distinct groups: a lack of pain referred to 0 mm, mild pain was characterized as a score exceeding 0 mm but not greater than 54 mm and encompassed the characterizations of faint, weak, and mild pain. Moderate pain was outlined as a score above 54 mm and lower than or equivalent to 114 mm and comprised the categories of strong, intense, and maximum possible pain. The dental anxiety was assessed before and after intervention by employing Corah's Dental Anxiety Scale-Revised (DAS-R), with an overall score between one and 20. A numerical rating of below nine suggests mild anxiety. A score of 9-12 indicates moderate anxiety, 13-14 suggests high anxiety, and 15-20 implies phobia/severe anxiety .Patients within the age group of 20-45 years with SIP and scheduled for root canal therapy in mandibular second molars were assessed for the following factors to be included in the study: a pretreatment HP-VAS score of above 54 suggesting moderate to severe pain, DAS-R score greater than nine, a positive response to electric pulp testing (EPT), and an extended response to cold test demonstrating pulp sensitivity. SIP was diagnosed for each tooth upon reviewing clinical and radiographic records. The exclusion criteria include those patients under the age of 19 years who disclosed pain on cold application, non-vital teeth, calcified root canals, teeth with periapical radiolucency or abscess with/without sinus opening, teeth with periodontitis, individuals with systemic disease, or with a history of previous endodontic treatment, allergies, or antibiotics intake in the preceding 48 hours.A digitally-generated randomization process was used to allocate recruited individuals to one of the four groups under study. The principal investigator and the individuals being studied were both blinded to the groups they were assigned to. The randomized sequence of numbers was made with unique codes for particular individuals and was hidden in consecutively assigned opaque, individualized confidential packages, which were unsealed by the clinician during treatment. The investigator was an endodontist with over 10 years of expertise. Age, gender, preoperative discomfort, and anxiety were documented and the entire procedure was standardized by using the same operator, materials, technique, and clinical conditions.Group 1 (negative control group)Conventional IANB was delivered employing a 27-gauge needle of 31 mm with 2 mL of 2% lignocaine hydrochloride with adrenaline 1:80000. The tip of the needle was placed slightly laterally in the center of the pterygomandibular raphe to approach the bone, gently retracted, and aspirated. The solution was delivered at an average rate of one mL/minute. The effectiveness of anesthesia was established by utilizing EPT after 15 minutes with two successive negative responses to a reading of 80 µA. The molars were isolated using a rubber dam and an access opening was carried out.Group 2 (positive controls)A conventional IANB was delivered as in group 1 following which small cold packs packed in sterile gauze were applied intraorally on the vestibular region. The study subjects were advised to maintain the ice packs for 5 minutes and take them out for a moment if they felt excessive cold or discomfort.Group 3 (Endo-ice group)The typical IANB was given as indicated in group 1. Endo-ice  was subsequently applied for a total of 10 seconds on the buccal, lingual surfaces (3 seconds each), and for 4 seconds on the occlusal surfaces preceding access opening.Group 4 (Endo-ice and ice sticks group)Endo-ice and intrapulpal ice sticks were employed as cold application methods. IANB was delivered as previously stated. Endo-ice was then applied as stated in group 3 before access opening followed by deroofing and locating the canals. Subsequently, two ice sticks were placed directly in the pulp chamber for 4 minutes Figure .Each ice sticks were with a diameter and length of 8 mm and 2 cm, respectively. The success of anesthesia was established after 15 minutes, as mentioned in group 1. The HP-VAS and DAS-R scales were used to assess the pretreatment pain and dental anxiety. A pretest comprising 20 patients was performed to determine the internal coherence of the questionnaires, exhibiting Cronbach's alpha of 0.84. Prior to the IANB, all groups received a 60-second application of 20% benzocaine anesthetic gel at the injection site. The endodontic therapy was commenced after evaluating lip numbness and two consecutive negative responses to the EPT following maximum activation. Those with positive responses had been excluded and received intraligamentary and/or intrapulpal injections. A rubber dam was used to isolate the teeth followed by an access opening. During the treatment procedure, individuals were prompted to rate their pain and anxiety at three different points, such as pre-treatment, during access opening, and immediately following pulpectomy.The canal length was determined using an apex locator . The periapical radiographs were used to ascertain the operating length. Nickel-titanium rotary files  were used for instrumentation of the canal using K-files up to #15 size. With the determined working length, the ideal size of the apical preparation has been estimated to be three sizes bigger than the initial file binding. During the process of instrumentation, each canal received one-minute irrigation using 2 mL of 17% EDTA with intermittent irrigation using 5 mL of distilled water. Obturation was performed with gutta-percha and interim restoration was placed. The endodontic therapy concluded a week thereafter.Statistical analysisThe IBM SPSS Statistics version 26.0 for Windows  was utilized for statistical analysis. Normal probability plots were used to check normality of data. Data were expressed as mean±SD and percentages for quantitative and categorical variables, respectively. The age, HP-VAS, and DAS-R score differences across groups were evaluated using one-way ANOVA paired with Tukey's post hoc tests for multiple comparisons. The chi-square test was applied to analyze the differences in gender and LA success rate between the groups.In this study, 200 subjects with SIP in mandibular second molars were assigned to one of four groups randomly, each with 50 subjects receiving conventional LA group 1), intraoral ice packs (group 2), Endo-ice (group 3), or a combination of Endo-ice and intrapulpal ice sticks (group 4). The mean age of the overall study sample was 30.8±2.08 years, and the differences in age distribution across the groups were reported to be statistically insignificant (p=0.5). The study included 102 males and 98 females, and the differences in gender distribution between study groups were also statistically insignificant with p=0.94 . The mean HP-VAS scores on access opening and pulpectomy, on the other hand, were determined to be statistically highly significant (p<0.001). Tukey's post hoc multiple comparison tests demonstrated statistically highly significant differences between negative controls, positive controls, and the use of combined Endo-ice and intrapulpal ice sticks  during access opening. Similarly, statistically highly significant differences during access opening were found between groups 2 and 4 and groups 3 and 4. During pulpectomy, the intergroup comparisons of HP-VAS score were shown to exhibit statistically significant differences across the groups, except for groups 2 and 3. The greatest reduction in HP-VAS score was observed in group 4, which received combined use of both Endo-ice and intrapulpal ice sticks during access opening and pulpectomy procedures. During access opening and pulpectomy, the HP-VAS scores for groups 2 and 3 were observed to be roughly comparable (Table The pretreatment DAS-R score was determined to be statistically insignificant between groups, indicating that the study subjects experienced similar degrees of anxiety before treatment Table . HoweverThis study assessed the adjunct techniques necessary for successful IANB in mandibular second molars with SIP. Age, gender, preoperative discomfort, and anxiety levels were comparable between groups. In mandibular molars with SIP, the effectiveness of IANB varies between 15% and 57%. It is crucial to consider that effective anesthesia of the IANB and associated tissues might be exhausting due to anatomical variances, inflammation, the technique employed, needle deflection, competence of the operator, and the patient's dental fear . FurtherAnalgesic effects are felt when cryotherapy is administered to the intended locations. Cryotherapy also delays brain signals and inhibits the production of chemical mediators involved in pain transmission by reducing the conduction rate of pain impulses ,18. ThesThe analgesic impact of cold application can be enhanced by prolonging the duration of cryotherapy. However, a tooth being subjected to excessive hot or cold temperature has been shown to induce thermal fatigue defects . AccordiThe success of IANB is frequently judged by the objective and subjective manifestations reported. Nevertheless, an EPT is a more reliable technique for evaluating pulpal anesthesia. A lack of response to EPT is the hallmark of profound pulpal anesthesia . The HP-According to a recent systematic review, while intracanal cryotherapy is a simple and affordable treatment for reducing postoperative endodontic discomfort, the degree of reliability of evidence demonstrated in the literature is inadequate . FurtherThe study findings showed that the combined use of Endo-ice and intrapulpal ice sticks used as an adjuvant to LA was significantly effective in lowering pain during endodontic therapy compared to the control groups. Cold administration before and during the procedure was more effective than conventional LA in lowering intraoperative anxiety. Furthermore, the use of Endo-ice and intrapulpal cold after IANB significantly improves the effectiveness of profound pulpal anesthesia in patients with SIP involving mandibular second molars. Additionally, more intensive studies in clinical settings are critical to evaluate the potential merits of this low-cost technique for routine therapeutic application in conditions where pain and inflammation are predicted.


Serotonin 1A (5-HT1A) autoreceptors located on serotonin neurons inhibit their activity, and their upregulation has been implicated in depression, suicide and resistance to antidepressant treatment. Conversely, post-synaptic 5-HT1A heteroreceptors are important for antidepressant response. The transcription factor deformed epidermal autoregulatory factor 1 (Deaf1) acts as a presynaptic repressor and postsynaptic enhancer of 5-HT1A transcription, but the mechanism is unclear. Because Deaf1 interacts with and is phosphorylated by glycogen synthase kinase 3β (GSK3β)—a constitutively active protein kinase that is inhibited by the mood stabilizer lithium at therapeutic concentrations—we investigated the role of GSK3β in Deaf1 regulation of human 5-HT1A transcription. In 5-HT1A promoter-reporter assays, human HEK293 kidney and 5-HT1A-expressing SKN-SH neuroblastoma cells, transfection of Deaf1 reduced 5-HT1A promoter activity by ~45%. To identify potential GSK3β site(s) on Deaf1, point mutations of known and predicted phosphorylation sites on Deaf1 were tested. Deaf1 repressor function was not affected by any of the mutants tested except the Y300F mutant, which augmented Deaf1 repression. Both lithium and the selective GSK3 inhibitors CHIR-99021 and AR-014418 attenuated and reversed Deaf1 repression compared to vector. This inhibition was at concentrations that maximally inhibit GSK3β activity as detected by the GSK3β-sensitive TCF/LEF reporter construct. Our results support the hypothesis that GSK3β regulates the activity of Deaf1 to repress 5-HT1A transcription and provide a potential mechanism for actions of GSK3 inhibitors on behavior. Major depressive disorder (MDD) is a debilitating psychiatric disorder characterized by persistent low mood and loss of pleasure . MDD has1A autoreceptors is associated with an increased risk of depression [Over the past decade, animal studies have increasingly implicated the serotonin system in processing emotionally salient stimuli ,15,16, fpression ,19,20, spression , and redpression . On the pression ,24.Sp100, AIRE-1, NucP41/75, DEAF1; aa 188–254) domain [Factors that differentially regulate pre- and post-synaptic 5-HT1A receptor expression are attractive drug targets to enhance antidepressant efficacy. The transcription factor deformed epidermal autoregulatory factor 1  is one such protein. Deaf1 regulates 5-HT1A expression by binding to a recognition site located in the 5-HT1A promoter region  via its ) domain . This si) domain ,28,29. D) domain ,31; thusTo identify regulators of Deaf1 activity, interacting proteins provide a useful lead. Interestingly, a yeast two-hybrid screen of proteins in the mTOR pathway implicated in antidepressant actions ,33 identhttps://www.phosphosite.org/ accessed on 12 July 2023). S176 and T179 phosphorylation was identified in large-scale cation exchange chromatography experiments [TPGPQSPPTP similar to the GSK3 phosphorylation consensus motif (www.phosphonetworks.org accessed on 12 July 2023) SPPPS/TPPXS/TP and is located immediately adjacent to the DNA-binding SAND domain of Deaf1  = 201.43, p < 0.0005). Moreover, the effect of lithium was concentration-dependent  = 60.28, p < 0.0005). Post hoc analysis revealed no significant variation in the Deaf1 effect for matching NaCl concentrations. LiCl treatments displayed a significant increase in 5-HT1A transcriptional activity in the presence of HisDeaf1 between the 2.5 mM to the 5 mM (p < 0.05), and between the 5 mM to the 10 mM dose (p < 0.0005). Furthermore, while at 2.5 mM and 5 mM of LiCl, HisDeaf1 shows a significant repression relative to pCDNA3 ; at 10 mM of LiCl, HisDeaf1 showed a significantly increased luciferase activity relative to pCDNA3 (p < 0.0005). Overall, these results suggest that the inhibition of GSK3β causes a reversal of Deaf1’s repressor activity at the 5-HT1A promoter.We addressed whether GSK-3 inhibition would affect Deaf1 activity. The transcriptional activity of HisDeaf1 at the 5-HT1A promoter was determined using luciferase reporter gene assays A. Compar = 109.02, p < 0.0005) in every case, except for 100 nM PMA (p = 0.075). There was a drug effect  = 298.04, p < 0.0005) that was a global decrease in normalized RLU values between the vehicle DMSO and the PMA treatments (p < 0.0005) and did not depend on PMA concentration  = 0.50, p = 0.616). Thus, although PKC affected 5-HT1A promoter activity, it did not affect Deaf1-dependent repressor activity at the promoter.Since there are also several putative protein kinase C (PKC) sites on Deaf1, we tested whether PKC activation could modulate Deaf1 activity using phorbol 12-myristate 13-acetate (PMA) to activate PKC C. There p < 0.01) (p < 0.01), confirming that lithium causes Deaf1-independent enhancement of the 5-HT1A promoter in vitro.A slight increase in basal 5-HT1A promoter activity with pCDNA3 control was observed between the 5 mM to 10 mM Li+ dose ( < 0.01) B, which  < 0.01) D. A dose = 43.2, p < 0.001) and also attenuated repression by Deaf1  = 20.2, p < 0.001)  = 73.6, p < 0.001). Interestingly, CHIR also reduced basal 5-HT1A promoter activity at the highest concentration, leading to a Deaf1-induced enhancer-like effect as observed with AR. While Deaf1 repressed 5-HT1A promoter activity normally in these experiments  = 22.1, p < 0.001), no effect of SB was observed on 5-HT1A promoter repression by Deaf1  = 0.50, p = 0.48). However, exploratory post hoc analysis showed a loss of significant Deaf1-induced repression at higher concentrations of SB, suggesting a partial effect. Taken together, these results suggest that GSK3β inhibitors can reduce Deaf1-induce repression of the 5-HT1A promoter, and even reverse this effect to reveal Deaf1-induced enhancer activity.Lithium had clear effects on Deaf1 activity, but it is difficult to make a causal link to GSK3β inhibition because lithium has many other targets. To provide further evidence, the concentration-dependent effects of three different GSK3β inhibitors, AR-014418, CHIR-99021, and SB216763 . In the < 0.001) A. Like l = 28.7, p < 0.001 and F = 10.6, p < 0.01, respectively), while sodium had little to no impact  = 49.6, p < 0.001), an effect that was significant at all but the lowest concentration tested in the post hoc (p < 0.01 in each case)  = 25.0, p < 0.001), indicating its effectiveness in the TCF-LEF reporter assay. Indeed, the post hoc analysis shows that SB has a significant effect on GSK3β activity at concentrations as low at 5 µM (p < 0.001). However, the maximal effect of SB was orders of magnitude less than for AR-014418, potentially explaining the lower efficacy to reduce Deaf1 inhibition  B. The coch case) C, a signhibition C. OveralThe finding that GSK3β mediates the phosphorylation of Deaf1 in two of three segments of the protein in vitro  lead us www.phosphonet.ca platform (accessed on 21 August 2023) revealed vascular endothelial growth factor (VEGF) receptors 1-3 as the top candidates for phosphorylating the Y300 site. VEGF receptors are tyrosine kinase membrane receptors with an intracytoplasmic catalytic domain [In contrast, the mutation of the Y300 site increased Deaf1 repressor activity at the 5-HT1A promoter in two cell lines, suggesting that Y300 phosphorylation inhibits Deaf1 activity. The Y300 site is located adjacent to the NLS of Deaf1 A and migc domain  that couc domain ,48. ThusOur findings are consistent with Deaf1 repressor activity at the 5-HT1A promoter in HEK-293 and SKN-SH neuroblastoma cells. We show that lithium and two selective GSK3 inhibitors AK and CHIR concentration-dependently inhibited Deaf1 repressor activity and revealed a Deaf1-dependent enhancer effect at the highest doses. Overall, these results are consistent with the existence of a GSK3β/Deaf-1/5HT1A pathway, except that SB did not significantly reduce Deaf-1 repression. However, under our conditions, the maximal GSK3β inhibition in the TCF-LEF assay by SB was approximately one order of magnitude lower than that of CHIR at 1 µM (the lowest concentration at which an effect on Deaf-1 repression is observed with this compound) . These dB4GAT1 and UBE2M genes but induces the RABEP2 gene [Perhaps the most intriguing finding was that at the highest concentrations, lithium, AR and CHIR revealed a paradoxical enhancer effect of Deaf1 transfection on the 5-HT1A promoter. At these high concentrations, it may be that GSK3β inhibition is not sufficient and that other targets are involved. However, using transfection of a Myc-DEAF1 construct in HEK293T cells, Pilot-Storck et al.  found thEP2 gene . Our stuHow Deaf1 exerts enhancer or repressor functions remains unclear, but Deaf1 functionally interacts with a variety of transcriptional regulators and signaling proteins in addition to GSK3β. These include the transcription factor LMO4 , methyl-In addition to their physical interaction and regulation of Deaf1 by GSK3β, the functional association of Deaf1 and GSK3β is supported by genetic studies in Drosophila. Deaf1 is required for the GSK3β-mediated repression of glycolytic genes in muscle . This fiComplementary to the role of GSK3β in the repression of 5-HT1A gene transcription, 5-HT1A receptors have been shown to inhibit GSK3β signaling to mediate hippocampal neurogenesis and behavioral improvement. Studies in rodents have addressed the effects of 5-HT1A receptors on GSK3β phosphorylation, which inhibits GSK3β. Like lithium, fluoxetine or selective 5-HT1A agonist 8OH-DPAT induces the phosphorylation of GSK3β in mouse and human Down’s syndrome neural precursor cells in vitro . The treLithium enhanced the basal activity of the 5-HT1A promoter through a mechanism independent of Deaf1, since similar results were obtained in MEF cells that lack Deaf1 D. SimilaThe stimulatory effect of lithium on 5-HT1A promoter activity at clinically relevant concentrations described in this study is consistent with findings in vivo. In male mice subjected to chronic social defeat stress, a two-week treatment with 2.4 mmol/kg/day lithium increased 5-HT1A RNA expression in the raphe by two-fold and had an anxiolytic effect . SimilarIn summary, we find that GSK3β inhibition by a variety of inhibitors including lithium prevents Deaf1-mediated repression of the 5-HT1A receptor, and at high concentrations can switch Deaf1 to increase 5-HT1A promoter activity. These studies suggest that GSK3β may mediate the brain-region-specific regulation of 5-HT1A receptors by Deaf1 and may account for at least some of the beneficial actions of lithium treatment on behavior.Lithium chloride and phorbol 12-myristate 13-acetate (PMA) were purchased from Millipore Sigma , while CHIR-99021, SB-216763, and AR-014418 were obtained from Cayman Chemicals . Lithium was administered as a 1 M solution in water, while specific inhibitors were administered dissolved in DMSO, with a final DMSO concentration of 0.1% or as indicated. Other reagents were obtained from Thermo Fisher Scientific  or Sigma, except where noted.The full-length human DEAF1 sequence  was scanned using phosphorylation site prediction algorithms NetPhos 2.0 , NetPhosDEAF1 gene. The tag was cloned in between Kpn1 and EcoR1 sites to produce a recombinant his-tagged DEAF1 gene inserted in the pCDNA3  vector. Mutations of the recombinant DEAF1 gene were produced by site-directed mutagenesis of the HisDEAF1 construct using the Stratagene QuickChange II XL site-directed mutagenesis kit . The 5-HT1A promoter construct used in this work was previously known as -1128-luc . The conAll cells  were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) 319-015-CL  supplemented with 10% fetal bovine serum  without antibiotics except for MEFs, for which penicillin and streptomycin were added. MEFs were generated from Deaf1−/− mouse fetuses [v/v heat-inactivated fetal bovine serum at 37 °C in 5% CO2. For luciferase assays, each transfection contained 1 μg of the pGL3-Basic construct (empty vector or containing 5-HT1A promoter), 1 μg of the pCDNA3 construct , and 1 μg of pCMV-β-gal. In experiments using the GSK3β inhibitor lithium chloride , equal concentration of NaCl was used as control. All drugs were administered at the time of transfection.Transfections in HEK293 cells or SKN-SH cells were conducted using Lipofectamine 2000 reagent (Invitrogen). For each reaction, 2 μL of reagent was used per 1 μg of plasmid. Cells were plated in 6-well plates and grown for 48 h in Dulbecco’s modified Eagle Medium (DMEM)  with 10% 5 cells/well and transfected the next day. A solution of 0.25 M CaCl2 containing 2 ng/µL of each plasmid was prepared. To this, an equal volume of 2× HEPES-buffered saline  was added drop-by-drop. A total of 200 µL of the resulting solution was added to each well of cells.For drug concentration dependence studies, HEK293 cells were transfected using the calcium-phosphate method. Cells were sown onto six well plates at a density of 1.5 × 105 cells/well the day before transfection. A total of 1 µg of each plasmid was used per well.MEF cells  were traFor all cells, the day after transcription the medium was removed, HBBS + EDTA was used to wash away any remaining transfection mix, and fresh medium was added. Cells were harvested and assayed for luciferase activity one day later, as described below.2HPO4 and 1.8 mM KH2PO4; pH 7.4) and then gently agitated in 300 µL of 1× Reporter Lysis Buffer (Promega) for approximately 20 min. The partially lysed cells were harvested and put through two freeze-thaw cycles to ensure complete lysis. Lysate was then centrifuged at 13,500× g for 2 min to separate out any particulate matter. The resulting supernatant was used in the luciferase and β-galactosidase assays. Luciferase assays were carried out using 100-µL aliquots of each sample loaded onto opaque, white 96-well plates. For the reading of luciferase activity, an equal volume of luciferase buffer  containing 5.46 mM luciferin potassium salt  substrate was injected, mixed for 2 s, and the luminance read for the next 5 s. The β-galactosidase activity was then measured on the SpectraMax M5  spectrophotometer at 570 nm. For HEK293 cells, 2 µL aliquots were used, while for MEFs, the volume was 10 µL. Reactions were carried out in Z-buffer  containing 0.53 mM chlorophenol red-β-D-galactopyranoside  substrate in a final volume of 125 µL. Absorbance at λ = 570 nm was read using a SpectraMax M5 (Molecular Devices) immediately after the last reaction was initiated and again at intervals. Reactions were mixed for 5 s before the initial reading, and for 2 s before each subsequent reading, using chlorophenol red galactopyranoside (CRPG) (Millipore Sigma) as a substrate. Normalized RLU were calculated by normalizing every luciferase activity value with the corresponding β-galactosidase absorbance. The % repression was calculated as 100 × (V − D)/V, where V is the average RLU of cells transfected with pcDNA vector, and D is the average RLU of cells transfected with Deaf1 plasmid.Two days after transfection, cells were washed with phosphate-buffered saline . A total of 7 cm IPG strips of pH ranges from 3 to 10 and from 4.7 to 5.9 (Bio-Rad) were rehydrated with 25 μg of protein in IEF buffer overnight. The first dimension (isoelectric focusing) was then conducted on Protean IEF Cell (Bio-Rad). Strips were then incubated in reducing solution , followed by incubation in alkylating solution . The second dimension was conducted by SDS-PAGE on an 8% gel, followed by transfer to a nitrocellulose membrane and Western blot.Proteins were extracted using isoelectric focusing (IEF) buffer , 1% DTT (Thermo Fisher Scientific), 4% CHAPS (Sigma), 0.001% bromophenol blue , in ddHA two-way ANOVA followed by Bonferroni-adjusted pairwise comparisons was used to compare the mean normalized RLU values in the luciferase reporter gene assays. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) 10.0 , and calculated using 95% confidence intervals.


With the increase of marine activities in the Arctic area, the demand for reliable design of marine structures is growing. Numerous publications can be found regarding simulations of ice action on structures using cohesive element models of the ice. However, previous studies have rarely discussed the influence of structural form, that is, the form of ice-structure interaction interface, on the ice load. Thus, a more comprehensive understanding of the ice load on structures with different interface geometries needs to be explored. In the present paper, three-dimensional finite element models with the cohesive element method are developed to investigate the ice load on different structures. The numerical results are validated based on in-situ testing data and the results of the previous numerical model. Parametric studies considering structure widths, inclination angles, ice velocity as well as structure roughness are conducted to explore the horizontal force and failure process of the ice sheet. The process of ice-structure interaction and ice loads on different structural forms were discussed and simplified diagrams of ice load distribution on the interface were developed. The growth of human activities in the Arctic and Subarctic regions leads to an increase of demands and challenges in engineering design of structures against ice-induced forces. Although researches related to ice-structure interactions have been carried out many years ago, engineering accidents induced by ice load still occur from time to time. Investigation on ice-structure interactions is hence an important prerequisite for the safety of human activities in these extreme cold regions.Sea ice in the Arctic and Subarctic regions poses a great risk to the safety of marine structures2. Many studies have been conducted to obtain the ice load on column structures and conical structures subjected to level ice8. ISO199006 shows the design method of ice load based on vertical structure and inclined structure. In the existing studies, the single structure form has received more attention, but the comparison of different structure shapes, especially the discussion of the difference of the distribution of ice load on the structure with finite size needs further investigation. When ice interacts with conical structures, different ice-structure interfaces can be formed. For instance, a sloping curved surface or a level sharp corner can be formed under different water levels. However, engineering structures have different geometric forms due to their functions or safety requirements, which results in diverse interfaces in ice-structure interactions. A more comprehensive understanding of the ice load on structures with different ice interfaces is required and should be explored.Prediction of the ice load on man-made structures, such as vessels, offshore structures, lighthouses, etc., is of particular concern for the structural design and Maintenance19. However, in-situ and model tests are time-consuming and expensive, and there are high requirements on test equipment and site conditions20. Development of simulation methods, e.g., finite element method (FEM), discrete element method (DEM) and lattice method, has been an interesting alternative that could save both time and cost, and offer a great number of test parameters under controlled conditions21. To effectively capture the fracture behavior, fracture mechanics was introduced in the ice-structure interaction23. Mulmule and Dempsey24 applied cohesive element to ice mechanics analysis to provide a new approach for sea ice fragmentation problems. The cohesive element method (CEM) relies on the mathematical framework of conventional FEM and can explicitly describe the viscous cracks and fracture process of ice25. As a convenient and reliable numerical method, the CEM has been widely used in fracture simulation of ice-structure interaction30.The in-situ and scaled model tests provide a large amount of ice load data in actual environmental conditions, which helps in understanding the progress of ice-structure interactions and failure of iceIn this paper, a three-dimensional (3D) finite element model with CEM was developed based on ABAQUS/EXPLICIT to investigate the ice load on various structures. The numerical model was validated with the in-site measured data of a load event on Norströmsgrund lighthouse. Three basic interfaces including cylindrical surface (CS), plate surface (PS) and corner interface (CI) were considered to investigate the ice load on structures with different widths and inclination angles. And simulation cases were performed to evaluate the influence of ice velocity and structure roughness. The process of ice-structure interaction and ice loads on different structural forms were explored. Simplified diagrams of ice load distribution on the interface were developed which would be a reference for engineering structure design.u of cohesive elements and the traction stress T26. A linear softening criterion  is shown in Fig. Tmax in Fig. uc, the cohesive element is deleted, and the crack is formed.In this study, the ice sheet is modeled using the CEM. The interaction region of the ice sheet is discretized with ice bulk elements which are connected by cohesive elements. The traction separation law (TSL) curve used in CEM depicts the relationship between the crack separation 31 to estimate the strength of ice rubble. However, it has been reported that MC model is not suitable for estimating the shear strength of ice at high internal friction angles32. In this study, an isotropic elastic–plastic material model , and the cohesive element (COH3D6) is inserted into the ice28. The section at sea level is selected for the lighthouse structure. Because of large overall stiffness difference of the structure, the lighthouse structure is considered as a rigid body.In order to verify the model reliability, the basic working conditions of the model are taken from simulation parameters reported by Hilding et al37.27, as follows:The normal displacement is constrained by smooth lateral boundaries of the ice, and the bottom of the structure is set to be fixed. Initially, the smooth ice front is tangent to the structure surface and it moves along the structure horizontally at a constant speed of 0.15 m/s. The action process is ended after reaching the horizontal displacement to 9 m. The initial velocity of 0.15 m/s is set on the ice body element to avoid extra stress due to inertia in the flat ice. The ice buoyancy force induced by sea water is calculated based on the volume of ice unit submerged in sea waterAs can be seen from Fig. 18 and Hilding33. The simulation results  for which the response of the structure matters and for which additional validation of the ice model would be neededv increases by 12 times (from 0.05 to 0.6 m/s) in the simulation, the average ice load and average peak load on the structure increase by 1.5 and 1.25 times, respectively. Growth of the sea ice velocity by 3.3 times (from 0.6 to 2 m/s) increases the average ice load and average peak load on the structure by 1.5 and 2.5 times, respectively. It is evident that the velocity impact on the ice load is more considerable for higher sea ice velocities. Combined with the results obtained in Fig. The impact of horizontal ice load on the structure under different ice velocities is also examined, as illustrated in Fig. Friction effect in the dynamic process is usually expressed by dynamic friction coefficient Figure 39,40. Interaction between sea ice and these three structural forms is compared in Fig. 41. And Gesa Ziemer43 observed intermittent crushing in model tests with different cross-section. which is considered to be superimposed by bending of the ice sheet, and the duration of intermittent crushing is longer in thicker ice.Various structures have different geometric forms due to their functions or safety requirements. Therefore, the ice-structure interaction is also different due to diversity of structure shapes facing the ice surface. The common structural forms in polar engineering applications can be simplified into three types: cylindrical surface (CS), plate surface (PS) and corner interface (CI), as depicted in Fig. 11In order to further explore the influence of different interfaces on ice load, parametric studies including the interface width, the inclination angle of interface and the angle of corner interface are conducted.Figure 27.The definition of structural inclination φ is presented in Fig. The definition of the interface corner angle 39:Similar to the above cases, when the projected width is constant, the larger value of It can be seen that the average line load of ice increases linearly with the increase of Figure The ice load distribution of the three structural geometries can be simplified to the form shown in Fig. Sea ice velocity has a significant influence on ice failure mode and ice load. With the increase of the sea ice velocity to 0.6 m/s or even 2 m/s, the effect of sea ice on the structure is “impact” resulting in obvious breakage and separation of the sea ice, or it can even exhibit “splash” in which a large number of isolated broken ice and floating ice pieces are formed.The static and dynamic friction coefficients show different effects on ice load. The ice load increases linearly with the growth of Ice load magnitude is related to the ice-structure contact area. Both larger width and larger inclination angle would increase the global ice load on the structure. Examination of cases with different interface corner angles indicates dependence of the ice load on the distance between the structure and the ice sheet.Different ice load distribution patterns were observed in various interfaces. In the CS case, the ice load on the structure is mainly distributed in the middle of the contact area which shows higher material utilization. The corner position of PS structure and CI structure is subjected to large ice load with less material, thus would be posed on higher risk.In this paper, the interaction between a moving level ice sheet and fixed structures with different interfaces was simulated by CEM. 3D numerical models were developed in ABAQUS and validated by in-situ testing data and results of a previous numerical model. The buoyancy and damping models were implemented through user-defined subroutines. Then, a series of simulations concerning ice velocity, structure roughness, interface width, inclination angle of interface and corner angle of interface were conducted. The ice load and its distribution on the structure were analyzed. The main conclusions can be expressed as follows:Supplementary Video 1.


Syndromic Logger) in the emergency room of a large hospital. The platform captured syndromic signals from audio, thermal imaging, and radar, while the ground truth data were collected from the hospital’s electronic health record. Our analysis revealed a significant correlation between the aggregated cough count and positive COVID-19 cases in the hospital . Notably, this correlation was higher than that observed with the number of individuals presenting with fever , a widely used syndromic signal and screening tool for such diseases. Furthermore, we demonstrate how the data obtained from our Syndromic Logger platform could be leveraged to estimate various COVID-19-related statistics using multiple modeling approaches. Our findings highlight the efficacy of aggregated cough count as a valuable syndromic indicator associated with the occurrence of COVID-19 cases. Incorporating this signal into syndromic surveillance systems for such diseases can significantly enhance overall resilience against future public health challenges, such as emerging disease outbreaks or pandemics.Syndromic surveillance is an effective tool for enabling the timely detection of infectious disease outbreaks and facilitating the implementation of effective mitigation strategies by public health authorities. While various information sources are currently utilized to collect syndromic signal data for analysis, the aggregated measurement of cough, an important symptom for many illnesses, is not widely employed as a syndromic signal. With recent advancements in ubiquitous sensing technologies, it becomes feasible to continuously measure population-level cough incidence in a contactless, unobtrusive, and automated manner. In this work, we demonstrate the utility of monitoring aggregated cough count as a syndromic indicator to estimate COVID-19 cases. In our study, we deployed a sensor-based platform ( The act of coughing is part of the body’s defense system that uses automatic reflex mechanisms to clear airways of foreign substances, irritants and excessive mucus. Cough and related complaints have been recognized as a cardinal symptom or syndromic signal of respiratory illnesses, such as the SARS-CoV-2, influenza and influenza-like illness, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Recent progress with cough assessment has led to improved diagnosis and more effective disease management for individual patients , 18. HowSyndromic Logger is the next-generation contactless sensing platform..https://Based on our findings, we propose the integration of a sensor-based, contactless, and unobtrusive platform as a valuable augmentation to existing approaches, such as body temperature-based screening, in diverse public environments. Our results demonstrate that sensor-based platforms have the ability to capture clinically relevant syndromic signals, specifically the aggregated cough count. This capability allows us to overcome the limitations of existing methods which include reliance on manual human logging and reporting, as well as associated reporting delays. By embracing the adoption of sensor-based platforms, we can enhance the effectiveness and efficiency of public health surveillance, providing a superior solution for detecting and monitoring public health situations to overcome potential challenges in the future.


Hippeastrum papilio (Amaryllidaceae) extracts using zebrafish brain homogenates. The biological assays show that the H. papilio bulb extracts present an interesting AChE inhibitory activity in comparison with the positive reference control galanthamine . The chemical profile of H. papilio shows that this species has a high amount of galanthamine, which may contribute to the inhibitory effect on AChE activity of zebrafish brains. Computational experiments were used to build the model for zebrafish AChE and to evaluate the interactions between galanthamine and the enzymic active site. This work suggests that zebrafish could represent an important model in the search for bioactive molecules from the Amaryllidaceae family for the treatment of Alzheimer’s disease.The Amaryllidaceae family constitutes an interesting source of exclusive alkaloids with a broad spectrum of biological activity. Galanthamine, the most relevant one, has been commercialized for the palliative treatment of Alzheimer’s disease symptoms since 2001 due to its potential as an acetylcholinesterase (AChE) inhibitor. In vitro screenings against AChE by applying different Amaryllidaceae species and alkaloids have been reported in the literature; however, they are usually carried out using purified market enzymes. The main goal of this work is to evaluate the AChE inhibitory potential of  The search for medicinal plants has always been of paramount importance for human health and wellbeing . NaturalAlkaloids, classified as secondary metabolites, are predominantly encountered in the plant kingdom. These compounds are distinguished by the presence of at least one nitrogen atom within a heterocyclic ring and exhibit an extensive range of structural diversity coupled with a broad spectrum of biological potential ,5. NotabAmaryllidoideae, a subfamily group of the Amaryllidaceae plant family, presents an exclusive and still expanding group of alkaloids known as the Amaryllidaceae alkaloids ,9. TheseAcetylcholinesterase (AChE) is a highly selective cholinergic enzyme involved in the hydrolysis of the acetylcholine (ACh) neurotransmitter that plays an important role in maintaining the cortical activity and cerebral blood flow, modulating cognition, learning, task and memory-related activities, the development of the cerebral cortex, and regulation of the sleep–wake cycle ,12. In 2Danio rerio) is a tropical freshwater fish native to Southeast Asia that has become an important organism for different areas of biological research, including neuroscience [Zebrafish  Van Scheepen (Amaryllidaceae) extract using zebrafish as a model. The alkaloid profile of this plant was analyzed by gas chromatography coupled to mass spectrometry (GC-MS). The inhibitory potential of H. papilio alkaloid extract against AChE was evaluated in vitro using zebrafish homogenized brain extract instead of the purified market enzyme. In silico experiments were carried out to better understand the interactions between galanthamine and the active site of zebrafish AChE.The aim of this study was to evaluate the cholinergic inhibitory activity of Hippeastrum papilio (Ravenna) Van Scheepen, belonging to the Amaryllidaceae family, were collected in Porto Alegre, situated in the state of Rio Grande do Sul, Brazil, during the month of April in the year 2019. Prior to this collection, the plant had undergone comprehensive identification by Dr. Julie H. A. Dutilh from the University of Campinas (Unicamp), located in Campinas, Brazil. A corresponding specimen voucher (ICN-149428) was subsequently deposited at the Institute of Botany, which is affiliated with the Federal University of Rio Grande do Sul (UFRGS) in Porto Alegre, Brazil.Bulbs of Hippeastrum papilio was subjected to a drying process at a controlled temperature of 40 °C for seven days. Subsequently, the resulting dried material, weighing 62.8 g, was triturated and underwent a maceration procedure. The maceration process was carried out with methanol (2 × 500 mL) and conducted at room temperature for a total of 48 h. Following the completion of the maceration, the combined macerate was subjected to filtration, facilitating the separation of solid particulates. The solvent was subsequently evaporated under reduced pressure until complete dryness was achieved. Upon reaching this stage, the resultant crude extract, weighing 20 g, was subjected to acidification by the addition of diluted sulfuric acid  until a pH of 3 was obtained. To further clean the extract, the neutral material was selectively removed using diethyl ether in five successive extractions, each employing 100 mL of the solvent. The remaining aqueous solution was then subjected to basification by the addition of ammonium  until a pH of 10 was achieved. This alkaline solution was then extracted with ethyl acetate in five sequential extractions, each one utilizing 100 mL of the solvent. The cumulative effect of these extractions resulted in the obtainment of the alkaloid extract, which weighed 303.6 mg.A quantity of 0.5 kg of fresh bulbs from v/v), and 1 μL of the sample was injected, using manual mode, into the GC-MS apparatus  operating in the electron ionization (EI) mode at 70 eV. A Durabond-DB-5 MS column  was used. The chromatographic conditions were adapted from Berkov and co-authors [A total of 2 mg of the alkaloid extract was dissolved in 1 mL of methanol and chloroform  spectra with those of authenticated Amaryllidaceae alkaloids. These authentic alkaloids had been previously isolated and identified using a range of spectrometric techniques such as nuclear magnetic resonance (NMR), ultraviolet (UV) spectroscopy, circular dichroism (CD), infrared (IR) spectroscopy, and mass spectrometry (MS). This rigorous identification process was conducted by the Laboratory of Natural Products at the University of Barcelona, Spain. In addition, a combination of the NIST 05 Database and relevant data from literature sources was also employed.The alkaloids present in the extract derived from 2 = 0.9899).To quantify the single constituents, a calibration curve of galanthamine  was used. The peaks areas were manually obtained considering selected ions for each compound . The ratio between the values obtained for galanthamine in each solution was plotted against the corresponding concentration of galanthamine to obtain the calibration curve and its equation  of both sexes , obtained from the department of Biochemistry at the Federal University of Rio Grande do Sul (UFRGS), were used. The water was kept at 26 ± 2 °C under a 14 h/10 h light–dark cycle photoperiod and fed twice a day with artemia and commercial flake fish food. All procedures presented in this study were performed in agreement with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the local ethics committee. Ten whole brains were removed by dissection for each sample and homogenized in a glass–Teflon homogenizer on ice in 1 mL of Tris-citrate buffer . Protein was measured using Coomassie blue as the color reagent [In essence, the AChE inhibition assay was based on the Ellman method  with mod reagent  and bovi2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, pH 7.5) was used in this assay. An amount of 2 μL of zebrafish brain protein and 48 μL of phosphate buffer were added to the plate. Then, 50 μL of the Amaryllidaceae alkaloid extract was added and the plate incubated for 30 min at room temperature. Following this, 100 μL of the substrate solution  was added. After 10 min, the absorbance of the reaction was read in a spectrophotometer at 405 nm. The enzyme activity was calculated as a percentage compared with the control using a buffer without any inhibitor. Galanthamine alkaloid was used as a reference control. The AChE inhibition activity of the H. papilio bulb alkaloid extract was calculated using a calibration curve  to determine the IC50 value of the plant sample (using the software Prism 9).The AChE inhibitory activity of the alkaloid extracts was evaluated in a 96-well microplate, and phosphate buffer (8 mM KDanio rerio AChE (Uniprot: Q9DDE3) based on the three-dimensional structure of AChE from Tetronarce californica  associated with rivastigmine. The protocol used to perform the comparative modeling experiments involved the generation of five models. All models were submitted to the evaluation of the energy function DOPE [The homology modeling approach, implemented in the MODELLER 9v23 program , was useion DOPE , aiming ion DOPE  and the ion DOPE  to verifDanio rerio AChE. The flexible docking simulations were performed using PyrX-0.9.8 [Molecular docking simulation was carried out to analyze the orientation and binding affinity of galanthamine into the binding site of rX-0.9.8 , where trX-0.9.8 . InitialThe project was approved by the Ethics Committee on Animal Use (CEUA/UFRGS), number 36120. The access authorization and collection for the plant species was emitted by the CGEN under AA621EE.Hippeastrum papilio utilizing gas chromatography–mass spectrometry (GC-MS). To determine the concentration of each individual constituent present in the chromatogram, a calibration curve for galanthamine was employed. The resulting quantification values were reported in micrograms of galanthamine per milliliter (µg GAL/mL). It is important to note that the quantification obtained through this methodology is not absolute; however, it provides a reliable means of comparing the relative amounts of specific alkaloids within Amaryllidaceae samples [A total of seven distinct structures were successfully detected and identified within the alkaloid extract derived from  samples .Hippeastrum papilio extract. Detailed information regarding these quantification and percentage values can be found in H. papilio and in In addition to quantification, the percentage composition of each constituent in the sample was calculated, shedding light on the relative distribution of these alkaloids within the H. papilio samples. De Andrade and co-authors [O-demethylmaritidine, vittatine, and 11-hydroxyvitattine in this species. Later, Guo and co-workers [O-demethyllycosinine B, and 3-O-demethyl-3-O-(3′-hydroxybutanoyl)haemanthamine, which were identified in H. papilio for the first time [H. papilio sample [Lapiedra martinezii species collected in different regions of Spain [Rhodophiala andicola species collected in different Chilean localities [The alkaloid profile obtained herein fits with other results for Brazilian -authors  describe-workers  also rep-workers ), along rst time . Althougo sample , and difof Spain , as wellcalities .H. papilio, galanthamine (1), was determined herein at a 1286.03 μg GAL/mL concentration, which corresponds to 67.3% of the total identified alkaloid in this species. According to the literature, this alkaloid was firstly isolated from Galanthus woronowii in 1952, and nowadays, it is still obtained by pharmaceutical companies from natural sources of Amaryllidaceae plants, such as Galanthus nivalis, Leucojum aestivum, Lycoris radiata, and different species of Narcissus [3) and 11β-hydroxygalanthamine (6), both belonging to galanthamine-type alkaloids, were also identified in H. papilio samples; however, these compounds were in lower quantities than galanthamine (1), see 3) has been detected in different members of the Amaryllidaceae genus, such as Chlidanthus, Galanthus, Hippeastrum, Narcissus, and Pancratium [6) has been reported in H. papilio samples [As shown in arcissus . Narwedincratium ,43,44,45 samples ,36.2) and 9-O-demethyllycosinine B (5) identified here (H. papilio by Guo and co-authors [H. breviflorum by Sebben and co-authors [4) is a homolycorine-type structure that is reported in The compounds papiline  undergoes hydrolysis through the action of two distinct cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) . The exish brain .Hippeastrum papilio on AChE, it was deemed important to replicate the physiological conditions. Consequently, homogenates of zebrafish brains, which inherently contain AChE, were employed as the enzymatic solution instead of purified AChE enzyme. This approach aimed to approximate the ideal conditions of a physiological medium for the evaluation of AChE inhibition exerted by the alkaloid extract from H. papilio. The utilization of a biologically derived enzymatic system further enhances the translational value of the findings, facilitating the extrapolation of results to potential physiological implications.To assess the potential inhibitory effects of the alkaloid extract from H. papilio presented a very high AChE inhibition activity, with IC50 values of 1.20 ± 0.10 µg/mL, whereas galanthamine (1), which was used as a positive control, showed IC50 values of 0.79 ± 0.15 µg/mL , 3.2% of narwedine (3), and 5.7% of 11β-hydroxygalanthamine (6), which suggested the potential of this plant extract as a great AChE inhibitor  in zebrafish brain homogenate and in human AChE. The study showed IC50 values of 39.0 ± 1.4 for the former and 20.0 ± 1.0 µM for the latter. The authors also used galanthamine as positive control and obtained IC50 values of 3.3 ± 0.5 µM in the zebrafish model [Applying a similar methodology, Pitchai and co-authors  evaluatesh model .50 galanthamine value necessary for activity against purified Electrophorus electricus AChE (eeAChE) is about 0.50 μg/mL, which correspond to approximately 1.7 μM [3) and 11β-hydroxygalanthamine (6) show inhibitory activity against eeAChE, with IC50 values of 9.38 and 3.49 µM, respectively [50 values of 1.61 and 1.07 μM, respectively [Previous investigations have described that when using the classical methodology of Ellman and co-workers  and Lópey 1.7 μM . Some auectively . Furtherectively .Hieronymiella marginata (Amaryllidaceae) extract against eeAChE, suggesting that its inhibitory activity  could be related to the presence of sanguinine in this sample; galanthamine, used as control, showed IC50 values of 1 ± 0.05 μM [According to the literature, different extracts of Amaryllidaceae species have presented anti-cholinesterase potential, which is usually correlated with the presence of galanthamine-type alkaloids in these samples . Researc 0.05 μM .According to the available literature, galanthamine exerts its effects on human acetylcholinesterase (AChE) in a dual manner, functioning as both a competitive and reversible inhibitor ,53. AddiDanio rerio (Uniprot: Q9DDE3) was constructed. This model serves as a valuable tool for simulating and investigating the specific binding interactions and molecular mechanisms underlying the galanthamine–AChE interaction within the zebrafish brain.To gain deeper insights into the interactions between galanthamine and the AChE enzyme within zebrafish brain homogenate, computational experiments were conducted. Consequently, a computational model of the AChE enzyme from D. rerio AChE active site is shown in H. papilio and the active site of DrAChE. The molecular docking of galanthamine on human AChE (PDB: 4EY7) shows that galanthamine presents a hydrogen bond with Ser203 interaction that plays an important role in this enzymic active site [According to the docking results, galanthamine binds to the zebrafish AChE active site with an estimated free binding energy of −9.3 kcal/mol see . We obseive site ,55. The Hippeastrum papilio, which demonstrated significant galanthamine content by GC-MS, exhibited a notable inhibitory effect in zebrafish brain homogenates.In summary, substantial efforts have been dedicated to exploring the acetylcholinesterase (AChE) inhibitory potential of Amaryllidaceae species in recent years by employing purified enzymes from various organisms. In this study, we aimed to replicate the ideal conditions of a physiological medium by utilizing zebrafish brain homogenate as a substitute for the purified AChE enzyme. The alkaloid extract obtained from Through the implementation of a computational model for zebrafish AChE, a deeper understanding of the interactions between galanthamine and the amino acid residues within the active site of this enzyme was attained. The insights gained from this computational analysis provided valuable information about the specific molecular interactions underlying the inhibitory activity of galanthamine in zebrafish AChE.These findings contribute to the broader understanding of Amaryllidaceae alkaloids and their interactions with AChE, highlighting the potential utility of zebrafish as an effective model for the preliminary evaluation of acetylcholinesterase inhibitory activity and paving the way for further investigations and subsequent preclinical studies.


The large Kuiper Belt object Eris is tidally locked to its small companion Dysnomia. Recently obtained bounds on the mass of Dysnomia demonstrate that Eris must be unexpectedly dissipative for it to have despun over the age of the solar system. Here, we show that Eris must have differentiated into an ice shell and rocky core to explain the dissipation. We further demonstrate that Eris’s ice shell must be convecting to be sufficiently dissipative, which distinguishes it from Pluto’s conductive shell. The difference is likely due to Eris’s apparent depletion in volatiles compared with Pluto, perhaps as the result of a more energetic impact. The dwarf planet Eris melted early in its history and still hosts a warm, convecting ice shell. New Horizons spacecraft and revealed a dynamic and variegated world  provide information on the rock:ice ratio . The rate at which the spin/orbital evolution happens depends on the Q is constant but relax this assumption below. The nominal values used are given in Rp , where Rp is the primary radius, as representative of the immediate postimpact situation; the results below are insensitive to this parameter unless very wide initial separations are assumed. The results are also insensitive to the value of Q assumed for Dysnomia.Equations describing the spin and orbital evolution of a binary system have been derived by various authors (Q/k2 is 3200. An earlier synchronization is possible but would require a lower (more dissipative) Q/k2.Numerical integration of the , where Ω is the spin frequency of Eris and n is the mean motion (see below). In this case, Eris will have become progressively more dissipative as it approached synchronous rotation. To obtain the same 4.5 Ga synchronization time scale as before but with Q varying with the forcing frequency, we need to set the initial Q/k2 to be 6300, when the forcing period is 165 hours.An advantage of the (Q for Eris varies linearly with forcing frequency. The spin and orbit curves (solid lines) are identical to the corresponding curves in (Q/k2) for Eris, showing that Eris becomes more dissipative as synchronous rotation is approached. As a result of the time-varying Q, the rate of outward evolution is nonmonotonic; the time-averaged Q/k2 is 5500, which is less than a factor of 2 different from the constant-Q value derived above. The advantage of the variable-Q model is that it is easier to interpret in terms of internal structure models, as discussed below.−3 and start at 9 Rp, then the time-averaged Q/k2 for Eris is 3500. Thus, our estimates of Eris’s dissipation are conservative.Because we have only an upper bound on Dysnomia’s mass, Eris could be more dissipative than our baseline calculations indicate. For instance, if we make Dysnomia’s density 500 kg m−3 , in particular in the deep past when heat production was higher. However, the central temperature is also about 500 K below the melting temperature of rock, which would therefore have a viscosity of order 1028 Pa s. Such high viscosities imply negligible dissipation.Since Q may be compared with Mars and the Moon, which have Q values of ≈80 and ≈35, respectively . The critical Rayleigh number for ice, which has a strongly temperature-dependent viscosity, is about 5 × 106 for a Cartesian geometry (−2) from the silicate interior. Our orbital inference of a warm ice shell is therefore consistent with the physical requirements for convection to occur.The natural way to explain a warm ice shell beneath a rigid lid is if the ice is convecting. In this situation, the bulk of the shell will be of a roughly constant viscosity, which must be sufficiently low that convection can occur. Quantitatively, the Rayleigh number  is roughly 10F = 5 mWm−2 and a mean ice thermal conductivity k = 4 Wm−1 K−1 (The presence of an assumed 30-km-thick lithosphere is consistent with the idea of ice shell convection (3) were present. Heat fluxes will have been higher in the past (due to enhanced radiogenic heating and stored energy of accretion). However, in the case of Pluto, it has been shown that ice shell convection can remove heat efficiently enough that an ocean never forms . In this case, the tendency of Q/k2 to decrease with time owing to the increasing forcing period  than that of Eris . Since Dysnomia’s small size leads to a smaller k2 (Q (it will be colder), this requirement is plausible.We have also neglected eccentricity evolution. A nonzero present-day eccentricity for Dysnomia was reported by  system for simplicity. If KBOs resemble comets, they could contain substantial fractions of carbonaceous material  would be hard to reconcile with our model of a convecting ice shell. Future measurements of the shape of Eris would thus be of great interest.Last, while our results do not require a subsurface ocean, one possible consequence of such an ocean would be ocean pressurization and possible cryovolcanism as freezing progressed  and j to the secondary (Dysnomia). We assume uniform density bodies such that the normalized C = 0.4. These equations are integrated using a simple first-order finite-difference scheme. For the frequency-dependent Q case, we specify Q at an initial angular forcing period and assume that Q−1 varies linearly with forcing period. The forcing period is calculated as 2π(Ω − n)−1.Here The Rayleigh number for a fluid with a strongly temperature-dependent viscosity is given by .Here ρ is density, α is thermal expansivity, ΔΤ is the temperature contrast across the fluid, Qloc is given by J1/2J, where we defineMcCarthy and Cooper (Ju is the unrelaxed compliance (=1/shear modulus), β and ϕ are rheological parameters, Γ is the gamma function, ω is the forcing frequency and η is the steady-state viscosity. For ice, we have β = 4.5 × 10−12 Pa−1 s−0.25, ϕ = 0.25, and the shear modulus E is 3 GPa.Here Qloc as a function of forcing period for Maxwell and Andrade rheologies, where the shortest period in our model  is uncertain but is roughly 40 .We wish to develop a simple expression for how the viscosity and iscosity  and thus


Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3′-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3′-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and ΔCOOLAIRFLC mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process.To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In  Long spells of cold winter weather may feel miserable, but they are often necessary for spring to blossom. Indeed, many plants need to face a prolonged period of low temperatures to be able to flower; this process is known as vernalization.COOLAIR, one of the rare genetic sequences identified as potentially being able to trigger vernalization. COOLAIR is a long noncoding RNA, a partial transcript of a gene that will not be ‘read’ by the cell to produce a protein but which instead regulates how and when certain genes are being switched on. COOLAIR emerges from the locus of the FLC gene, which is one of the main repressors of flowering, and it gradually accumulates in the plant when temperatures remain low for a long period. While some evidence suggests that COOLAIR may help to switch off FLC, other studies have raised some doubts about its involvement in vernalization.While the molecular mechanisms which underpin vernalization are well-known, it is still unclear exactly how plants can ‘sense’ the difference between short and long periods of cold. Jeon, Jeong et al. set out to explore this question by focusing on FLC gene in a range of plants closely related to A. thaliana, and in which COOLAIR also accumulates upon cold exposure. This helped them identify a class of proteins, known as CBFs, which could bind to sequences near the FLC gene to activate the production of COOLAIR when the plants were kept in cold conditions for a while. CBFs were already known to help plants adapt to short cold snaps, but these experiments confirmed that they could act as both short- and long-term cold sensors.In response, Jeon, Jeong et al. examined the COOLAIR levels increase as the cold persists, until changes in the structure of the FLC gene prevent CBF from binding to it and COOLAIR production drops. Unexpectedly, examining the fate of mutants which could not produce COOLAIR revealed that these plants could still undergo vernalization, suggesting that the long noncoding RNA is in fact not necessary for this process.This work allowed Jeon, Jeong et al. to propose a model in which CBF and therefore COOLAIR in vernalization; they also give insight into how coding and noncoding sequences may have evolved together in various members of the A. thaliana family to adapt to the environment.These results should prompt other scientists to further investigate the role of  Appropriate timing of flowering provides evolutionary advantage of plant reproductive success. As sessile organisms, plants have evolved mechanisms through which seasonal cues coordinate the transition to flowering. One of the significant environmental factors affecting flowering time of plants adapted to temperate climates is the temperature changes during the seasons, and plants have evolved complex sensory mechanisms to monitor the surrounding temperature to properly control the timing of flowering and tolerate thermal stress .Arabidopsis thaliana  (cold-regulated (COR) genes by binding to CRT/DREs in their promoters  ; the clo(CAMTAs) ; and the(CAMTAs) . In addi(CAMTAs) , and col(CAMTAs) . Further(CAMTAs) . Low-temromoters . Diverseg stress .A. thaliana is mainly achieved by silencing the floral repressor gene, FLOWERING LOCUS C (FLC)  and FLOWERING LOCUS T , by directly binding to their promoter regions  ecotypes, flowering is prevented by the high expression of FLC before exposure to winter cold (FLC by the FRIGIDA (FRI) supercomplex, which recruits general transcription factors and several chromatin modifiers (FLC chromatin is highly enriched with active histone marks such as histone H3 acetylation and trimethylation of Lys4 or Lys36 at H3 (H3K4me3/H3K36me3) , in the nucleation region around the first exon and intron of FLC (FLC chromatin region by LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which ensures stable FLC suppression and renders plants competent to flower  . FLC enc regions . In Arabter cold . This isodifiers . Prior t3K36me3) . In cont the FLC . Addition of FLC . Subsequo flower .FLC. VERNALIZATION INSENSITIVE 3 (VIN3) family genes, which are upregulated by prolonged cold, encode plant homeodomain (PHD) proteins that recognize H3K9me2 enriched in FLC chromatin during vernalization , and CCA1/LHY act as positive regulators of VIN3 during the vernalization process  domain-containing proteins that can bind to CRT/DRE and are usually present in the promoters of cold- and drought-responsive genes (Arabidopsis cistrome database and the genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) results from a previous study suggested that CBFs bind to the 3′-end sequence of FLC containing CRT/DREs -fused CBF3  failed to compete (FLC locus. Two (DREa and b) were present in the FLC promoter, while the other two (DREc and d), were present in the first and last exons, respectively (FLC locus (We performed an electrophoretic mobility shift assay (EMSA) to confirm this binding using probes harboring CRT/DREs (named DRE1 or 2) from the sed CBF3 . The ban compete . We alsoectively . Only DRectively . The preLC locus .CBF3-overexpressing transgenic plant, pSuper:CBF3-myc , indicating that CBF3 protein can activate FLC under warm conditions, as previously reported  assay using a CBF3-myc , was con located . Withoutreported . Howeverlization .CBF genes are rapidly and transiently induced upon exposure to cold (COOLAIR contains CBF3-binding sites in its promoter (COOLAIR shows CBF regulon-like expression. The expression of genes assigned to the CBF regulon is up- or down-regulated by CBF overexpression under warm conditions (COOLAIR increased in CBF3-overexpressing transgenic plants (pSuper:CBF3-myc) grown at room temperature (22 °C) compared to that in the wild type, Columbia-0 (Col-0)  and distal (type II) variants of COOLAIR were higher in the CBF3 overexpressor than in the wild type, it is likely that COOLAIR transcription, instead of the 3′-processing events, is affected by CBF3 . Such results imply that the three CBFs regulate COOLAIR transcription subtly differently. to cold . CBF reg to cold . As COOLpromoter , we analnditions . Similar (Col-0) . Such up by CBF3 . It has milarity . Thus, w 2, or 3 . Overall plants with 0, 0.5, 1, 1.5, 2, 3, 6, and 24 hr of cold (4 °C), then measured the levels of total COOLAIR. The results showed that cold treatment for less than 3 hr failed to induce COOLAIR expression, although CBF transcripts reached a peak at 3 hr of cold exposure , suggesting that both short-term and vernalizing cold treatments upregulated CBF expression  plants were collected every 4 hr under a short-day photoperiod  under both NV and 40V conditions. As shown in CBF was variable, the overall transcript levels of all three CBFs were much higher at 40V than under NV. Consistent with the transcript level, the level of CBF3 protein in vernalized plants was higher than that in NV plants during all circadian cycles , although their levels remained high until the end of the vernalization period  or long-term (14V) cold, which supports our results , throughout the vernalization period , and then suppressed during the late phase. This result implies that both the transcriptional and post-transcriptional regulations of CBFs are involved in the long-term cold response of COOLAIR.Consistent with the reduced n period . It is nCOOLAIR expression  during the winter season strongly induces pression . Therefoild type . However and 20V . In partFLC (DRE1 and 2)  was used as a reporter. Additionally, co-transfection of the 35S:CBF3-HA effector and pFLC:LUC reporter construct, which harbors the 1 kb sequence with the promoter and first exon of FLC, did not show increased luciferase activity. These results strongly indicate that CBF3 can activate COOLAIR transcription through DRE1 and 2 located in the COOLAIR promoter, but not via the first exon of FLC.Since CBF3 could bind to the CRT/DREs in the first exon (DREc) and the 3′-end of 1 and 2) , we invepromoter . In contFLC is required for cold-induction of COOLAIR expression, we measured COOLAIR expression before and after vernalization in the ΔCOOLAIRFLC mutant (ΔCOOLAIR-1FLC), that lacks a 324 bp portion of the COOLAIR promoter region  and a 13 bp sequence replacement downstream of CRT/DREs, showed a normal vernalization-induced increase in all COOLAIR variants, similar to the wild type. This strongly suggests that CRT/DREs located at the COOLAIR promoter are critical for CBF-mediated COOLAIR induction during vernalization. Notably, the low-abundance convergent antisense transcripts  was still slightly detected by quantitative PCR (qPCR), probably due to alternative TSSs   . Taken tCOOLAIR induced by CBFs in the vernalization-triggered FLC silencing process. COOLAIR was reported to facilitate the removal of H3K36me3 from FLC chromatin during vernalization, whereas the other lncRNAs, COLDAIR and COLDWRAP, are required for H3K27me3 deposition , Wassilewskija-2 (Ws-2), and Col FRISf2 (FRI Col) were used as the wild types in this study. “Wild type” in this paper mainly refers to FRI Col unless otherwise specified. The FRI Col . For the vernalization treatment, the plants were grown at 22 °C for the prescribed period and then transferred to a 4 °C growth chamber under an SD cycle. To adjust the developmental stage, the plants vernalized for 40  days (40V), 30  days (30V), 20  days (20V), 10  days (10V), 4  days (4V), and 1  day (1V) were transferred to 4 °C after being grown at 22 °C for 10, 12, 14, 15, 15, and 16  days, respectively. Non-vernalized (NV) plants were grown at 22 °C for 16  days. For 0.5, 1, 1.5, 2, 3, 6, and 24 hr of cold treatment, the plants were grown at 22 °C for 16 days before the 4 °C treatment. For the freezing treatment, NV, 10V, and 20V plants were transferred to −1 °C under dark condition for 8 hr (F). To measure flowering time of vernalized plants, plants were transferred to 22 °C under a long-day (LD) cycle (16 hr light/8 hr dark) directly after the vernalization treatment.For all experiments using plant materials, seeds were sown on half-strength Murashige and Skoog (MS) medium  containing 1% sucrose and 1% plant agar (Duchefa) and stratified at 4 °C for 3  days. Plants were germinated and grown at 22 °C under a short-day (SD) cycle (8 hr light/16 hr dark) with cool white fluorescent lights , and sgRNA-Cas9 cassettes were introduced into Arabidopsis by floral dipping. Homozygous deletion mutants were screened in T2 generation, and sgRNA-Cas9 free mutants were further isolated in T3 generation.The construction of escribed . Briefly35S:CBF3-HA construct, the entire coding sequence of CBF3 was amplified using PCR and cloned into the HBT-HA-NOS plasmid , and the last exon of FLC was amplified by PCR and cloned into the LUC-NOS plasmid  strain. Cell extracts were isolated with a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 200 mM NaCl, 10% glycerol, 1 mM DTT, and 1 mM PMSF. Proteins were purified from cell extracts using MBPTrap HP column  with ÄKTA FPLC system . The Cy5-labeled probes and unlabeled competitors were generated by annealing 25 bp-length oligonucleotides. Electrophoretic mobility shift assay (EMSA) was performed as previously described with a few modifications  and MBP-CBF3 recombinant fusion proteins were induced by 500 μM isopropyl β-D-thiogalactoside (IPTG) in the ications . For eac2, 0.1 mM EGTA, 15 mM NaCl, 60 mM KCl, 0.5% Triton X-100, 5 mM β-mercaptoethanol, and 1×cOmplete EDTA-free Protease Inhibitor Cocktail  as described previously with a few modifications  were used to detect CBF3 protein enrichment at the FLC locus, and anti-H3K36me3 , anti-H3K4me3 , and anti-H3K27me3  were used to detect histone modifications on the FLC chromatin. DNA was extracted with phenol:chloroform:isoamyl alcohol  or Chelex 100 Resin according to the manufacturer’s instruction.Approximately 4 g of seedlings grown on MS plates was collected and cross-linked using 1% (v/v) formaldehyde. Nuclei were isolated from seedlings using a buffer containing 20 mM PIPES-KOH (pH 7.6), 1 M hexylene glycol, 10 mM MgClications . Isolate18. Quantitative PCR analysis was performed using iQ SYBR Green Supermix  and a CFX96 Real-Time PCR system (Bio-Rad).Total RNA was isolated from –100 mg of seedlings grown on MS agar plate using TRI Reagent  or TS Plant Mini Kit . cDNA was generated using 4  μg of total RNA, 5 units of recombinant DNase I , 200 units of RevertAid reverse transcriptase , and buffer containing 0.25 mM dNTP and 0.1 μg oligo(dT)COOLAIR expression in the COOLAIR promoter deletion lines, Total RNAs were extracted using the RNeasy Plus Mini Kit  according to the manufacturer’s instructions, followed by the digestion of residual genomic DNA by the gDNA wiper . 1.0 µg RNA from each sample was taken for cDNA synthesis using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme) with the transcript-specific primers . Fifty micrograms of total protein were loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were transferred to PVDF membranes (Amersham Biosciences) and probed with an anti-myc  antibody overnight at 4 °C. The samples were then probed with horseradish peroxidase (HRP)-conjugated anti-mouse IgG  antibody at room temperature. The signals were detected with ImageQuant LAS 4000  using WesternBright Sirius ECL solution .Total protein was isolated from –100  mg of seedlings using a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgClcbfs plants in a Sweden-ecotype (SW) background  data deposited in the National Center for Biotechnology Information (NCBI) under BioProject accession codes PRJNA416120 and PRJNA732005. PRJNA416120 contains raw reads from NV, 1V, and 14V wild-type and ckground . PRJNA73ckground . The rea2, 5 mM β-mercaptoethanol, and 0.1 g bovine serum albumin. For protoplast transfection, 200 μg of plasmid DNA and isolated protoplasts were incubated in a buffer containing 0.1 M D-mannitol, 50 mM CaCl2, and 20% (w/v) PEG. Luciferase activity in the protoplasts was measured using the Luciferase Assay System  and MicroLumat Plus LB96V microplate luminometer .Protoplast isolation and transfection were performed as previously described with some modifications . Protopl This important work advances our understanding of the systems that plants evolved to coordinate developmental processes such as the timing of flowering with seasonal change, particularly with respect to the regulation and role of long non-coding RNAs (lncRNAs) complementary to genes encoding proteins that regulate developmental switches. The evidence supporting the conclusions is solid. The work will be of interest to those interested plant development as well those interested in the role and regulation of lncRNAs. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Richard Amasino as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jürgen Kleine-Vehn as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Iain Searle (Reviewer #2); Chris A. Helliwell (Reviewer #3).Thank you for submitting your article "Vernalization-triggered expression of the antisense transcript COOLAIR is mediated by CBF genes" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:I would like you to respond to the following points of one of the reviewers.1. It wasn't clear why the pCBF3:CBF3-myc lines were not used for ChIP assays to look at the binding of CBF3 to the COOLAIR promoter, this would allow a more realistic assessment of CBF3 interaction with the COOLAIR promoter under native conditions.2. Figure 4. Why isn't the same scale used for the COOLAIR expression levels in parts A and B of this Figure? This would allow more direct comparison between the CBF3 over-expressors, wild type, and cbf1 mutants.3. Figure 5. Do the authors have a qPCR assay for the various COOLAIR transcripts rather than the semi-quantitative gel-based assay used in this figure?4. Figure 5. The conclusions of the deletion analysis would be stronger if specific deletions of the actual CRT/DRE elements were made, and this is likely to be possible with gene editing techniques.5. I wasn't sure that Figure 8 really added anything to the paper as there isn't any new information on the actual function of COOLAIR.Reviewer #1 (Recommendations for the authors):Nothing to add. The work seems solid. I understand it has been submitted before so in this version the authors have addressed previous concerns.Reviewer #2 (Recommendations for the authors):There is very compelling data presented to support the manuscript's conclusions. Unusually, I have no suggestions for further experiments to support the paper's conclusions.I found the manuscript very clearly written, the data elegantly presented, and no typographical errors.The authors should be commended on the quality of the presented experimental data, and for challenging a widely accepted model by the research community.Reviewer #3 (Recommendations for the authors):1. It wasn't clear why the pCBF3:CBF3-myc lines were not used for ChIP assays to look at the binding of CBF3 to the COOLAIR promoter, this would allow a more realistic assessment of CBF3 interaction with the COOLAIR promoter under native conditions.2. Figure 4. Why isn't the same scale used for the COOLAIR expression levels in parts A and B of this Figure? This would allow more direct comparison between the CBF3 over-expressors, wild type, and cbf1 mutants.3. Figure 5. Do the authors have a qPCR assay for the various COOLAIR transcripts rather than the semi-quantitative gel-based assay used in this figure?4. Figure 5. The conclusions of the deletion analysis would be stronger if specific deletions of the actual CRT/DRE elements were made, this is likely possible with gene editing techniques.5. I wasn't sure that Figure 8 really added anything to the paper as there isn't any new information on the actual function of COOLAIR.6. Given the differences in responses observed between this study and other it would have been informative if experiments (particularly to measure FLC and flowering time) had been carried out in different growth conditions such as in common gardens or cabinets programmed to mimic natural conditions. This may have been helpful in identifying conditions where COOLAIR transcription is required or reinforced the conclusion that it is not an essential component of the vernalization response. Essential revisions:I would like you to respond to the following points of one of the reviewers.1. It wasn't clear why the pCBF3:CBF3-myc lines were not used for ChIP assays to look at the binding of CBF3 to the COOLAIR promoter, this would allow a more realistic assessment of CBF3 interaction with the COOLAIR promoter under native conditions.COOLAIR promoter during vernalization. The cold-triggered changes in CBF3 transcript level in pCBF3:CBF3-myc would make us difficult to simply address this. We, therefore, thought the pSuper:CBF3-myc line is more proper than pCBF3:CBF3-myc for ChIP assays to check the CBF3 protein binding to the COOLAIR promoter regardless of the changes in CBF3 transcript levels. To make this point clear, we have added this statement into the Result section (line no. 164-167).We aimed to show the binding affinity of CBF3 protein to the 2. Figure 4. Why isn't the same scale used for the COOLAIR expression levels in parts A and B of this Figure? This would allow more direct comparison between the CBF3 over-expressors, wild type, and cbf1 mutants.cbfs mutant is in the FRI Col background, while the pSuper:CBF3-myc line is in Col. The basal COOLAIR level is known to be affected by the presence/absence of a functional FRI allele . Thus, it is not directly comparable for the COOLAIR levels among cbfs, wild type (Col and FRI-Col), and pSuper:CBF3-myc.The 3. Figure 5. Do the authors have a qPCR assay for the various COOLAIR transcripts rather than the semi-quantitative gel-based assay used in this figure?COOLAIR variant levels in ΔCOOLAIRFLC lines. However, as the COOLAIR levels in the ΔCOOLAIR-1FLC and ΔCOOLAIR-3FLC lines were too low to reliably detect by RT-qPCR and did not increase by 14-d cold treatment, we presented the data as DNA gel images. Instead, we have added the RT-qPCR results for CAS and unspliced FLC levels in ΔCOOLAIRFLC lines as shown in Figure 5—figure supplement 2.We performed RT-qPCR assays to assess 4. Figure 5. The conclusions of the deletion analysis would be stronger if specific deletions of the actual CRT/DRE elements were made, and this is likely to be possible with gene editing techniques.We agree that the specific mutations of CRT/DREs would provide stronger evidence. However, unfortunately we were unable to generate the mutant and test its vernalization response on time. Instead, the results of luciferase assay using the reporter with specific mutations in CRT/DREs shown in Figure 5A may partially address the concern.5. I wasn't sure that Figure 8 really added anything to the paper as there isn't any new information on the actual function of COOLAIR.We agree that Figure 8 does not have any new information. But we believe the model in Figure 8 summarizing our results would allow readers to catch the take home message more easily and clearly.Reviewer #1 (Recommendations for the authors):Nothing to add. The work seems solid. I understand it has been submitted before so in this version the authors have addressed previous concerns.We appreciate for the positive assessment.Reviewer #2 (Recommendations for the authors):There is very compelling data presented to support the manuscript's conclusions. Unusually, I have no suggestions for further experiments to support the paper's conclusions.I found the manuscript very clearly written, the data elegantly presented, and no typographical errors.The authors should be commended on the quality of the presented experimental data, and for challenging a widely accepted model by the research community.Thank you for the positive evaluations and encouragement.Reviewer #3 (Recommendations for the authors):1. It wasn't clear why the pCBF3:CBF3-myc lines were not used for ChIP assays to look at the binding of CBF3 to the COOLAIR promoter, this would allow a more realistic assessment of CBF3 interaction with the COOLAIR promoter under native conditions.COOLAIR promoter throughout the vernalization period. The vernalization-induced increase of CBF3 transcript level in pCBF3:CBF3-myc would make us difficult to simply address this. We, therefore, thought the pSuper:CBF3-myc line is more proper than pCBF3:CBF3-myc for ChIP assays. To make this point clear, we have added this statement into the Result section (line no. 164-167).We deeply agree with your concern that using endogenous promoter would provide a more realistic assessment of CBF3 interaction. However, what we actually aimed to show in Figure 1C was a change in the binding affinity of CBF3 protein to the 2. Figure 4. Why isn't the same scale used for the COOLAIR expression levels in parts A and B of this Figure? This would allow more direct comparison between the CBF3 over-expressors, wild type, and cbf1 mutants.cbfs mutant in Figure 4A is in FRI Col background, while pSuper:CBF3-myc line shown in Figure 4B is in Col background. It is already known that the presence/absence of a functional FRI allele affect the basal COOLAIR level . Hence, COOLAIR levels among three lines were not directly comparable.The 3. Figure 5. Do the authors have a qPCR assay for the various COOLAIR transcripts rather than the semi-quantitative gel-based assay used in this figure?COOLAIR variants expressed in ΔCOOLAIRFLC lines. However, as COOLAIR levels in the ΔCOOLAIR-1FLC and ΔCOOLAIR-3FLC lines were too low to reliably detect by RT-qPCR and did not increase by 14-d cold treatment, we presented the data as DNA gel images. Instead, we have added the RT-qPCR results for CAS and unspliced FLC levels in ΔCOOLAIRFLC lines as shown in Figure 5—figure supplement 2.Yes, we performed RT-qPCR assays for the 4. Figure 5. The conclusions of the deletion analysis would be stronger if specific deletions of the actual CRT/DRE elements were made, this is likely possible with gene editing techniques.Thank you for the valuable suggestion. We agree that the specific mutations of CRT/DREs would provide stronger evidence to our claims. However, we were unable to generate the mutant and test its vernalization response on time. Instead, the result of luciferase assays using the reporter with specific mutations in CRT/DREs shown in Figure 5A may partially address the concern.5. I wasn't sure that Figure 8 really added anything to the paper as there isn't any new information on the actual function of COOLAIR.COOLAIR during vernalization, summarizing all the results shown in the paper. Of course, this figure does not provide any additional information, as you pointed out. But we believe this will enable a broad range of readers to understand this paper more clearly.Figure 8 is presenting a model of how cold-induced CBFs activate 6. Given the differences in responses observed between this study and other it would have been informative if experiments (particularly to measure FLC and flowering time) had been carried out in different growth conditions such as in common gardens or cabinets programmed to mimic natural conditions. This may have been helpful in identifying conditions where COOLAIR transcription is required or reinforced the conclusion that it is not an essential component of the vernalization response.COOLAIR for vernalization by carrying out the experiments under a natural condition or natural environment-mimicking conditions. However, under our current situation, such environmental conditions cannot be implemented. We are sorry but we have to leave it as a future interest.We appreciate this insightful comment. As we briefly described in the Discussion section of the manuscript, it would be worthwhile to reexamine the role of