This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly Since then, studies have been conducted extensively to elucidate its structure along with function3. This 459-amino acid transporter protein has 12 transmembrane domains (TMDs) with N- and C-termini located inside the cell5. It has two N-glycosylated Asn residues (Asn58 and Asn68), which are located in the first extracellular loop between TMDs 1 and 25. The first intracellular loop (IL) between TMDs 2 and 3 has been suggested to be a functionally important reentrant loop having a β-turn structure formed by a DXXGRR sequence6, which begins at Asp109. In the loop, Asp109, Gly112, and Arg113 have been identified to be of particular importance10. For many of the TMDs, their roles in the structure and/or function of hPCFT have been identified, which include TMDs 3 and 6 that play the major role in its homo-oligomerization13, TMDs 2 and 4 that assist the oligomerization by the function of GXXXG motifs (G93XXXG97 in TMD2 and G155XXXG159 in TMD4)13, TMDs 2, 4, and 5 that are involved in substrate binding and translocation14, and TMDs 1, 2, 7, and 11 that forms an extracellular gate15. In addition, it has been suggested that TMDs 1, 3, 4, and 6 may potentially form a hydrophobic cavity involved in substrate binding10. Topologically, Gly40/Pro41 in TMD 1, Gly158/Gly159 in TMD 4, Pro314 in TMD 8, and Gly361/Tyr362/Gly363 in TMD 10 have been indicated to be potential break points, which are likely to play an important role in the conformational change2. Amino acid residues that are of particular importance have also been identified in TMDs, which include Glu185 in TMD 5 for proton coupling16, His281 in TMD 7 for proton and substrate binding17, Arg376 in TMD 10 for assisting proton and substrate binding18. Intracellularly located Ser172 (IL2) and His247 (IL3) have been suggested to be in interaction and together positioned to impact on hPCFT function17. There are also quite a few amino acid residues of which the mutations result in the impairments of hPCFT function linked to hereditary folate malabsorption2.Human proton-coupled folate transporter (hPCFT/SLC46A1) was identified about a decade ago as the transporter that operates for the epithelial uptake of folate (vitamin B9) and analogues in the intestine21. The extensive and sustained inhibition of hPCFT by this flavonoid, which is widely present in vegetables and fruits22, raises a concern that it could potentially lead to harmful impairment of the intestinal absorption of those PCFT substrates. In beverages rich with myricetin, its concentration could be high enough, as typically reported to be 60 μM in red grape wine22, to induce the sustained inhibition, in which almost complete inhibition could be attained at its concentration of around 50 to 100 μM21. Thus, attention needs to be paid to the myricetin-induced hPCFT inhibition as one of potential drug interaction cases. Myricetin could be one of those flavonoids that act as perpetrators to inhibit enzymes or transporters involved in the disposition of drugs and nutrients27.hPCFT has recently been found to be extensively inhibited by myricetin by a sustaining mechanism that persists after its removalmax) was reduced20. It is notable, however, that the Michaelis constant (Km) was reduced at the same time, attenuating the inhibition at lower folate concentrations. Since the expression of hPCFT, which was tagged with green fluorescent protein (GFP) for detection and stably introduced into MDCKII cells, was unchanged, being highly localized to the cellular membrane, it could be hypothesized that hPCFT undergoes some kind of myricetin-induced modulation to be less efficient for the translocation of folate bound with increased affinity. It should also be noted that the inhibition is of a sustained type, but can be reversed in a few hours20. Moreover, the inhibitory action of myricetin was suggested to be quite specific to hPCFT, as its orthologue in rat (rPCFT) and human riboflavin transporter 3 (hRFVT3/SLC52A3), which operates for the epithelial uptake of riboflavin (vitamin B2) in the intestine, were found not to be inhibited21.Although the sustaining mechanism of the myricetin-induced inhibition is yet to be fully clarified, kinetic analyses of the myricetin-induced inhibition of the uptake of folate in stably hPCFT-transfected Madin-Darby canine kidney II (MDCKII) cells and in the Caco-2 cell line, which is a well recognized intestinal epithelial cell model, consistently indicated that the maximum transport rate  cells at the low and high folate concentrations of 5 nM and 5 μM, respectively, in the absence of myricetin after preincubation for 60 min in its presence at 100 μM. Those folate concentrations were previously determined to evaluate the approximate estimates of Vmax/Km, the transport coefficient in the linear phase of transport kinetics, and Vmax, respectively, for hPCFT and rPCFT, based on their Km values of about 2 μM for folate30. Those estimates were used to assess myricetin-induced shifts in Vmax and Km, where the latter could be estimated by dividing Vmax by Vmax/Km. The uptake period was set to be 2 min, which was previously determined for the evaluation of the initial folate uptake by those PCFTs, and the medium condition was set to be pH 5.5, which was also determined previously for their optimal operation30.To assess the sustained effect of myricetin with the minimal and essential kinetic characterization, the initial uptake of folate  to start uptake in the absence of myricetin. All the procedures were conducted at 37 °C. After uptake assays were stopped by addition of ice-cold modified Hanks’ solution (2 ml), the cells were washed with the same solution and, then, solubilized to determine the associated radioactivity by liquid scintillation counting for the evaluation of folate uptake. The specific uptake of folate was estimated by subtracting its uptake in mock cells from that in transfectant cells. The uptake was normalized to cellular protein content, which was determined by the bicinchoninic acid (BCA) method , using bovine serum albumin as the standard.Uptake assays were conducted as described previouslyt-test was used for statistical analysis, where the difference in means was considered significant when the p-value was less than 0.05.Student’s Supplementary information


Brassica vegetables. Among 11 tested compounds, phenyl isothiocyanate and its derivatives showed the most promising inhibitory activity. 2-Methoxyphenyl ITC showed best inhibition on acetylcholinesterase with IC50 of 0.57 mM, while 3-methoxyphenyl ITC showed the best inhibition on butyrylcholinesterase having 49.2% at 1.14 mM. Assessment of the antioxidant efficacy using different methods led to a similar conclusion. The anti-inflammatory activity was also tested using human COX-2 enzyme, ranking phenyl isothiocyanate, and 3-methoxyphenyl isothiocyanate as most active, with ∼99% inhibition at 50 μM.Finding a new type of cholinesterase inhibitor that would overcome the brain availability and pharmacokinetic parameters or hepatotoxic liability has been a focus of investigations dealing with the treatment of Alzheimer’s disease. Isothiocyanates have not been previously investigated as potential cholinesterase inhibitors. These compounds can be naturally produced from their glucosinolate precursors, secondary metabolites widely distributed in our daily  Brassica vegetables which encompass many species used in daily diets .Alzheimer’s disease is the most common neurodegenerative disorder in western societies, mostly affecting elderly population. The most prominent symptom includes decrease in cognitive function, which in turn leads to changes in the behavioural patterns of an individual. Both acetylcholinesterase and butyrylcholinesterase still represent the only pharmacotherapy able to affect the increase of the acetylcholine neurotransmitter in the brain,in vivo.Oxidative stress is most commonly associated with the development of many diseases, including neurodegenerative disordersvia the activation of Nrf2 and inhibition of NF-κB,,,,,Antioxidant and anti-inflammatory effects of ITCs are likely mediated In the present study, 11 commercially available ITCs were tested for their cholinesterase inhibitory (using Ellman’s method), and antioxidant  activities as well as anti-inflammatory activity via PGE-2 synthesis suppression through COX-2 inhibition.1–11 were purchased from Sigma-Aldrich GmbH . Acetylcholinesterase , butyrylcholinesterase , acetylthiocholine iodide (ATChI), butyrylthiocholine iodide (BTChI), and 5,5-dithiobis(2-nitrobenzoic acid) , were also purchased from Sigma-Aldrich. The human COX-2 inhibitor screening assay kit was purchased from Cayman Chemical . Indomethacin (purity 99%) was obtained from Fluka . Absorbance and fluorescence measurements were performed on a Synergy HTX S1LFA multi-mode microplate reader .All reagents and solvents used were of analytical grade. Isothiocyanates Assessment of the antioxidant activity was carried out using five methods: scavenging ability of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙), Oxygen Radical Absorbance Capacity (ORAC), Briggs–Rauscher (BR) oscillating reaction, Rancimat assay and Ferric Reducing/Antioxidant Power (FRAP) method.50 values (where possible) and inhibition percentages of DPPH radical (% inhibition).DPPH scavenging ability of the samples was measured according to a recently reported procedureORAC assay was performed according to the slightly modified procedure described by Prior et al.ITCs ability to stop the Briggs–Rauscher (BR) oscillations was assayed as described previouslyT = 1.4 °C) and a constant air flow of 20 L/h. The conductivity was measured as a function of time and the results are expressed as Protection Factor (PF), calculated according to the equation PF = sample induction time/control induction timeFish oil was investigated using a Rancimat 743  instrument in order to monitor the progress of accelerated oxidation. The fish oil samples (3 g) were exposed to a temperature of 120 °C -s-triazine (TPTZ) to form a Fe2+-TPTZ complex absorbing at 593 nm.The reducing potential of ITCs was measured as described by Benzie and StrainAChE/BChE inhibitory activity measurements were carried out by a slightly modified Ellman assay as described before for AChE inhibitory activityTo analyse the inhibition of the COX-2 enzyme activity mediated by the selected ITCs, the COX-2 (human) inhibitor screening assay kit from Cayman was used. In summary, the COX-2 enzyme was incubated with the samples  for 10 min at 37 °C and subsequently the reaction was initiated by adding arachidonic acid and incubating the mixture for 2 min at 37 °C. The background tubes correspond to inactivated COX-2 enzyme obtained after keeping the tubes containing enzymes in boiling water for 3 min along with sample solvent. Enzyme catalysis was blocked by adding saturated stannous chloride solution. Prostaglandin (PGF2α) release was quantified using ELISA at 412 nm. Indomethacin solution  was used as a positive control.Structures of isothiocyanates (ITCs) tested on different biological activities are shown in 50) was calculated for phenyl ITC (1), 3-methoxyphenyl ITC (8), 4-methoxyphenyl ITC (6), 4-methylphenyl ITC (5), and 2-methoxyphenyl ITC (7) having IC50 of 1.08, 1.16, 1.25, 1.45 and 3.90 mM, respectively. 4-Methylsulfanylphenyl ITC (4) had to be tested at lower concentration due to turbidity problems, with 1.19 mM  showing 9.9% of DPPH inhibition. Montaut et al.50 of 9.43 mM. This compound, having OH group as a strongly activating substituent on the aromatic ring, showed a much weaker activity than phenyl ITC (1). 3-Methoxyphenyl ITC (8), 4-methoxyphenyl ITC (6), 4-methylphenyl ITC (5), and 2-methoxyphenyl ITC (7), bearing a substituent on the aryl ring, also exhibited a decrease in the DPPH activity, thus indicating a negative effect of substitution. When the ITC group was not directly attached on the aryl ring like in benzyl ITC (2) and 2-phenylethyl ITC (3), the free radical scavenging ability also rapidly decreased.The free radical scavenging potential of ITCs was determined by DPPH, ORAC, and BR antioxidant assays . For praS-addition of the ITC, hence increasing the overall efficiency of the cascade reaction. In connection with the results of Benati et al.5–8) bearing electron-donating groups showed a decrease in free radical scavenging ability when compared to “group free” phenyl ITC (1).Benati et al.11), allyl ITC (9), and isopropyl ITC (10) were also tested and they were found to be very inefficient scavengers at 4.76 mM having 12.6, 10.1 and 4.2%, respectively. Montaut et al.50 of 57.1 mM. 4-(Methylsulfanyl)but-3-enyl ITC (raphasatin) was reported to have a high reactivity toward the DPPH radical, presumably due to hydrogen atom abstraction either in allylic position, or in alpha to the –NCS group or both50 of 3.8 mM3-(Methylsulfanyl)propyl ITC (11) in the side-chain and arylalkyl ITCs (like 2 and 3) are expected to react with radicals by H-atom transfer from the methylene group neighbouring the S-atom or the phenyl ring,2, 3 and 11 showed very insignificant antiradical activity. Similarly, ITC 3 was reported by Sarkar et al.Alkyl ITCs bearing a sulfanyl group . The activity of this compound was not detected by DPPH method due to turbidity problems, while the ORAC method circumvents turbidity problems since it is based on measurement of fluorescence. Direct connection of the –NCS group to the aromatic ring resulted in good activity of the ITCs, as previously observed, while in the case of compounds 2 and 3 a significant decrease in the activity was detected. The lowest activity was found for 2-methoxyphenyl ITC (7) and isopropyl ITC (10), as well as for arylalkyl ITCs 2 and 3. Among methoxyphenyl ITCs  the highest free radical scavenging activity was detected for 3-methoxyphenyl ITC (8) while among alkyl ITCs bearing a sulfanyl group, a remarkable activity was observed for 3-(methylsulfanyl)propyl ITC (11) (20.3 mM TE).ORAC assay is a method that combines both inhibition percentage and inhibition time of the reactive species action by antioxidants into a single quantity6) that prolonged oxidation for 9180 s at final concentration of 0.81 mM. However, the ortho- (7) and meta- (8) regioisomers of 6 did not exhibit activity at four times higher concentration. Chen et al.The Briggs–Rauscher assay gave more or less no activity for any of the ITCs at the highest tested concentration except for 4-methoxyphenyl ITC (2 (III) and any species able to reduce it to Fe(TPTZ)2 (II), making this species an antioxidantITCs were also tested using FRAP assay, which is presumed to estimate the “total antioxidant power”. The relevant chemical reaction of the FRAP method represents the “total reducing power” as it involves a single electron reaction between Fe(TPTZ)1) with PF = 2.5 at 3.23 mM. For ITCs in which the –NCS group is not directly attached to the aromatic ring, the activity is decreasing (compared to 1) as seen for compounds 2 (PF = 1.5) and 3 (PF = 1.3) at the same concentration. Some of the ITCs – either alkyl or aryl – exhibited activities close to that of 2, i.e. allyl ITC (9), 3-methoxyphenyl ITC (8), 2-methoxyphenyl ITC (7), with PF = 1.7, and 3-(methylsulfanyl)propyl ITC (11) with PF = 1.6 at 3.23 mM. 4-Methylsulfanylphenyl ITC (4), 4-methylphenyl ITC (5), 4-methoxyphenyl ITC (6), and isopropyl ITC (10) did not show any protective effect.The antioxidant activity of ITCs was also tested by means of a Rancimat apparatus, using a lipophilic system at 120 °C with an air flow rate of 20 L/h. The results are expressed as protection factors (PF), taking PF = 1 for the control sample. The best activity was found for phenyl ITC . The best activity was shown by 3-methoxyphenyl ITC (8) with 61.4% of AChE inhibition at 1.14 mM. Para- (6) and ortho- (7) isomers of ITC 8 also showed relatively good activity with 30.4 and 57.0%, respectively, at the same concentration. The inhibitory activity of 4-methylphenyl ITC (5) on AChE was comparable to that of 7 and 8 with 58.4% at 1.14 mM. Electron donating groups  seem to contribute to the increase of inhibitory activity when compared to phenyl ITC (1) which showed only 17.9% at the same concentration. In the case of arylaliphatic ITCs 2 and 3, the inhibitory activity was also relatively good with 37.2 and 48.1%, respectively, at 1.14 mM.To the best of our knowledge, the present paper reports the first investigation on cholinesterase (ChE) inhibitory activity of ITCs. The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was assessed for 6 aromatic, 2 arylaliphatic, and 3 aliphatic ITCs and results are reported in 1 and 11. As observed in the case of AChE, the inhibitory activity of the aromatic ITC 8 towards BChE was shown to be the best with 49.2% at 1.14 mM. Closely related aryl ITCs 1 and 5 also showed notable activity with 42.1 and 40.7%, respectively, at the same concentration. In comparison to 1, the inhibitory activity towards BChE is similar  or inferior when substituents are present on the aromatic ring , or when the –NCS group is not directly attached to the aromatic ring (2). Aliphatic ITCs 9–11 showed very weak activity or no inhibition at all tested concentrations on both enzymes.In general, all tested compounds showed lower inhibitory activity towards BChE, except for compounds 1), 2-methoxyphenyl ITC (7), and 3-(methylsulfanyl)propyl ITC (11) exhibited significant inhibitory activity of COX-2 enzyme with 98.9, 99 and 48%, respectively, at final concentration of 50 μM. At the same concentration, ITCs 2, 6, and 8 did not show any inhibitory activity on prostaglandin production. On the other hand, ITCs 9, 3, 4, 5 and 10 showed moderate to low inhibitory activity having 19.5, 17.8, 15.5, 13.6 and 9.4%, respectively, at final concentration of 50 μM. The effective inhibitory activity of 1 is in accordance with previously reported results on suppression of PGE2 synthesis9, the most common and most studied naturally occurring ITC, showed no expected inhibitory activity against COX-2. However, studies of in vitro ITC inhibitory activity on COX-2 enzyme are scarce. The data presented in 7 showing strong inhibitory activity contrary to its regioisomers 6 and 8 which proved inactive at the tested concentration. The effective inhibition of prostaglandin synthesis by ITCs 1, 7 and 11 calls for further investigation of their effects on COX-2 protein and mRNA expression.Results for the inhibition of prostaglandin biosynthesis, by ITCs as determined using COX-2 assay, are given in Herein, we tested 11 ITCs for their biological activity . Generally, among the tested ITCs, it can be suggested that aryl ITCs show the most promising potential. These results can be used as a basis for future investigation of other phenyl ITC derivatives bearing electron-donating and -withdrawing groups in order to better understand the influence of diversified substituents towards different biological activities.


Asn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNA The fidelity of protein translation depends on the accurate pairing of cognate tRNAs to their cognate amino acids. The ligation of the amino acid to its tRNA is catalyzed by a highly specific group of enzymes known as the aminoacyl-tRNA synthetases (aaRSs) ,2,3. UndAsp to produce Asp-tRNAAsp  . T. TAsp toectively B  , glutamic acid, L-  and adenosine triphosphate (ATP) [γ-32P] were purchased from American Radiolabeled Chemicals . Ampicillin, chloramphenicol, kanamycin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), isopropyl β-d-thiogalactopyranoside (IPTG), phenylmethanesulfonyl fluoride (PMSF), and tris(hydroxymethyl) aminomethane (Tris) were from GoldBio Biotechnology, Inc., . Potassium chloride (KCl), sodium chloride (NaCl), trichloroacetic acid (TCA), and ammonium acetate (NH4OAc) were purchased from Fisher Scientific .Potassium dihydrogen phosphate (KHHp ND-AspRS [Hp tRNAGln-specific glutamyl-tRNA synthetase (GluRS2) [Mycobacterium smegmatis (M. smegmatis or Ms) ND-AspRS (vector provided by Dr. Babak Javid), and Escherichia coli (E. coli or Ec) AspRS and GluRS (vectors provided by Dr. Takuya Ueda [Ec Bl21 (DE3) RIL in Luria-Bertani medium  inoculated with a saturated overnight culture grown from a single colony. The cultures were incubated at 37 °C, 200 rpm. IPTG (1 mM) was used to induce overexpression in each case. For Hp ND-AspRS and GluRS2 overexpression, the medium was supplemented with ampicillin (100 μg/mL), chloramphenicol (100 μg/mL), and glucose (0.5%). IPTG was added when the OD600 reached 0.8–1.0. Cells were collected after a 1 h induction period. For Ms ND-AspRS, the only difference was that kanamycin (25 μg/mL) was added instead of ampicillin. The two discriminating Ec enzymes were grown in the same medium as the two Hp enzymes. IPTG induction was initiated when the OD600 reached between 0.4 and 0.6 and continued for 4 h. Cells were harvested by centrifugation and frozen at −80 °C until ready for use.ND-AspRS , the Hp (GluRS2) , Mycobacuya Ueda ) were eaSa ND-AspRS (vector provided by Dr. Kelly Sheppard) was overexpressed and purified as previously described [2PO4, pH 7.4, 300 mM NaCl, 10 mM imidazole) and the cells were lysed with lysozyme (2 mg/mL) and sonication. Saturated PMSF (15 μL/mL) was added every 20 min to reduce proteolysis. Typically, a cell extract was added to a polyprep column that contained high-density cobalt agarose beads  pre-washed with lysis buffer. The lysate was incubated with the resin by rotating at 4 °C for 1 h. The His6-tagged aaRSs were eluted according to the manufacturer’s protocol.The escribed . In all 2PO4, pH 7.2, 0.5 mM Na2EDTA, 5 mM β-mercaptoethanol) at 4 °C for 1–2 h and then against fresh dialysis buffer overnight. The aaRSs were adsorbed onto a HiTrapTM DEAE FF column . The proteins were eluted with a stepwise gradient of 20–300 mM KH2PO4, pH 7.2, supplemented with 0.5 mM Na2EDTA, 5 mM β-mercaptoethanol. The proteins were concentrated with 30 kDa molecular weight spin filters . After this two-column purification procedure, all enzymes were judged to have been purified to near homogeneity by SDS-PAGE . The eluted tRNA sample was isopropanol precipitated. The resultant pellet was dissolved in water, folded, and quantified by aminoacylation assay, as previously described [Ec tRNA. Separately, total Ec tRNA was isolated from a saturated Ec MV1184 culture that had been grown at 37 °C in LB medium supplemented with glucose (0.5%) and purified as described [Ec tRNA was calculated by UV-Vis spectroscopy at 260 nm assuming 1 OD = 1.6 μM. tRNAAsn  and tRNAAsn [Gln  were oveescribed . After described . These pescribed . The con2, 2 mM ATP, 0.1 mg/mL BSA, 20 μM amino acid, 50 μCi/mL 3H-labeled amino acid, and 10 μM Hp tRNAAsn or tRNAGln as noted. Assays were conducted at 37 °C and were initiated with the addition of 200 nM Hp ND-AspRS or GluRS2. Aliquots were removed and quenched on Whatman filter pads containing TCA. The pads were washed 3 times for 15 min with chilled 5% TCA. Pads were dried and counted in 3 mL ECOLITE (+) scintillation fluid . Aminoacylation assays with total Ec tRNA were carried out in the same buffer containing 50–100 μM total tRNA. Hp ND-AspRS (100 nM or 500 nM) or Hp GluRS2 (100 nM or 500 nM) were used in these assays as noted.Each tRNA was pre-folded as previously described . AminoacHp ND-AspRS and GluRS2 used in these assays were only purified by cobalt affinity purification. The aminoacylation assays were conducted at 37 °C in buffer containing 20 mM HEPES-OH, pH 7.5, 4 mM MgCl2, 2 mM ATP, 100 μM amino acid, and 25 μCi/mL 3H-labeled amino acid. All aaRSs were added to a final concentration of 1 μM and overexpressed tRNA isoacceptor was added to a final concentration of 10 μM.v/v, pH 4.5). The tRNAs were ethanol precipitated and the resultant pellets were dissolved in buffer containing 10 mM NaOAc, pH 4.5, 1 mM Na2EDTA. Acid gel electrophoresis was used to separate deacylated tRNAs from their aminoacylated counterparts. The tRNA samples were quantified by UV-Vis spectroscopy at 260 nm, and 0.05 OD260 units were loaded per lane for overexpressed tRNAs. This amount was increased to 0.25 OD260 units for total Ec tRNA. Isoacceptor-specific oligonucleotides were used in Northern blots to visualize each tRNA  was added to each reaction and they were quenched with phenol/chloroform  catalyzed by chniques . We contoduction ,31. The ubstrate D, we hypHp ND-AspRS does not aminoacylate Hp tRNAGln with either aspartate or glutamate (Ec tRNAs (compared to the production of Glu-tRNAGln by Hp GluRS2 in Hp ND-AspRS aminoacylates Ec tRNAGlu with glutamate to produce Glu-tRNAGlu (Asp and tRNAAsn. We considered the possibility that E. coli glutamyl-tRNA synthetase (Ec GluRS) had inadvertently co-purified with Hp ND-AspRS, however, no evidence of this contamination was observed by SDS-PAGE  were conducted with all four enzymes: The discriminating AspRS from Ec and the ND-AspRSs from Hp, Ms, and Sa  and glutamate (B) by different bacterial aaRSs: Ec GluRS was used as a control. The results of panel A reveal that each AspRS attaches aspartate to tRNAAsp and the three ND-AspRSs also attach aspartate to tRNAAsn, as expected  than the tested AspRSs (~200–300 mM), such that any GluRS that survived the affinity purification step would have been removed by DEAE chromatography. Furthermore, no evidence of Ec GluRS contamination was visible by using SDS-PAGE gel  and glutamate to tRNAGlu without cross misacylation of these tRNAs? Our results suggest that communication between the amino acid and tRNA binding sites must occur to achieve this specificity. Further research is needed to understand this apparent tRNA-induced conformational change.This GluRS-like activity of AspRSs is remarkable and unexpected because it is accurate and yet outside the normal purview of an AspRS. It truly is GluRS activity as each AspRS specifically attaches glutamate only to tRNAEc tRNAAsp, the GUC anticodon, the G73 discriminator base, the G2:C71 base pair in the acceptor stem, C38 in the anticodon loop, and G10 in the D arm of the tRNA serve as the positive identity elements for Ec AspRS [Ec GluRS recognizes the G1:C72 and U2:A71 base pairs in the acceptor stem, A37 in the anticodon loop, U11:A24 base pair in D arm, and the U13:G22-A46 tertiary base pair in Ec tRNAGlu. Further, the lack of a nucleotide at position 47 and the modified uridine (s2U) in the UUC anticodon of tRNAGlu are also known identity determinants [Ec tRNAGlu does contain several tRNAAsp identity elements: The G73 discriminator base, G10 in the D arm, and C38 in the anticodon loop. But are these shared elements enough for recognition of Ec tRNAGlu by Ec AspRS? Or is an alternative, expanded identity set recognized in such a way as to favor glutamylation of Ec tRNAGlu? To answer these questions, the identity elements in Ec tRNAGlu that allow recognition by Ec AspRS would have to be specifically evaluated.AaRSs almost always recognize discrete molecular features in their tRNA substrates, termed identity elements. These determinants can be positive and favor recognition or negative and disfavor interactions. For Ec AspRS ,35,36. Irminants ,37,38,39Glu, at least in vitro. The relevance of this reaction in vivo is very difficult to demonstrate as it is unlikely that this activity is robust enough to enable deletion of the native Ec GluRS, especially in the presence of the cognate tRNAAsp substrate. It is possible that this activity exists as a backup in case the cognate GluRS becomes damaged, for example. It is also possible that it is a remnant of early AspRS evolution. The early genetic code was likely sloppy with ancestral aaRSs capable of aminoacylating multiple tRNAs with different amino acids [In conclusion, we have demonstrated that bacterial AspRSs have GluRS activity and are capable of producing Glu-tRNAno acids . Evidencno acids ,41, with


Population studies from the African continent have observed a marked increase in the prevalence of allergy‐related diseases over the past few decades, but the cause of this rise is not fully understood. The most investigated potential risk factor has been the relationship between exposure to helminths and allergy‐related outcomes. Immunologically, parallels exist between responses to helminths and to allergens as both are associated with elevated levels of immunoglobulin E, increased numbers of T helper 2 cells and other immune cells. However, epidemiological studies from the African continent have found inconsistent results. In this review, observations from population studies carried out in Africa over the last decade that focus on the relationship between helminth infections and allergy‐related outcomes are examined. How these findings advance our understanding of the complex interactions between helminths and allergies at the population level is also explored as well as some of the underlying immune mechanisms involved. This knowledge is important for better diagnosis, treatment and prevention of allergy‐related diseases and has wider global significance. Both are associated with T helper 2 (Th2) cell induction, high levels of immunoglobulin E (IgE) and the involvement of immune cells such as mast cells, basophils and eosinophils.We identified relevant publications through searches conducted in PubMed using keywords related to “allergy” or “hypersensitivity” in combination with “helminths,” “parasites,” or “worms” and “Africa (South of the Sahara and Northern Africa).” Our search was restricted to human studies reported in the English language that were published between January 2008 and March 2018. The search was limited to the last decade to highlight new and recent findings in the literature given that older observations have been covered extensively elsewhere.In this review, we first explore findings on helminths and reported/clinical allergy outcomes from epidemiological studies conducted in the past decade. Our reported/clinical allergy outcomes included asthma, wheeze, exercise‐induced bronchospasm, rhinitis, conjunctivitis, eczema, reported adverse reactions to peanut and skin prick test (SPT) reactivity to allergens. We then examine population study findings related to immune mechanisms including helminths and IgE sensitization, cellular immune mechanisms and other pathways through which helminths may protect or increase susceptibility to allergy‐related diseases.22.1Epidemiological studies that investigated the relationship between helminths and allergy‐related outcomes , between January 2008 and March 2018, are shown in Table 2.2Schistosoma mansoni, and urine filtration for the detection of S. haematobium, a few studies used other methods such as formol‐ether concentration. In a few studies, methods used were not specified. The type of helminth infections among participants varied from study to study, but the most common helminths were Schistosoma, hookworm, Ascaris and Trichuris species. The most commonly studied allergy‐related outcomes were reported wheeze, eczema and asthma, all with a prevalence of less than 10%. The percentage of positive SPT responses varied from 2%As shown in Table 2.3Ascaris and respiratory symptoms of wheezeTrichuris and SPT.Ascaris larva through the lungs is mostly asymptomatic, on rare occasions this may be associated with respiratory symptoms such as wheezing, dyspnoea and bronchospasm.A. lumbricoides infection and exercise‐induced bronchospasms, but an inverse association between Ascaris and SPT positivity,Information on associations between helminth infections and allergy‐related outcomes is summarized in Table Of the six studies that showed an inverse association between helminths and allergy‐related outcomes in Table , five ofTables Secondly, misclassification of helminth status may have occurred since many of these studies relied on the examination of a single parasitological sample using microscopy, which is not very sensitive.Lastly, there is evidence that the risk of allergy‐related outcomes is established early in life,2.4Trichuris) were associated with a reduced risk of eczema in later childhood.P‐value: 2.72 (1.11‐6.63), P = 0.03] but not if the mother had hookworm during pregnancy .At the time of this review, only one study in Africa had investigated the possible role of exposure to helminths in utero and early life on allergy‐related disease risk. This study, conducted in Uganda, was a randomized placebo‐controlled trial of albendazole vs placebo and praziquantel vs placebo  in pregnancy, followed by albendazole vs placebo for the offspring between 15 months to 5 years of age.33.1P = 0.007].Observations from Africa have shown that allergen‐specific IgE and SPT are not as strongly associated with allergy‐related outcomes as seen in high‐income countries and that this association is even weaker in rural compared to urban areas. For example, a study in Ghana found that the association between mite‐specific IgE and mite SPT positivity was strongest among individuals from an urban high socioeconomic status (SES) background [aOR (95% CI): 15.58 (7.05‐34.43)], followed by urban low SES participants [10.44 (5.60‐19.47)], and least among rural individuals [5.43 (3.83‐7.69); test for heterogeneity 3.2A. lumbricoides were found to have 28 times higher levels of total IgE compared to Swedish children of the same age.The role of helminths in stimulating the production of IgE antibodies was first postulated in a study published in the late 1960s in which a group of Ethiopian pre‐school children infected with the helminth With regard to helminths and protein IgE cross‐reactivity, a number of allergens from invertebrate sources such as house dust mite, shrimp and cockroach have been shown to cross‐react with helminth antigens.Ascaris antigen and SPT reactivity to aeroallergens among adolescents in South Africa.Ascaris proteins such as tropomyosin or GST and their corresponding homologues in house dust mite and cockroach may explain the positive SPTs. Additionally, cross‐reactivity between the filarial nematode Onchocerca volvulus tropomyosin (OvTrop) and house dust mite tropomyosin (Der p 10) has been demonstrated in vitroAlthough there are very few studies examining IgE cross‐reactivity between proteins from helminths and homologues from invertebrate allergen sources in African individuals, cross‐reactivity may explain some observations made in African studies in the past decade. For example, Levin et al observed a positive association between specific IgE to The sugar components of insect and plant glycoproteins known as CCDs are central to IgE carbohydrate cross‐reactivity.S. haematobium infection was positively associated with peanut IgE sensitization and a strong correlation was observed between IgE to CCDs and IgE to whole peanut extract. In a subset of children in that study, inhibition assays showed that S. haematobium soluble egg antigen and the CCD marker bromelain were strong inhibitors of IgE binding to peanut extract.Schistosoma infection was linked to IgE cross‐reactivity involving the core xylose glycan motif.In Africa, a role for helminths in cross‐reactivity involving carbohydrates was demonstrated by an investigation conducted among schoolchildren in Ghana in which 18% were peanut IgE‐sensitized, but 92% of those sensitized were peanut SPT negative.Although further investigations are needed to examine how CCD‐specific IgE may inhibit allergic effector responses, Doenoff and colleagues have explored the role of IgG antibodies as blocking antibodies.Amblyomma americanum) in the Southeastern United States,Ixodes ricinus) in Sweden,Ixodes holocyclus) in AustraliaHaemaphysalis longicornis) in Japan.Generally, IgE to CCDs are not thought to be biologically active, but in recent years, responses to the mammalian carbohydrate epitope galactose‐α‐1,3‐galactose  have been of interest in the field of allergy since this epitope has been linked to two forms of anaphylaxis.3.3S. mansoni‐mediated protection against experimental ova albumin‐induced allergic airway inflammation was dependent on IL‐10 producing B cells in mice.hi B cells were observed in Gabonese children infected with S. haematobium. Based on these findings, the authors posited that IL‐10‐producing regulatory CD1dhi B cells are induced in both humans and mice during chronic schistosomiasis. In mice, these IL‐10‐producing regulatory B cells have an important role in protection against experimental allergic inflammation.Studies on immune mechanisms from population studies illustrate the role of helminths in protection from or susceptibility to allergy‐related outcomes. The anti‐inflammatory environment associated with helminths in the human host is characterized by elevated levels of the cytokines IL‐10 and TGF‐β, and general immune hyporesponsiveness.S. mansoni in Lake Victoria, Uganda, found that in a subset of participants, reported wheeze was negatively associated with S. mansoni‐specific cytokine responses.Other recent studies have also looked at how helminth‐induced cytokine profiles may influence allergic immune responses. For example, the investigation conducted on islands endemic for S. haematobium.S. haematobium may modify the development of the allergic immune response by modulating the expression levels of innate immune genes such as TLR2 and SOCS‐3.When it comes to other immune mechanisms, a study among Ghanaian schoolchildren found that high expression of innate immune gene Toll‐like receptor 2 (TLR2) and suppressor of cytokine signalling (SOCS)‐3 messenger RNA (mRNA) was positively associated with SPT reactivity to house dust mite.P = 0.032).Allergen‐specific IgG4 is often considered as a marker of immune modulation while allergen‐specific IgE as a marker of allergic disease.S. haematobium transmission community as well as in a high transmission community.Schistosoma infection intensity was negatively associated with Der p 1‐specific IgE to IgG4 ratio in the high transmission area.In a study conducted in Zimbabwe, Rujeni and colleagues looked at the ratio of mite‐specific (Der p 1) IgE to IgG4 in a low S. haematobium prevalence, Rujeni et al hypothesized that the levels of soluble CD23 would be negatively associated with allergen‐specific IgE titres as well as SPT wheal size in schistosome‐infected individuals.The low‐affinity CD23 receptor is thought to be involved in the regulation of IgE synthesis.S. mansoni and hookworm were examined.S. mansoni.Another immune mechanism that has been examined is the association between helminth infection and basophil cell suppression. A study in Uganda investigated the hypothesis that immunoregulatory responses that characterize helminth infections reduce the response of IgE effector cells, such as basophils, to IgE‐mediated activation resulting in suppression of responses to parasite and non‐parasite antigens.Studies conducted in Africa have observed a strong correlation between helminths and rural environments. For instance, studies that have reported an inverse association between helminths and allergy‐related outcomes were predominantly from rural areas.4Studies from Africa investigating the association between helminths and allergy‐related outcomes have found a number of inconsistent observations, some showing inverse, positive or no associations. Since most of these studies are observational and conducted among school‐age children, establishing causation is impossible. However, these studies have illustrated how IgE cross‐reactivity between environmental or food allergens and helminth antigens has limited the diagnostic value of measuring IgE responses to whole allergen extracts in populations from helminth‐endemic areas. They also raise pertinent questions about the classification of asthma as “atopic”/”allergic” or not based on the measurement of allergen‐specific IgE to whole allergen extracts.Although not consistent, studies on immune mechanisms have illustrated how helminth infections can induce immune regulatory responses that may protect against allergic immune responses. The observed lower burden of allergy‐related outcomes in rural compared to urban areas deserves further investigation. There is also evidence to suggest that the critical period worth investigating is early life. Therefore, studies in early life, either as anthelmintic trials during pregnancy and early childhood or as birth cohorts investigating a broad range of risk factors in both rural and urban areas, are needed. The current epidemiological transition in Africa offers an excellent opportunity to conduct such studies, so as to identify the primary factors driving the global rise in allergic disorders.None.


Terminalia catappa leaves are used in managing both diabetes mellitus and its complications in Southwest Nigeria. However, its inhibitory activity on enzymes implicated in diabetes is not very clear. This study investigated the in vitro inhibitory properties and mode of inhibition of T. catappa leaf extracts on enzymes associated with diabetes. The study also identified some bioactive compounds as well as their molecular interaction in the binding pocket of these enzymes. Standard enzyme inhibition and kinetics assays were performed to determine the inhibitory effects of aqueous extract (TCA) and ethanol extract (TCE) of T. catappa leaves on α-glucosidase and α-amylase activities. The phytoconstituents of TCA and TCE were determined using GC-MS. Molecular docking of the phytocompounds was performed using Autodock Vina. TCA and TCE were the most potent inhibitors of α-glucosidase (IC50 = 3.28 ± 0.47 mg/mL) and α-amylase (IC50 = 0.24 ± 0.08 mg/mL), respectively. Both extracts displayed a mixed mode of inhibition on α-amylase activity, while mixed and noncompetitive modes of inhibition were demonstrated by TCA and TCE, respectively, on α-glucosidase activity. The GC-MS analytic chromatogram revealed the presence of 24 and 22 compounds in TCE and TCA, respectively, which were identified mainly as phenolic compounds, terpenes/terpenoids, fatty acids, and other phytochemicals. The selected compounds exhibited favourable interactions with the enzymes compared with acarbose. Overall, the inhibitory effect of T. catappa on α-amylase and α-glucosidase may be ascribed to the synergistic action of its rich phenolic and terpene composition giving credence to the hypoglycaemic nature of T. catappa leaves. Diabetes mellitus (DM) is an endocrine, chronic, noncommunicable disease plaguing the world populace with a rapid increase. A reported 425 million individuals were globally affected by DM, while 629 million people have been projected to be affected by 2045 . DM is cTerminalia catappa Linn, commonly known as Indian almond, belongs to the Combretaceae family and grows in the tropics of Asia, Africa, and Australia [T. catappa extracts such as hepatoprotective effects, anticancer property, antimutagenic activity, and antiaging property [T. catappa methanolic leaf extract on diabetic-linked enzymes. Despite this antidiabetic claim by the locals, the elaborate antidiabetic mechanism is far from clear. This study assessed the inhibitory properties of T. catappa leaf extracts on α-glucosidase and α-amylase, the mode of enzyme inhibition, as well as identified phytocompounds present and proposed the molecular mechanism of binding in the active sites of the enzymes.ustralia . In urbaustralia . Severalproperty . Divya aproperty  have alsα-Glucosidase, α-amylase enzymes, and their substrates were acquired from Solarbio Life Sciences, Beijing, China. Other chemicals were products of Sigma-Aldrich, St. Louis, USA.T. catappa leaves were sourced between October and December 2016, from Covenant University compound. They were identified by Dr. J. O. Popoola of Biological Sciences Department and voucher specimen deposited at Biological Sciences Department herbarium, Covenant University, Ota, Ogun State, with herbarium number TC/CUBio/H809. Aqueous T. catappa (TCA) and ethanol T. catappa (TCE) leaf extracts were prepared as reported by Iheagwam et al. [Mature α-Glucosidase inhibitory activity of the extracts was evaluated according to the method described by Ibrahim and Islam [μL) were incubated at 37°C for 15 min with α-glucosidase solution . ρ-Nitrophenyl-α-D-glucopyranoside (pNPG) solution  was thereafter added, and the resulting mixture was incubated for 20 min at 37°C. The reaction was terminated by adding Na2CO3 , and absorbance was measured at 405 nm. Phosphate buffer (100 mM) was used as control in place of inhibitors. Inhibitory activity was calculated using the following equation:As = absorbance in the presence of sample and Ac = absorbance of control. All solutions were prepared in 0.1 M phosphate buffer (pH 6.8).nd Islam  with sliα-glucosidase inhibitory kinetics. Extract  was preincubated with α-glucosidase solution  for 10 min at 25°C. Varying pNPG concentrations  were added and incubated for 10 min at 25°C to both sets of reaction mixtures to start the reaction. Thereafter, Na2CO3  was added to stop the reaction. For the control kinetic reaction, 100 mM phosphate buffer  was used in place of the extract. Reaction rates (v) were calculated, and double reciprocal plots of α-glucosidase inhibition kinetics were determined.The method of Sabiu and Ashafa  was adopα-Amylase inhibitory activity of the extracts was evaluated by adopting the method described by Ibrahim and Islam [μL) and acarbose were incubated at 37°C for 20 min with amylase solution . Starch solution  was later added to the reaction mixture and incubated at 37°C for 1 h. Dinitrosalicylic acid (DNS) colour reagent (1 mL) was added to stop the reaction. The resulting mixture was boiled for 10 min, and absorbance was measured at 540 nm. Phosphate buffer (100 mM) was used as control in place of inhibitors. The α-amylase inhibitory activity was calculated using the following formula:As = absorbance in the presence of sample and Ac = absorbance of control. All solutions were prepared in 100 mM phosphate buffer (pH 6.8).nd Islam  with sliα-amylase inhibitory kinetics. In brief, extract  was incubated with α-amylase  for 10 min, before the addition of various substrate concentrations . The reaction proceeded as highlighted for α-glucosidase. α-Amylase inhibition kinetics was determined from the Lineweaver–Burk double reciprocal plot.The method of Sabiu and Ashafa  was adopT. catappa extracts was carried out using GCMS-QP2010SE SHIMADZU, Japan, fused with the Optima 5 ms capillary column (30 × 0.25 mm) of 0.25 μm film thickness following the described method of Ajiboye et al. [μL in the splitless mode with a split ratio of 1 : 1 and injector temperature set at 200°C. Mass spectrophotometer conditions were as follows: ion source temperature (230°C), interface temperature (250°C), solvent delay at 4.5 min, and acquisition in a scan range of 50–700 amu. Electron ionization mode and multiplier voltage were set at 70 eV and 1859 V, respectively. Retention time, fragmentation pattern, and mass spectral data of the unknown components in the extracts were compared with those in Wiley and National Institute of Standards and Technology (NIST) libraries for compound identification.The GC-MS analysis of e et al.  with sliα-glucosidase and α-amylase was modelled using the crystal structures with PDB codes 5kzw and 1b2y, respectively, obtained from RCSB protein data bank as templates in SWISS-MODEL [The structures of the GC-MS identified compounds with ≥5% abundance were prepared as reported by Iheagwam et al. . The 3D SS-MODEL .α-glucosidase and α-amylase, respectively. Inhibition constant (Ki) of docked ligands were calculated by using the following formula:Virtual screening of selected identified ligands, analysis of drug-likeness using the rule of five (RO5), and molecular docking were carried out according to the methodology of Iheagwam et al. . Howeverp < 0.05.Data were analysed using SPSS version 25  and subjected to one-way analysis of variance (ANOVA) using the Duncan multiple range post hoc test. Values were reported as mean ± standard deviation (SD) of three (3) replicates and considered significantly different at α-glucosidase inhibitory activity of TCA and TCE as shown in p < 0.05) lower inhibition by the extracts was observed at all concentrations relative to control. TCA exhibited a significantly (p < 0.05) higher inhibition of α-glucosidase activity compared to TCE. Nonetheless, at lower concentrations (1–3 mg/mL), there was no difference between the inhibitory activities of TCA and TCE. These were further supported by a lower IC50 value (2.23 ± 0.21 mg/mL) for acarbose when compared with TCA (3.28 ± 0.47 mg/mL) and TCE  higher than TCA and acarbose at all concentrations. TCA elicited inhibitory effects that competed favourably with the standard drug (acarbose). These results were supported with an IC50 of 0.24 ± 0.08, 0.75 ± 0.14, and 0.85 ± 0.18 mg/mL recorded for TCE, TCA, and acarbose, respectively - were higher in TCE. It was also observed that there was no much difference in the abundance of vitamin E in both extracts.The identified phytochemicals present in TCE and TCA are shown in Tables For TCE, 9, 26, 13, 30, and 25% of the identified compounds were classified as carbohydrates, fatty acids, hydrocarbons, phenolics, and terpenes/terpenoids, respectively , while fα-amylase. However, only vitamin E (−82.91) and ethyl-α-D-glucopyranoside (−78.11) were relatively comparable with the standard  and vitamin E (−89.64), n-hexadecanoic acid (−81.89) and phytol (−80.87) binding affinities were also comparable with acarbose (−115.55).As illustrated in When the hit compounds were screened for their drug-likeness, they all obeyed Lipinski's RO5. However, phytol and vitamin E, on the one hand, violated only the octanol-water partition coefficient due to higher values than the RO5 threshold as presented in α-amylase and α-glucosidase, respectively. These values though lower were comparable with acarbose where −8.3 was recorded for α-amylase and −7.4 for α-glucosidase. Concomitantly, 1.39 to 40.51 μM was the α-amylase inhibition constant (Ki) recorded for the compounds compared to 0.84 μM for acarbose, while 47.95 to 184.70 μM was the α-glucosidase Ki recorded for the compounds compared to 3.83 μM for acarbose.The binding affinity of the selected compounds as shown in in vitro results as the majority of the ligands favoured active site binding compared to the allosteric site. Hydrogen, van der Waals, and π bonds were the common interactions displayed between the compounds and amino acids present in the binding sites of the enzymes. Trp 73, Trp 74, Tyr 77, Tyr 166, and Ile 250 were common amino acids stabilising the binding of vitamin E and acarbose in the binding pocket of α-amylase, while in the α-glucosidase binding pocket, Ala 284, Asp 616, and Trp 481 were common amino acids stabilising phytol, vitamin E, and acarbose  leaf extracts were higher than our extracts except for A. lanata aqueous leaf extract α-amylase inhibitory activity which was reported to be lower than ours [T. catappa leaf extracts was higher on α-amylase than on α-glucosidase at the varied concentrations and may be attributed to the different mechanism of action on these enzymes. This was further buttressed by the kinetic studies, where the TC extracts exhibited a mixed mode of inhibition on α-amylase, while mixed and uncompetitive inhibition mechanisms were observed for TCA and TCE, respectively, on α-glucosidase. The mixed mechanisms exhibited by TCA and TCE may suggest the bioactives present in the extracts may bind in the active site of these enzymes thereby reducing the affinity of the substrate [α-glucosidase active site affecting catalysis without having an effect on substrate binding in the active site [Smilax aristolochiifolia root extract and its compounds where the number of hydrogen bonds did not affect binding affinity. Vitamin E had the lowest free energy and Ki in amylase and glucosidase binding pockets which was comparable to acarbose. Consequently, it exhibited a more stable affinity with only a small concentration required to inhibit these enzymes [in vitro inhibitory mechanisms as more identified compounds bound to the active site than the allosteric site signifying a preference for the (E) to elicit their potential pharmacological action [lycaemia , 16. Reslycaemia . Promisit manner . Nonetheacarbose , 19. It extracts , 21. Thehan ours , 22. Conhan ours  and Wan han ours , the inhubstrate , 26. Binubstrate , 26. Theive site . The obsive site . Compounive site , 30, varive site , hexadecive site  have beeive site , 32, 33.ive site . In leadive site , 36. Comive site . Since aive site  reported enzymes . Molecull action . The coml action –41.T. catappa leaf extracts on glucosidase and amylase is being reported, making it an effective agent in managing postprandial hyperglycaemia. These extracts preferably bind to the active site of these enzymes where their various identified compounds synergistically illicit their inhibitory action. From the different GC-MS identified compounds, vitamin E was the most potent ligand that qualified as a potential drug candidate after docking studies. These plants can be leveraged upon as a natural source of not only vitamin E but other antidiabetic compounds for drug formulation. On the other hand, isolation and characterisation of these identified phytocompounds in addition to in vivo studies are still required to confirm these findings.This is the first time, to the best of our knowledge, the inhibitory mechanism of


There is increasing evidence that consumption of polyphenol and phenolic-rich foods and beverages have the potential to reduce the risk of developing diabetes type 2, with coffee a dominant example according to epidemiological evidence. One of the proposed mechanisms of action is the inhibition of carbohydrate-digesting enzymes leading to attenuated post-prandial blood glucose concentrations, as exemplified by the anti-diabetic drug, acarbose. We determined if the phenolic, 5-caffeoylquinic acid, present in coffee, apples, potatoes, artichokes and prunes, for example, and also selected free phenolic acids , could inhibit human salivary α-amylase and rat intestinal maltase activities, digestive enzymes involved in the degradation of starch and malto-oligosaccharides. Using validated assays, we show that phenolic acids, both free and linked to quinic acid, are poor inhibitors of these enzymes, despite several publications that claim otherwise. 5-CQA inhibited human α-amylase only by <20% at 5 mM, with even less inhibition of rat intestinal maltase. The most effective inhibition was with 3,4-dimethoxycinnamic acid (plateau at maximum 32% inhibition of human α-amylase at 0.6 mM), but this compound is found in coffee in the free form only at very low concentrations. Espresso coffee contains the highest levels of 5-CQA among all commonly consumed foods and beverages with a typical concentration of ~5 mM, and much lower levels of free phenolic acids. We therefore conclude that inhibition of carbohydrate-digesting enzymes by chlorogenic or phenolic acids from any food or beverage is unlikely to be sufficient to modify post-prandial glycaemia, and so is unlikely to be the mechanism by which chlorogenic acid-rich foods and beverages such as coffee can reduce the risk of developing type 2 diabetes.  •Chlorogenic and phenolic acids weakly inhibit human α-amylase activity.•Chlorogenic acids weakly inhibit rat maltase activity.•Free phenolic acids exhibit no inhibition of rat maltase activity. In foodin vitro studies  and caffeic acid were alleged to be inhibitors of both α-amylase (porcine) and α-glucosidase enzymes with IC50 values <100 μM  and 3 mL (60 mg) were purchased from Waters Ltd., Milford, MA, U.S.A. All the reagents were of the highest purity and standards were ≥98 %. The colour reagent was prepared by mixing 20 mL of 96 mM of 3,5-dinitrosalicylic acid with 8 mL of 5.3 M (12 g in 8 mL of 2 M sodium hydroxide) and 12 mL Millipore water. Human salivary amylase type IX-A stock concentration of 1.25 U/mL was prepared in PBS  to give 0.5 U/mL in the assay according to the optimized assay .2.2Amylose (1 mg/mL) was used as the substrate and the assay was conducted according to the optimized assay . A total% Inhibition = ((Abs Control − Abs sample)/Abs control) x 100.50 was calculated graphically by dose-dependent inhibition.Where inhibition was obtained above 50%, IC2.3The method used to assess rat α-glucosidase inhibition was adapted from 2.4p ≤ .05). The data are expressed as the mean ± standard deviation (n = 3).Statistical analysis was performed by one-way analysis of variance using the Number Cruncher Statistical System version 6.0 software . Significant differences were assessed with Tukey-Kramer multiple comparison test  tested concentration A. As a pectively B and C, ectively .Fig. 2Inp ≥ .05). Caffeic acid and ferulic acid both showed inhibition of <20% at the highest concentration tested of 1 mM. None of the free phenolic acids inhibited rat intestinal maltase activity , did not attenuate post-pranidal blood glucose after consumption of bread as a carbohydrate-rich food (The aim of the present study was to determine whether the antidiabetic properties attributed to drinking coffee (and possibly to other foods or beverages containing chlorogenic acids) are due to an acarbose-like action, a drug used to attenuate hyperglycaemia through inhibition of carbohydrate-digesting enzymes. In a detailed survey of espresso coffees, the strongest form of coffee normally consumed and commercially available in a major city of the UK, a typical serving of espresso was in the range of 24–422 mg per serving in an average serving size of 43 mL . Since 5ich food . Accordiich food , filter ich food , at ~2 mich food . Raw potich food . Free phich food  and in mich food .50 value of 0.08 mM for 5-CQA  rather than a synthesized dye-linked substrate, which would have markedly different affinity for the enzyme, have also used a human source of α-amylase, and have ensured that the phenolic acids do not interfere in the DNS product determination. Several studies using the porcine pancreatic enzyme  have repor 5-CQA  and of 0or 5-CQA . It is nor 5-CQA  that theor 5-CQA  can yielin vivo. We therefore conclude that the anti-diabetic effects of coffee consumption are not due to inhibition of carbohydrate-hydrolysing enzymes. However, several studies have shown that consumption of coffee reduces postprandial blood glucose levels. There was a significant reduction in total area under the glucose curve in a rat model after consumption of a standardised meal containing carbohydrate with chlorogenic acid (via modulation of glucose metabolism, other mechanisms may be involved which include inhibition of intestinal glucose transport. In this respect, when consumed with a glucose bolus, coffee exhibited some effect on post-prandial glycemia, although not directly on plasma glucose concentration (For inhibition of rat intestinal maltase, 5-CQA was a weak inhibitor, but none of the free phenolics showed inhibition. Other research (Iwai, Kim, Onodera, & Matsue, 2006nic acid . In healnic acid . It was nic acid  and in anic acid , that contration , and thi


Rhus verniciflua is widely known for its antioxidant, antibacterial, anticancer, and antiaging efficacy and α-glucosidase inhibition. This study was designed whether Rhus verniciflua extracts inhibit the IgE-antigen-mediated allergic reaction in RBL-2H3 mast cells, and it further investigated the FcεRI- and arachidonate-signaling by which Rhus verniciflua extracts exert its antiallergic effects. IgE-antigen-sensitized RBL-2H3 mast cells were investigated for the cytotoxicity of Rhus verniciflua extracts and β-hexosaminidase release, and inflammatory mediators  were then assessed. Additionally, we examined expressions of genes involved in arachidonate- and FcεRI-signaling pathway in RBL-2H3. Rhus verniciflua extracts inhibited β-hexosaminidase release and production of the inflammatory mediators in RBL-2H3. Rhus verniciflua extracts reduced amounts of histamine and expressions of FcεRI signaling-related genes such as Lyn and Syk and phosphorylation of extracellular signal-regulated kinase in mast cells. Finally, in late allergic responses, Rhus verniciflua extracts reduced PGD2 release and COX-2 and cPLA2 phosphorylation expressions from IgE-antigen-mediated mast cells. Lastly, 250–500 mg/kg RVE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. These findings provide novel information on the molecular mechanisms underlying the antiallergy properties of Rhus verniciflua extracts in FcɛRI-mediated allergic reaction. Rhus verniciflua (RV) has been used in traditional Asian medicine in many countries including Korea and China to prevent gastritis, stomach cancer, atherosclerosis, aging, and hypertension and to promote blood circulation [α-glucosidase-inhibition effects. Several studies reported that it inhibits proinflammatory cytokines and the vascular endothelial growth factor in fibroblast-like synoviocytes in in vivo models of rheumatoid arthritis [εRI-mediated cell signaling mechanism.culation . Recentlrthritis . It was rthritis . HoweverεRI receptors, which are known as immunoglobulin E (IgE) receptors, in the plasma membrane [εRIs on the mast cell surface consist of α, β, and γ subunits, of which the β and γ subunits are involved in cell signaling. When an allergen crosslinks with IgE, the receptor is phosphorylated by the Src-kinase combined with the β subunit, initiating cell signaling [β and γ subunits are phosphorylated by Lyn kinase combined with the receptor [Mast cells contain Fcmembrane . When mamembrane . These cmembrane , 6. FcεRignaling , 8. Addireceptor . The phoreceptor . In the receptor , 11. As receptor . As explRhus verniciflua extract (RVE) can demonstrate inhibitory effects on the allergic reaction of mast cells, which are mediated by FcεRIs. Therefore, we measured the β-hexosaminidase activity and assessed the degree of degranulation of FcεRI-mediated RBL-2H3 mast cells, in order to examine the antiallergic effect of RVE. Then, the effect of RVE on inflammatory cytokines such as TNF-α, IL-4, and IL-6 was assessed in RBL-2H3 mast cells. Finally, the effect of RVE on the antiallergic mechanism associated with the FcεRI signaling was investigated.We assumed that −1 . Gallic acid was obtained from Tokyo Chemical Industry Co., Ltd. . Protocatechuic acid, methyl gallate, caffeic acid, ethyl gallate, and quercetin were purchased from Sigma-Aldrich . Butein was obtained from Faces Biochemical Co., Ltd. . The purity of all standards was above 97%. Minimum essential medium alpha modification (MEM-α), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and antibiotics were purchased from GE Healthcare Life Sciences . Dinitrophenyl-human serum albumin (DNP-HSA), DNP-immunoglubulin E (DNP-IgE), 4-methyl-umbellyferyl-N-acetyl-β-d-glucosaminidase (p-NAG), dimethyl sulfoxide (DMSO), and dexamethasone were acquired from Sigma-Aldrich . Antibodies were obtained from Cell Signaling . The ethanolic extracts of Rhus verniciflua (RVE) were produced in Korea Institute of Oriental Medicine.HPLC-grade methanol was purchased from Fisher  and HPLC-grade acetic acid obtained from Sigma-Aldrich Co. . Ultrapure water was prepared by using the Puris-Evo UP water system with Evo-UP Dio VFT and Evo-ROP Dico20 . Ultrapure water (UW) was prepared with a resistivity of 18.2 MΩ·cmRV was obtained as a dried herb from Yeongcheon Oriental Herbal Market  and was authenticated by the Korean Medicine Application Center, Korea Institute of Oriental Medicine. RV (50 g) was extracted using a 70% ethanol at 40°C for 24 h in a shaking incubator. Then, the extraction was filtered using a 150 mm filter paper and concentrated using a rotary vacuum evaporator . The sample was freeze-dried and stored in desiccators at 4°C until use. The RVE powder was dissolved in 50% DMSO for the experiments.μg/mL), respectively. The seven standards were mixed from stock solutions and then diluted at final concentrations of 100 μg/mL, respectively. RV 70% ethanol extracts (20 mg/mL) were prepared in 100% methanol. Then, the standard solutions and sample were filtered through a 0.22 μm membrane filter  prior to injection into the HPLC-DAD system. The injection volume was 5 μL.The seven standard stock solutions were prepared by dissolving accurately weighed in 100% methanol , and the column oven temperature was kept at 40°C. The mobile phase consisted 2% acetic acid (v/v) in water (A) and methanol (B). The gradient elution system, to improve the chromatographic separation capacity, was programmed as follows: 5–20% B, 0–10 min; 20–35% B, 10–50 min; 35–50% B, 50–70 min; 50% B, 70–75 min; 50–80% B, 75–80 min; 80% B, 80–85 min; 80–5% B, 85–86 min; 5% B, 86–95 min at a flow rate of 1.0 mL/min. The detection wavelengths for seven components were set at 230, 254, 280, and 360 nm.To identify seven components in RV, high-performance liquid chromatography (HPLC) analysis was performed using a Dionex UltiMate 3000 system  equipped with a binary pump, an autosampler, a column oven, and a diode array UV/VIS detector (DAD). System control and data analysis were processed with Dionex Chromeleon. The separation was carried out using a Xbridge C18 column  in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The growth medium was replenished every two days.RBL-2H3 mast cell line was acquired from the American Type Culture Collection  and grown in MEM-4 cells were seeded on a 96-well plate and grown to confluence overnight. The cells were rinsed with fresh DPBS and cultured in MEM-α with 0.1 μg/mL DNP-IgE for 24 h. The cells were pretreated with various concentrations of RVE  and/or 100 nM dexamethasone for 1 h and treated with 0.1 μg/mL DNP-HSA for 4 h. Cell viability was analyzed after the addition of 0.5 mg/mL MTT reagent in each well and an another additional incubation for 40 min at 37°C. After removal of the medium, cells were lysed with DMSO. The cell viability was measured using a microplate reader at 570 nm.Prior to the experiments, 2 × 104/wells) were cultured into 96-well plate and preincubated with RVE. After 4 h of DNP-HAS (0.1 μg/mL) treatment, cell culture supernatant of each well was allowed to react with 4-methyl-umbellyferyl-N-acetyl-β-d-glucosaminidase (10 mM p-NAG) for 1 h at 37°C. The reaction was terminated by adding sodium carbonate buffer , and the absorbance was measured with a microplate reader at 405 nm.RBL-2H3 mast cells  were used to detect COX-2, β-actin and the phosphorylated form of Lyn, Syk, Fyn, PLCγ1, cPLA2, ERK, and Akt, respectively.The cells were scraped from the plates with RIPA lysis buffer containing a protease and phosphatase-inhibitor cocktail . The lysates were quantified using the BCA protein assay kits . Equal amounts of protein were resolved on 10% SDS-PAGE and then transferred to a PVDF membrane. Anti-COX-2, anti-n = 5), Ag/IgE group , Ag/IgE treated with 10 mg/kg dexamethasone group , Ag/IgE treated with 250 mg/kg RVE group , and Ag/IgE treated with 500 mg/kg RVE group . RVE was prepared in saline, and CTL and Ag/IgE groups received equivalent volumes of saline. All experiments were approved by the Committee on Animal Experimentation and Ethics of KIOM.Male ICR mice, 5 weeks of age, were randomly assigned to five groups after 1 week adaptation period: control group  antibody was subcutaneously injected into the ears of mice. On day 2, IgE-sensitized mice were administered with oral RVE (250 or 500 mg/kg) or dexamethasone (10 mg/kg). One hour later, DNP-HSA (300 μg/mL) containing 1% Evans blue was injected into the tail veins. One hour later, the mice were anesthetized with CO2, and tissues from the treated ears were obtained. The Evans blue dye was removed by the ear tissue, which were then incubated with 0.4 mL formamide at 63°C for 16 h. Absorbance at 620 nm wavelength was measured using a microplate reader.On day 1, anti-DNP-IgE .The results are presented the mean ± SE of from three independent experiments. All data were statistically evaluated by one-way ANOVA with Bonferroni's post hoc test using Graph Pad PRISM software . Differences were considered statistically significant at The identification of the seven components in RVE was based on comparisons of their retention times (tR), UV spectra, and chromatograms pattern with those of each standard using HPLC analysis system. As shown in μg/ml in RBL-2H3 cells . Concentrations of Evans blue were significantly lower in the RVE 250 group , the RVE 500 group , and Dex group  (The concentration of Evans blue significantly increased from 3.69 ± 0.15  0.0005) .2, and leukotrienes from granules, and the mast cells have high affinity with Fc receptor for IgE on their cell surface [The mast cell is a critical source of studies on inflammatory and allergic diseases such as allergic rhinitis, asthma, and autoimmune disease . Mast ce surface . RBL-2H3 surface . In thisα, IL-4, IL-6, histamine, and PGD2) in IgE-antigen-mediated mast cells. β-Hexosaminidase is used as an indicator of degranulation and is useful for monitoring allergy inhibitory effects of unknown substances [β-hexosaminidase releases. As undergoing degranulation, secretion of histamine and expression of FcεRI signaling-related protein promote release of the inflammatory cytokines such as TNF-α, IL-4, and IL-6 [α, which is originally isolated in granules or is newly produced by activated mast cells themselves, is secreted by stimulating with IgE in mast cells. TNF-α released in mast cells affects the endothelial cells and mediates allergic inflammatory reaction. It is not only the inducer of another inflammatory cytokine but also a self-sustaining agent [Antiallergic reactions of RVE result from its ability to suppress both the degranulation process and the production of inflammatory mediators  and extracellular signal-regulated kinase phosphorylation in mast cells (in early phase). Also, RVE reduced PGD2 release via regulation of COX-2 and cPLA2 in FcɛRI-mediated mast cells (in late phase). RVE decreased the risk of allergic reactions related to the FcɛRI-mediated process, which may be a potentially beneficial effect of antiallergic drugs.In conclusion, we have shown that RVE reduces the risk of IgE-antigen-mediated allergic reactions by inhibiting degranulation in mast cells and modulating inflammatory cytokine release. RVE reduced Fc


Neurospora circadian clock mutants. Mutations that specifically affect the FRQ-CK1 interaction lead to severe alterations in circadian period. The FRQ-CK1 interaction has two roles in the circadian negative feedback loop. First, it determines the FRQ phosphorylation profile, which regulates FRQ stability and also feeds back to either promote or reduce the interaction itself. Second, it determines the efficiency of circadian negative feedback process by mediating FRQ-dependent WC phosphorylation. Our conclusions are further supported by mathematical modeling and in silico experiments. Together, these results suggest that the FRQ-CK1 interaction is a major rate-limiting step in circadian period determination.Circadian clock mechanisms have been extensively investigated but the main rate-limiting step that determines circadian period remains unclear. Formation of a stable complex between clock proteins and CK1 is a conserved feature in eukaryotic circadian mechanisms. Here we show that the FRQ-CK1 interaction, but not FRQ stability, correlates with circadian period in  Neurospora.Circadian clocks control daily rhythms of molecular and physiological activities. Here, the authors show that the interaction between proteins FRQ and CK1, rather than FRQ stability, is a major rate-limiting step in circadian period determination in the model fungus  Neurospora, Drosophila, and mammalian cells, the circadian oscillators consist of autoregulatory transcription- and translation-based negative feedback loops5. In these negative feedback loops, Per-Arnt-Sim (PAS)-domain transcription factors act as positive elements that activate the transcription of the negative elements, and the latter feedback to repress their own transcription by inhibiting the activity of the positive elements. How circadian clocks generate endogenous and temperature compensated periodicity of ~24 h has always been a central question in chronobiological research. Although the mechanisms of eukaryotic circadian clocks have been extensively investigated in the past four decades, the main rate-limiting steps that determine circadian period length remains unclear7.Circadian clocks control daily rhythms of molecular and physiological activities to allow organisms to adapt to daily environmental changes. In 17, which led to the proposal that degradation rates of the negative elements is the major factor that determines the period length. However, protein stability of the negative elements frequently does not correlate with circadian period length in some mutants19. In addition, impairments in the ubiquitin proteasome pathway that is required for PERIOD (PER) degradation only result in modest period changes in animal cells22. Recently, it was shown that in Neurospora mutants with a defective FREQUENCY (FRQ) degradation pathway, FRQ degradation rate and circadian period length can be uncoupled23. This result suggested that FRQ phosphorylation but not protein stability plays an important role in period determination. How FRQ phosphorylation regulates circadian period length is unclear.Post-translational modification of clock proteins by phosphorylation is a highly conserved feature in eukaryotes. A major function of phosphorylation is to regulate the stability of the negative elements. Mutations of clock protein phosphorylation sites or of kinases alter protein stability and result in changes in period lengthNeurospora and PER in animal systems. Among these kinases, CK1 has been shown to play a major role in FRQ and PER phosphorylation. Unlike typical kinase-substrate interactions, which are weak and transient, CK1 forms a tight stoichiometric complex with FRQ in Neurospora and with PER in Drosophila and mammals29, suggesting that this conserved feature is critical for eukaryotic clock functions.Multiple conserved kinases, including casein kinase 1 (CK1), CK2, and protein kinase A (PKA), phosphorylate FRQ in Neurospora circadian negative feedback loop, the PAS domain-containing factors WHITE COLLAR (WC) 1 and 2 form a heterodimeric complex that activates frq transcription by binding to the C-box of its promoter32. FRQ forms a complex with FRQ-interacting RNA helicase (FRH) to act as the negative element33. After its synthesis, FRQ becomes progressively phosphorylated by CK1a , CK2, PKA, and other kinases at about 100 sites37. CK1a is a major FRQ kinase in vivo, but, unlike CK2 and other FRQ kinases, CK1a forms a tight complex with FRQ through two FRQ-CK1a interaction domains (FCD). Mutations of either FCD can completely eliminate the FRQ-CK1a interaction and abolish CK1a-mediated FRQ phosphorylation in vivo and in vitro39. Extensive FRQ phosphorylation triggers its degradation mediated by F-box/WD-40 repeat-containing protein-1  that interacts with phosphorylated FRQ41. FRQ phosphorylation has opposing roles in period determination: Mutations of FRQ phosphorylation sites can result in short or long period mutants, and, in many cases, the period length correlates with FRQ stability15. However, it was recently shown that in the fwd-1 mutant background, the FRQ degradation rates and its circadian period length are uncoupled, challenging the long proposed role of FRQ degradation in circadian clock function23.In the core 47. When the FRQ-CK1a interaction is abolished, WCs remain hypophosphorylated and active. Therefore, the physical interaction between FRQ and CK1a has two functions in the clock: this interaction mediates FRQ phosphorylation, and it is required for the circadian negative feedback process. In this study, by using a combination of genetic, biochemical, mathematical modeling and in silico experiments, our results support that the formation of the FRQ-CK1a complex is the major rate-limiting process in circadian period determination.To close the negative feedback loop, the FRQ-FRH complex interacts with the WC complex and recruits CK1a to phosphorylate WC proteins, a process that inhibits WC DNA binding15. Two mutants at FRQ phosphorylation sites either downstream of the FCD-2 domain (M9) or within the PEST-1 domain (M10) both exhibited very long period phenotypes   Fig. . Comparily) Fig. , indicat48, we examined the FRQ-CK1a interaction in these strains. Because FRQ phosphorylation is rhythmic in constant darkness (DD), we performed immunoprecipitation experiments using cultures grown in constant light (LL) which has constant FRQ phosphorylation profiles. Using a CK1a antibody that specifically recognizes the endogenous CK1a protein  and FCD2 (488–495), were previously shown to mediate the FRQ-CK1a complex formation Fig. . Mutatioion Fig. . These ahms Fig. , suggestNeurospora.Immunoprecipitation of CK1a from extracts of mutants showed that the period length negatively correlated with the FRQ-CK1a interaction: The longer the period length of the mutant, the weaker the interaction between FRQ and CK1a Fig. . In contComparison of FRQ phosphorylation profiles for cultures grown in constant light (LL) showed that FRQ became hypophosphorylated in strains carrying mutations in the FRQ FCDs Fig. . In addiWe compared the FRQ rhythmic phosphorylation profile between the WT and one of the FCD mutants (Q494N) in DD . Consistent with a previously reported Mass spectrometry data14, our IP experiments showed that the relative amount of FRQ associated with CK1a exhibited a circadian rhythm  resulted in long period phenotypes  was more sensitive to trypsin digestion than was hypophosphorylated FRQ (DD16)  Fig. . This refrq transcription, closing the negative feedback loop48. This latter process should occur independently of the effect of the FRQ-CK1a interaction on FRQ stability. As expected, the 4A12 mutation, which completely abolished the FRQ-CK1a interaction, resulted in hypophosphorylation of both FRQ and WC-2  assays using a WC-2 antibody. The relative WC-2 enrichment at the  DD Fig. . As expeain Fig. , consistfrq transcription. To allow side-by-side comparison and to avoid the interference from the endogenous FRQ protein, we inducibly expressed FRQ  in a frq9 strain from a construct induced by quinic acid (QA); this allowed control of FRQ expression in a QA concentration-dependent manner55. The frq9 strain has a frame-shift mutation in the frq open reading frame that results in a truncated non-functional FRQ protein and constant high frq RNA level due to the loss of circadian negative feedback process49. FRQ protein was induced to higher levels as QA concentration increased, and the FRQ levels were similar at each QA concentration in the three qa-FRQ strains  into FRQ protein, which interacts with CK1a (α1). The formation of FRQ-CK1a complex leads to subsequent phosphorylation of FRQ (α2) followed by a physical interaction with WCC (α3). Additional phosphorylation of FRQ and WC proteins (α4) leads to the inactivation of WCC (WCCi). Finally, the CK1-FRQpp-WCCi complex dissociates (α5), and each protein is either degraded or recycled. We assumed that there are two forms of WCC (WCCa and CK1-FRQ-WCCa) that activate the transcription of frq , but either one alone is sufficient to generate autonomous oscillations. Each species is degraded with a rate of δi (1–10). In addition, we incorporated the following steps based on our experimental observations that FRQ phosphorylation has opposing roles on FRQ-CK1a interaction: (1) the initial FRQ phosphorylation events (CK1-FRQp) promote the association of FRQ and CK1a (σ3), forming a positive feedback loop, and (2) hyperphosphorylation of FRQ (CK1-FRQpp) promotes the dissociation of FRQ-CK1a (σ4), forming an additional negative feedback loop. This molecular wiring diagram was converted into a set of ordinary differential equations for in silico analyses .Our experimental data established that the physical interaction between FRQ and CK1a is a critical molecular step in determining the period of the WCC Fig. . Briefly1, α2, αc) as the top three parameters and the rates of frq mRNA and FRQp-CK1a degradations . Our representative data using the parameter set 1 shows that the rate of association between FRQ and CK1a, α1, was the most sensitive parameter; reductions in α1 caused periods of more than 60 h  and the rate of phosphorylation FRQ-CK1a complex (α2) also increased the period to more than 40 h  and FRQ degradation after phosphorylation (δ5) moderately influenced the period, up to 36 h in case of δ1 and only 27 h for δ5 , showed a large region of oscillatory domain with minimal changes in period  occurs because the system is approaching to a bifurcation point  at which an unstable steady state with a stable limit cycle collides with a saddle point 59, generating an oscillation with an infinite period , which triggers positive feedback loop, are very low, resulting in a critical slowdown of the system. In other words, a certain amount of CK1-FRQp has to accumulate to trigger the positive feedback loop promoting the association of FRQ and CK1a. Therefore, if α1, α2, or αc are low, then it takes much longer time to accumulate a critical concentration of CK1-FRQp that will induce the positive feedback loop. In contrast, this unique dynamical behavior is not observed with other parameters in the model. Bifurcation analysis of δ1 showed that the oscillatory domain is bounded by two Hopf bifurcation points  depends on the prior interaction between these two proteins (α1). Therefore, our computational analyses revealed that the regulated complex formation between FRQ and CK1a is the major parameter in determining the period of the Neurospora circadian clock. In addition, our in silico analyses suggest that the molecular wiring diagram is one of the critical constraints that determine appropriate parameter space for robust oscillations.The level of CK1 is known to be relatively constant, and the phosphorylation of FRQ by CK1a  interaction domain of PER abolishes the ability of PER to be phosphorylated by CK1 and severely impairs the function of PER in transcriptional suppression61. In addition, DBT is required for the PER-dependent phosphorylation of CLOCK (CLK), which is associated with repression of E-box-dependent transcription63. The PER-CK1 interaction domain appears to be conserved in mammals64. In addition, the overexpression of the CK1δ/ε binding domain of PER2 abolishes circadian rhythms in cells65, indicating the critical importance of PER-CK δ/ε binding in clock function. Moreover, CK1δ phosphorylation of CLOCK-BMAL1 in the complex was recently suggested to promote the dissociation of CLOCK-BMAL1 from DNA26. These results suggest that, as in Neurospora, the complex formation between PER and CK1 in Drosophila and mammals regulates both PER phosphorylation and the circadian negative feedback process, and thus the PER-CK1 interaction may have a similar role in period determination in animal circadian systems.The formation of a tight stoichiometric complex between clock proteins and CK1 is conserved in bd, a) strain or a frq complementation strain (KAJ120) was used as the wild-type strain in this study. S513R, S519A, M9, M10, M13, M17, and ckbRIP strains were created previously35. The frqKO  strains were used as host strains for constructs with his-3-targeting vectors. The control strain was a frqKO strain transformed with KAJ120, a vector containing the wild-type frq gene. All constructs bearing FCD1 and FCD2 mutations were transformed into the frqKO strain. Constructs with the qa-2 promoter driving expression of Myc.His.CK1a and pBT6 were co-introduced into the KAJ120, S513R, S519A, and 4A12 strains by homologous recombination. Positive transformants were verified by western blot analyses. Constructs with the qa-2 promoter driving expression of Myc.His.CK1a.hph were introduced into the 87–3, frq1, frq7, and ckbRIP strains at the csr locus by homologous recombination. Constructs with the qa-2 promoter driving expression of Myc.His.CK1a.bar were introduced into the KAJ120, V320I and Q494N strains at the csr locus by homologous recombination. pBA50 constructs with the qa-2 promoter driving expression of WT, Q494N, or 4A12 was introduced into the frq9  strain at the his-3 locus by homologous recombination. Positive transformants were identified by western blot analyses, and homokaryon strains were isolated by microconidia purification using 5-µm filters .The 87–3 , 1× Vogel’s, 0.1% glucose, and 0.17% arginine. For rhythmic experiments, Neurospora was cultured in petri dishes in liquid medium for 2 days. The Neurospora mats were cut into discs and transferred into medium-containing flasks and were harvested at the indicated time points.Liquid cultures were grown in minimal media . When QA  was used to activate the The medium for race tube assays contained 1× Vogel’s salts, 0.1 or 0.03% glucose, 0.17% arginine, 50 ng/mL biotin , and 1.5% agar with or without QA. After entrainment of 24 h in the LL condition, race tubes were transferred to the DD conditions and marked every 24 h.66. Briefly, tissue was ground in liquid nitrogen with a mortar and pestle and suspended in ice-cold extraction buffer (50 mM HEPES (pH7.4), 137 mM NaCl, 10% Glycerol) with protease inhibitors at final concentrations: Pepstatin A (1 μg/mL), Leupeptin (1 μg/ml), PMSF (1 mM). For certain experiments with PPase inhibitors (made fresh): 25 mM NaF, 10 mM Na4P2O7.10H2O, 2 mM Na3VO4, 1 mM EDTA. After centrifugation, protein concentration was measured using protein assay dye . For western blot analyses, equal amounts of total protein (40 μg) were loaded in each protein lane. After electrophoresis, proteins were transferred onto PVDF membrane , and western blot analyses were performed. To analyze the phosphorylation profile of WC-2, 7.5% SDS-PAGE gels containing a ratio of 149:1 acrylamide/bisacrylamide were used. Otherwise, 7.5% SDS-PAGE gels containing a ratio of 37.5:1 acrylamide/bisacrylamide were used.Protein extraction, quantification, and western blot analyses were performed as previously described67. For northern blot analyses, equal amounts of total RNA (20 μg) were loaded onto agarose gels. After electrophoresis, the RNA was transferred onto nitrocellulose membrane . The membrane was probed with [32P] UTP  -labeled RNA probes specific for frq 5′UTR. RNA probes were transcribed in vitro from PCR products by T7 RNA polymerase (Ambion) as per the manufacturer’s protocol. The frq primer sequences used for the template amplification were frq Northern F, 5′-TAATACGACTCACTATAGGGGAAGCCTGCGATGCTACTCAC-3′, and frq Northern R, 5′-CATCGCATCCACTTCGCACAC-3′. The list of primers used in this study is shown in Supplementary Table RNA was extracted with Trizol  in accordance with the manufacturer’s protocol, and then further purified with 2.5 M LiCl bed with 2P UTP , 1 μL WC-2 antibody43, or 1 μL CK1a antibody were used. After incubation with antibody for 3 h, 40 μL GammaBind G Sepharose beads  were added, and samples were incubated for 1 h. For the FLAG pull-down assay, 4 mg purified FLAG-CK1a fusion protein and 2 μL FLAG antibody  were incubated with 2 mg Neurospora extracts. After incubation with antibody for 3 h, 40 protein GammaBind G Sepharose beads were added, and samples were incubated for 1 h. Immunoprecipitated proteins were washed three times using extraction buffer before Western blot analysis. IP experiments were performed using cultures harvested in constant light (LL) except for the rhythmic experiments. Quantification of relative FRQ-CK1a or FRQ-WC-2 interaction levels are based on the ratio of IP to Input and normalized with CK1a or WC-2 levels, respectively.IP analyses were performed as previously describedFor CHX  treatments, discs made from 2-day-old mycelial mats of individual strains were grown in liquid media for 1 day under constant light conditions. CHX was then added to a final concentration of 10 μg/mL, and tissues were harvested at the indicated time points.For the trypsin-sensitivity assay, protein extracts were diluted to a protein concentration of 2.5 μg/μL. A 100-μL aliquot was treated with trypsin  at 25 °C. A 20 μL sample was taken from the reaction at each time point  after addition of trypsin. Protein samples were mixed with protein loading buffer and resolved by SDS-PAGE. To compare trypsin sensitivity of FRQ from different strains, experiments were performed side by side, and the protein samples were transferred to the same membrane for western blot analysis.68. Briefly, Neurospora tissues were fixed with 1% formaldehyde for 15 min at room temperature with shaking. Glycine was then added at a final concentration of 125 mM. The cross-linked tissues were ground and resuspended in lysis buffer . Chromatin was sheared using sonication to 500–1000 base-pair fragments. For each immunoprecipitation reaction, 1 mg protein was used. WC-2 antibody was generated previously43. The ChIP reaction was carried out with 2 μL WC-2 antibody. Immunoprecipitated DNA was enriched using GammaBind G Sepharose beads and washed sequentially with low-salt washing buffer , high-salt washing buffer , LNDET buffer , and TE buffer . Elution buffer  was used to retrieve protein-DNA complex. Finally, eluted DNA was purified and quantified by real-time qPCR. The primer sets used were frq C-box F, 5′-GTCAAGCTCGTACCCACATC-3′, and frq C-box R, 5′-CCGAAAGTATCTTGAGCCTCC-3′. Occupancies were normalized by the ratio of ChIP to Input DNA.ChIP assays were performed as previously described69.The GST-CK1a fusion protein (containing CK1a amino acids Q299-E384) was expressed in BL21 cells, and the recombinant proteins were purified and used as the antigens to generate rabbit polyclonal antiserat-test.Quantification of western blot and northern blot data were performed using Image J software. All studies were performed at least three independent experiments (different days and different preparations). Error bars are standard deviations for IP assays and standard error of means for race tube assays. Statistical significance was determined by Student’s Variables used are:Fm] = concentration of frq mRNA[F] = concentration of FRQ[C] = concentration of CK1[F:C] = concentration of complex FRQ:CK1[Fp:C] = concentration of complex FRQp:CK1[Wa] = concentration of WCCa[Wi] = concentration of WCCi[Fp:C:Wa] = concentration of complex FRQp:CK1:WCCa[Fpp:C:Wi] = concentration of complex FRQpp:CK1:WCCi[Fpp] = concentration of double-phosphorylated FRQ[Parameters used are listed below:α1, α2, α3, α4: forward reaction ratesα5: dissociation rateβ1, β2, β3, β4: backward reaction ratesαf: synthesis rate of FRQ proteinαc: synthesis rate of CK1αw: synthesis rate of WWCaδ1, δ2, δ3, δ4, δ5, δ6, δ7, δ8, δ9, δ10: degradation ratesσ1,σ2: transcription ratesσ3: activation rate of autocatalysisσ4: Fpp:C:Wi dependent dissociation of F:Cσ5: conversion of Wi to WaK1, K2, K3, K4: thresholdsSystem of ordinary differential equations are listed below:frq gene expression using a luciferase reporter fused with the frq promoter in constant dark (DD) conditions in three independent experiments  estimation. In the statistical inference, we view the experimental measurement of τ as 0.1, so that the fitted values of i = 1, 2, 3. After 1000 iterations of burn-in period, we store the MCMC draws up to 4000 iterations, resulting in numerous sets of model parameter estimates. Distribution of parameters is summarized in Supplementary Fig. 5 shows a large spread of parameter space ranging from close to 0 to about 11 with a mean value close to 0. This indicates that α5 is not a critical parameter in the system, because oscillations can be generated even when its value is close to 0 . In contrast, δ1 and δ5 show tighter region of parameter space, which suggest that they may play critical roles in generating autonomous oscillations. However, distribution of these parameters does not indicate how the circadian period is altered as a function of each parameter.We put a simple assumption that the deviation of frq gene expression levels (solid curves) determined by the randomly selected three different sets of estimated parameters are similar to each other. Supplementary Table frq curves) are identical.Supplementary Fig. Neurospora circadian clock system such as changes in period and molecular levels. Bifurcation diagrams were obtained by varying a single parameter, while the rest of the parameters were held fixed as their default values; see Fig. For each parameter set, we performed one-parameter bifurcation analyses to investigate the dynamical changes in the P0 is the period of default parameter values and ΔP is the difference between the maximum period and minimum period as each parameter varies from 0 to 100% more than the default parameter value. For each parameter set, the ranking of period sensitivity is listed in Supplementary Table We also evaluated the period sensitivity of each parameter for three parameter sets as follows:entclass1pt{minimaFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary