Herein, we evaluated CBD’s inhibitory effects on α-glucosidase using in vitro assays and computational studies.Cannabidiol (CBD) has been reported to have anti-diabetic effects in pre-clinical and clinical studies but its inhibitory effects on α-glucosidase activity was evaluated in a yeast enzymatic assay and by molecular docking. The stability of CBD in simulated gastric and intestinal fluids was evaluated by high-performance liquid chromatography analyses.CBD’s inhibitory effect on α-glucosidase activity with inhibition of 17.1, 20.4, 48.1, 56.6, 59.1, 63.7, 74.1, and 95.4%, respectively. Acarbose, the positive control, showed a comparable inhibitory activity (with 85.1% inhibition at 608 μM). CBD’s inhibitory effect on α-glucosidase was supported by molecular docking showing binding energy  and interactions between CBD and the α-glucosidase protein. CBD was stable in simulated gastric and intestinal fluids for two hours (maintained ≥ 90.0%).CBD, at 10, 19, 38, 76, 152, 304, 608, and 1216 μM, inhibited α-glucosidase activity and was stable in gastric and intestinal fluids. However, further studies on CBD’s anti-α-glucosidase effects using cellular and in vivo models are warranted to support its potential application for the management of type II diabetes mellitus.CBD showed moderate inhibitory effect against yeast  Our group has had a long research interest in the identification of α-glucosidase inhibitors from medicinal plants  based in vitro assay. In addition, CBD’s inhibitory effect on α-glucosidase was studied by computational docking experiment and its stability was evaluated in simulated gastric and intestinal fluids.Increasing legalization and social perceptions of medical use of cannabis raises the issue of patients using cannabis and its derivatives, such as cannabidiol (CBD), to self-medicate for certain medical conditions including diabetes. Claims that cannabis derivatives have beneficial effects on diabetic conditions are easily accessed by the public. However, these claims of cannabis combating diabetes are not substantially supported by medical research published in peer-reviewed journals. To date, only one randomized, double-blind, placebo-controlled, parallel group pilot study reported the investigation of CBD’s efficacy on glycemic parameters in patients with type II diabetes  from Saccharomyces cerevisiae, p-nitrophenyl-α-D-glucopyranoside (pNPG), methanol , were obtained from Sigma Aldrich . Simulated gastric fluid (CAT# 7108–16) and intestinal fluid (CAT# 7109.75–16) were obtained from RICCA Chemical Company .Cannabidiol (CBD), acarbose, dimethyl sulfoxide (DMSO), α-Glucosidase inhibition assay was carried out using previously reported method  was incubated in a 96-well plate at room temperature for 10 min followed by adding pNPG  to each well. The reaction mixtures were incubated at room temperature for 30 min before the absorbance was recorded at wavelength of 405 nm with a micro-plate reader . α-Glucosidase inhibitory activity was expressed as inhibition% as compared to the control group. Each sample was tested three times, each in three replicates (n = 3), and data were shown as mean ± standard deviation (S.D). Statistical analysis was performed using GraphPad Prism 7  using one-way analysis. A p-value that less than 0.001 (***) or 0.0001 (****) was considered as a statistical significance between the control group and the experimental groups.α-glucosidase from Saccharomyces cerevisiae were retrieved in PDB format from the RCSB protein data bank  . AutodockTools 1.5.6 was used to perform molecular docking using Autodock 4.2 algorithm . Files of CBD and 3A4A were converted into pdbqt format and then a grid parameter file was prepared as a glg file in AutoGrid 4 software. Docking parameter file was prepared for the final docking and Autodock 1.5.6 software was used to perform molecular docking.The 3D structure of 18 column  and the HPLC solvent system consisted of 0.1% trifluoroacetic acid in water (B)/methanol (A) linear gradient as follows: 1–19 min,80% A; 19–20 min, 80–100% A; 20–30 min, 100% A; 30.1–36 min, 80% with a total run time of 36 min, a flow rate of 0.75 mL/min, and an injection volume of 10 μL. The CBD content in the solutions were presented as the area under curve (AUC) value.CBD was prepared in simulated gastric and intestinal fluids according to a previously reported method , CBD’s anti-α-glucosidase activity was comparable to the positive control, acarbose  with inhibition rates of 46.8, 76.5, and 85.1% at concentrations of 19, 152, and 608 μM, respectively. The inhibitory effect of CBD on yeast α-glucosidase enzyme activity was supported by molecular docking. The binding site of CBD was predicted to be at several helix and coil structures of α-glucosidase protein  and π-alkyl bond (between benzyl ring and proline 312)  vale of CBD was 142,944,597, 128,856,052, and 129,986,500 in the control group and in the experimental groups including simulated gastric and intestinal fluids, respectively. CBD was fairly stable in both simulated gastric and intestinal fluids for two hours with a remaining CBD level of 90.1 and 90.9%, respectively, as compared to the control group . Although yeast α-glucosidase enzyme is a well-accepted experimental model to evaluate the potency of α-glucosidase inhibitors (Hakamata et al. α-glucosidase inhibitors showed weaker activity when they are evaluated in assays with α-glucosidase obtained from mammalian sources (Yuan et al. α-glucosidase enzymatic assay showed promising anti-α-glucosidase activity but its efficacy as a α-glucosidase inhibitor in animals or human remains unknown. Therefore, further evaluations of CBD’s inhibitory effects on α-glucosidase using cellular and in vivo models are warranted. In addition, given that there are hundreds of phytocannabinoids in cannabis, they may also exert inhibitory effects on α-glucosidase enzyme and therefore investigations of ‘whole extracts’, beyond just CBD alone, is warranted. This is also important since other ‘non-phytocannabinoids’ types of compounds in cannabis extracts may also contribute to their overall anti-glycemia effects via the modulation of α-glucosidase enzyme activity. For instance, flavonoids including apigenin, luteolin, quercetin, and kaempferol have been identified in cannabis extracts and they have been reported as potent α-glucosidase inhibitors (Proença et al. A large number of α-glucosidase activity in vivo is its stability in a physiologically relevant environment. This is because α-glucosidase enzymes are secreted in the brush border of the small intestine and CBD would have to survive drastic pH conditions, including acidic gastric and basic intestinal environments, to reach the small intestine for desired biological functions. Our current study supported that CBD is fairly stable after exposure to simulated gastric and intestinal fluids for two hours. This is in agreement with previously reported pharmacokinetic studies showing that CBD did not convert to tetrahydrocannabinol in humans (Crippa et al. α-glucosidase is also expressed in other organs including lung and epididymis at lower levels (Peña et al. A factor that could play an important role in CBD’s anti-α-glucosidase enzyme, which was supported by molecular docking experiments. In addition, CBD was stable in simulated gastric and intestinal fluids for two hours. Further cellular and in vivo studies are warranted to evaluate CBD’s anti-α-glucosidase and anti-hyperglycemia activity. Overall, the findings from this study add to the growing body of evidence supporting the potential use of CBD for type II diabetes management.In summary, CBD showed moderate inhibitory effect on the activity of yeast


Accounting for internal variability reveals insights into mechanisms underlying an Arctic–mid-latitude teleconnection riddle. Arctic sea ice extent in autumn is significantly correlated with the winter North Atlantic Oscillation (NAO) in the satellite era. However, questions about the robustness and reproducibility of the relationship persist. Here, we show that climate models are able to simulate periods of strong ice-NAO correlation, albeit rarely. Furthermore, we show that the winter circulation signals during these periods are consistent with observations and not driven by sea ice. We do so by interrogating a multimodel ensemble for the specific time scale of interest, thereby illuminating the dynamics that produce large spread in the ice-NAO relationship. Our results support the importance of internal variability over sea ice but go further in showing that the mechanism behind strong ice-NAO correlations, when they occur, is similar in longer observational records and models. Rather than sea ice, circulation anomalies over the Urals emerge as a decisive precursor to the winter NAO signal. Autumn Arctic sea ice loss, especially over the Barents-Kara Sea, is associated with a negative North Atlantic Oscillation (NAO) the following winter . This obThe ice-NAO relationship, simulated by climate models from the Coupled Model Intercomparison Project Phase 5 (CMIP5), differs substantially from that observed over recent decades, as reported in a number of previous studies. Long, preindustrial control simulations show very weak correlations between autumn/winter sea ice and the NAO (or Arctic Oscillation) compared to observations  climate variability, along with ensembles of shorter transient simulations to examine the influence of transient climate forcing. This analysis allows for a quantitative assessment of whether climate models capture the observed ice-NAO relationship and leads to new insights into the underlying physical mechanisms.The statistical relationship between autumn sea ice and the late-winter NAO is, at first glance, very weak in coupled climate models compared to reality. To put the comparison on a more equal footing, we use a bootstrapping test in which we randomly sample 40 years from a preindustrial control simulation  and repeat this 10,000 times . The distribution of correlations from the 10,000 bootstrap samples may then be compared to observations directly.The bootstrapped ice-NAO correlations show a large spread encompassing both negative and positive values throughout the cold season  that mimic reality in the period of interest. To account for internal variability, one can run a transient simulation multiple times with slightly different initial conditions to produce a multimember ensemble. One such “large ensemble” is the 40-member CESM1–Large Ensemble (LENS) [see Materials and Methods; . SimilarThe results to this point indicate that internal variability strongly modulates the ice-NAO relationship on time scales of the satellite-era observational record, regardless of the presence of transient external forcing. Within the large range of possible ice-NAO behavior, the ice-NAO correlation over the past 40 years is a rare occurrence. It is also unusual in the context of observational products covering the 20th century  turbulent heat fluxes through the winter in the satellite era . In this way, we identify a group of samples that are most similar to the observations , but we find no support for this. Causality would require a physical linkage from ice to surface turbulent heat flux anomalies , indicatThe fact that climate models and longer observational records yield comparable spread in ice-NAO correlations on a 40-year time scale suggests that the strong negative correlation over the most recent 40-year period is indeed unusual. However, it is possible that sea ice can drive a winter NAO response through teleconnection pathways that global climate models fail to capture . These periods are marked by circulation signals that are consistent with proposed teleconnection mechanisms, including atmospheric blocking over the Urals and a weakening of the polar vortex. However, the winter circulation changes are primarily driven by atmospheric variability rather than autumn sea ice variability, with Urals blocking emerging as a decisive precursor. Barnes and Screen  ERA5 reanalysis dataset . We removed the climatological monthly/daily means in all data from all reanalysis and simulations. We further detrended all data from the reanalysis and transient simulations. The results are not sensitive to the detrending method  or to whether decadal detrends in preindustrial simulations are removed or not. Here, we used the monthly sea ice concentration and sea level pressure to calculate the following:1) Barents-Kara sea ice (ice): sea ice area fraction area-averaged over 65°N to 85°N, 10°E to 100°E (2) NAO: difference in area-averaged sea level pressure between two regions, 55°N to 85°N, 40°W to 20°E and 25°N to 55°N, 50°W to 10°E (Nov) and the monthly NAO index for each month from October to February (NAOOct–Feb) in the observations and the preindustrial control simulations. In the simulations, we first examined the iceNov-NAOOct–Feb correlations over the entire simulation period to assess the long-term relationship. Then, we examined these correlations within 10,000 bootstrap samples, each comprising 40 winter seasons (October to February) randomly drawn from the entire simulation without replacement. The bootstrapping approach generates a large number  of iceNov-NAOOct–Feb correlations based on periods that are equivalent in length to the observations, providing an estimate of the range of ice-NAO correlations due to internal climate variability. The random sampling assumes that every year is equivalent, which is reasonable for unforced preindustrial control simulations. Similar analyses were repeated for the consecutive 40-year periods, and the result was found to hold, meaning that it is not sensitive to the presence of lower-frequency internal variability.We calculated lagged correlations between the Barents-Kara sea ice index in November . The CESM1-LENS experiment provides some measure of the range of internal variability over its 40 members.Nov-NAONov–Feb correlations , we calculated the root mean square error (RMSE) to quantify the difference between the ERA5 reanalysis (observations) and the models. Bootstrap samples with smaller RMSE values were determined to better resemble the observations. We repeated the procedure for each bootstrap sample to identify the 100 bootstrap samples (1%) with an ice-NAO relationship most and least similar to the observations. Similar results were obtained when defining groups according to iceNov-NAODec–Feb or iceNov-NAOJan–Feb correlations and when using alternative methods for identifying the least similar  samples.Bootstrap samples were classified according to how well they reproduce the observed relationship between autumn sea ice and the late-winter NAO to investigate the proposed underlying mechanisms. Considering the monthly iceTo investigate proposed mechanisms in these samples, we defined a selection of indices that represent the key features of the teleconnection pathways (1) Turbulent heat flux: sum of surface sensible and latent heat flux area-averaged over 65°N to 85°N, 10°E to 100°E, with positive defined as heat flux from the ocean to the atmosphere;2) Urals sea level pressure: sea level pressure area-averaged over 45°N to 70°N, 40°E to 85°E ;3) Polar cap height at 50 hPa: monthly geopotential height at 50 hPa area-averaged from 65°N poleward;4) Polar cap height in all levels: daily geopotential height from 10 to 1000 hPa, area-averaged from 70°N poleward at each level.Several metrics were used to compare the indices from the selected bootstrap samples with those from the observations: lagged correlations with ice (monthly), gridpoint regressions onto ice (monthly) to investigate spatial signatures, and lagged height-time correlations between ice and polar cap height (daily) to investigate the downward influence of the stratosphere on the troposphere. To further investigate the underlying mechanisms, we partitioned the 40-year bootstrap samples according to hypothesized linkage mechanisms on the basis of correlations between November ice and November to December turbulent heat flux, and correlations between November Urals sea level pressure and January to February NAO.


O-glucuronide) was examined using ELISA microtiter assay. Among them, only smilagenin and kokusaginine displayed inhibitory action against AChE . BChE was inhibited by only methyl rosmarinate and kokusaginine . IC50 values for galantamine as the reference drug were 1.33 ± 0.11 µg/mL for AChE and 52.31 ± 3.04 µg/mL for BChE. Molecular docking experiments showed that the orientation of smilagenin and kokusaginine was mainly driven by the interactions with the peripheral anionic site (PAS) comprising residues of hAChE, while kokusaginine and methyl rosmarinate were able to access deeper into the active gorge in hBChE. Our data indicate that similagenin, kokusaginine, and methyl rosmarinate could be hit compounds for designing novel anti-Alzheimer agents.Cholinesterase (ChE) inhibition is an important treatment strategy for Alzheimer’s disease (AD) as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the pathology of AD. In the current work, ChE inhibitory potential of twenty-four natural products from different chemical classes (i.e., diosgenin, hecogenin, rockogenin, smilagenin, tigogenin, astrasieversianins II and X, astragalosides I, IV, and VI, cyclocanthosides E and G, macrophyllosaponins A-D, kokusaginin, lamiide, forsythoside B, verbascoside, alyssonoside, ipolamide, methyl rosmarinate, and luteolin-7- Neurodegenerative diseases, most of which are incurable, are characterized by progressive neuronal damage in the brain and peripheral nervous system occurring by complicated heterogenous mechanisms. Among them, Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease defined as the most common type of dementia. In fact, AD has become a major global health concern due to its increasing prevalence particularly in elderly population affecting health care expenses of many countries ,2. AD isN-methyl-D-aspartate (NMDA) receptor antagonism [Neuropathology of AD is correlated with two main hypotheses generally accepted as “cholinergic hypothesis” and “amyloid hypothesis” ,5,6. Curtagonism ,8,9. ChEtagonism . Since Atagonism . Howevertagonism .Galanthus woronowii Losinsk., Amaryllidaceae) [N-norgalanthamine as a promising dual ChE inhibitor of herbal origin [O-glucuronide using ELISA microtiter assays  ,14. Recel origin . Similarr assays . The tes50 values of 43.29 ± 1.38 and 70.24 ± 2.87 µg/mL. Kokusaginine (IC50 = 61.40 ± 3.67 µg/mL) and methyl rosmarinate (IC50 = 41.46 ± 2.83 µg/mL) were able to inhibit BChE effectively. IC50 values galantamine as the reference drug were calculated as 1.33 ± 0.11 µg/mL for AChE and 52.31 ± 3.04 µg/mL for BChE. Rest of the compounds tested had AChE inhibition ranging between none to 25.36 ± 1.11%, while they exerted none to 22.24 ± 2.54% of inhibition against BChE  and hBChE (PDB: 4TPK) active site using the Glide module implemented in Schrödinger Small-Molecule Drug Discovery Suite.50 value of 43.29 µg/mL, occupied the region between the oxyanion hole and the PAS in AChE active site forming a water-mediated hydrogen bond with TYR124, the PAS residue. Besides, a hydrogen bond stabilizing the inhibitor-protein complex was formed between its 3-hydroxyl group and SER293  comprising residues . Smilaged SER293 A. Kokusad TYR341 B. The bed TYR341 C. Hecoged TYR341 D,E. The d TYR341 F.The binding energies of the docked compounds and the residues interacted in hAChE active site were given in 50 value of 41.46 µg/mL, was positioned toward the region between acyl binding site and oxyanion hole of catalytic site  isolated from Asparagus racemosus was reported 9.9 μM and 5.4 μM for AChE and BChE, respectively, whereas we found only 15.04 ± 3.01% of inhibition by the same compound at 100 μg/mL. Besides, it was revealed to possess neuroprotective effect through anti-amyloidogenic effect against Aβ42 and H2O2-mediated cytotoxicity on PC12 cells. In addition to its ChE inhibitory effect, smilagenin earlier displayed neuroprotection in rat cortical neurons and SH-SY5Y cells [Nature has afforded many efficaciously ChE-inhibiting natural products isolated from plants, marine organisms, and microorganisms having different chemical core structures ,17,18. I60 μg/mL . Consistp et al. , IC50 va5Y cells  and rais5Y cells . Althoug5Y cells . ConsideAstragalus species, particularly in A. membranaceus. During our literature survey, we have not so far come across a study relevant to ChE inhibition by astragalosides. Nevertheless, Santoro et al. lately described low inhibitory activity of three commercially available A. membranaceus root extracts along with the extract of the same species they prepared [25–35-induced cytotoxicity and synaptotoxicity as well as blocking mitochondrial dysfunction [Astragalosides are the cycloartane type of major saponins in prepared . Their ifunction ,32. No nOlea europea L. has been demonstrated to show no inhibition against both ChEs [Calceolaria talcana J.Grau & C.Ehr. was reported with IC50 values of 189.9 μg/mL for AChE and 105.9 μg/mL for BChE, when compared to galanthamine (reference compound) having consequent IC50 values of 13.2 and 7.3 μg/mL against AChE and BChE [50 = 27.6 μg/mL) and no AChE inhibition. Relevantly, verbascoside from Pseuderanthemum carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb. was identified to have a rather weak AChE inhibition (<50%) at 100 μg/mL [Verbascum xanthophoeniceum Griseb. and V. mucronatum Lam. indicated low ChE inhibition below 30% at 100 μg/mL, which comply with the current data on this compound [Verbascoside as a phenylpropanoid derivative isolated from oth ChEs . In anotand BChE . In the 00 μg/mL . Our precompound ,37.Esenbeckia leiocarpa Engl. and Evodia lepta (Spreng). Merr. [Kokusaginine, a furoquinoline alkaloid tested herein, was identified with moderate AChE and prominent BChE inhibition, closer to that of the reference . Previou). Merr. ,39.Although rosmarinic acid was earlier reported to be the prominent inhibitor of ChEs by different researchers including us ,41,42,43Digitalis cariensis Boiss. (Scrophulariaceae). The isolation procedure of tigogenin was described elsewhere [Haplophyllum myrtifolium Boiss. (Rutaceae), whose isolation was reported by Sener et al. [Thymus pseudopulegioides Klokov & Des. -Shost. (syn. Thymus nummularius M. Bieb.) collected from Ormanustu plateau, Macka district, Trabzon province, August, in 2014 at the altitude of 1850 m. The plant was identified by Prof. Dr. Zeki Aytac  and preserved at the Herbarium of Pharmacy Faculty of Ankara University (AEF 23176). The air-dried and powdered aerial parts of the plant (300 g) were extracted with methanol (MeOH) (2000 mL × 3) under reflux at 40 °C for 3 h and combined MeOH extracts were concentrated under reduced pressure. MeOH extract (64 g) was suspended with H2O:MeOH . This mixture was partitioned with CHCl3 (300 mL × 3) and ethyl acetate (EtOAc) (300 mL × 3), respectively. CHCl3, EtOAc, and aqueous phases were evaporated at reduced pressure at 40 °C. EtOAc extract (6.5 g) was subjected to Sephadex LH-20 column by eluting with MeOH. Five fractions (A-E) were obtained. Fraction C (1.3 g) were subjected to silica gel column by eluting with CHCl3:MeOH:H2O (70:30:3→50:50:5). Fractions 13–45 (160 mg) were combined and then were subjected to VLC. The combined fraction of 96–107 was subjected to Sephadex LH-20 column by eluting with MeOH, which yielded methyl rosmarinate (20 mg). The structure of the compound was elucidated by using by spectroscopic methods using 1H-NMR and 13C-NMR, which the NMR data of the compound agreed well with the reported data in the literature [Diosgenin (CAS number 512-04-9), hecogenin (CAS number 467-55-0), rockogenin (CAS number 16653-52-4), and smilagenin (CAS number 126-18-1) were obtained from Sigma Aldrich . Tigogenin was earlier isolated from the roots of lsewhere . Kokusagr et al. . Methyl Astragalus oleifolius DC. [Astragalus cephalotes var. brevicalyx Eig [Astragalus melanophrurius Boiss. [Isolation of the tested cycloartane-type triterpene saponins was formerly achieved. Macrophyllosaponins A-D were obtained from the roots of lius DC. , while calyx Eig ,49,50. Is Boiss. ,50.Inhibition of AChE and BChE was determined by Ellman’s method with slight modifications . To the E value expresses the effect of the test sample or the positive control on AChE/BChE/TYR enzyme activity articulated as the percentage of the remaining activity in the presence of test sample or positive control. C value is the absorbance of the control solvent (blank) in the presence of enzyme, where T is the absorbance of the tested sample (plant extract or positive control in the solvent) in the presence of enzyme. Data are expressed as average inhibition ± standard deviation (S.D.) and the results were taken from at least three independent experiments performed in triplicate.The measurements and calculations were evaluated by using Softmax PRO 4.3.2.LS software. Percentage of inhibition of AChE/BChE/TYR was determined by comparison of rates of reaction of test samples relative to blank samples. Extent of the enzymatic reaction was calculated based on the following equation: I% = (C-T)/C × 100, where I% is the activity of the enzyme as percent inhibition. The molecular docking studies were carried out using Glide protocol implemented in the Schrödinger Small-Molecule Drug Discovery Suite . The compounds which were built via builder panel in Maestro were subjected to ligand preparation by LigPrep  using default conditions. The x-ray crystal structures of the hAChE (PDB: 4EY7)  and the According to our results obtained from our screening on twenty-four natural products; smilagenin, kokusaginine, and methyl rosmarinate the compounds came into prominence as the promising ChE inhibitors. The active inhibitors selected for molecular docking studies displayed similarity in interaction with PAS region of hAChE. For BChE, the compounds were located deeply in the catalytic site due to larger active site of hBChE compared to hAChE. The molecular docking simulations performed may suggest that being capable to interact with the key residues highlighted is in full agreement with their inhibitory potency against AChE and BChE. Thus, smilagenin (a saponin derivative), kokusaginine  and methyl rosmarinate (a phenolic acide ester) out of twenty-four natural products tested herein could be evaluated as the hit molecules for further anti-Alzheimer research. Our research also supports the previous findings that natural compounds having diverse chemical structures still represent a hope for discovering novel candidate ChE inhibitors.


In pathway association studies, forty significant pathways had FDR  and or FWER (family-wise error rate) ≤ 0.001: spermatogenesis, leukocyte trans-endothelial migration, cell adhesion, glycoprotein, membrane lipid, steroid metabolic process, and insulin signaling pathways were among the most significant pathways that associated with the plasma cystatin C levels. We also performed Genome-wide association studies for eGFR, top associated genes were largely overlapped with those for cystatin C.As a marker for glomerular filtration, plasma cystatin C level is used to evaluate kidney function. To decipher genetic factors that control the plasma cystatin C level, we performed genome-wide association and pathway association studies using United Kingdom Biobank data. One hundred fifteen loci yielded  Chronic kidney disease (CKD) is a global public health problem with high morbidity , which pCST3 on 20p11.21), as an endogenous biomarker of kidney function  in European-ancestry subjects   was applSubjects were originally collected to United Kingdom Biobank, which is an open access prospective cohort study in the United Kingdom. In 2006–2010, a total of 502,665 subjects from across the United Kingdom with age between 40 and 69 were recruited to participate in United Kingdom Biobank . Of thes® Array  or the Affymetrix United Kingdom BiLEVE Axiom® Array  and the methods have been described in detail , SNPs were also preselected with an LD-pruning method and those SNPs were deleted in the same LD block (r2 > 0.6). IBD checking and adjustment of MDS component were performed in 5,000 randomly chosen Caucasian subjects. We also performed GWASs for eGFR, creatinine, and urea, respectively. Besides GWAS, we also conducted gene-based association study for cystatin C that implemented in MAGMA  procedure was conducted to control the fraction of expected false-positive findings and family-wise error rate (FWER) ensured that there were no false positive gene sets in the output list. SNPs with minor allele frequencies (MAF) < 0.05 or Hardy-Weinberg equilibrium (HWE) < 0.001 were excluded and SNPs mapped at 20bp upstream or downstream of the gene region are regarded as part of the gene. A total of 377,863 SNPs that covered 21,787 genes were selected for Pathway-based association analyses. Meanwhile, 1,347 pathways/gene sets with size between 5 and 200 genes were selected from Kyoto Encyclopedia of Genes and Genomes (KEGG), BioCarta, and Gene Ontology (GO) databases. Empirical p values ≤0.001, the false-discovery rate (FDR) and or family-wise error rate (FWER) ≤ 0.001 were considered as significant.Pathway-based association studies were carried out by the GenGen program  based onWe collected phenotype data of plasma cystatin C from subjects in the United Kingdom Biobank, 460,858 participants were included in our study after excluding outliers (>3 SD). The average level of cystatin C was 0.899 ± 0.137 mg/L . Distributions of cystatin C were shown in p values less than 1 × 10−100, including significant associations in three gene regions: the CST gene cluster on 20p11.21, the SHROOM3 -CDCC158 region on 4q21.1, and the SH2B3-ATXN2 region on 12q24.11. The gene regions are shown in the Manhattan plots . However, no association was found for other renal function related phenotypes  for CST cluster SNPs.Quantitative GWAS analyses were performed for cystatin C in United Kingdom Biobank data. One hundred fifteen SNPs had an plots . Analysean plots . Many SNan plots . The mosSHROOM3 gene, one of the most replicated loci for renal function, reached a p < 1 × 100−200  with cystatin C  in the  100−200 . The SNPectively . rs90744statin C . A SNP rstatin C .We carried out pathway associations based on the GSEA algorithm. A total of 1,347 Kyoto Encyclopedia of Genes and Genomes (KEGG), BioCarta, and Gene Ontology (GO) pathways were tested. Plasma cystatin C, as a superior endogenous biomarker of kidney function and healthy aging, is reported to be produced at a near constant rate and not to be affected by age, gender, or weight . HoweverCST3, UMOD, ATXN2, and STC1 were identified. Of note, CST3, located in cystatin (CST) superfamily gene cluster on chromosome 20 and encoding the most abundant extracellular inhibitor of cysteine proteases, has associated with plasma cystatin C levels. SNPs that significantly related to cystatin C were identified, including rs1158167  have been reported . From ths1158167 , rs13038s1158167  and rs91s1158167  on the CSH2B3-ATXN2 region on chromosomal 12q24.11 is another locus that we found related to cystatin C, which had associated with a wide spectrum of complex diseases, such as hematopoietic disorders of red and white blood cells, rheumatoid arthritis, retinal microcirculation problems, chronic kidney disease, cardiovascular infarction, and neurodegenerative diseases in previous studies . Indeed, several independent GWASs reported that rs653178 displayed significant associations with renal function phenotypes  and CKD in populations of European and African ancestry  which is in the pleckstrin homology (PH) domain of LNK and induces changes in the structure and hydrophilic properties. At the same time, the missense variant also introduces a new phosphorylation site in the PH domain which may influence signaling pathways mediated by SH2B3. High conservation in mammals of the R262 residue in LNK suggests that it may have irreplaceable function in biological activities (SH2B3 seems to be a powerful pleiotropic variant involved in multiple complex diseases or traits including cystatin C. in mice . The SNPancestry . SNPs intivities . This maSHROOM3) also appeared to be very significant in our GAWS studies on cystatin C. It plays an important role in mammalian kidney development and human kidney disease including CKD and ESRD through estimated glomerular filtration rate based on creatinine (eGFRcrea) (p = 5.32 × 10−124) in SHROOM3 gene showed a strong correlation with cystatin C levels, which has not been reported by association research. However, the intronic SNP rs17319721 located in a highly conserved region in SHROOM3 on chromosome 4 proved to be related to glomerular filtration rate  in our study. The consistency of renal traits suggests that it may be an important functional site that interacts with other gene sites to perform its function. Another significant cystatin C associated SNP in SHROOM3 was rs9992101  and both closely associated with CKD (Shroom family member 3 (GFRcrea) . SHROOM3GFRcrea)  and partGFRcrea) , which mGFRcrea) . Remarkaion rate  and impafibrosis . It is wwith CKD .CCDC158) gene SNPs were significantly associated with cystatin C levels. CCDC158, located on 4q21.1, contains a conserved coiled-coil domain, which are found in important proteins involved in crucial interactions such as transcription factors (leucine zippers), molecular motors , receptors, and signaling molecules  . AbundanIn summary, recent technological advancements resulted in large collections of whole-genome genotyping data which provides better coverage and increased imputation quality. It was the largest GWAS for cystatin C in sample size, and the first genome wide pathway association study for cystatin C that ever carried out. We have also found novel associations and pathways for plasma cystatin C levels. However, under current conditions, we are unable to verify biological functions of results from our pathway research, further verifications in the transcriptome dataset are needed in the future.We performed genome wide association and pathway association studies on cystatin C in an open access prospective cohort study. We have identified three major gene loci and 40 novel biological pathways, including cell adhesion and insulin signaling pathways, that associated with the plasma cystatin C levels. Our results provided more genetic bases for further understanding the phenotypic variations of cystatin C.


Here, we report the complete genome sequence of infectious bronchitis virus (IBV) strain JP/KH/64, which is the reference strain for the JP-I genotype in Japan. This information should be useful for an in-depth understanding of the evolution of the JP-I genotype. Gammacoronavirus in the family Coronaviridae of the order Nidovirales (–Avian infectious bronchitis virus (IBV) belongs to the genus ovirales . This hiovirales . IBV hasovirales –4.In Japan, seven genotypes of IBV  have been confirmed, based on the partial nucleotide sequence of the S1 gene , 6. AnalThe JP/KH/64 strain was isolated from chickens with respiratory symptoms in 1964 in Japan and was first confirmed as the JP-I genotype in Japan . The virThe sequenced fragments were assembled using ATGC-Mac v.5 . All tools were run with default parameters unless otherwise specified. The length of the complete genome of JP/KH/64 was 27,672 nucleotides (nt), with a G+C content of 38.20%, excluding the poly(A) tail. The open reading frames (ORFs) were identified using Genetyx-Mac v.18 (Software Development) and were confirmed by alignment with previously published IBV genome sequences. The genome has the typical genetic structure of all IBV strains  and contAY296744) (87.6% nucleotide identity), which was isolated in Japan (IBV is classified into six main genotypes (GI to GVI), comprising 32 viral lineages (lineages 1 to 32), based on the complete nucleotide sequences of the S1 glycoprotein and its typical geographical distribution . Phylogein Japan  Fig. 1)AY296744)in Japan , 13, thiLC634083.The genome sequence of IBV strain JP/KH/64 was deposited in GenBank under accession number


N. ashei (including other Naja sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of Naja sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-Naja sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control . Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant . At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in  Naja nigricollis, Naja mossambica, Naja katiensis, Naja pallida, and others. Naja ashei belongs to the African spitting cobras, which are distributed widely across sub-Saharan Africa. N. ashei, also called the large brown spitting cobra was in 2007 considered a distinct species and classified accordingly [N. ashei is known to induce mainly cytotoxic effects caused by the predominant presence of 3FTx proteins and phospholipases A2 [N. ashei is a medically important snake species in Kenya, Ethiopia, Uganda, Somalia, and Tanzania, and thus, is implicated in snakebites and associated morbidity, mortality, and disability [Snakebite envenoming constitutes a major public health challenge in developing countries. It contributes significantly to cases of morbidity and mortality, particularly in poorly developed regions including sub-Saharan Africa and Asia . The Worordingly . As is tpases A2 ,6. N. asN. nigricollis [N. ashei revealed that 3FTx proteins accounted for 60–80% [Proteomic analysis of the venom of spitting cobras shows that the venoms are made up largely of three-finger toxins (3FTxs), accounting for about 67–80%. For instance, 3FTx proteins were found to constitute 73.3% of the total venom load of r 60–80% ,6. Threer 60–80% ,10, and r 60–80% . Three-fr 60–80% . They alr 60–80% .Among other things, improved diagnostic methods are crucial in discriminating between various venoms . With adAn inhibition ELISA assay was developed, optimized, and evaluated for the detection of 3FTx proteins in African spitting cobras. The assay was able to detect 3FTxs in both spiked samples and venom-challenged mice samples.p-value = 0.0207). Consequently, these parameters were considered optimal and thus adopted for subsequent assays.Following optimization of ELISA parameters, the highest signal-noise ratio of the conjugate was obtained at a dilution of 1:8000. Also, significant levels of detection were attained at 1 µg/mL of the coating antigen concentration. A two-tailed t-test analysis of substrate sensitivity showed that OPD produced significantly higher ODs compared to TMB (N. ashei and N. nigricollis (18.80 and 21.00% respectively). On the other hand, a% inhibition less than 16% was observed among the heterologous samples across all the inhibitor concentrations tested  and heterologous (different genera) venom samples. At all the inhibitor concentrations tested, a percent inhibition of greater than 18% was observed across all homologous venoms, with the pattern more closely related between s tested . On the N. ashei venom at various concentrations. Negative controls made up of pre-immune mice serum samples were also analyzed. With a mean and standard deviation (of the negative control) of 0.835 and 0.207 respectively, the limit of detection (LOD) of the assay was determined to be approximately 0.01 µg/mL  particularly in wells containing high concentration of the antigen. Suffice to say, in these wells, there were more 3FTxs antigen, resulting in more binding between the primary antibody and the antigen, hence the high percent inhibition. From the six-point three-fold dilution of the sample antigen, the percent inhibition was observed to be highest (77.70%) at 27.00 µg/mL and lowest (36.56%) at 0.04 µg/mL of the antigen, respectively . In termp-value < 0.0001) within the sample antigen-induced inhibition in terms of the means. Nearly 72% of the variation in inhibition caused by the sample antigen therein in the crude venoms is explained by the analysis (R-squared = 0.7185).The results of the inhibition ELISA were used to confirm, first of all, the presence of three-finger toxins in the crude venoms assayed and secondly, to determine the % inhibition caused by the different sample antigens present in the crude venoms at the different concentrations tested. The ANOVA summary  shows thNaja species and their non-Naja counterparts, with the difference being particularly significant between N. nigricollis vs. B. arietans, N. nigricollis vs. D. polylepsis, N. haje vs. B. arietans, and N. haje vs. D. polylepsis . Also, there was no significant difference in the 3FTx-induced inhibition between all three spitting cobra species. The multiple comparisons test also show that the sample antigens-induced inhibition (if any) between B. arietans vs. D. polylepsis was not significant .From the post hoc results , it can Naja species were low. The ODs, however, increased with decreasing inhibitor concentrations. As low as 0.008 µg/mL inhibitor concentration, the sample antigens induced varying levels of inhibition . Conversely, increasing inhibitor concentrations resulted in an increase in the percent inhibition for the same species.It was also be observed that at high inhibitor concentrations , the ODsB. arietans and D. polylepsis suggest that there was no or minimal inhibition mainly because the venoms either contain little or none of the antigen of interest. As such, the ODs are comparable to those of the NAC  produced the highest OD values, an indication of inhibition when compared with wells containing different inhibitor concentrations. ODs obtained for  the NAC . N. ashei 3FTxs-containing venom at all the dilution points. In contrast, the negative control showed no inhibition. The % inhibition was largely similar across the different sample collection times, but varied significantly relative to the negative control. One-way ANOVA results showed no significant difference among the means  in terms of sample collection times. Similarly, no significant difference was found between the negative control and the other samples collected at the respective times. The results nonetheless demonstrate that the inhibition ELISA is capable of detecting 3FTxs in blood samples spiked with crude venom as depicted in the The utility of the inhibition ELISA was further tested for its capacity to detect 3FTxs in blood samples spiked with crude venom at different times. The spiked samples were assayed alongside previously collected pre-immune serum samples as control. An analysis of the results shows that there was inhibition of the coated antigen (purified 3FTxs) by the N. ashei venom. The results demonstrate that 3FTxs were detected, evidenced in the observed inhibition in the sample-containing wells. Albeit varied inhibition between the samples and negative control were observed, the differences were not statistically significant  as determined by one-way ANOVA. Similarly, multiple comparison analysis showed that the inhibition between the negative control and the samples collected at different times were not significantly different. It is worthy to note that regardless of the differences, the assay was able to detect 3FTxs in the samples tested as shown in the observed inhibition , particularly in samples containing high concentrations of the antigen. Suffice to say, in these wells, there were more 3FTxs antigen, resulting in more binding between the primary antibody and the 3FTxs antigen, hence the high percent inhibition. ODs obtained for B. arietans and D. polylepsis suggest that there was no or minimal inhibition mainly because the venoms either contain little or none of the antigen of interest. As such, the ODs are comparable to those of the NAC  and second, the venom proteomes of Naja sp. contain more 3FTxs. This finding is consistent with Hus et al. [N. ashei venom, while the same toxin was found to constitute 67–73% of N. nigricollis venom [Naja species are made up of largely 3FTxs. Also, the similarities in the inhibition among the three Naja snakes is supported by Casassola et al. [Hemachatus haemachatus, an African spitting cobra-like species found predominantly in Southern Africa was reported to contain 63.3% 3FTx proteins [Naja genus phylogenetically.The results of the inhibition ELISA indicate that the 3FTxs present in the crude  the NAC . The casr venoms . D. polys toxins . Howevert et al. . Thus, treported ,29. The s et al. ,6 and Pes et al. , who fous et al. ,6 found is venom . Thus, ta et al.  who obseA variety of methods are available for determining the analytical sensitivity of assays ,33,34. TIn both blood samples spiked with crude venom and mice challenged with the same, the inhibition ELISA was able to detect the presence of 3FTx proteins, as demonstrated in the observed differences in % inhibition when compared to the negative control. This finding is significant to the extent that such an assay could be useful not only in discriminating between venoms , but alsN. ashei venom was obtained from snakes maintained at the Bioken snake farm in Watamu, Kenya. The venom was reconstituted in distilled water and stored at −20 °C until use. Three-finger toxin (3FTx) fraction was purified using size-exclusion chromatography as described elsewhere [Naja nigricollis, Naja haje, Bitis arietans, and Dendroaspis polylepis were obtained from Baringo Snake Park, Kenya, in liquid form.Lyophilized crude lsewhere . Venoms ad libitum.Eight-week-old adult female Balb/c mice were challenged with 3FTxs (50 and 100 µg). Animals were sampled in February 2021 fortnightly in the tail vein for antibody screening for about 14 weeks. Blood samples were centrifuged to obtain serum, which were then stored at −20 °C until use, which antibodies were used as primary antibodies in all experiments. Animals were kept at the animal facility of the Kenya Medical Research Institute (KEMRI) and provided with food and water Approval for the study was obtained from the KEMRI Center for Biotechnology Research and Development (CBRD), KEMRI’s Animal Care and Use Committee (ACUC), and KEMRI Scientific and Ethical Committee Unit (SERU) with protocol number KEMRI/SERU/CBRD/229/4340.Protein content of all venoms was determined using the BCA kit in line with the manufacturer recommendations .Goat anti-mouse HRP conjugated antibody, antigen coating concentration, and substrate sensitivity were optimized using an indirect ELISA method described by Islam and Jones . N. ashei, N. nigricollis, N. haje) and two heterologous (B. arietans and D. polylepsis) venom samples  prepared to a final concentration of 27 µg/mL in blocking buffer  was added to row A of a separate plate (2). One hundred microliters (100 µL) of blocking buffer was added to rows B to H. A three-fold serial dilution was then performed by transferring 50 µL from row A to B and so on until G. The final 50 µL was discarded. Serum sample containing primary antibodies was prepared in blocking buffer at 1:1000 and 100 µL added to the duplicate wells of the same plate (including row H). The plate was then incubated at 37 °C for 60 min. The coating buffer in the earlier plate (1) was then aspirated and the plate washed three times with the washing/blocking buffer. The plate was then blocked with 200 µL of blocking buffer per well for 60 min at room temperature, following which washing was done as described previously. The content of the plate (2) containing the sample antigen and primary antibody was then transferred to the coated plate (1) and incubated at 37 °C for 60 min. The plate was washed again three times and 100 µL of goat-anti mouse IgG conjugated to horseradish peroxidase  added to each well. After a one-hour incubation at 37 °C, the washing step was repeated (with an extra wash) and 200 µL of 5 mg OPD substrate  added to the wells. The plate was again incubated for 60 mins at 37 °C for color development and then stopped with 50 µL per well of 3 M H2SO4. The plate was then read at 492 nm with a plate reader . Row H was considered the ‘No Antigen Control’ (NAC) and had its wells containing the blocking buffer and primary antibody without sample antigen. The NAC wells recorded the highest ODs. Subsequently, the percentage inhibition of the different wells containing different concentration of the sample antigen was determined against the absorbance of the NAC as per the formula belowThe method described by Sharma and colleagues  with modNaja sp. (N. nigricollis and N. haje) and two non-Naja sp. (B. arietans and D. polylepsis). The inhibition ELISA protocol described in The inhibition ELISA described above was tested for its ability to discriminate between venoms containing 3FTxs and those without using crude venoms from two other N. ashei venom. To spike samples, blood samples were collected from mice and then mixed with crude venom. Whereas the components of the assay were similar to the one described earlier, the sample collection and preparation were slightly different. In this experiment, six different Swiss mice were bled through the tail vein at different times—30 min, 1 h, 2 h, 6 h, 8 h, and 24 h. Immediately after sample collection, blood samples were spiked with equal volume of reconstituted crude N. ashei venom. Samples were then stored at −20 °C until assayed as earlier described. Pre-immune samples were added as negative control. The % inhibition was determined taking into account the NAC and the individual OD values.The inhibition ELISA described earlier was also tested for its capacity to detect 3FTxs proteins in blood samples spiked with crude N. ashei venom. Post the challenge, mice were bled in the tail vein at 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. Pre-immune samples were added as negative control. The samples were processed, subjected to the inhibition ELISA procedure and % inhibition determined based on the OD values.The applicability of the inhibition ELISA in snakebite envenoming diagnostics was tested in mice challenged with crude p-value < 0.05 cut-off, all comparisons were considered statistically significant.Data analysis was done using GraphPad version 8.4.3 software  and Microsoft Excel 2013. Differences in group means was determined by one-way ANOVA. In comparing the means within the comparisons, the one-way ANOVA was proceeded by Tukey’s or Dunnett’s post hoc multiple comparison test. At The study reports an inhibition ELISA model and its potential application in snakebite envenoming, especially in relation to cobra bites. Albeit not for diagnostic purposes, the inhibition ELISA described herein demonstrates the possibility of detecting three-finger toxins present in the venoms of cobra snakes. Venoms of all five snakes tested with the assay are considered ‘category 1’, constituting the greatest threat to public health . The det


FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML. The internal tandem duplication (ITD) mutation of the class III receptor tyrosine kinase FMS-like Tyrosine Kinase 3 (FLT3) is found in 25% of all AML cases and is associated with a dismal prognosis . DespiteThe FLT3 TKIs midostaurin and gilteritinib were recently approved for the treatment of FLT3-mutated AML , 13. The2. Midostaurin was a kind gift from Novartis . Sorafenib was purchased from American Chemicals Custom Corporation . Gilteritinib was purchased from Selleckchem .Ba/F3, MOLM-13, MV4–11, and THP-I were cultured in Roswell Park Memorial Institute 1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin, for Ba/F3 additional 2 ng/ml IL-3. OCI-AML3 were cultured in alpha-MEM with 20% FBS and penicillin/streptomycin. Phoenix Eco  were cultured in Dulbecco’s Modified Eagle Medium with 10% FBS and periodically selected with hygromycin 300 mg/ml and diphtheria toxin 2 µg/ml. MV4–11, MOLM-13, and OCI-AML3 cells were kindly provided by M. Lübbert . Cell lines were generated via retroviral transduction via Phoenix Eco cells. Human cell lines expressed the murine ecotropic receptor Slc7a1. All Cells were incubated at 37 °C with 5% COHuman CSF2RB cDNA was kindly provided by M. Martinez-Moczygemba  and subcloned into pMSCV. Human FLT3 constructs were expressed using puromycin-IRES-GFP (PIG). FLT3 and FLT3-ITD 598/599  were kinImmunoblotting and immunoprecipitation were performed as previously described . FollowiEscherichia coli BL21, purified, and immobilized on glutathione-sepharose beads . Cytoplasmic fragments of FLT3 and CSF2RB used for T7 in vitro translation  were cloned into pcDNA3.1. GST-tagged peptides captured by glutathione-sepharose beads were incubated with in vitro translated target protein overnight at 4 °C. Interacting complexes were recovered by collecting washed beads. Analysis and detection of retained target proteins were performed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting.Sequences of FLT3 and CSF2RB were cloned into pGEX-4T-1. GST fusion proteins were expressed in U test.Oligo-coupled primary antibodies (1-PLA) were used to restrict the detection of a nanoscale protein:protein interaction to a 10–40 nm range as previously described . As primCell viability was measured using an MTS-based method by absorption of formazan at 490 nm . Cells were plated at a density of 6000 or 10000 cells per well in a 96-well plate and treated with inhibitors at indicated concentrations. Measurements were taken in triplicates after 48 or 72 h.All animal experiments were conducted in accordance with national and local ethical regulations and were approved by the Regierungspräsidium Freiburg, Germany (Protocol number: G-16/164). Mice used for experiments were between 8 and 12 weeks old.tm1.1Flv Il2rgtm1.1Flv/J knockout mice (JAX stock #014593) . Th. Th33].  598/599 , involvi 598/599 . SurprisWe next asked whether the inserted ITD sequence or the localization of the ITD is critical for FLT3 downstream signaling or CSF2RB phosphorylation. Most FLT3-ITD variants occur within the JMD whereas ITDs in the tyrosine kinase domain are rare , 36. We Taken together our findings demonstrate that CSF2RB directly interacts with FLT3 and is phosphorylated in an FLT3-ITD-dependent fashion. It participates in FLT3-ITD induced activation of STAT5 and plays a pivotal role in promoting the oncogenic potential of FLT3-ITD in vitro and in vivo.In our previous work, we demonstrated that activating mutations in JAK1, 2, and 3 mediate resistance to FLT3 inhibitor treatment in FLT3-mutated AML and lead to reactivation of CSF2RB . We hereSTAT5 is a critical mediator of myeloid transformation by FLT3-ITD . The mecTo better understand the mechanism of FLT3-ITD-dependent CSF2RB activation, we performed interaction mapping experiments. CSF2RB activation required the presence of FLT3-ITD. FLT3 also interacted with but failed to activate CSF2RB, even after stimulation with FLT-ligand. Of note, although our in vitro studies did not demonstrate a difference between FLT3 and FLT3-ITD in CSF2RB binding, we detected less CSF2RB/FLT3 than CSF2RB/FLT3-ITD interactions in our PLA experiments suggesting that within a membrane structure of cells, the differential conformation of FLT3-ITD might favor interaction with CSF2RB. Inhibition of FLT3-ITD kinase activity did not interfere with the interaction but abrogated CSF2RB phosphorylation. In addition, the interaction of CSF2RB with the corresponding alpha chain  was not We could demonstrate that phosphorylation of at least one of the two tyrosines 598 and 591 within the JMD of FLT3-ITD is required for phosphorylation of CSF2RB, and that neither the sequence nor the insertion site of the ITD affects the ability to activate CSF2RB. In addition, we demonstrated that only tyrosine 599 is phosphorylated in FLT3 expressing cells after treatment with FLT-ligand, whereas phosphorylation of tyrosines 589 and 591 was restricted to FLT3-ITD-expressing cells and thus constitutes a pathological feature of FLT3-ITD. Our observations are in agreement with a working model of FLT3 activation by ITDs proposed by Chan et al. . AccordiWe here demonstrate the pivotal role of CSF2RB in FLT3-ITD oncogenic signaling and cellular transformation in vitro and in vivo, suggesting it to be a rational drug target in FLT3-ITD positive AML.Supplementary


Background: Diabetic retinopathy (DR) is one of the most important microvascular diseases of diabetes. Our previous research demonstrated that bile acid G-protein-coupled membrane receptor (TGR5), a novel cell membrane receptor of bile acid, ameliorates the vascular endothelial cell dysfunction in DR. However, the precise mechanism leading to this alteration remains unknown. Thus, the mechanism of TGR5 in the progress of DR should be urgently explored.Methods: In this study, we established high glucose (HG)-induced human retinal vascular endothelial cells (RMECs) and streptozotocin-induced DR rat in vitro and in vivo. The expression of TGR5 was interfered through the specific agonist or siRNA to study the effect of TGR5 on the function of endothelial cell in vitro. Western blot, immunofluorescence and fluorescent probes were used to explore how TGR5 regulated mitochondrial homeostasis and related molecular mechanism. The adeno-associated virus serotype 8-shTGR5 (AAV8-shTGR5) was performed to evaluate retinal dysfunction in vivo and further confirm the role of TGR5 in DR by HE staining, TUNEL staining, PAS staining and Evans Blue dye.Results: We found that TGR5 activation alleviated HG-induced endothelial cell apoptosis by improving mitochondrial homeostasis. Additionally, TGR5 signaling reduced mitochondrial fission by suppressing the Ca2+-PKCδ/Drp1 signaling and enhanced mitophagy through the upregulation of the PINK1/Parkin signaling pathway. Furthermore, our result indicated that Drp1 inhibited mitophagy by facilitating the hexokinase (HK) 2 separation from the mitochondria and HK2-PINK1/Parkin signaling. In vivo, intraretinal microvascular abnormalities, including retinal vascular leakage, acellular capillaries and apoptosis, were poor in AAV8-shTGR5-treated group under DR, but this effect was reversed by pretreatment with the mitochondrial fission inhibitor Mdivi-1 or autophagy agonist Rapamycin.Conclusion: Overall, our findings indicated that TGR5 inhibited mitochondrial fission and enhanced mitophagy in RMECs by regulating the PKCδ/Drp1-HK2 signaling pathway. These results revealed the molecular mechanisms underlying the protective effects of TGR5 and suggested that activation of TGR5 might be a potential therapeutic strategy for DR. Diabetic retinopathy (DR) is one of the most frequent microvascular complications of diabetes mellitus (DM) and the leading cause of blindness in the working-age population . The numIn peripheral blood vessels, vascular endothelial cells directly come into contact and interact with high glucose (HG). Persistent hyperglycaemia induces metabolic dysfunction in endothelial cells . MitochoUnder physiological states, mitophagy eliminates damaged mitochondria through the induction of lysosome-dependent degradation . MitophaThe mitochondrial quality is crucially linked to energy metabolism. Recently, bile acids have emerged as pivotal signaling molecules for the regulation of glucose metabolism, lipid homeostasis, as well as energy metabolism, which exerts multiple biological effects by binding to its receptors, the bile acid nuclear receptor Farnesoid X receptor (FXR) and the membrane G-protein-coupled bile acid receptor (TGR5). Moreover, dysregulation of signal molecules in bile acid can result in lipid, glucose dysmetabolism and the gut dysbiosis. TGR5 has been reported to play a critical role in energy metabolism, while the direct relationship of bile acids and microvascular abnormalities in DR is rarely reported The bile acid G-protein-coupled membrane receptor (TGR5), a novel cell membrane receptor of bile acid, has been reported to play a critical role in energy metabolism . MitochoMdivi-1, 3-bromopyruvic acid, INT-777, 3-methyladenine, Rapamycin, BAPTA-AM, and Z-VAD (OMe)-FMK were procured from MedChemExpress . MitoBright LT Red was obtained from DOJINDO . Glucose, streptozocin (STZ) and Evans Blue were obtained from Sigma-Aldrich . Fluo-4 AM, Cell counting kit-8 (CCK8), JC1 assay kit and cell mitochondria isolation kit were obtained from Beyotime . The TUNEL assay and haematoxylin-eosin (HE) staining kits were obtained from Roche  and BOSTER , respectively. The primary antibodies used were as follows: PKC δ, Phospho-PKCδ, HK2, LC3B, P62, DRP1 ; Parkin, PINK1, Phospho-DRP1, Cleaved caspase-3 , Bax, β-actin, COX4 ; and TOMM20 . information on reagents and antibodies is shown in 2 in complete DMEM medium containing FBS (10% v/v), penicillin (1% v/v) and streptomycin (1% v/v).Human retinal microvascular endothelial cells (HRMECs) were obtained from the Institute of BeNa Biotechnology . RMECs were cultured at 37°C and 5% CORMECs were transfected with TGR5 or PKCδ siRNA  at 80% confluence by using the riboFECT CP Transfection Kit  in accordance with the manufacturer’s protocol. Briefly, 120 μL Opti-MEM medium containing 12 μL riboFECT CP, 100 nM TGR5 siRNA, 100 nM PKCδ siRNA or 100 nM NC was premixed in a 6-well plate. Next, the cells were transduced for 48 h, and silence efficiency was evidenced by Western blot. The detailed siRNA information is provided in Male Sprague-Dawley rats of 6 weeks (220–250 g) were obtained from Changzhou Cavans Experimental Animal Co., Ltd. (China). All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Nanjing Medical University before implementation 2020-0010).The rats were conducted by intraperitoneal injection of streptozotocin . Diabetic rats were successfully established when blood glucose greater than 16.7 mM.The diabetic retinopathy model was successfully established by streptozotocin (STZ) administration as previously described . In shorAfter 2 weeks, when the diabetic model was stably established, drug administration was initiated. The Mdivi-1 dose (30 ng/μL) and Rapamycin dose (5 ng/μL) were chosen according to the literature and preliminary studies . Rats we12 viral genomes/mL) gradually by intravitreal injection 2 weeks before streptozotocin (STZ) administration. The detailed shRNA information is provided in .Diabetic rats were randomly assigned into four groups: AAV8-vector, AAV8-shTGR5, AAV8-shTGR5 + Mdivi-1, and AAV8–shTGR5 + Rapamycin.The AAV8 was constructed and packaged by the Vigene Biosciences and targeted the RNAi against TGR5 (AAV8-shTGR5) by controlling the albumin promoter. A 33-gauge needle was used to inject 5 μl AAV8-shTGR5 . The cell suspension was inoculated in a 96-well plate (100 μL/well) at a minimum density of at least 1,000 cells per well. Then, cells were pretreated with drugs for 2 h and stimulated with or without HG (33 mM) for 5 days. Finally, 100 μL CCK-8 solution was added into each well for 1.5 h at 37°C, and absorbance values were measured at 450 nm by using a microplate reader.4) were incubated in 24-well plates for 5 days after drug treatment. The cells were stained with pre-warmed MitoTracker Red  at 37°C and 5% CO2 in the dark. After washing three times with PBS, live confocal imaging was performed to evaluate the mitochondrial morphology by confocal microscopy  and the length of mitochondria was quantified with ImageJ software as described previously . Cells (1 × 10eviously .4) were incubated in 24-well plates for 3 days after drug treatment. Intracellular Ca2+ were measured with Fluo-4 AM  for 1 h and then mitochondria were stained with the MitoTracker Red  for 30 min according to manufacturers’ instructions. The fluorescent intensity was obtained Colocalization of intracellular Ca2+ and mitochondria was detected by Laser scanning Confocal Microscopy .Cells (1 ×104) were incubated into 24-well plates for 5 days after drug treatment. The cells were The JC-1 staining solution was added for 20 min, incubated with JC-1 dye at 37 °C for 20 min, washed twice with dilution buffer, and placed in the culture medium. and the fluorescence intensity was obtained by laser scanning confocal microscopy  Lastly, images were obtained by laser scanning confocal microscopy  at 490 nm excitation and 530 nm emission for green and at 525 nm excitation and 590 nm emission for red. Mitochondrial membrane potential is expressed as the ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers) using the ImageJ software.The JC-1 assay kit  is universally used to detect the mitochondrial membrane potential. JC-1 aggregated in the mitochondrial matrix at high membrane potential and could form red fluorescent aggregates. However, JC-1 cannot aggregate to form a polymer at low membrane potential and produce green fluorescent monomers. Briefly, cells (1 × 104) were incubated in 24-well plates for 5 days after drug treatment. Cells or tissue sections were fixed with 4% paraformaldehyde for 30 min. After washing three times with PBS, triton-100 (0.2%) was added to samples for permeabilisation, and TUNEL detection solutions were reactions were incubated on ice for 5 min. After washing three times, TUNEL reaction mixture was prepared by mixing 50 µL Enzyme solution to 450 µL Label solution, 50 µL of TUNEL reaction mixture was added to sample and incubated for 1 h at 37°C in the dark. The samples were again washed three times with PBS and then nuclei were labelled with DAPI . TUNEL-fluorescent images were obtained by laser scanning confocal microscopy . The TUNEL assay quantification was performed using the ImageJ software.RMECs , permeabilised with Triton X100  and blocked with QuickBlock™ Blocking Buffer (1 h). The primary antibodies p-Drp1 (1:100), HK2 (1:100), TOMM20 (1:50), PINK1 (1:100), isolectin B4 , LC3B (1:500) and cleaved-caspase3 (1:400) and secondary antibody labelled by Alexa 488/555 were incubated into the samples. Nuclei were labelled with DAPI . Fluorescence images were observed using confocal microscopy .The assay followed a documented protocol . In brieRMECs were lysed with lysis buffer  containing protease, phosphatase inhibitors  and PMSF . The supernatant was obtained by centrifugation at 12,000 ×g for 15 min. Protein was quantified by a BCA kit . Protein samples were boiled in 1 × SDS-PAGE loading buffer . The samples were electrophoresed and transferred onto PVDF, and the membranes were blocked with 5% milk. The primary antibodies TGR5 , Drp1 , p-Drp1 , PKCδ (1:2000), p-PKCδ , PINK1 , Parkin , HK2 , LC3B , P62 , BCL-2 (1:2000), Bax (1:2000), cleaved-caspase3 , Cox4  and β-actin  were incubated overnight at 4°C. Then, horseradish peroxidase-linked antibodies were added for 2 h. Signal detection was visualised with a chemiluminescence kit .The total RNA was extracted using Trizol, and reverse transcription was performed using the HiScript III RT SuperMix kit . The relative mRNA expression levels of OPA-1, Mfn-1, Mfn-2, Fis-1, and MFF were quantified by RT-PCR and normalised by GAPDH mRNA. Primer sequences are detailed in p < 0.05 was considered statistically significant.Results were expressed as mean ± SD of three independent experiments. Datasets were compared by the Student t-test or single-factor ANOVA with Tukey’s post-hoc analysis. The GraphPad Prism version 8.00  was used for data analysis. Endothelial cell dysfunction is closely associated with the development of DR . Our prein vitro.To further confirm the inhibitory effect of INT-777 on endothelial cell apoptosis, Western blot was performed to detect the expression levels of apoptotic-related proteins, Bax and cleaved-caspase3. In the HG-treated group, the protein levels of Bax and cleaved-caspase3 were obviously high-expressed, compared with the control group. However, INT-777 could effectively protected part of cells from apoptosis caused by HG, suppressed the expression levels of apoptotic-related proteins. . SimilarDynamic mitochondrial processes, especially mitochondrial fission and mitophagy, play a key role in the pathogenesis of DR . FirstlyMitochondria were damaged by excessive mitochondrial fission, and damaged mitochondria were degraded by mitophagy. Next, we wondered whether TGR5 participated in the autophagy-dependent clearance of damaged mitochondria. As shown in 2+ concentration and PKC activation  was used in this study to further confirm that TGR5 regulated PINK1/Parkin signaling pathway through HK2. We found that the upregulation of PINK1 and Parkin induced by INT-777 was reversed by 3-BP . In para2+-PKCδ/Drp1-HK2 signaling pathway To further confirm the role of TGR5 in DR, we performed an in vivo experiment by using AAV8-shTGR5 in DR rat to verify the hypothesis. As shown in The above results indicated that TGR5 inhibited mitochondrial fission and enhanced mitophagy by regulating the Cain vivo. Our study showed that the AAV8-shTGR5 group decreased the protein levels of PINK1 and Parkin compared with AAV8-vector group  and mitochondria. The mechanism by TGR5 decreased mitochondrial calcium levels is less clear and perhaps ER calcium homeostasis play distinct functional roles in it, which need for further studies. Secondly, we have preliminarily found that TGR5 can affect mitochondrial autophagy, but the specific molecular mechanism was not deeply studied and a number of gaps are yet to be filled. Thirdly, the main conclusion of our study was only based on the role of TGR5 on endothelial cells, and further in-depth research is obviously required to elaborate DR pathogenesis precisely in future. Despite these limitations, we believe that our study helps to understand the imbalance of mitochondrial homeostasis is involved in DR, and TGR5 may serve as a potential therapeutic target for DR.Taken together, we showed that TGR5 inhibited mitochondrial fission and enhanced mitophagy in RMECs by regulating the PKCδ/Drp1-HK2-PINK1/Parkin signaling pathway, which revealed the molecular mechanisms underlying the protective effects of TGR5. However, our study also had the following limitations. Firstly, our results indicated that TGR5 decreases intracellular Ca


Cymbopogon schoenanthus (L.) Spreng., Litsea cubeba (Lour.) Pers., Melissa officinalis L., and Verbena officinalis L. However, only L. cubeba EO has been investigated for use as a potential skin-whitening agent. This work evaluates the in vitro tyrosinase inhibitory activity of these EOs and studies, using bio-assay oriented fractionation, whether their differing chemical compositions influence the overall EO inhibitory activities via possible synergistic, additive, and/or competitive interactions between EOs components. The inhibitory activity of C. schoenanthus EO and that of M. officinalis EOs, with negligible (+)-citronellal amounts, were in-line with their citral content. On the other hand, L. cubeba and V. officinalis EOs inhibited tyrosinase to considerably greater extents as they contained β-myrcene, which contributed to the overall EO activities. Similar observations were made for M. officinalis EO, which bears high (+)-citronellal content which increased citral activity.Excessive melanin production causes serious dermatological conditions as well as minor aesthetic problems . The downregulation of tyrosinase is a widespread approach for the treatment of such disorders, and plant extracts have often proven to be valuable sources of tyrosinase inhibitors. Citral  is an important fragrance ingredient that has shown anti-tyrosinase potential. It is highly concentrated in the essential oils (EOs) of  Although there are other enzymes involved in melanogenesis, only the tyrosinase-catalyzed reactions cannot occur spontaneously, whereas the remaining steps can proceed without enzyme activity at physiological pH [Tyrosinase is the key enzyme in the biosynthesis of melanin pigments in several bacteria, fungi, plants, animals, and humans. In humans, tyrosinase catalyzes the rate limiting steps in the melanin biosynthetic pathway. This biosynthesis is characterized by several enzymatic and chemical reactions that lead to melanin formation from the amino acid L-tyrosine, with tyrosinase catalyzing its hydroxylation to gical pH . For thigical pH .In the EU market, the tyrosinase inhibitors that are employed as skin-whitening agents can be grouped into two main categories: those banned under EU cosmetic regulation 1223/2009  due to their severe side effects, but that are still used to treat hyperpigmentation under medical supervision; and tyrosinase inhibitors that are permitted for use in cosmetics products  [Plants have been valuable sources of skin-whitening agents, and three out of five of the most employed agents, both medically and cosmetically, are plant specialized metabolites . To date, phenolic compounds have principally been investigated as potential tyrosinase inhibitors, and these include flavonoids , stilbecis- and trans-3,7-dimethyl-2,6-octadienal , which have been proven to block the in vitro enzymatic activity of mushroom tyrosinase [Citral is among the limited number of terpenoid derivatives with anti-tyrosinase properties that have been studied. It is a mixture of two isomers, rosinase . In addirosinase ,10,11. Mrosinase ,13,14,15Cymbopogon schoenanthus (L.) Spreng., Litsea cubeba (Lour.) Pers., Melissa officinalis L., and Verbena officinalis L. To the best of the authors’ knowledge, only L. cubeba EO has been investigated for its tyrosinase inhibitory activity [C. schoenanthus, L. cubeba, M. officinalis, and V. officinalis EOs, using an in vitro colorimetric assay, to assess whether the different chemical compositions influence the overall EO inhibitory activities via any possible synergistic, additive and/or competitive interactions between their components. This study uses a bioassay-guided fractionation approach to evaluate comprehensively the EOs constituents and their enantiomers, when chiral, that contribute to the EO inhibitory activity against a mushroom source of tyrosinase, which is a good model system for the preliminary screening of tyrosinase inhibitors [Citral is obtained from the essential oils (EOs) of several botanical species, including activity . Therefohibitors .In our attempt to comprehensively characterize all of the potential EO components that contribute to the considered biological activity, the investigated EOs were analyzed by GC, with both FID and MS detection. The normalized relative percentage abundances  of acis-3,7-dimethyl-2,6-octadienal) and geranial , which are the most abundant compounds. The neral/ geranial ratio was very similar in all the investigated EOs and corresponded to 0.74 ± 0.05. The C. schoenanthus and L. cubeba EOs display the highest neral and geranial content, which accounts for, on average, 60% of their entire EO compositions, and which is 1.5-times greater than in V. officinalis EO and in the three M. officinalis EOs . The additional oxygenated compounds that are common to all the EOs are 6-methyl-5-hepten-1-one, linalool and citronellal. The latter is significantly more abundant in the M. officinalis EO 1 than in the other investigated EOs, including the M. officinalis EO 2 and 3. All of the investigated EOs are rich in neral , containing both monoterpenes  and sesquiterpenes  in moderate amounts. In the L. cubeba and V. officinalis EOs, the hydrocarbon fraction accounts for 20% of the total EO and limonene is the most abundant compound , followed by α-pinene, β-pinene, sabinene, trans-β-caryophyllene, β-myrcene, camphene, and α-copaene. Finally, M. officinalis EO 2 and 3 are characterized by the highest hydrocarbon fraction content . In both samples, the hydrocarbon fraction mainly contains sesquiterpenes, namely trans-β-caryophyllene , and α-humulene (3.0% and 2.6%), and a reduced monoterpene fraction that is mainly characterized by limonene . The abundance of the hydrocarbon fraction varies significantly in the different EOs. L. cubeba, V. officinalis, and C. schoenanthus Eos produced in different years as well as three samples of M. officinalis EOs from distinct manufactures were investigated. GC-MS analyses of C. schoenanthus, L. cubeba, M. officinalis, and V. officinalis did not reveal significant qualitative and quantitative differences in the chemical composition of the three samples of different years of production. This may be ascribed to optimal storage conditions, i.e., in an amber-glass container at 4 °C in the dark with a negligible head space. On the other hand, GC-MS analyses showed significant differences in the abundances of citronellal and trans-β-caryophyllene in the three investigated M. officinalis EOs. Citronellal amounted to 19.6%, 0.26%, and 0.31% in the M. officinalis EO 1, 2, and 3, respectively. On the contrary, as previously described, trans-β-caryophyllene is considerably more abundant in the M. officinalis EOs 2 and 3 than in M. officinalis EO 1. These results are in agreement with the findings reported by Seidler-Lozykawska et al., who highlighted significant differences in citral, citronellal, and trans-β-caryophyllene abundances in the EOs obtained from 22 selected M. officinalis genotypes originating from European botanical gardens [Three samples of  gardens . m/z) used for SIM-MS quantitation of the marker compounds under investigation together with the calibration range, the calibration curve equation, correlation values, and regression standard error of each analyte and the quantitation results, respectively. A true quantitation was performed by the external standard calibration to accurately evaluate the abundance of potential bioactive specialized compounds  and did not generate solubility issues in the reaction mixture. Citral concentration–response curve was studied by plotting the observed inhibitory activity as a function of its concentration in the reaction mixture. All of the EOs were tested at a concentration of 166.7 μg/mL, which provided, irrespective of the EO, a resulting citral concentration within its concentration-response curve linearity range . In regard to L. cubeba and C. schoenanthus EOs, these outcomes are in good agreement with the results obtained from the quantitative GC-MS analyses that revealed an almost identical citral amount in the EOs of different years of production. The batch 2020 of V. officinalis EO contains a slightly higher citral amount than the batches 2019 and 2018. However, according to citral concentration–response curve, the citral excess in batch 2020 is not sufficient to determine a statistically significant higher percentage of enzymatic inhibition considering the random error associated with the measurements. For additional details, see M. officinalis EOs, provided by distinct manufacturers, inhibited mushroom tyrosinase to different extents, which will be further described in the following paragraphs. The greatest inhibitory activities were observed for the EOs of L. cubeba, M. officinalis 1, and V. officinalis, which inhibited 59 ± 6%, 55 ± 7%, and 52 ± 6% of tyrosinase activity, respectively, when tested at a concentration of 166.7 μg/mL. Statistically significant (p < 0.05) lower activities were observed for the EOs of C. schoenanthus and M. officinalis 2 and 3 whose enzyme inhibitory activity was 42 ± 5%, 40 ± 5%, and 38 ± 6%, respectively. 50) for each investigated inhibitor . All of the EOs effectively inhibited mushroom tyrosinase and displayed an inhibitory activity that was, on average, 100-times lower than that of kojic acid, which was used as the positive control. The box plot reported in C. schoenanthus, M. officinalis 2, and M. officinalis 3 displayed inhibitory activities that were in-line with their citral content, while L. cubeba, M. officinalis 1, and V. officinalis EOs inhibited mushroom tyrosinase to a greater extent than expected. The histogram reported in L. cubeba, M. officinalis 1, and V. officinalis EOs were isolated by flash chromatography and individually tested for their mushroom tyrosinase inhibitory activities. The fractions phytohemical compositions are reported in the A bio-guided approach was adopted to identify the additional compounds that contribute to citral activity. The oxygenated and hydrocarbon fractions of the L. cubeba and V. officinalis EOs, both the oxygenated and hydrocarbon fractions inhibited mushroom tyrosinase, although to different extents. The activities of the oxygenated fractions  account for most of the EOs anti-tyrosinase potential and were in-line with the respective citral content, suggesting that the compounds that contribute to citral activity belong to the hydrocarbon fractions. The hydrocarbon fractions of the L. cubeba and V. officinalis EOs present quite similar chemical compositions. Limonene , trans-β-caryophyllene , α-pinene , β-pinene , sabinene , and β-myrcene  are the most abundant compounds in both fractions and are present in rather similar amounts, except for α-pinene and β-pinene, which prevail in the V. officinalis EO hydrocarbon fraction. As for trans-β-caryophyllene (>99% in both EOs), limonene  and sabinene (87% in both EOs), while different enantiomeric excesses were observed for α-pinene ((-)-enantiomer: 38% in L. cubeba EO and 73% in V. officinalis EO) and β-pinene ((-)-enantiomer: 67%in L. cubeba EO and 88% in V. officinalis EO). In both EOs, (-)-limonene accounts for more than 50% of the entire fraction. However, although previous studies have reported an inhibitory activity against mushroom tyrosinase because of its high abundance [trans-β-caryophyllene, (±)-α-pinene, and (±)-β-pinene. Sabinene was not tested as it had already been proven to have negligible mushroom tyrosinase inhibitory effects [L. cubeba and V. officinalis EOs, β-myrcene activity bridged the gap between the EOs’ expected inhibitory effects if citral was the only active compound and the experimental results. Contrary to the observations by Matsuura et al. [50 was almost ten times lower (13.3 μg/mL vs. 121.8 μg/mL). This difference may be ascribed to the different substrates used; Matsuura et al. investigated mushroom tyrosinase diphenolase activity only, as they used L-DOPA as the substrate, whereas, in this study, L-tyrosine was used. The current findings suggest that β-myrcene may be more effective at inhibiting mushroom tyrosinase monophenolase activity than the diphenolase one. The chiral recognition revealed high enantiomeric purities in favor of the (-)-configured enantiomers for bundance ,23, (-)-M. officinalis EO 1 displays a small hydrocarbon fraction that accounts for less than 3% of the total, and has no tyrosinase inhibitory activity. However, the M. officinalis EO 1 oxygenated fraction inhibited mushroom tyrosinase to a greater extent than would be expected from its citral content  Imbach, L-tyrosine, kojic acid, citral, citronellal, β-myrcene, (+)-limonene, (-)-limonene, (±)-limonene, (±)-α, and β pinene were purchased from Merck Life Science S.r.l. . Litsea cubeba, Verbena officinalis, and Cymbopogon schoenanthus EOs were supplied by Erboristeria Magentina S.r.l. . Three batches of different years  were tested for each. Three samples of Melissa officinalis EOs were investigated; one was provided by Agronatura , one by Erboristeria Magentina S.r.l., while the last was purchased from a local shop and was from Specchiasol S.r.l. . In the text, the authors refer to the different EOs of Melissa officinalis as M. officinalis EOs 1, 2, and 3, respectively. The provided EOs were obtained following the procedures described in the European Pharmacopoeia [Melissa officinalis and Verbena officinalis EOs were produced by hydrodistillation from the leaves and plants aerial parts, respectively; similarly, Litsea cubeba and Cymbopogon schoenanthus EOs were obtained by steam distillation of the fresh fruits and fresh aerial parts, respectively. Each EO was individually analyzed by GC-MS as soon as it was purchased/provided by the corresponding manufacturer, every storage year, and just before the study of its mushroom tyrosinase inhibitory activity.Dimethyl sulfoxide (DMSO), mushroom tyrosinase from acopoeia . MelissaAgaricus bisporus (J.E. Lange) Imbach was selected for this study. L-Tyrosine was used as the substrate, meaning that the overall tyrosinase inhibitory activity was investigated without distinguishing between tyrosinase monophenolase and diphenolase activity. Photometric measurements at 475 nm were performed on a Thermo spectronic Genesys 6 and kojic acid was used as the positive control inhibitor. The solutions of the investigated potential inhibitors  were prepared in DMSO. 475 (Control) or (Sample) − A475 (Control Blank) or (Sample Blank).The tyrosinase inhibitory activities of the EOs and isolated compounds were investigated in vitro using a colorimetric readout-based enzyme assay optimized by Zengh et al. , with sl50 values for the inhibitors were obtained by interpolation from the concentration–response curve. The concentration-response curve for each inhibitor was determined by plotting the inhibitory activity as a function of inhibitor concentration in the reaction mixture. IC®-Sphera Cartridge Stationary) was from Sepachrom; mobile phase: petrolether (A) and ethyl acetate (B); flow-rate 25 mL/min. Linear gradient elution was adopted from 100% of A to 80% of A and 20% of B over 20 min. EO fractionation was carried out on a flash column chromatography system PuriFlash 450 by Sepachrom , equipped with both UV and ELSD detectors. The amount of EO fractionated: 900.0 mg. Stationary phase: spherical silica gel particles, 50 μm, 25 mg  1.0 mg/mL was used as the internal standard to normalize the analyte signals. GC-MS analyses were carried out using a Gerstel MPS-2 multipurpose sampler  installed on an Agilent 6890 N GC coupled to a 5975 MSD and equipped with a ChemStation Version E.02.02.1431 data processing system . GC conditions: injector temperature: 250 °C; injection mode: split; ratio: 1/20; carrier gas: helium; constant flow rate: 1 mL/min; columns: Mega 5  df 0.25 µm, dc 0.25 mm, length 25 m, from MEGA . Temperature program: 50 °C//3 °C/min//180 °C//10 °C/min//250 °C (5 min). MSD conditions: MS operated in EI mode (70 eV); scan range: 35 to 350 amu; dwell time 40 ms; ion source temperature: 230 °C; quadrupole temperature: 150 °C; transfer-line temperature: 280 °C. EO markers were identified by comparing both their linear retention indices (ITs), calculated versus a C9-C25 hydrocarbon mixture, and their mass spectra either against those of authentic samples, or from commercially available mass spectral libraries . EO chiral analyses were performed by adopting the same analysis conditions on a 2,3-di-O-methyl-6-O-t-butyldimethylsilyl-β-CD  df 0.25 µm, dc 0.25 mm, length 25 m from MEGA. Temperature programs: 40 °C (1 min)//2 °C/min//220 °C (5 min).GC-FID analyses were carried out on the same instrument. GC conditions: injector temperature: 250 °C; injection mode: split; ratio: 1/20; carrier gas: hydrogen; flow rate: 1 mL/min. Temperature programs: 40 °C (1 min)//2 °C/min//220 °C (5 min). Cymbopogon schoenanthus, Litsea cubeba, Melissa officinalis, and Verbena officinalis EOs and (2) to determine whether their biological activity is ascribed to their citral content only or if there are additional bioactive monoterpenes that contribute to the investigated biological activity by using a bioassay-guided fractionation approach. This study has identified that in L. cubeba and V. officinalis EOs, the β-myrcene contributes to the EOs inhibitory activities despite its little amount and it has been shown to have a greater inhibitory power to citral. The second major finding was that (+)-citronellal enhanced citral mushroom tyrosinase inhibitory power, potentially via synergistic interaction as it displayed no activity on its own. The latter finding explained why in M. officinalis EOs that bear negligible (+)-citronellal amounts, the inhibitory activities were in-line with their citral content while the contrary was true for the M. officinalis EO with relatively high (+)-citronellal abundance.The purposes of this investigation were (1) to examine comprehensively the in vitro mushroom tyrosinase inhibitory activities of the Even though further studies are still required to accurately define the type of interactions that occur in between β-myrcene and citral and in between citronellal and citral, and to assess the inhibitory activities of these EOs and individual compounds on human tyrosinase, the results of this study may help to rationally design mixtures of EOs or enriched EOs that improve their biological efficacy and increase their potential as adjuvants in the treatment of hyperpigmentation.