Purposively, the established molecular docking virtual screening method is not limited to exploring flavonoid structure activity relationships to anti-thrombin activity but also usefully discovering other natural active constituents.Thrombin, a key enzyme of the serine protease superfamily, plays an integral role in the blood coagulation cascade and thrombotic diseases. In view of this, it is worthwhile to establish a method to screen thrombin inhibitors  as well as investigate their structure activity relationships. Virtual screening using molecular docking technique was used to screen 103 flavonoids. Out of this number, 42 target compounds were selected, and their inhibitory effects on thrombin assayed by chromogenic substrate method. The results indicated that the carbon-carbon double bond group at the C2, C3 sites and the carbonyl group at the C4 sites of flavones were essential for thrombin inhibition, whereas the methoxy and O-glycosyl groups reduced thrombin inhibition. Noteworthy, introduction of OH groups at different positions on flavonoids either decreased or increased anti-thrombin potential. Myricetin exhibited the highest inhibitory potential against thrombin with an IC In recent years, thrombotic disease has been a major threat to human health worldwide. As mentioned earlier, thrombin, a principal serine protease, plays a critical role in blood coagulation cascade response by activating blood platelet and catalyzing the conversion of soluble plasma-protein fibrinogen to insoluble fibrin ,2. This O-β-d-glucopyranoside and 5,6,2′,4′-tetrahydroxy-7,5′-dimethoxyflavone have been proven to have inhibitory effect on thrombin [Typha pollen, Salvia miltiorrhizae, Radix et rhizoma, Chuanxiong rhizoma and Platycladi cacumen are enriched with natural flavonoids and have been used for treating thrombotic diseases for centuries [In recent research, plant-derived flavonoids, namely, kaempferol, quercetin and its glycosylated derivatives, (+)-catechin, (-)-epicatechin, cyanidin, cyanidin-3-glucoside, eupatilin-7-thrombin . Notablyenturies ,10. Structurally, 2-phenylchromanone and C6-C3-C6 form the backbone of flavonoids. Some well-known flavonoids include but are not limited to flavonols, isoflavones, flavones, flavanonols, anthocyanidins, chalcones, dihydrochalcone and aurones. ,12. ThesMolecular docking technology, an algorithm that predicts the putative geometry of a protein–ligand complex has been successfully applied in the studies of SAR of compounds and TCM as well as predicting binding affinities of ligands ,20,21,22To verify the feasibility of the established docking protocol, the original ligand molecule was extracted from the co-crystallized thrombin complex  and re-docked into 2GDE using CDOCKER. The conformation of the re-docked co-crystallized thrombin complex (2GDE and SN3) is shown in One hundred and three flavonoids compounds were docked into the active site of thrombin model and identified by CDOCKER module in discovery studio 4.5 software . The results showed 62 hit compounds with -CDOCKER_energy above 20.0 kcal/mol and 54 hit compounds with -CDOCKER_INTERACTION energy above 40.0 kcal/mol see . The docBased on the above results, the anti-thrombin activities of 42 target flavonoids were selected for further research using a chromogenic substrate in vitro method. Selection of flavonoids was done according to three principles. The first rule was the value of -CDOCKER_energy should exceed 25 kcal/mol or -CDOCKER_INTERACTION energy should exceed 45 kcal/mol in order to improve the accuracy of measurement results. Secondly, flavonoids including flavonols, flavones, flavanonols and catechins with comparable structural features were given preference. Lastly, naturally derived and easily obtained compounds were chosen. The chemical structures of the 42 flavonoids are shown in 50 values and docking values -CDOCKER_energy and -CDOCKER_INTERACTION energy of the evaluated flavonoids are listed in 50 values less than 500 μM. The IC50 value of the positive control, Argatroban was 1.86 μM whereas the test flavonoids, myricetin, scutellarein, isorhamnetin, myricitrin, baicalin, apigenin and hydroxygenkwanin were 56.5 μM, 70.8 μM, 72.2 μM, 79.5 μM, 88.6 μM, 96.2 μM and 99.7 μM, respectively. These flavonoids were classified as strong inhibitors. Moreover, compounds with IC50 value ranging from 100 μM to 200 μM were considered as moderate inhibitors. Kaempferol (107 μM), hispidulin (126 μM), diosmetin (131 μM), herbacetin (133 μM), luteolin (146 μM), luteoloside (155 μM), galangin (159 μM) and fisetin (178 μM) were considered as moderate inhibitors. Compounds such as quercetin, genkwanin, astragaline, morin, baicalein, rutin and naringin were classified as weak thrombin inhibitors since their IC50 values ranged from 200 μM to 500 μM.IC50, -CDOCKER_energy and -CDOCKER_INTERACTION energy of the 22 flavonoids with IC50 values less than 500μM. Results showed that IC50 was negatively correlated with -CDOCKER_energy (p(corr) = −0.55, p = 0.008) and positively correlated with -CDOCKER_INTERACTION energy (p(corr) = 0.45, p = 0.035). It was demonstrated that the target compound shows high anti-thrombin activity when the -CDOCKER_energy was higher and -CDOCKER_INTERACTION energy lower.Pearson correlation analysis was established using SPSS 21.0  based on the anti-thrombin ICBinding mechanisms of flavonoids and thrombin of strong inhibitors and moderate inhibitors were investigated using Discovery Studio (DS). The theoretical binding sites of these compounds interaction with thrombin model (2GDE) are shown in detail . 3 on B ring improved bioactivity. Lastly, 2GDE residues of thrombin were bound by hydroxyl groups at C7 and C3′ sites and O-glycosyl groups on C3 site of myricitrin. The introduction of O-glycosyl group at the C3 site increased the interaction between the glycoside and protein; however, this hindered some interactions between other sites and the target.For a detailed observation, the top four flavonoids with the IC50 values less than 100 μM  were selected. The binding conformations of the 4 target flavonoids at the active site of thrombin are shown in 50 values and –CDOCKER_ and –CDOCKER_INTERACTION energies, the potential SARs of these structurally diverse flavonoids on the inhibitory effect of thrombin were analyzed as follows:Based on IC50=205 μM) vs. dihydroquercetin  and epicatechin . Myricetin  vs. dihydromyricetin  and (-)-epigallocatechin . Apigenin  vs. naringenin . Diosmetin  vs. hesperetin . The presence of C2=C3 and C4=O bonds of flavones was essential for inhibition of thrombin. Lack of either led to the loss of thrombin inhibition, which was consistent with the published report . For ins50 = 96.3 μM) vs. kaempferol . Luteolin  vs. quercetin ). Notwithstanding the above, there was an exception with galangin (IC50 = 159 μM). It had a single hydroxyl group at C3 site which displayed moderate inhibition potential on thrombin compared to chrysin . The absence of steric hindrance effects on B ring on galangin without any substituents made it easy for the flavonoid to enter the interior of the thrombin and bind the C3-OH to the protein.Moving forward, flavonoids with single hydroxyl group at C3 site were observed to have slightly decreased inhibitory activity. For example, apigenin  vs. myricitrin (IC50 = 79.5 μM); kaempferol (IC50 = 107 μM) vs. astragaline (IC50 = 217μM); quercetin (IC50 = 205 μM) vs. rutin (IC50 = 273 μM) and quercitrin (IC50 > 500 μM) and quercetin-3-O-β-d-glucoside (IC50 > 500 μM). Moreover, Isorhamnetin (IC50 = 72.2 μM) vs. isorhamnetin-3-O-neohespeidoside (IC50 > 500 μM) and typhaneoside (IC50 > 500 μM). Furthermore, flavonoid docking into the active center of 2GDE was hindered due to steric effects on the C3 site caused by the introduction of hydroxy group and glycosyl group at the C3 site. Introduction of O-glycosyl groups at the C3 site on flavonoids weakened or led to loss of thrombin inhibitory activity. For instance, myricetin (IC50 = 70.8 μM) vs. apigenin  vs. hispidulin  vs. genkwanin . Also, baicalein  vs. chrysin  vs. wogonin . In another instance, luteolin  vs. 6-methoxyluteolin . There was an exception with hydroxygenkwanin  with an OCH3 at the C7 site that had a higher activity than luteolin . Moreover, a hydroxyl at the C5 site weakened the anti-thrombin activity in fisetin  vs. quercetin . This could be attributed to hydrogen-bond interaction between C5-OH and C4 = O on the quercetin. Moreover, an ortho-hydroxyl substitution on the C7 site near the C8-OH site had a moderate influence on inhibitory activity; for instance, kaempferol  vs. herbacetin  with the IC50 values 107 μM and 133 μM, respectively. Thrombin inhibition was higher with trihydric phenol hydroxyl groups at the C5, C6 and C7 sites compared to meta-phenolic hydroxyl at the C5, C7 sites. In addition, more OH groups in the A-ring are beneficial for inhibitory effect . However50 values of 88.6 μM and >500 μM, respectively. In the case of flavonol, there was an exception. Kaempferol-7-O-β-d-glucopyranoside with an O-glycosyl groups at the C7 site was inactive to thrombin with IC50 > 500 μM compared with kaempferol (IC50 = 107 μM). Glycosylation at the 7 position on the ring system might have affected activity in a different way to glycosylation at C3. The thrombin inhibitory activity of flavones and flavanones increased when OH group at the C7 site was replaced by O-glycosyl group. For example, baicalin vs. baicalein with IC50 values of Kaempferol (C4′-OH) vs. galangin (without C4′-OH) were 107 μM vs. 159 μM whereas scutellarein (C4′-OH) vs. baicalein (without C4′-OH) had IC50 values 70.8 μM vs. 249 μM. The 3D binding conformation of scutellarein (C4′-OH) indicated that C4′-OH interacted with residues ASPH189 (Flavonoids with single hydroxy group at the C4′ site showed relative high activity of thrombin inhibition activity. IC ASPH189 B.50 = 96.2 μM) vs. luteolin . In another example, kaempferol  vs. quercetin  vs. morin . However, there was an exception with hydroxygenkwanin . It had diphenol hydroxyl groups at the C3′, C4′ sites which exhibited higher activity compared with genkwanin . This exception may be due to the influence of C8-OCH3, compared with three hydroxyl substitutions (apigenin), the additional introduction of OCH3 (hydroxygenkwanin) increased the anti-thrombin activity. Triphenol hydroxyl groups at the C3′, C4′, C5’ sites were very beneficial for effecting thrombin inhibition activity. Myricetin  vs. kaempferol (C4′-OH) vs. quercetin  reported IC50 values, 56.5Μm, 107 μM and 205 μM, respectively. Compared to the single hydroxy group at the C4′ site, ortho-diphenol hydroxyl groups of ortho-position at the C3′, C4′ sites and meta-diphenol hydroxyl groups at the C2′, C4′ sites reduced thrombin inhibition activity. For instance, apigenin -epigallocatechin, (-)-epicatechin gallate, proanthocyanidin B1 with 98% purity on the basis of HPLC analysis were obtained from Chengdu Herbpurify Co., Ltd. . Herbacetin and morin were obtained from Chengdu Must Bio-Technology Co., Ltd. . chrysin, apigenin, diosmetin and kaempferol-7-O-β-d-glucopyranoside were purchased from Chengdu Desite Bio-Technology Co., Ltd. .Chromogenic substrate S-2238 was purchased from Chromogenix ; bovine thrombin and argatroban were purchased from Sigma-Aldrich . The phosphate buffer saline (PBS)  and dimethyl sulfoxide (DMSO) were purchased from Solarbio . Standard substance of galangin, kaempferol, astragaline, fisetin, quercetin, quercitrin, isoquercitrin, rutin, isorhamnetin, isorhamnetin-3-The crystal structure of thrombin  and target protein were downloaded from the PBD database and pre-processed by Discovery Studio (DS) V 4.5  including addition of hydrogen atoms, protonation treatment, and deletion of water molecules, original ligand molecule (SN3) and other unrelated protein conformations. Then the crystal structure was energy optimized by adding CHARMm force field. The active pockets (sphere) were defined and radius modified to 9.0 Å based on its original ligand position.All the collected compounds (103 flavonoids) from traditional Chinese herb medicines were sketched by ChemDraw V10.0 . These compound structures were imported into DS, and then, CHARMm force field was added to minimize the energy.According to the above conditions, the compound structures and 2GDE were imported into DS for screening the potential flavonoids components which could act on thrombin. Docking results of 2GDE and ligand molecule were graded by “-CDOCKER_energy” and “-CDOCKER_INTERACTION energy”.blank was the absorbance of the blank group; Asample was the absorbance of the sample group, and Abackground was the absorbance of background group. IC50 values for active compounds were determined on three replicates of six concentrations. Statistical analyses and IC50 values were calculated using GraphPad Prism version 5.01 .Activity of thrombin was measured on Multiskan™ FC microplate photometer  based on published method with modification to suit our requirements ,19,23. BIn this study, potential active flavonoids were screened from traditional Chinese medicine by molecular docking; the activity was studied by enzymatic bioassay, and SARs of natural flavonoids against thrombin were revealed. Molecular docking provided a method of screening target compounds with specific structure from a large molecular dataset quickly and easily. The enzymatic bioassay focused on functional verification of thrombin inhibitors from the perspective of molecular biology. The combination of the two methods provided a reference to accurately explore the structural and activity characteristics of flavonoids against thrombin. In this paper, the results showed that the C2, C3 and C4 sites on C ring and C5, C7 sites on A ring as well as C3′, C4′ and C5′ sites on B ring were the key sites of flavonoids for activity against thrombin. Moreover, C=C, C=O bonds and OH groups were the indispensable substituent group for flavonoids against thrombin. In conclusion, these are the key aspects revealed through the SAR analysis of natural flavonoids on anti-thrombin activity. These findings would promote the study on activity mechanism of flavonoid-type compounds as well as aid medicinal chemists to design and develop more potent flavonoid-type inhibitors against thrombin.


Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) sometimes occurs in mildly fibrotic livers, while HCC incidence in NASH-related cirrhosis is lower than and less predictable than in hepatitis C virus (HCV)-related cirrhosis. Transforming growth factor (TGF)-β signaling in hepatocytic nuclei is implicated in fibrosis and carcinogenesis. TGF-βtype I receptor (TβRI) and c-Jun N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3, resulting in 2 distinct phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). In mature hepatocytes, oncogenic signaling via the JNK/pSmad3L pathway antagonizes signaling via the tumor-suppressive TβRI/pSmad3C pathway. We immunohistochemically examined domain-specific Smad3 phosphorylation in liver biopsy specimens from 30 NASH patients representing different fibrotic stages and 20 chronically infected hepatitis C patients as controls, correlating Smad3 phosphorylation with clinical course. HCC occurred during follow-up in 11 of 12 NASH patients with abundant pSmad3L and limited pSmad3C but in only 2 of 18 with limited pSmad3L. In contrast, HCC developed in 12 of 15 NASH patients with limited pSmad3C but only 1 of 15 with abundant pSmad3C. Two of fourteen NASH patients with mild fibrosis developed HCC, their hepatocytic nuclei showed abundant pSmad3L and limited pSmad3C. Five of sixteen patients with severe fibrosis did not develop HCC, their hepatocytic nuclei showed limited pSmad3L and abundant pSmad3C. Smad phospho-isoforms may represent important biomarkers predicting HCC in NASH and potential therapeutic targets for preventing NASH-related HCC. Hepatocellular carcinoma (HCC) ranks third among causes of cancer-related death worldwide . ChronicINK4B and p21CIP1 and repression of c-Myc genes /[(PLT (x109/L) × √ALT (IU/L)]. We also studied 20 randomly chosen patients at each stage of HCV-related fibrotic liver disease (F1–F4) as controls. Patients were enrolled in the study after informed consent and following the approval and recommendations of the Ethics Review Board of Kansai Medical University (code: 2019067).From January 2003 to December 2007 at the Department of Gastroenterology and Hepatology of Kansai Medical University Hospital, 100 patients were diagnosed histologically with NASH based on liver biopsy findings in the absence of other liver diseases such as viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, and drug-induced liver disease. Among patients diagnosed with NASH, 30 could be followed up continuously for 10 years or more until onset of HCC. Ultrasound (US) or computed tomography (CT) was performed for every 6 months to detect HCC. Laboratory tests included blood cell counts and measurements of serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, albumin, and prothrombin, using standard clinical laboratory techniques. Body mass index (BMI) was calculated as weight (kg)/[height (m)]All enrolled patients underwent percutaneous liver biopsy under US guidance. The liver specimens were embedded in paraffin and stained with hematoxylin-eosin. The specimens were evaluated by two hepatopathologists (K.T. and M.I.) who were blinded to clinical findings. Histologic grading and staging used the Brunt Criteria, an internationally accepted standard in pathologic practice .Polyclonal anti-phospho-Smad3 [anti-pSmad3L (Ser. 208/213) and anti-pSmad3C (Ser. 423/425)] respectively were raised against phosphorylated linker and C-terminal regions of Smad3 by immunizing rabbits with synthetic peptides. Relevant antisera were affinity-purified using phosphorylated peptides. We confirmed that these domain-specific Abs selectively distinguished between phosphorylated linker regions and phosphorylated C-terminal regions of Smad3, as described previously .Immunohistochemical analyses were performed as described previously. Primary Abs used in this study included rabbit polyclonal anti-pSmad3L (2 μg/mL) and rabbit polyclonal anti-pSmad3C (0.5 μg/mL) as described above.For immunohistochemical analyses, sections exposed to primary Abs then were incubated with peroxidase-labeled polymer conjugated to anti-rabbit immunoglobulin G . Finally, staining was developed with 3,3′-diaminobenzidine tetrahydrochloride , counterstained with Mayer’s hematoxylin , and mounted under coverslips. We counted and scored pSmad3C/L positivity in the nuclei of hepatocytes. Hepatocytic Smad3 phosphorylation was scored as follows; 0, no positivity; 1, <25% Smad3 phosphorylation; 2, 25% to 50% Smad3 phosphorylation; 3, 50% to 75% Smad3 phosphorylation; and 4, >75% Smad3 phosphorylation, as described previously .HCC incidence during the follow-up period after liver biopsy was determined using the Kaplan–Meier method. HCC occurrence curves were compared between abundant (score 3–4) and sparse (score 0–2) Smad3L/C phosphorylation by means of the log-rank test. For continuous variables, the optimal cutoff threshold for defining the group was set using receiver operating characteristic curves. All parameters with a P value less than 0.10 in univariate analysis were subjected to multivariate analysis using a Cox proportional hazards model, and those with P values below 0.05 were considered significant. The Mann-Whitney U test was used to identify significant differences between hepatocytic pSmad3L and pSmad3C positivity according to fibrosis stage, with or without HCC occurrence.During progression of NASH-related liver disease, hepatocytic Smad phospho-isoforms come to reflect liver fibrosis progression and carcinogenesis, shifting from tumor-suppressive pSmad3C to carcinogenic pSmad3L. HCC was rarely developed in NASH livers with severe fibrosis but limited Smad3L phosphorylation. On the other hand, the risk of HCC was likely to increase in those with mild fibrosis but limited Smad3C phosphorylation. Thus, abundant phosphorylation at Smad3L and limited phosphorylation at Smad3C may be important risk factors for HCC in NASH. Smad phospho-isoforms may serve as important new biomarkers for HCC risk assessment in NASH.


Optimum hatchability and fertility in the goose industry is inevitable without emphasizing on reproductive health of ganders. Determining stages of spermatogenesis is a critical factor in evaluating the age of maturity and reproductive health of animals. The current study was designed to evaluate the steps of spermiogenesis and stages along with morphological changes in seminiferous epithelium in Yangzhou goose ganders. The results of the study revealed that inside seminiferous epithelium, initial steps of spermiogenesis are depicted by changes in acrosomic granules, whereas further stages are identified by nuclear morphological changes. Furthermore, ten steps of spermiogenesis, nine stages of seminiferous epithelium and four types of spermatogonia Ad, Ap1, Ap2 and B were identified. In Yangzhou goose ganders, recommended age of sexual maturity is 227 days of age.The past three decades revolutionized the goose industry in the world. China holds the world’s largest goose breeds stock by 95% of the global total. To optimize the goose industry and cope with ever increasing poultry meat and egg demands, there is a dire need to focus on reproduction, as most geese breeds exhibit poor reproductive performance. The present study was conducted with the aim to add a contribution in the goose industry and research by the histological visualizing step wise development of germ cells during spermatogenesis by microscopy and a histological technique. Yangzhou goose is a synthetic breed developed by using local goose germplasm resources of China. It is popular in the Chinese goose industry due to high productivity and adaptability. This research evaluated the steps of spermiogenesis and stages along with morphological changes in the seminiferous epithelium in Yangzhou goose ganders. For the assessment of various stages of the seminiferous epithelium cycle, testis sections were embedded in molten paraffin wax. The initial steps of spermiogenesis were depicted by changes in acrosomic granules, whereas further stages were identified by nuclear morphological changes. Ten steps of spermiogenesis and nine stages of seminiferous epithelium were identified. Four types of spermatogonia Ad, Ap1, Ap2 and B were recognized. The results depicted a clear variation in the diameter of seminiferous tubules (ST), epithelium height (EH), luminal tubular diameter (LD), number of seminiferous tubules per field and the Johnsen score. Microscopy indicated that the stages of seminiferous epithelium were similar to other birds and mammals and the ST diameter, EH, LD and Johnsen score are positively correlated while the number of seminiferous tubules per field is negatively correlated with the ST diameter, EH, LD and Johnsen score. Fertility in Yangzhou ganders can further be improved by visualizing the histological development of germs cells in testis tissues during spermatogenesis after onset of breeding season and maturity. Our results suggest that Yangzhou ganders reach complete sexual maturity at 227 days of age.  The goose industry in China is especially prominent with an annual output accounting for approximately 95% of the world’s total production: with over 600 million geese slaughtered and more than 250 million tones of meat produced . Due to The main morphological features of testes in three different phases of development and/or activity are sexually matured testis, sexually matured and active testis and sexually matured but resting testis . HistomoAnser domesticus) is the synthetic breed developed by using the local goose germplasm resources of China. This breed is widely used in China due to its high production traits for fertility, hatchability, egg production and meat production [In birds, the cellular association in the seminiferous epithelium occupies only a small area of the seminiferous tubule. Therefore, it is difficult to determine the composition of the associations and consequently the cycle of the epithelium and spatial arrangement of stages of the cycle in the seminiferous tubule . Overalloduction . This brThe housing of Yangzhou ganders and experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institutional Animal Care and Use Committee of Nanjing Agricultural University, China . The experiments were carried out at Sunlake Swan Farm , Henglin Township, Changzhou, Jiangsu Province, China. Changzhou lies 31.76463° N and 119.9409° E. Temperature varies from 0 to 32 °C in winter and summer respectively. Rainfall is monomial with annual rainfall ranging by 1000–1100 mm and peaks from June to July.Anser anser) at 161 days of age, having same genetic origin, were used for experiments. The experiment was conducted in April-June 2016. Birds were individually identified using tags placed through their inner wings to prevent detection by other birds and to avoid pecking. Birds were maintained at ambient temperatures between 25 and 32 °C until the end of the experiment. The ganders had free access to drinking water. They were fed ad libitum with a mixed feed of 12.5% crude protein, supplemented with green grass whenever possible. Feed was offered during daytimes. The lighting schedule was 11 h light and 13 h dark until the end of the experiment. A flock 30 Yangzhou ganders  from the left testis of each gander was taken and immediately fixed in 10% buffered neutral formalin solution for 24 h and used for histological evaluation by using an automated tissue processor (LEICA RM 2235). The fixed tissues were dehydrated in alcohol of ascending concentrations, i.e., 70%, 80%, 90% and 100%, and absolute alcohol, respectively, cleared in xylene (two changes), unfiltered and embedded in molten paraffin wax. Tissue sections (5 μm) were cut perpendicular to the longest axis of the testis, mounted on glass slides and stained with hematoxylin and eosin . Stained sections were individually examined under a bright field Olympus BX63 light microscope  at 10× and 40× magnification for changes in the diameter of the seminiferous tubule, numbers of spermatogonia, spermatocytes and elongated spermatids.A sliced piece of testicular tissue  were cut perpendicular to the longest axis of the left testis, mounted on glass slides and stained with hematoxylin and eosin . Stained sections were individually examined under a bright field Olympus BX63 light microscope  at 10× and 40× magnification to observe microscopic changes in the diameter of the seminiferous tubule (ST) and lumen (LD), epithelium height (EH) and number of seminiferous tubules per field (no. ST/field). The height of the seminiferous epithelium was determined by the mean of two diametrically opposed measures EH1 and EH2. While the luminal tubular diameter was determined by the difference between the total diameter (TD) and the sum of both measures of the epithelium height (EH1 + EH2). Ten histological fields were counted on each testicular region from each group using the Image J public domain software .The maturation of germ cells in the seminiferous epithelium was categorized according to the Johnsen score. It applies the numbers from 1 to 10 to a cross section of each tubule according to the following criteria: 10 = complete spermatogenesis and perfect tubules; 9 = many spermatozoa present with disorganized spermatogenesis; 8 = only a few spermatozoa present; 7 = no spermatozoa, but many spermatids present; 6 = only a few spermatids present; 5 = no spermatozoa or spermatids, but many spermatocytes present; 4 = only a few spermatocytes present; 3 = only spermatogonia present; 2 = no germ cells present, but Sertoli cells present and 1 = no germ cells and no Sertoli cells present. The mean Johnsen score was calculated by randomly selecting 10 seminiferous tubules per Yangzhou goose gander.p-values of <0.05 were considered significant.Computations were carried out with SPSS (Version 20.0) and Graph Pad Prism (Version 5.0). All values were expressed as the mean ± standard error of the mean (SEM). The differences across groups were calculated with a one-way analysis of variance (ANOVA), followed by a Turkey’s post hoc test and two-way ANOVA by considering Bonferroni post-tests to compare the means of the replicates, where The steps of spermiogenesis and cellular associations in the seminiferous epithelium of Yangzhou ganders are shown in Step 1: a new generation of spermatids that have just emerged from the second meiotic division touching the luminal and subluminal border of the epithelium. These spermatids had an oval shaped nucleus containing a number of centrally located stained chromatin bodies. The faintly stained area represents the Golgi complex in the cytoplasm. A few proacrosomal granules can be seen in the Golgi complex during this stage.Step 2: the nuclear chromatin begins to decondense and appear uniformly distributed in the nucleoplasm. A large centrally located mass of acrosomal granules was observed connecting to the peripheral junctions by thin filamentous chromatin material. Small stained proacrosomal granules were located in the cytoplasm between the plasma and nuclear membranes.Step 3: stage 3 spermatids had spherical nuclei, but chromatin decondensation had advanced in this stage and fewer small clumps of the chromatin were present. Acrosomic granules were attached to the nuclear membrane in this stage. The cell became oval and the nucleus seemed inconspicuous in shape.Step 4: in this stage, the chromatin of the spherical nucleus continues to decondense along the nuclear membrane. The central chromatin mass started disappearing and the nucleus became eccentric within the cytoplasm spermatogonia, Ap2 , B type B spermatogonia, L (leptotene primary spermatocytes), P (pachytene primary spermatocytes), Z (zygotene primary spermatocytes), Dp (diplotene primary spermatocytes), Pe (peritubular myoid cell) and S (Sertoli cells).Step 5: now the pear shaped nucleus contained the granular nucleoplasm. The cell also became elongated in shape. Step 6: spermatids at this stage had a further elongated cytoplasm and the cell was in a wavy shape. The nucleus seemed to be dividing into two lobes. Light microscopy observations indicated that the spermatid nucleus during elongation is coiled within the cytoplasm.Step 7: the cell at this stage had divided into two equal halves. At the apex, the head of the spermatid became tapered. The nuclear chromatin became deeply stained and condenses to become granulofilamentous. Step 8: the deeply stained spermatid further elongated in shape. The nucleus elongated and became inconspicuous accompanied by a decrease in the dense chromatin material. The nucleoplasm seemed to be vanished and the spermatid head was observed pointing out.Step 9: the nucleus was absent at this stage and the maximum length of the spermatid was seen at this stage. There was a marked reduction in the cytoplasm of the cell. Step 10: the spermatid at this stage is cylindrical in shape and maintains a curvature accompanied with a cytoplasm that completely disappeared. This was the final stage of sperm before releasing in lumen.Nine cellular associations were found in the seminiferous epithelium of Yangzhou ganders shown in Histological diagrams in The Johnsen score was analyzed as depicted by the criteria mentioned above. In The present study is the first to our knowledge that elaborates the steps of spermiogenesis and the classification of the seminiferous epithelium cycle, which measures histological parameters of seminiferous tubules development in matured and active testis in Yangzhou goose ganders. Normal development of germ cells is inevitable for spermatogenesis to attain high fertility in ganders, which can be seen by observing their morphological development. Thus we can speculate that germ cells  act as one of the important factors affecting fertility and ultimately producing high quality and a high quantity of semen. Ten steps of spermiogenesis and nine stages of seminiferous epithelium were observed in Yangzhou ganders. Periodic alterations in Yangzhou ganders’ seminiferous epithelium were similar to other birds and mammals. Four types of spermatogonia, dark type A (Ad), two pale types Ap1, Ap2 and B spermatogonia were identified. In histological sections of the Yangzhou ganders’ testis, the first five stages of spermatids development was facilitated by the acrosomal position while the later five steps were identified by nuclear morphological changes. Similar observations were seen in the quail . In thisNuclear morphological changes in spermatids started from step 5 at which the nucleus elongated and began to show a spiral shape in the later stages. Several stages of seminiferous epithelium were present in the single seminiferous tubule cross section. These observations were similar to the duck  and JapaIn this study Ad spermatogonia were undifferentiated stem cells that were present in all stages of the seminiferous epithelium and were mostly found near the basement membrane. A similar observation was found  in JapanAging is not only the factor for testis development in birds, several factors are also involved in it. One of the major factors is the Sertoli cell that is responsible for testicular development. Sertoli cells acts as a central regulator of testis development ,42. In tSeminiferous epithelium with an increasing age corresponds to the reduction in the number of Sertoli cells and germs cell composing the seminiferous epithelium . As the In the present study, the diameter of ST, EH and lumen diameter were positively correlated. With the progression of spermatogenesis, the testes weight accelerated, the diameter of the seminiferous tubules increased accompanied with higher EH and LD on days 200. However there was a slight decrease in the ST diameter, EH and LD on days 227. These findings are in accordance with Jamieson et al.  that itsIn conclusion, ten steps of spermiogenesis and nine stages of seminiferous epithelium were identified in Yangzhou goose ganders. Four types of spermatogonia Ad, Ap1, Ap2 and B were recognized. Taking in consideration the steps of spermiogenesis, stages of seminiferous epithelium, testicular weight, diameter of ST, EH, LD, Johnsen score and the number of ST per field, Yangzhou ganders seemed in the culmination phase. This information could be used to determine the age at which the ganders become morphologically and functionally prepared for reproductive activity. From our previous and present study, it is clear that body maturation in Yangzhou ganders is different from testis maturation. The body matured at 160–170 days of age while the testis underwent complete sexual maturation at 227 days of age. Histological evaluation of the testis proved that the testis underwent sexual maturation in the summer months in Yangzhou ganders.


Eukaryotic elongation factor-2 kinase (eEF2K) is a Ca 2+ /calmodulin (CaM)-dependent protein kinase that inhibits protein synthesis. However, the role of eEF2K in cancer development was reported paradoxically and remains to be elucidated.Herein, A549 cells with eEF2K depletion or overexpression by stably transfected lentivirus plasmids were used in vitro and in vivo study. MTT and colony assays were used to detect cell proliferation and growth. Extracellular glucose and lactate concentration were measured using test kit. Immunoblot and co-immunoprecipitation assays were used to examine the molecular biology changes and molecular interaction in these cells. LC-MS/MS analysis and [γ- 32 P] ATP kinase assay were used to identify combining protein and phosphorylation site. Nude mice was utilized to study the correlation of eEF2K and tumor growth in vivo.We demonstrated that eEF2K inhibited lung cancer cells proliferation and affected the inhibitory effects of EGFR inhibitor gefitinib. Mechanistically, we showed that eEF2K formed a complex with PKM2 and STAT3, thereby phosphorylated PKM2 at T129, leading to reduced dimerization of PKM2. Subsequently, PKM2 impeded STAT3 phosphorylation and STAT3-dependent c-Myc expression. eEF2K depletion promoted the nuclear translocation of PKM2 and increased aerobic glycolysis reflected by increased lactate secretion and glucose.Our findings define a novel mechanism underlying the regulation of cancer cell proliferation by eEF2K independent of its role in protein synthesis, disclosing the diverse roles of eEF2K in cell biology, which lays foundation for the development of new anticancer therapeutic strategies. Samples were taken at 5, 10, 15, and 30 min by spotting 8-μl aliquots from the 40-μl assay mixture onto squares of Whatman P81 paper (2 cm by 2 cm), which were washed three times (5 min each) in 75 mM phosphoric acid followed by methanol before drying in air and scintillation counting.Recombinant GST-tagged eEF2K prepared in escribed . GST-tagescribed  were incFollowing in-vitro eEF2K kinase assay and SDS-PAGE run, PKM2 bands were excised from the gel. Excised bands were destained with 50 mM Triethyl Ammonium Bicarbonate (TEAB) (50%)/acetonitrile (50%) overnight and subsequently 30 min on a rotation device. Gel plugs were dehydrated for 30 min with 100% acetonitrile. Dehydrated gel plugs were reduced with 10 mM Tris (2-Carboxyethyl) phosphine (TCEP) for 45 min at 55 °C and alkylated with 55 mM iodoacetamide at room temperature in the dark for 30 min. Gel pieces were washed 3 times with 50 mM TEAB for 10 min each on a rotation device before they were dehydrated with 100% acetonitrile. Dehydrated gel plugs were digested with trypsin (Sigma# T7575) dissolved in 25 mM TEAB at 37 °C overnight. Digested tryptic peptides were freeze-dried and resuspended in 0.1% (v/v) formic acid and analyzed by LC-MS/MS using a Q-Exactive plus mass spectrometer (Thermo Scientific) fitted with nanoflow reversed-phase-HPLC . The nano-LC system was equipped with an Acclaim Pepmap nano-trap column  and an Acclaim Pepmap RSLC analytical column . Typically for each LC-MS/MS experiment, 5 μL of the peptide mix was loaded onto the enrichment (trap) column at an isocratic flow of 5 μL/min of 3% (v/v) acetonitrile containing 0.1% (v/v) formic acid for 6 min before the enrichment column is switched in-line with the analytical column. The eluents used for the LC were 0.1% (v/v) formic acid (solvent A) and 100% acetonitrile/0.1% formic acid (v/v) (solvent B). The gradient used was 3% B to 25% B for 23 min, 25% B to 40% B in 2 min, 40% B to 80% B in 2 min and maintained at 85% B for the final 2 min before equilibration for 9 min at 3% B prior to the next analysis. All spectra were acquired in positive mode with full scan MS spectra scanning from m/z 375–1400 at 70000 resolution with AGC target of 3e6 with maximum accumulation time of 50 ms. Lockmass of 445.120024 was used. The 15 most intense peptide ions with charge states ≥2–5 were isolated with isolation window of 1.2 m/z and fragmented with normalized collision energy of 30 at 35000 resolution with AGC target of 1e5 with maximum accumulation time of 120 ms. Underfill threshold was set to 2% for triggering of precursor for MS2. Dynamic exclusion was activated for 30s. Mass spectrometric raw data were searched using Mascot search algorithm against human SwissProt database. Cysteine carbamidomethylation was searched as a fixed modification, whereas oxidation of methionine, phosphorylation of serine, threonine and tyrosine were searched as variable modifications.pRK7-FLAG-Rheb was described in previously published paper . pWZL-FL3 for 1 h at room temperature. And then lysed in 2 × loading buffer (0.125 M Tris (pH 6.8), 3% SDS, 10% glycerin, 0.02% BPB, and 5% β-mercaptoethanol) at 4 °C for 30 min. Levels of PKM2 dimerization and tetramerization were analyzed by immunoblotting.Cells were washed twice with PBS (pH 8.0) before treated with BS6 cells/per site). Cancer growth was periodically monitored after post-injection for 40 days. Cancer volume was estimated using the following formula: cancer length × (cancer width)2 × 0.5. Half of the dissected cancer issues were fixed with formalin for subsequent immunohistochemistry (IHC) analysis to detect PCNA (proliferating cell nuclear antigen) expression, and the other half were lysed on ice, followed by immunoblotting analysis of indicated proteins.Nude mice were inoculated subcutaneously with A549 shNC and A549 sheEF2K stable cells . Figure To explore the mechanism underlying eEF2K-mediated cell proliferation, we examined major signaling pathways related to cell proliferation. We found that eEF2K depletion prominently increased STAT3 phosphorylation, but not the phosphorylation and expression of EGFR, PDGFR, PI-3 K, Src, AKT, JAK1, JAK2, and ERK  PKM2 and enhanced phosphorylation of STAT3 Fig. f, These 3 volume earlier than the cells without eEF2K depletion (Table To determine the role of eEF2K in tumor growth in mice, we subcutaneously injected A549 cells with or without eEF2K depletion in athymic nude mice. As shown in Fig. on Table , highligon Table b. Immunoon Table c, supporeEF2K belongs to a small family of protein kinases, which are termed ‘α-kinases’ on the basis of its active-site geometry and is different from typical protein kinase superfamily . However− 37. Our studies showed that eEF2K impaired the activation of STAT3. As we know that frequent STAT3 activation in cancer cells is largely due to the fact that STAT3 is a point of convergence for numerous tyrosine kinases, including PDGFR, EGFR, AKT, SRC and JAK family, etc. [STAT3 is constitutively activated in diverse cancer cell types and have been demonstrated to be important for the proliferation and survival of cancer cellsin vivo [. PKM2 exists as both tetrameric and dimeric forms [PKM2 is highly expressed in cancer cells and promotes aerobic glycolysis and cell proliferation in vitro and in vivo , 20. PKMic forms . Increasic forms . While tic forms . We reveic forms , 39. Impic forms , which iThr129 is highly conserved among species, we found that phosphorylation of PKM2Thr129 regulates PKM2 activation by blocking dimerization of PKM2, thus, an approach to retain eEF2K could be alternative therapy strategy in intervening formation of dimer PKM2. In fact, eEF2 shouldn’t have been recognized a unique substrate of eEF2K, AMPK and alpha4 were reported to be new substrates of eEF2K before [In recent years, non-canonical localizations of PKM2 has been extensively studied. PKM2 was found in the nucleus, mitochondria, and extracellular secretion, which related to novel biological functions in tumor progression , 40, 41.K before , suggestIn summary, our findings constitutes an additional paradigm of eEF2K as a suppressor of carcinogenesis other than a role of cytoprotection in lung cancer cells, and delineate a novel mechanism underlying regulation of proliferation by eEF2K-mediated PKM2 phosphorylation and subsequent dimerization, leading to inhibit STAT3 phosphorylation and c-Myc expression accompanied by collapse of aerobic glycolysis Fig. d. These Additional file 1: Figure S1. The phosphorylation level and activity of eEF2K in A549 when cultured with glucose-free or serum-free mediun. Figure S2. eEF2K inhibition by A484954 alleviates the effects of gefitinib. Figure S3. eEF2K positively regulates the glycolytic enzyme activity of PKM2. Figure S4. eEF2K inhibition increases STAT3 phosphorylation in H1299 cells. Figure S5. eEF2K affects protein tyrosine kinase activity of PKM2 in the cytoplasm and nucleus extracts. Figure S6. Phosphorylation kinetics of known eEF2K substrates.


In theory, the same sea-ice models could be used for both research and operations, but in practice, differences in scientific and software requirements and computational and human resources complicate the matter. Although sea-ice modeling tools developed for climate studies and other research applications produce output of interest to operational forecast users, such as ice motion, convergence, and internal ice pressure, the relevant spatial and temporal scales may not be sufficiently resolved. For instance, sea-ice research codes are typically run with horizontal resolution of more than 3 km, while mariners need information on scales less than 300 m. Certain sea-ice processes and coupled feedbacks that are critical to simulating the Earth system may not be relevant on these scales; and therefore, the most important model upgrades for improving sea-ice predictions might be made in the atmosphere and ocean components of coupled models or in their coupling mechanisms, rather than in the sea-ice model itself. This paper discusses some of the challenges in applying sea-ice modeling tools developed for research purposes for operational forecasting on short time scales, and highlights promising new directions in sea-ice modeling. Broadly speaking, there are three overlapping communities of sea-ice modelers: those who are interested in understanding and accurately simulating the detailed physical processes, climate modelers who are interested in computationally efficient representations of large-scale sea-ice characteristics and processes, and the operational forecasting community, who are interested in models that produce efficient, skillful predictions for a range of spatio-temporal scales. A fourth community does not perform the modeling work itself but uses the results: policy makers and other stakeholders, such as mariners.Numerical weather forecasting centers traditionally have used relatively simple sea-ice modelsWhile they share a common subject, research and operational communities have different goals, requirements, and needs. For the research community , understanding and properly representing the physical processes are paramount; forecasters need products that are both skillful and valuable to stakeholders, particularly with respect to hazards and risk . For insSea ice occurs in many forms that present different types of hazards, from solid, stationary shelves of landfast ice to large, brittle plates, to loose mixtures of smaller floes with slushy brash ice and icebergs. Critical elements of a physics-based sea-ice model framework include necessary, first-order physical representations such as basic thermodynamics and dynamics with sound subgrid-scale parameterizations of processes essential to major feedback cycles, relevant coupling mechanisms with the atmosphere and ocean, system memory , awareness of sea ice’s multiscale character, and, for longer time scales, suitably conserved properties such as heat, momentum, and mass of water and salt. As described below, first-order physical processes are currently represented in large-scale Earth system models, but whether they are necessary or sufficient now or in the future depends on the application. For instance, mariners say that area fractions of multiple ice thicknesses or floe sizes are not especially useful for navigation, while locations of high sea-ice pressure and ridged ice are . ClimateSince the inaugural voyage of the icebreaker Yermak in 1899, mathematical models of sea ice were conceived to serve a dual purpose of short-term prediction and climatic signal detection. In , early cModelers must balance the physics that can be included in sea ice component models against their computational expense. Internal variability makes the atmosphere difficult to predict , and modAnother practical consideration is availability and reliability of modeling technologies. Well-vetted, community-developed modeling tools offer attractive options for operational forecasting centers when upgrading their models. While sea-ice processes that are primarily restricted to action in a vertical column are easy to share, dynamical cores are fundamentally linked with the underlying code structure  and therefore are more difficult to share. Fortunately, much of sea-ice physics is already represented within column descriptions, and therefore our recommendations focus on dynamics and accelerating community-wide progress through shared model frameworks.3, )The dynamics components of sea-ice models capture the spatial character and evolution of the ice pack through processes that affect its momentum, strength, and deformation, and are thus directly relevant to operational forecasting of ice drift, compression, and ridging. The computed velocity is used to conservatively transport the ice horizontally, and divergence and convergence create areas of open water and closely packed, often ridged ice, which affect the large-scale sea-ice state. The ice pack can generally be divided into two regimes: the consolidated pack, in which ice is composed of large, brittle plates that behave as a plastic material, and the marginal ice zone (MIZ), in which ice floes tend to be smaller and less concentrated. The transition region between these two regimes is not well defined and changes in both time and space as the ice pack freezes, melts, and moves around. Vessels operating in the ice pack encounter less resistance in the MIZ than in the consolidated pack, although it is still a very hazardous area; the consolidated pack generally requires icebreaking capabilities, and identifying paths of weaker or less consolidated ice is helpful for transiting the pack. In the Arctic, the MIZ has traditionally been a relatively narrow region around the edges of the consolidated ice pack, but this distinction is changing as the Arctic pack becomes more seasonal and similar to the Antarctic .There are a number of different approaches for sea-ice dynamics  and the applied forcing required to move it. Most large-scale models use the isotropic, viscous-plastic (VP) model of  or an exIn the consolidated ice pack, recent research has focused on capturing its brittle and/or anisotropic character e.g., , 50–53) –53 50–53A majority of current operational sea ice models assume constant, neutral transfer coefficients of momentum, heat, and moisture between the air-ice and ice-ocean interfaces. These transfer coefficients, which depend on the large-scale roughness of these interfaces, e.g., from pressure ridges and keels, are expected to vary spatially and temporally. Numerical models , 56 suggCurrent models also do not account for floe rotation, pitching, rolling, and flexing. In promising work that can span both consolidated and MIZ regimes, some researchers are examining the subgrid-scale interaction and evolution of floes. For continuum models, this takes the form of floe size distributions that evolve in response to freezing, melting, and damage by waves and tides through flexure. Wave energy reaches deeper into the interior Arctic Ocean now than previously , which cLarge-scale motion and deformation products derived from remote sensing  provide Operational forecasts often need information such as sea-ice pressure at subgrid scales, e.g., in the vicinity of vessels Fig. . The RisApproaches used to objectively evaluate models’ representation of sea-ice characteristics and evolution also depend highly on the application. Seasonal sea-ice prediction skill is significantly affected by the choice of the verification product , 64. ForData assimilation offers a mathematically robust framework for integrating observations in models for many purposes, including state estimation, initialization, and parameter calibration . It can Short-term forecasts are considered an initial value problem, for which data assimilation provides an optimal initial state. When the lead time of a forecast is increased, the significance of the initial condition decays at varying rates. For example, the “memory” of ice thickness is long, and so ice thickness is sometimes assimilated for seasonal ice prediction e.g., , 71–73).–73.71–73A primary issue for short-term forecasts based on coupled ocean and sea ice models is bias and uncertainty in the atmospheric forcing, along with a lack of data to properly constrain the models. This is particularly problematic where satellite data is ambiguous, near the coasts, and in the marginal ice zone, areas with numerous marine forecast users.Ensembles can be used for data assimilation to determine the uncertainty of a model prediction. The ensemble spread represents all uncertainties originating from forcing and physical parameterizations within a modeling tool; missing processes create immense uncertainties in forecasts. For instance, before landfast ice was parameterized in models,  had to dA critical consideration for operational tools is their complexity in terms of maintenance and computational costs, validation of upgrades, and characterization of uncertainty. Greater model complexity is associated with increased human and computational resources and tends to feature larger state vectors and more free parameters across a greater variety of parameterizations. Operational centers require a stable code base, and often prefer simpler codes because of the number, length, and frequency of runs performed each day. Increased complexity is warranted if it improves the atmospheric forecast, or to meet demand for other sea-ice parameters as products. One aim of added complexity is to provide model output that is comparable with observations, in terms of variables’ definitions and their simulated quality of mean sea-ice state and variability.Another reason to include more detailed sea-ice parameterizations is to better capture the physical processes themselves with more faithful feedback and sensitivity to perturbations . For insBoth continuum and discrete element models face computational challenges associated with communication in a parallel computing environment and storage of ever-increasing amounts of output data, with clear impacts on the generation of sea-ice diagnostics needed for applications. Physically based statistical analyses and reduced-order modeling can decrease the data volume in a comprehensible way. Machine learning can also be used to train parameterizations within physical models, such as the floe-contact models needed for DEM, and holds great promise for revolutionizing the data-intensive aspects of Earth system prediction.Model complexity in the form of multiple options for representing the physics adds value for users, who can choose the level of complexity relevant for their problem. For example, simpler configurations with coarse resolution are preferred for long  simulations and large ensembles, and simpler sea-ice model configurations might also be appropriate if data assimilation dominates the sea-ice response. While detailed, complex physical descriptions are fundamental for understanding Earth system processes and their interactions, being able to reduce the model to a simpler version enables experimentation, and mechanisms can be prioritized for further modeling and observational study. Moreover, developing a common set of integrated modeling tools entrains capability from the whole community, including research model development and operational validation resources.The next generation of sea-ice models and parameterizations is being developed while application groups utilize and continue to improve existing models. This approach includes three phases: operational applications using older, well-tested codes; research groups using newer, released versions of codes and updating them with incremental development; and model developers building novel algorithms and modeling frameworks.A hierarchy of models (or model configurations) is necessary to address the variety of scientific questions spanning operational needs, process understanding, and climate research. A key question is whether to create a simple model to address each scientific problem, or one all-purpose modeling framework that can address a range of problems by selecting relevant parameterizations.Sea ice is a highly complex material, and implementing numerous options for every process into a single model framework can become unwieldy. Thus, a diversity of model frameworks is useful to better understand alternative mechanisms and balances. The modeling paradigm for coupled, Earth system models has long been modular, allowing components such as the ocean model to be swapped with other options. Now, the components themselves are moving toward mix-and-match subcomponents. For instance, the Discrete Element Model for Sea Ice (DEMSI ) uses thAlthough a single model framework is unlikely given the diversity of funding agencies, research interests, and user needs, a common set of metrics and data assimilation tools could be jointly developed and deployed online. Modeling tools necessarily are configured to meet the demands of the problem at hand, but model-agnostic analysis tools that incorporate recommended sea-ice metrics , standarThese types of shared resources foster communication across the modeling and observational communities. To build the modeling, observational, and deployed capabilities that meet society’s needs, dedicated resources are needed to engage the entire community of developers and users in a co-design “value cycle” in which each community contributes and responds to the needs of the others , 85. A kAlthough the same models can be used for both research and operational applications, in practice this is complicated, with different products and parameters needed, or needed on very different spatial scales. Computational resources present considerable constraints for both research and operations, driving choices of included physics as well as spatial and temporal resolution, simulation length, and ensemble size. Data assimilation is necessary for initializing short-term forecasts, for which sea-ice physics parameterizations that mainly influence longer term feedbacks are likely irrelevant. The main parameters desired by mariners are already available in large-scale sea-ice models, but validation metrics and probabilistic risk information need to be developed. These parameters may not be accurate enough for operational use, requiring additional research and development in new or refined approaches.A community framework for sea-ice modeling would seek to leverage the whole community for model development, verification, and validation toward a goal of mutually useful, flexible, robust sea-ice modeling tools. With a strong foundation in physics, computer science, and observations, we could use models to make recommendations for targeting observations as well as predicting the sea-ice state and future change. We recommend creating an international benchmarking product for model intercomparisons and forecasts, as an initial, coherent, directed strategy toward a sea-ice modeling and observations co-design cycle valuable for both research and operations.Shared code is broadly exercised and understood, but a mix of code frameworks is likely to continue. For instance, data assimilation methods are usually tightly intertwined with the physical models, making the code difficult to share. We encourage code sharing when-, where-, and however it makes sense, recognizing that code diversity is also valuable. Within a common conceptual framework, this can take the form of multiple model frameworks  with shared elements . Defining the same basic output is crucial, along with a variety of common and unique metrics for applications.Thus, we need revolution, evolution, and the status quo, simultaneously. Because of limitations in the inherent assumptions of continuum models, short-range forecasting needs DEM, but big changes are not needed for standard climate scales—evolution is sufficient. In particular, VP models continue to be useful because they produce desirable features , although some of the features are not realistic . We can continue to use continuum models for climate studies, but high-resolution simulations need an alternative. DEM is a promising, revolutionary candidate for the heterogeneous, anisotropic ice pack at fine spatial scales.


Despite attempts to halt it, the prevalence of food allergy is increasing, and there is an unmet need for strategies to prevent morbidity and mortality from food-induced allergic reactions. There are no known medications that can prevent anaphylaxis, but several novel therapies show promise for the prevention of food-induced anaphylaxis through targeting of the high-affinity IgE receptor (FcϵRI) pathway. This pathway includes multiple candidate targets, including tyrosine kinases and the receptor itself. Small molecule inhibitors of essential kinases have rapid onset of action and transient efficacy, which may be beneficial for short-term use for immunotherapy buildup or desensitizations. Short courses of FDA-approved inhibitors of Bruton’s tyrosine kinase can eliminate IgE-mediated basophil activation and reduce food skin test size in allergic adults, and prevent IgE-mediated anaphylaxis in humanized mice. In contrast, biologics may provide longer-lasting protection, albeit with slower onset. Omalizumab is an anti-IgE antibody that sequesters IgE, thereby reducing FcϵRI expression on mast cells and basophils. As a monotherapy, it can increase the clinical threshold dose of food allergen, and when used as an adjunct for food immunotherapy, it decreases severe reactions during buildup phase. Finally, lirentelimab, an anti-Siglec-8 antibody currently in clinical trials, can prevent IgE-mediated anaphylaxis in mice through mast cell inhibition. This review discusses these and other emerging therapies as potential strategies for preventing food-induced anaphylaxis. In contrast to other food allergy treatments which largely focus on individual allergens, blockade of the FcϵRI pathway has the advantage of preventing clinical reactivity from any food. Approximately 15 million people in the United States have food allergy and are at risk for anaphylaxis, a potentially life-threatening systemic allergic reaction , 3. Therde novo cytokine production, which collectively cause clinical symptoms  is a humanized antibody that binds to the C3 domain of IgE, thereby sequestering free IgE away from FcϵRI  is another humanized anti-IgE mAb which binds to free IgE with higher affinity than omalizumab. Ligelizumab effectively prevents passive systemic anaphylaxis (PSA) in human FcϵRIα transgenic mice . InteresThe low-affinity IgE receptor, FcϵRII or CD23, appears to regulate IgE homeostasis . Anti-CDvia apoptotic depletion of IgE-producing B cells, but does not bind to circulating IgE or IgE already bound to FcϵRI. In early clinical trials, a single dose of quilizumab reduced serum IgE levels in patients with allergic rhinitis or asthma for approximately 6 months (Quilizumab (Genentech) is a humanized afucosylated anti-IgE antibody in development that binds to M1’, a domain specific to membrane-bound IgE in B cells. In this way, it reduces circulating IgE 6 months . Unfortu6 months . The futMEDI4212 is a high-affinity antibody for IgE which can both bind to circulating IgE as well as membrane-bound IgE, thus depleting IgE-producing B cells through antibody-dependent cell-mediated cytotoxicity . Becausein vivo, though it has not yet been tested in clinical trials.Eggel et al. created a non-immunoglobulin ankyrin repeat protein (DARPin) inhibitor of IgE, E2_79, which not only blocks IgE from binding to FcϵRI, but is also able to rapidly dissociate preformed IgE-FcϵRI complexes . IntraveNumerous kinases are involved in FcϵRI pathway signaling, including spleen tyrosine kinase (Syk), Bruton’s tyrosine kinase (BTK), Lyn, Fyn, phospholipase Cγ (PLCγ), PI3 kinase (PI3K), and others . All of in vivo in humans is still preliminary, but ibrutinib has been shown to suppress IgE-mediated ex vivo basophil activation and skin prick testing to both aeroallergens and foods in allergic subjects , which was approved in 2018 for chronic refractory immune thrombocytopenic purpura, prevented ex vivo basophil activation after a single dose in humans  recruit phosphotyrosine phosphatases (SHP‐1 and SHP‐2) and inositol phosphatases (SHIP‐1), which then provide direct inhibitory feedback on the FcϵRI pathway.Expressed on basophils and some tissue mast cells as well as other cells, the low-affinity IgG receptor FcγRIIb (CD32b) is a potential target for the prevention of allergen-mediated anaphylaxis . Co-aggrCD300a is an inhibitory receptor expressed on numerous human immune cells including mast cells and basophils. Cross-linking of CD300a recruits SHP-1 and SHIP-1 to elicit strong inhibitory signals on the FcϵRI pathway . Monocloin vitro and showed efficacy in murine models of allergic asthma  are transmembrane receptors found on the surface of immune cells, so-called because they bind to sialic acid-containing ligands. Most Siglecs have ITIMs or ITIM-like motifs, which upon ligand binding, enable inhibitory signaling to counteract the actions of tyrosine kinases in the FcϵRI pathway , 57. TheIn vitro, pre-incubation of human CD34-derived mast cells with anti-Siglec-8 mAb markedly shifted the anti-FcϵRI antibody-induced secretion dose response curve for histamine and PGD2 secretion  is expressed on most human myeloid cells, including mast cells and basophils. Duan et al. showed that dual targeting of CD33 and specific IgE on mast cells with liposomes expressing CD33L and a synthetic antigen (TNP) could prevent IgE-mediated anaphylaxis . These l2 completely prevented degranulation and partially prevented PGD2 release and GM-CSF production  which only counteract the effects of a few of many mediators that participate in causing allergic reactions.Both short-term and long-term protection strategies to prevent anaphylaxis are needed to improve the quality of life of food allergic patients. Short-term therapies which can facilitate food OIT build-up to prevent adverse reactions would allow more patients to reach maintenance dose. Alternatively, short-term treatments which could protect against accidental exposures during isolated high risk situations (e.g. a family vacation abroad or birthday parties) would reduce morbidity and mortality and help alleviate anxiety for those suffering from food allergies. Perhaps more importantly, therapies are needed which could be used chronically to reliably prevent reactions from accidental food exposures.Many questions remain regarding the utility and safety of the above therapies, most of which are still experimental or in clinical development. Each therapy’s risk-benefit ratio and mechanism of action will determine its specific indication Table 1.MD conceived of the concept and wrote the manuscript. BB and DM contributed sections and provided feedback on the manuscript. All authors contributed to the article and approved the submitted version.This work was funded in part by a K23 mentored career award from NIH to MD (grant AI143965), a U19 grant from NIH to BB (AI136443), the Ludwig Family Foundation, and the Johns Hopkins School of Medicine Clinician Scientist Career Development Award.MD has received research funding from Acerta Pharma/AstraZeneca in addition to consultant fees from AlphaSights, BVF Partners, and DAVA Oncology. BB has also received research funding from Acerta Pharma/AstraZeneca, as well as consultant fees from Regeneron, Sanofi, and GlaxoSmithkline. Finally, BB receives remuneration for serving on the scientific advisory board of Allakos, Inc. and owns stock in Allakos. He is a co-inventor on existing Siglec-8–related patents and thus may be entitled to a share of royalties received by Johns Hopkins University during development and potential sales of such products. BB is also a co-founder of Allakos; the terms of this arrangement are being managed by Johns Hopkins University and Northwestern University in accordance with their conflict of interest policies. DM consults for Boehringer-Ingelheim.


The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an integrative molecular sensor for detecting environmental irritant compounds, endogenous proalgesic and inflammatory agents, pressure, and temperature. Different post-translational modifications participate in the discrimination of the essential functions of TRPA1 in its physiological environment, but the underlying structural bases are poorly understood. Here, we explored the role of the cytosolic N-terminal residue Ser602 located near a functionally important allosteric coupling domain as a potential target of phosphorylation. The phosphomimetic mutation S602D completely abrogated channel activation, whereas the phosphonull mutations S602G and S602N produced a fully functional channel. Using mutagenesis, electrophysiology, and molecular simulations, we investigated the possible structural impact of a modification (mutation or phosphorylation) of Ser602 and found that this residue represents an important regulatory site through which the intracellular signaling cascades may act to reversibly restrict or “dampen” the conformational space of the TRPA1 channel and promote its transitions to the closed state. Phosphorylation is supposed to be one of the most important post-translational mechanisms reversibly regulating the Transient Receptor Potential Ankyrin 1 (TRPA1) channel under various physiological and pathophysiological conditions . This chFor effective phosphorylation by PKA and PKC, TRPA1 needs the presence of the scaffolding A-kinase anchoring protein 79/150 (AKAP) . AKAP di2+/calmodulin-dependent protein kinase families. Interestingly, the side chain of this serine is exposed to the cytoplasm in TRPA1 structures obtained in the ligand-free state  or in tD: 6PQQ; ), benzylD: 6PQQ; ), and JTD: 6PQQ; ). In conD: 6PQQ; ), the sin = 10), the S602D construct was nonfunctional in all cells tested within a recording period of at least 120 s (n = 9). To investigate if the S602D mutation affects the surface expression of TRPA1, we expressed tGFP-tagged wild-type TRPA1 or S602D in HEK 293T cells and performed confocal microscopy by using CellBriteTM Fix 640 to visualize plasma membrane , the region around Trp605 may represent not only a putative phosphorylation site but also a putative consensus binding site for Ca2+/calmodulin [p = 0.905, 0.682 and 0.620 at +120 mV and p = 0.970, 0.676 and 0.845 at −100 mV; n = 4). The currents through S602N were not significantly different from wild-type channels , which indicates structural involvement of the tryptophan 605 in the process of channel activation. These data together suggest that the introduction of a negatively charged amino acid at position 602 mimics the phosphorylated state of the channel and that possible phosphorylation at Ser602 may cause the channel to be irresponsive to voltage and chemical stimuli.The structural role of Ser602 in TRPA1 channel functioning was next investigated by substituting this residue with asparagine, a polar amino acid of a similar size as negatively charged aspartate. We also evaluated the possibility that the disruptive effect of S602D may be caused by salt-bridge formation with proximal residues Lys603 or Arg604. Moreover, we tested the structural role of Trp605, a residue contained in the allosteric nexus. According to various prediction servers  simulations using the published cryo-electron microscopic structure of human TRPA1 captured in the open, ligand-occupied state . The siThe molecular dynamics (MD) trajectory reached 42–44-ns and the root mean square deviation (RMSD) measured for individual subunits and for the channel complex indicated that the models were stable . ObviousOur present data show that the single mutation of Ser602 to aspartate renders the TRPA1 channel inactive, whereas the replacement of this serine with asparagine, a residue of a similar side-chain as aspartate but lacking its negative charge, or with a flexible glycine, fully retains channel functionality. The data show further that the phosphomimetic mutant S602D is expressed on the plasma membrane, and thus appears to retain key protein functions. These results raise the possibility that Ser602 represents a potential phosphorylation site through which specific intracellular signaling cascade(s) may act to promote the transition of the channel to the closed state.Although phosphomimetic/phosphonull substitution studies have proved to be instrumental and highly informative in obtaining new insights into the mechanisms of TRPA1 regulation by phosphorylation ,36,43,44More than 900,000 different protein phosphorylation sites, including predicted, have been reported in various literature-curated databases and numerous bioinformatics resources and tools are available for the prediction of phosphorylation sites  or protein kinase C  has been already shown to up-regulate TRPA1 ,25,26, wTRPA1 gene have been detected at positions immediately preceding and following these two residues: T598M (rs147715599), I599V (rs145600263), I599N (rs745749488), I600V (rs1236229851), R601S (rs148585412), R601T (rs1347666288), R604I (rs377764138), R604T, W605R (rs748788882), D606N, D606Y (rs1352827754), E607K (rs755681762), C608Y (rs747708313), and C608R (rs955642376). In particular, some of these polymorphisms may affect the phosphorylation consensus or gating and deserve further investigation to elucidate their potential involvement in human pathophysiology.The enormous variability of the mechanisms found in relation to the role of TRPA1 in various physiological processes is a major challenge for the future. Most of the kinases described so far act on TRPA1 to functionally up-regulate the channel’s function. The region around Ser602, on the contrary, might represent a potential site from which the activity of the channel can be effectively down-regulated under some pathophysiological conditions. Future studies, perhaps using a co-expression of TRPA1 with the predicted kinases see  or examil-lysine and collagen  with 200 ng of green fluorescent protein (GFP) plasmid  and with 400 ng of cDNA plasmid-encoding wild-type or mutant human TRPA1  or control plasmid. The cells were used 24–48 h after transfection. At least three independent transfections were used for each experimental group. The wild-type channel was regularly tested in the same batch as the mutants. The mutants were generated by PCR by using a QuikChange II XL Site-Directed Mutagenesis Kit , and they were confirmed by DNA sequencing .Human embryonic kidney 293T cells  were cultured in Opti-MEM I media  supplemented with 5% fetal bovine serum. The magnet-assisted transfection  technique was used to transiently co-transfect the cells in a 15.6 mm well on a 24-well plate coated with poly-2+-free and contained: 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid), 10 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 10 mM glucose, and pH 7.4 was adjusted by tetramethylammonium hydroxide. The intracellular solution contained 125 mM Cs-gluconate, 15 mM CsCl, 5 mM EGTA, 0.5 mM CaCl2, 10 mM HEPES, 2 mM adenosine 5′-triphosphate magnesium salt, 0.3 mM guanosine 5′-triphosphate sodium salt hydrate, adjusted with CsOH to pH 7.4. The chemicals were purchased from Sigma-Aldrich . The current-voltage relationships were recorded by using 50-ms steps ranging from holding potential of –70 mV to –100 mV, followed by a 220-ms ramp from –100 mV to +120 mV.Whole-cell membrane currents were filtered at 2 kHz by using the low-pass Bessel filter of the Axopatch 200B amplifier, and they were digitized (5–10 kHz) with a Digidata 1440 unit and the pCLAMP 10 software . Patch electrodes were pulled from borosilicate glass and heat-polished to a final resistance of between 3 and 5 MΩ. Series resistance was compensated by at least 60%. The experiments were performed at room temperature (23–25 °C). Only one recording was performed on any one coverslip of cells to ensure that recordings were made from cells that had not been previously exposed to chemical stimuli. Extracellular bath solution was CaTM Fix 640  according to the manufacturer’s protocol. Briefly, the cells were incubated in phosphate-buffered saline (1 mL) containing the dye (1 µL) for 20 min at 37 °C in a 5% CO2. After treatment, cells were washed and imaged in Opti-MEM I media  supplemented with 5% fetal bovine serum. The colocalization analysis was performed using the Colocalization Finder plugin in ImageJ and fluorescence intensity profiles measured along a rectangle drawn across the cell membrane.HEK 293T cells transfected with wild-type or S602D mutant of human TRPA1 tagged with tGFP  were stained with the cytoplasmic membrane labeling dye CellBriteTo elucidate the possible structural mechanism through which the substitution S602D produces inactivation of the channel and compare the impact of phosphorylation at Ser602, we used the human TRPA1 structure with PDB ID: 6V9X, determined by cryo-electron microscopy . First, Our data identify an important N-terminal serine residue from which the channel functioning can be abrogated by a phospho-mimetic mutation. Beyond that, our data suggest that conformational changes produced by a substitution of Ser602 affect not only electrophilic- but also non-electrophilic- and voltage-dependent activation. This indicates that these activation mechanisms may converge and involve changes in the coordination between Ly671 and the TRP-like helix. Whether this serine residue indeed represents a phosphorylation site in vitro and in vivo is an interesting topic for future studies.


Ground-motion models (GMMs) are a key driver for the results of probabilistic seismic hazard analyses and their uncertainty. GMMs that bridge seismological and empirical approaches are an effective tool to represent the distribution of ground motion and its uncertainty in seismic hazard assessment. A methodology is presented that uses ground-motion data recorded at seismograph sites in eastern North America and shows how they can be used to calibrate simple scalable seismological models of ground-motion generation and propagation. Such GMMs can directly account for the gross features of source scaling (magnitude and stress parameter), attenuation, site response, and kappa effects. It is shown that, by application of appropriate GMM strategies, sigma  could be greatly reduced, resulting in lower calculated hazard for nuclear plants founded on rock. This reduction in sigma requires that high-quality seismic monitoring  be installed and operated over a period of years (in addition to strong-motion stations), and that an ongoing investment be made in data analysis and targeted GMM development using the data. Ground-motion models (GMMs), also referred to as ground-motion prediction equations, are a key component in probabilistic seismic hazard analysis (PSHA), and often the most important uncertainty affecting PSHA results. GMMs provide median estimates of ground-motion amplitudes as a function of explanatory variables such as magnitude, distance, and site conditions, along with estimates of variability. Empirical GMMs are commonly used in data-rich regions such as California and Japan; For instance, the second-generation Pacific Earthquake Engineering Research–Next Generation Attenuation-West (NGA-W2) project , in comparison with the underlying NGA-W2 data used in model development. The generic GMM matches the data as well as strictly empirical GMMs developed from the NGA-W2 database and has the added benefit of being parameterized by simple seismological parameters.Yenier and Atkinson , b develDrup):Y) is the  logarithm of a ground-motion intensity measure, such as peak ground acceleration (PGA), velocity (PGV), and 5 %-damped pseudospectral acceleration (PSA) at a selected oscillator frequency. FE, FZ, and FS are the model components for earthquake source, geometrical spreading, and site amplification, respectively. The anelastic attenuation (γ) and empirical calibration (C) coefficients are frequency dependent. The C term is an empirical constant that scales the simulation amplitudes to match the amplitude of the observations. The source (FE) and geometrical spreading  terms are constrained in their scaling behavior by the equivalent point-source simulations that were validated using the rich empirical database from California , site amplification model (C).Atkinson et al.  used theFE) isolates the effects of magnitude and stress parameter on the ground-motion amplitudes:FM represents the magnitude scaling term, ignoring near-distance-saturation effects, and FM term is a function of moment magnitude (M) and is defined using a hinged-quadratic functional form that follows an empirical form from data-rich regions . The values of eσΔ as determined from the simulations have a variability in magnitude and frequency that is rather complicated, and the shape of the function differs depending on whether one is upscaling or downscaling the stress parameter. The shape can be described by a polynomial:s0 to s9 are frequency-dependent coefficients.High-frequency ground-motion amplitudes relative to low-frequency amplitudes are controlled by the stress parameter Boore . The strh is a pseudodepth term that accounts for distance saturation effects. The pseudodepth term is adopted from inversion results for active regions , and the geometric spreading is that of a classic point source.It is important to note that, because we model geometric spreading using an equivalent point-source distance, the geometric spreading term implicitly includes the near-distance saturation effects attributable to finite-fault effects for large events. For small to moderate events, FZ) isZ represents the geometrical attenuation of Fourier amplitudes, whilst the multiplicative component, (b3 + b4M)ln(R/Rref), accounts for the change in the apparent attenuation that occurs when ground motions are modeled in the response spectral domain rather than the Fourier domain. Rref is the reference effective distance, given as Z is a hinged bilinear model that provides for a transition from direct-wave spreading to surface-wave spreading of reflected and refracted waves, beyond the critical distance for reflections from the Moho:Rt represents the transition distance (= 50 km), and b1 (= − 1.3) and b2 (= − 0.5) are the geometrical attenuation rates of Fourier amplitudes at R ≤ Rt and R > Rt, respectively. Note that the coefficients describing geometric spreading and anelastic attenuation can be determined in ENA from empirical data for small to moderate earthquakes.The geometrical spreading function (VS30 ~ 2000 m/s); this is the site condition corresponding to most seismograph records in eastern Canada. The approach taken in Atkinson et al.  on the response spectral domain ground-motion amplitudes (PSA) as well as on the ground-motion peak amplitudes (PGA and PGV):Hassani and Atkinson  further κ0)  for ∆σ = 100 bar and different κ0 values, for the adjustment model of Hassani and Atkinson , for an event of M = 6 having a stress parameter of 300 bar, median 20-Hz PSA at 10 km would be ~ 490 cm/s2 for a very hard rock site with κ0 = 0.002. This scenario is the type of event that contributes significantly to hazard for nuclear sites in ENA situated on hard rock. However, it has been suggested that kappa values on some rock sites may be significantly higher than on others. For the same scenario event at a site for which κ0 = 0.01, the median PSA would be only ~ 330 cm/s2. Thus, kappa is an important high-frequency site parameter for rock sites.Figure Atkinson , over a M, stress parameter), path effects (geometric spreading and anelastic attenuation), and site effects (near-surface amplification and kappa), without the need to repeat simulations. It can be calibrated to limited regional ground-motion observations to provide a complete and robust GMM that follows scaling constraints in magnitude–distance space as established by empirical GMMs in data-rich regions. As such, it forms a practical and effective bridge between empirical and simulation-based modeling approaches.The generic GMM model as presented in the foregoing is a useful way to encapsulate simple seismological models into a convenient functional form that facilitates the modeling of key source effects  and uncertainty regarding the true median values (epistemic uncertainty) (Bommer and Scherbaum τ) and within-event variability (φ). τ reflects the fact that some events are stronger than others due to their source attributes, such as a higher or lower stress parameter, whilst φ reflects deviations from the median attenuation curve within a single event. φ is sometimes further subdivided into components expressing the within-event variability component for a single station (φSS) and the site-to-site variability (φS2S); φS2S represents the systematic deviation of the ground motion at a specific site from the median event-corrected ground motion predicted by the GMM . The reader is referred to Al Atik et al.  – ln(predicted) amplitudes]. The total sigma can be partitioned into components that express between-event variability  should be recorded at each station. This will suffice to obtain the linear amplification for each site; the nonlinear component is typically determined separately based on either an empirical or analytical model , recorded at distances to 500 km; this is significantly lower than the corresponding values for GMMs that do not model amplification on a site-specific basis  for each site, relative to the GMM. To ensure stable site-specific amplifications, a minimum of five regional events —which in ENA is a more important site variable than VS30—then we reduce the sigma by about 0.1 ln units at high frequencies. Most of this reduction comes from the φS2S component for soil sites. If we consider only rock-like sites (those with VS30 > 1000 m/s), and model the site response at each specific station based on the seismographic data recorded at the site, the total sigma drops to values in the range of 0.5–0.6 ln units, or a factor of 1.7 about the median, again due largely to reduction of φS2S. Finally, if the GMM uses a magnitude scale based on high-frequency amplitudes , instead of moment magnitude, we can reduce the τ component at higher frequencies  is installed and operated over a period of years, and investment is also made in data analysis and targeted GMM development using the data. This has not been the approach taken in most parts of ENA to date. It should also be noted that, whilst reduction of sigma will lead to reductions in computed hazard for rock sites, and other sites with relatively low site amplification, the computed hazard may increase at sites where it is currently being underestimated by non-site-specific approaches.Epistemic uncertainty in median GMMs has often been modeled using alternative equations , with model weights in a PSHA logic tree being used to represent the relative confidence in each alternative. However, this is not necessarily the best way to model epistemic uncertainty in GMMs  could be largely modeled as epistemic, reflecting uncertainty in the source characteristics that are expected to be realized in future events through the alternative GMM branches. In this case, the aleatory uncertainty would represent just the variability of observations about a median event-specific prediction equation for a single station . Considering the variability components of Fig. To some extent, the details of the method used to represent epistemic uncertainty may not be of critical importance. Sensitivity tests indicate that it is the range covered by the GMM models and their relative weights that are important to the PSHA results, not the mechanics of how they are treated (Atkinson and Adams φSS. This approach would allow PSHA to be more site specific in its application of epistemic and aleatory uncertainties.Such an approach presupposes that we can model epistemic uncertainty in ground motions from future events through definition of the distribution of earthquake source, path, and site parameters of the GMM model. The generic GMM approach outlined here is a practical way to implement such distributions in Monte Carlo PSHA software, in which simulated earthquake catalogs and their generated ground motions at a site are used to calculate the ground-motion distribution at a site Musson . For eacThis paper has dealt with the use of conventional GMMs to define ground motion and its epistemic and aleatory uncertainty, within the context of contemporary PSHA methodology. In the longer term, a truly site-specific PHSA would be based on simulations that fully consider the source, path, and site attributes that govern ground motions for all potential future earthquake scenarios, eliminating the need for GMMs altogether (e.g., Atkinson


Among entire fractions, the chloroform soluble fraction showed excellent activity against α-glucosidase with % inhibition 90.8 with IC50160.2 ± 1.85 μg and daturaolone (1) with 98.7% inhibition with IC50840.4 ± 1.74 μM, respectively. Similarly, extract and daturaolone (1) also exhibited significant activity against the β-secretase enzyme (BACE1) with % activities 88.27 and 95.19 and with IC50 values 304.21 ± 2.98 μg and 260.70 ± 1.87 μM, respectively, as compared to the standard inhibitor -amyloid-β/A4 precursor protein 770 fragments 662-675) with % activity 94.21 and IC50 value 289.24 ± 1.60 μM. This finding encourages and opens a new window for further detail phytochemical investigation on D. metel in order to isolate novel compounds with promising enzyme inhibitory potential.This study deals with  Datura metel belongs to family Solanaceae which grows throughout the year and is normally known as devil's trumpet +440.3710 found 440.3700. The spectroscopic data were compared to the literature and found identical with literature reported data [1) was confirmed by X-ray crystallography data  were screened for α-glucosidase inhibitory activity; the tested extracts, fraction, and isolated daturaolone (1) were found active on α-glucosidase.The crude methanolic extract and various fractions and also daturaolone  are potent antidiabetic agents. Several α-glucosidase inhibitors including voglibose and acarbose obtained from a natural source can sufficiently control blood glucose levels and have been used clinically in the cure of diabetes mellitus. Among several natural products, only a few α-glucosidase inhibitors are industrially available [Diabetes mellitus is a dominant disease in developed as well as developing countries; it is a prominent metabolic turmoil or in other words an abnormal postprandial increment of blood glucose level. The controller of postprandial hyperglycemia is accepted to be imperative in the cure of diabetes mellitus. vailable –24.50 value 160.2 ± 1.85 μM followed by ethyl acetate fraction 82.7% with IC50 of 86.2 ± 2.00. The methanolic extract also showed promising activity with % inhibition of 80.2% with IC50 value 110.6 ± 2.66 μg, respectively. The daturaolone isolated from most active frication showed potent activity 98.7% with IC50 value 830.4 ± 2.01 μM as compared to the standard drug having 90.2% inhibition with IC50 value 840.4 ± 1.74 μM  is the 1st step in a pathway leading to the formation of Aβ (amyloid); therefore, it is a major target for the development of a drug for the cure of Alzheimer's disease (AD). The β-secretase enzyme (BACE1) is important for the production of ab. It has been documented that BACE1 level is high in Alzheimer's disease (AD); therefore, the study BACE1 inhibition is helpful in developing new Alzheimer's disease (AD) therapies [1) were screened β-secretase inhibitory potency. Among tested fractions, the chloroform fractions showed excellent activity against β-secretes (BACE1) enzyme with % inhibition 88.27% with IC50 value of 304.21 ± 2.98 μg followed by methanolic extract 78.42% with IC50246.51 ± 1.98 μg, respectively. The ethyl acetate fractions also showed good inhibition with 67.98% activity with IC50 value 87.93 ± 2.87 μM, while n-hexane fraction was found the least active (1) was found significantly active against BACE1 enzyme with % inhibition 95.19 and IC50 value 260.70 ± 1.87 μM as compared to standard having % activity 94.21 and IC50 value 289.24 ± 1.60 μM.herapies . Keepingt active . The dat1) toward α-glucosidase and β-secretase suggests that the bioactivity is due to the keto moiety at position C-3 and a hydroxyl group at position C-6 which may bind with residues in the active sites of selected enzymes.The bioactivity of daturaolone (1) exhibited excellently α-glucosidase inhibition activity. Similarly, the extract/fractions and isolated daturaolone (1) showed potent and β-secretase activity. In this study, the extracts and natural daturaolone (1) inhibit α-glucosidase and β-secretase significantly and can be used as an excellent template compound for the new drug used in diabetes and AD.Phytochemicals can provide an excellent pharmacophore template for novel drug discovery used for the cure of various diseases. The crude extract/fraction and daturaolone (