The highest numbers of these pelagic diatoms were observed in Barrow Strait. Spatial patterns of fatty acid profiles and stable isotopes indicated two source populations for C. centralis: a western origin with low light conditions and high nutrients, and a northern origin with lower nutrient levels and higher irradiances. Fatty acid analysis revealed that pelagic diatoms had significantly higher levels of polyunsaturated fatty acids (mean ± SD: 50.3±8.9%) compared to ice-associated producers (30.6±10.3%) in our study area. In particular, C. centralis had significantly greater proportions of the long chain omega-3 fatty acid, eicosapentaenoic acid (EPA), than ice algae . Thus, C. centralis represented a significantly higher quality food source for local herbivores than ice algae, although feeding experiments did not show clear evidence of copepod grazing on C. centralis. Our results suggest that C. centralis are able to initiate growth under pack ice in this area and provide further evidence that biological productivity in ice-covered seas may be substantially higher than previously recognized.Pelagic primary production in Arctic seas has traditionally been viewed as biologically insignificant until after the ice breakup. There is growing evidence however, that under-ice blooms of pelagic phytoplankton may be a recurrent occurrence. During the springs of 2011 and 2012, we found substantial numbers (201–5713 cells m Arctic marine food webs are classically viewed as being supported by two temporally and ecologically distinct types of primary production: ice-associated algae and pelagic open water phytoplankton Chaetoceros spp. and Thalassiosira spp. that follow ice melt/breakup Contrary to the classical view, increasing evidence suggests that pelagic blooms occur under sea ice throughout the Arctic, sometimes hundreds of kilometers from open water Coscinodiscus centralis Ehrenberg is a large centric diatom with a mean cell size of 180–200 µm in diameter, although cell size ranges from 100 to 370 µm in diameter C. centralis is reported as cosmopolitan, found in every marine biogeographic region C. centralis has been found under the ice at the North Pole in the late summer and early fall Coscinodiscus are often found at the base of the euphotic zone, and there is evidence to suggest that they are adapted to very low light irradiances C. centralis is poorly studied and little is known about its growth requirements or general population dynamics Chaetoceros spp. or Thalassiosira spp. and were linked to increasing light levels due to melt ponds C. centralis under sea ice in the Canadian High Arctic before the onset of melt, challenging our current understanding of Arctic marine food web dynamics. We examined the environmental conditions associated with the presence of C. centralis and used fatty acid and stable isotope analyses to provide insights into:Most observations of phytoplankton growth under ice have been of assemblages dominated by The origins of these large-celled diatoms.C. centralis to sustain growth under sea ice.The potential for C. centralis as a food source for pelagic zooplankton and their potential impact on Arctic food webs.The role of The permit for this study was received from the Nunavut Impact Review Board (NRIB). We confirm that the study did not involve any endangered or protected species. This study did not involve vertebrates. Maps for this manuscript were generated using Ocean Data View v 4.6.1 −1 in the spring with speeds increasing up to 60 cm s−1 during the tidal flux Sampling was conducted in a region of first-year sea ice in the Canadian High Arctic, Nunavut, Canada. A total of 47 stations were visited between 1–18 May, corresponding to the ice algal bloom period Snow depth and ice thickness were measured at each station. Measurements of photosynthetically active radiation  were obtained under the sea ice using a LICOR Li-192S underwater quantum sensor held at the ice-water interface 1m from the hole using a horizontal under-ice arm. All PAR readings were conducted under undisturbed snow cover.C. centralis were collected using a zooplankton net  fitted with a flowmeter  to allow calculation of volume sampled. Three vertical hauls, from 5 m above bottom to the surface, up to a maximum depth of 150 m, were obtained at each station and pooled. Material was then separated by size using 500 µm and 250 µm sieves. Cells of C. centralis were isolated from the 250 µm size fraction by letting 500 ml of sample settle for 25 min. This procedure was repeated three times and any remaining zooplankters were removed by handpicking using a Pasteur pipette and a dissecting microscope. These samples, composed predominately of C. centralis cells, were then filtered onto pre-combusted Whatman GF/F filters, which were immediately placed into chloroform  and stored at −20°C until analysis. Isolated samples of C. centralis could not be obtained for fatty acid analysis in 2011; rather, qualitative estimates of abundance were made as explained below.Bottom ice and pelagic samples were collected at each station. Sea-ice samples were collected using a manual ice corer . The bottom 3 cm of each core was cut and put in a sterile bag  and melted overnight with the addition of 500 ml filtered seawater  to avoid osmotic stress C. centralis in 2011 were made on a 0–3 scale. A value of 3 characterized a sample that, if shaken in a scintillation vial, was completely opaque, a value of 2 indicated a medium level of opacity when the vial was shaken , a value of 1 meant that few specimens were present, and 0 indicated no diatoms were present. In 2012 quantitative subsamples were taken from each site, preserved in Lugol's solution, and counted under a dissecting microscope.Qualitative abundance estimates of C. centralis could be a suitable food source for copepods, zooplankton, C. centralis and ice algae were collected at a single first-year sea ice station in Resolute Passage on 14 May 2011. Calanoid copepods larger than 500 µm in length were retained from pooled vertical hauls taken from 30 m depth to the surface. The copepods were placed in 12 experimental enclosures  containing 450 ml of FSW, which were placed in a seawater bath at 0°C with light levels of 2.5–3.5 µE m−2 s−1 during 24 h. The enclosures were randomized into two treatments, and fed either ice algae or C. centralis. Two controls, with ice diatoms and C. centralis respectively, were incubated with no copepods. After five days each bottle was sieved through 500 µm mesh and copepods were retained for fatty acid analysis. The bottle content from which copepods had been removed was then filtered  for chlorophyll a (chl a) analysis. Samples were extracted in 90% acetone during 24 h at 4°C in the dark. Fluorescence was read on a Turner Designs 10 AU fluorometer before and after acidification, according to Parsons et al. To test whether C. centralis were extracted using a modified Folch procedure in situ method similar to Park and Goins Lipids were extracted using two different methods. Lipids from C. centralis were freeze-dried at −40°C for 48 h, after lipids were extracted in 2∶1 chloroform and methanol 13C and δ15N stable isotopes at the Great Lakes Institute for Environmental Research   interfaced to an Elemental Analyzer via the Conflo III. Ice algae samples were analyzed at the Stable Isotopes in Nature Laboratory (SINLAB), Canadian Rivers Institute, University of New Brunswick, Canada. Isotopic signatures are expressed as a deviation (δX =  [(Rsample/Rstandard) −1] ×1,000) from international standards calibrated against Vienna-PeeDee Belemnite (VPDB) for carbon, and atmospheric N2 (AIR) for nitrogen. Canada) . Ice algC. centralis based on 13 fatty acids * 100 = 1/16*100 = 6.25% C. centralis15N between clusters. Regressions were used to assess relationships between stable isotopes, environmental variables and lipid abundances. Unless otherwise noted, all fatty acids and stable isotope values are reported as mean ± one standard deviation.Statistical analyses were done using R statistical software −2 s−1 in 2011 and 1056.1±181.9 µE m−2 s−1 in 2012 while under-ice PAR measured at the stations was lower and less variable in 2011 (mean  = 4.90±3.03 µE m−2 s−1) than in 2012 (mean  = 9.36±8.76 µE m−2 s−1). Pennate diatoms dominated the species composition of the sea ice community with Nitzschia frigida being the most abundant.Mean ice thickness in 2011 was 133.0±19.5 cm (range  = 96.2 to 167.6 cm). Near the end of the study there was open water in Barrow Strait east of Cornwallis Island and several polynyas were present in the northern portion of Wellington Channel. In 2012, the ice was thicker, ranging from 121.4 to 235.0 cm (mean 163±35 cm), and there were no signs of melting or open water in our study area. Mean snow depth at the stations was higher in 2011  compared to 2012 . Downwelling PAR was similar both years, mean  = 1010.0 ±285.3 µE m15N isotopic signatures for C. centralis showed a broader range of values . C. centralis δ13C values were significantly more depleted and less variable  relative to ice-associated δ13C values . Pelagic samples had significantly higher abundances of both EPA  and total PUFA  than ice algal communities (versus 26.4±6.3%) whereas C. centralis had higher levels of all 18-carbon fatty acids with the exception of 18∶3n-6 , despite the C. centralis sample being closer to cluster 2 based on the first two principal component axes (Cluster 2 (red) in the HCPC comprised 12 pelagic samples, with samples in this cluster further from each other in Euclidean distance compared to cluster 3 (green), which contained 11 samples of e sample . Differee sample . Sites oent axes .C. centralis correlated significantly with δ15N values. Pelagic samples from Barrow Strait (cluster 3) showed significantly lower δ15N values than those from Wellington Channel (cluster 2) . Fatty acid concentrations of C. centralis were negatively correlated with δ15N . PUFA concentrations were also negatively correlated with δ15N (2 = 0.38 p = 0.001). Three stations in cluster 3 had high values of δ15N; two of these were located on the eastern half of the McDougall Sound transect while the other was from the north-west in Wellington Channel . Qualitative patterns observed in 2011 closely followed the quantitative cell counts obtained in 2012  occurred in the north of Wellington Channel , and EPA, which decreased in C. centralis fed copepods . Chlorophyll a concentrations were significantly higher in bottles that had both copepods and C. centralis than control bottles that contained only C. centralis . Conversely, there was a no significant difference in chl a concentration for ice algae replicates that were incubated with copepods relative to copepod-free controls .No significant differences were observed between the fatty acid profiles of copepods fed C. centralis under sea ice in the Canadian High Arctic contributes to this evidence and supports indications that biological productivity in this region may be underestimated C. centralis may be initiating growth both in polynyas and under pack ice and may represent a source of high quality omega-3 fatty acids for Arctic food webs. Despite this, the large cell sizes observed may preclude the consumption of C. centralis by herbivorous pelagic zooplankton, specifically calanoid copepods.Emerging evidence of substantial under-ice pelagic production has the potential to fundamentally change our understanding of Arctic marine food webs C. centralis had significantly higher levels of PUFA, specifically the essential fatty acid EPA, compared to ice algae. Although PUFA levels in C. centralis varied, presumably depending on the origin of the cells (32–62%)  .B, PUFA 13C values for C. centralis samples were more depleted and less variable than those of ice algae, as expected due to the higher levels of dissolved inorganic carbon available in the water column compared to the ice/water interface during growth  indicates that C. centralis cells were uniformly distributed throughout the water column. Barrow Strait (cluster 3), an area of mixed ice cover and moving pack ice, showed higher concentrations of fatty acids  and cell abundances than McDougall Sound and Wellington Channel (cluster 2)   would therefore be located north of Cornwallis Island and had much lower associated abundances.During the two years of our study, cells of en years . The clo mg m−3) . Based oC. centralis cells can be made based on the combination of fatty acid and stable isotope values. In both years under-ice PAR levels were high enough to support photosynthesis (2–9 µE m−2 s−1) 15N and high PUFA  advected from Viscount Melville Sound had high nitrogen availability and low light conditions. Furthermore, the similarity between individuals within the cluster indicates that conditions were relatively consistent during growth  of C. centralis could preclude nauplii or juvenile copepods from effectively grazing on these cells, which may explain the lack of any observable grazing in our feeding experiment. Despite C. centralis and ice algae having significantly different fatty acid profiles, no difference was observable in copepods fed either of the treatments. Based on turnover rates of lipids in calanoids, a dietary switch should have been evident via modification in fatty acids within the duration of the experiment if grazing of C. centralis did take place C. centralis cells still appear to be healthy at the end of the experiment but chl a concentrations significantly increased in bottles containing zooplankton. As irradiance levels were between 2.5 and 3.5 µE m−2 s−1 during the incubation, this provides a direct observation of shade adaptation in C. centralis collected under the ice, a condition necessary for a life history strategy alternate to spring bloom conditions The large cell size of C. centralis cells were present in the Canadian High Arctic under sea ice concurrently with the ice algal bloom, and therefore spring primary production is likely being underestimated in this region. Fatty acid and stable isotope signatures indicated that growth was likely occurring primarily in polynyas and under moving pack ice where light conditions are more favorable than under landfast ice. Given the ability of C. centralis to grow under a variety of conditions C. centralis occurs regularly in early spring in this area and may have been overlooked in the past. The presence of C. centralis under sea ice represents a source of high quality PUFA during a period when most other pelagic producers are limited; however it is not clear if they can be effectively grazed by copepods. Our results support growing evidence indicating that pelagic primary production may play a biologically important role during periods of ice cover and highlight the need for further investigation of under-ice processes and their impacts on Arctic marine food webs.Our results showed that healthy Table S1C. centralis cell abundances, sampling locations, stable isotope values (δ15N and δ13C) and fatty acids .(XLSX)Click here for additional data file.


O-GlcNAc cycling contributes to the etiology of major human diseases of aging, such as diabetes, cancer, and neurodegeneration. Recent advances in tools to study O-GlcNAcylation at the individual site level and specific inhibitors of O-GlcNAc cycling have allowed more rapid progress toward elucidating the specific functions of O-GlcNAcylation in essential cellular processes.Even though the dynamic modification of polypeptides by the monosaccharide, O-linked N-acetylglucosamine (O-GlcNAcylation) was discovered over 30 years ago, its physiological significance as a major nutrient sensor that regulates myriad cellular processes has only recently been more widely appreciated. O-GlcNAcylation, either on its own or by its interplay with other post-translational modifications, such as phosphorylation, ubiquitination, and others, modulates the activities of signaling proteins, regulates most components of the transcription machinery, affects cell cycle progression and regulates the targeting/turnover or functions of myriad other regulatory proteins, in response to nutrients. Acute increases in O-GlcNAcylation protect cells from stress-induced injury, while chronic deregulation of  N-acetylglucosamine moieties in cells of the murine immune system , based upon tritiated UDP-GlcNAc as the donor and synthetic peptide acceptors, was developed and OGT activity was identified and characterized ? Clearly, several different mechanisms are involved. In vitro, OGT has remarkable specificity for peptide subtrates, which appears to change with UDP-GlcNAc concentrations  What are the specific roles of O-GlcNAcylation in nutrient regulation of transcription? While it is now clear that O-GlcNAcylation is fundamentally important in nearly every aspect of transcription, we currently know almost nothing with respect to its protein-specific or site-specific roles on individual transcription regulatory proteins. This area will remain an enormous challenge for some time to come.Many important questions remain with respect to O-GlcNAcylation. (1) How does trations . Most imtrations , 45, 80.O-GlcNAc antibodies; (2) A molecular biology approach to either mimic O-GlcNAcylation or to generate site-specific O-GlcNAcylation on proteins; (3) Methods are need that can raise or lower O-GlcNAcylation on individual proteins or at individual sites to evaluate functions. Unfortunately, current methods either based upon inhibitors or genetic approaches to alter O-GlcNAcylation, all act globally. (5) There continues to be a need for better methods to both detect and site-map O-GlcNAc on proteins. The challenges in this field are large but so is the pay off for our understanding of cellular physiology and chronic disease.Finally, while the tools to study O-GlcNAcylation have advanced substantially in the last three decades, there remains an acute need to develop better methods and approaches that can be applied by biologists. These include: (1) The development of many site-specific The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose  and starch  as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.Human pancreatic  It is often accompanied with disturbances of carbohydrate, fat and protein metabolism and severe diabetic complications such as retinopathy, neuropathy, nephropathy, cardiovascular complications and ulceration –4. WHO p by 2030 –7.An eff by 2030 . This su by 2030 ,11. By iA useful model system to study the inhibition of secreted HPA is the rat pancreatic acinarAR42J cell line, derived from azaserine-induced malignant nodules from rat pancreas. The cell line is an amphicrine model with exocrine and endocrine functions and is characterized by the presence of digestive enzyme-containing dense core vesicles . InducinThe currently available treatments have side effects such as hypoglycemia, weight gain and other complications which necessitate the need for development of new antidiabetic targets and therapies for glycemic control –16. The wonder tree’ Neem , native to Indian subcontinent but cultivated throughout the tropics is well-known for its diverse medicinal uses for more than 2000 years. Earlier studies have shown that the aqueous leaf extract of Neem resulted in hypoglycemia in normal rats and lowered blood sugar level in streptozotocin induced diabetic rats [A. indica leaves, and swietenine from Swietenia macrophylla have been reported to exhibit α-amylase inhibitory activity in streptozotocin induced diabetes in mice [in vitro cytotoxicity on AR42J cell line (2) to reveal their mode of action and underlying molecular interactions with respect to molecular docking, inhibition kinetics and ligand binding.The ‘tic rats . It is otic rats ,23. Limotic rats ,24.Very  in mice –27. On t in mice ,29. The Human pancreatic α-amylase (HPA), maltopentaose, were purchased from Sigma Aldrich, USA. 3,5-dinitrosalicylicacid (DNSA), Ham’s F-12K with L-glutamine culture media, fetal bovine serum, antibiotics streptomycin, penicillin, Trypsin -2,5-diphenyltetrazoliumbromide (MTT) were obtained from HiMedia Laboratories, Mumbai, India. Porcine pancreatic α-amylase (PPA) was procured from SRL, Mumbai, India. AR42J rat pancreatic cell line was from ATCC-CRL142. All other chemicals and solvents from local manufacturer were of AR or HPLC grade.Rt) 28.3 min), azadirachtin B (Rt 29.7 min), nimbin (Rt 47.9 min), salannin (Rt 52.2 min), azadiradione (Rt 46.0 min), azadirone (Rt 16.2 min) and epoxyazadiradione (Rt 56.2 min), respectively as mentioned in our earlier studies [1H, 13C, DEPT-135) studies.The chemical structure of neem limonoids isolated and characterized from Neem fruits (fruit coat/seed) are presented in  studies ,28,29. SThe inhibition assay was performed using the chromogenic DNSA method ,31. PPA In brief, the total assay mixture consisting of 0.02 M sodium phosphate buffer, pH6.9, (containing 6 mM NaCl), 0.2–0.25 U of PPA and inhibitors (13.8–214.3 μM) in DMSO was incubated at 37°C for 10 min. After pre-incubation, the substrate starch  solution was added to the reaction mixture and further incubated for 15 min. The control PPA (0.21 U) without any inhibitor represented 100% enzyme activity. One unit of enzyme activity is defined as the amount of enzyme required to release one micromole of maltose from starch/maltopentaose per min under the assay conditions. The other quantifiers calculated were:50 values were determined from plots of percent inhibition versus inhibitor concentration and calculated by logarithmic regression analysis. To eliminate the absorbance produced by the compound, appropriate controls were included. The known PPA/HPA inhibitor acarbose was used as a positive control at a concentration range of 10–50 μM. The compounds exhibiting PPA inhibition were subjected to docking simulations and for studies on AR42J cell line.The IC-1 streptomycin, 100 unitsml-1 penicillin) at 37°C under a humidified condition of 95% air and 5% CO2. Cells were seeded at 2×104 cells well-1 onto 96 wells culture dish. After overnight attachment of the cells, the medium was replaced with the medium containing 10 nM dexamethasone, and the compounds at a concentration of 1.7–36.3 μM in 1% DMSO for 24 h [-1) added and incubated for 3 h at 37°C. Metabolically active cells reduce MTT to insoluble purple formazan dye crystals, which were dissolved by addition of 300 μl DMSO solution at the end of the incubation period and quantitated at 550 nm [AR42J rat pancreatic acinar cells were used to screen the compounds exhibiting PPA inhibition for cytotoxicity and bioactivity. The cell lines were cultured in Ham’s F-12K with L-glutamine culture media supplemented with nonessential amino acids, sodium pyruvate, 7.5% sodium bicarbonate, 20% fetal bovine serum and antibiotics (100 μgmlfor 24 h . Approprt 550 nm . In ordeDocking simulations of inhibitors with HPA (PDB ID: 1HNY), were performed using AutoDockVina , 35. StaThe lead inhibitors azadiradione and gedunin, were subjected to HPA inhibition at concentrations exhibiting minimum cytotoxicity and maximal bioactivity. Inhibition assay was performed similar to that of PPA with 0.2–0.25 U of HPA and individual inhibitors azadiradione (22.2–133.3 μM), gedunin (20.7–124.3 μM). The control HPA (0.25 U) without inhibitors represented 100% enzyme activity. In order to demonstrate the stoichiometry of HPA inhibition, the enzyme (14.85μM) in buffer was added to a solution of varying concentrations of azadiradione (22.2–133.3 μM) and gedunin (20.7–124.3 μM), incubated for 10 min and assayed as mentioned above and percent inhibition determined.-1) and maltopentaose (0.05–0.4mM) were incubated with inhibitors-HPA for 10 and 2.5 min, respectively and the residual enzyme activity determined by DNSA method for starch and Nelson-Somogyi’s method for maltopentaose [Ki) were determined on the basis of mode of inhibition.The mode of inhibition of HPA by the lead inhibitors azadiradione and gedunin were determined using Michaelis-Menton and Lineweaver-Burk equations. Starch (1–5mg mlpentaose .The inhiLigand binding studies using fluorescence measurements of HPA were carried as described in our earlier report . BrieflyKa), dissociation constant (Kd) and Gibbs free energy (ΔG°) of binding for azadiradione and gedunin were determined at various temperatures 25°C to 60°C by fluorescence quenching studies.The thermodynamic parameters, association constants  and far–uv (195 to 250 nm) regions. The quartz cuvette (0.1-cm) contained 50–100 μg of HPA in 0.02 M sodium phosphate buffer (pH 6.9). For ligand binding analysis, ligands at the concentration exhibiting maximum bioactivity in the cell lines was pre-incubated along with HPA for 10 min and CD scans recorded.R factor of 17% showed an electron density pattern corresponding to the binding of oligosaccharides at the active site and at three other surface binding sites. Blind docking, encompassing the PPA-oligosaccharide complex with the grid box, parameters (92x126x120 points), and grid box center set at x = 33.255, y = 30.824, and z = 14.790 with the grid box spacing of 0.5 Å for both azadiradione and gedunin were performed individually. The interactions of inhibitor-PPA-oligosaccharide were analyzed and visualized in Discovery Studio 4.0 client [Docking simulations of the ligand to the enzyme-substrate  complex were performed to reveal the binding of limonoids in presence of substrate using Autodock Vina. The structural X-ray map of a PPA crystal soaked (and flash-frozen) with maltopentaose substrate (PDB: 1UA3) was taken as the enzyme-substrate complex  to dock 0 client .Each experiment was repeated as 3 independent sets with each set in triplicates. Results are expressed as means S.E.M. with number of observations (n). The best-fit values were achieved by applying either linear fit or non-linear least square regression using the software, Microcal Origin 6.0 . Data was analyzed by student’s t-test using SPSS statistical package SPSS 17.0.Differences were considered statistically significant for p < 0.05.A. indica were screened for PPA inhibition. Limonoids azadiradione, epoxyazadiradione and gedunin exhibited PPA inhibition with IC50 values of 138.4, 100.2, and 72.2 μM, respectively. No inhibition was exhibited by other six limonoids viz., azadirachtin A, azadirachtin B, salannin, nimbin, azadirone and 17β-hydroxyazadiradione.Porcine pancreatic α-amylase (PPA) an enzyme having a high degree of sequence, structural and functional similarity with HPA as mentioned earlier was used for preliminary screening of compounds possessing α-amylase inhibitory property ,32,33. A50 values of 11.1, 13.4 and 4.1 μM for azadiradione, gedunin and epoxyazadiradione, respectively while exhibiting amylase inhibition of 70.1, 69.2 and 53.2%. At concentrations of 3.5, 1.7 and 3.3 μM, lower cell death of 8.6, 2.7 and 8.6% with reasonably high pancreatic amylase inhibition of 41.8, 31.8 and 53.4%was seen for azadiradione, epoxyazadiradione and gedunin, respectively (Cytotoxicity and bioactivity of azadiradione (3.5–17.7 μM), epoxyazadiradione (1.7–8.5 μM) and gedunin (3.3–16.5 μM) on the amylase secretory cell line AR42J was carried out. These compounds were found to be cytotoxic at ICectively . The bioectively .In silico screening of Neem limonoids azadiradione, epoxyazadiradione and gedunin docked onto HPA was carried out with the validated docking parameters as reported in our earlier study [-1) for azadiradione, epoxyazadiradione and gedunin respectively. The interactions with azadiradione include pi-alkyl interaction between, Trp 58, Tyr 62 with C29 of A ring, and Trp 59 with C28 with bond lengths of 4.83, 4.94, and 4.84 Å, respectively. Conventional hydrogen bonding with a bond length of 3.12 Å was observed between oxygen of acetyl group at C7 position and His 305. The oxygen at C3 keto was observed to have a conventional hydrogen bonding with H2O 641 with a bond length of 3.2 Å. Epoxyazadiradione forms pi-alkyl interaction between, Trp 58 with C29 of A ring with a bond distance of 4.98 Å and a conventional hydrogen bond between C3 keto and H2O 641 with a bond distance of 3.28 Å. The principle interaction of gedunin with HPA involves pi-alkyl interaction between, Trp 58 and C29 methyl of A ring with a bond length of 5.30 Å. The five membered furan ring is also involved in the pi-alkyl interaction with Ile 235 and bond length 5.04 Å. Conventional hydrogen bond with a bond length of 3.19, 3.36, 3.08, 3.19, 3.22 Å, is observed between (C3 keto oxygen and H2O 641), (C3 keto oxygen and H2O 729), (acetoxy oxygen at C7 and His 305), (oxygen of the furan ring and Lys 200) and (oxygen of furan ring with H2O 650) respectively.er study . Initial50 value of (74.17 and 68.38 μM) respectively. Acarbose used as a positive control, showed an IC50 value of 15 μM against HPA. The stiochiometry of inactivation of HPA with azadiradione and gedunin is shown in Ki determined using secondary plots suggested an apparent Ki′ of 75.8 and 37.4 μM for starch as substrate and Ki of 42.2 and 18.6 μM with maltopentaose for azadiradione and gedunin, respectively.Based on their lower cytotoxicity and higher bioactivity values, the lead molecules azadiradione and gedunin were carried forward for further studies. A concentration dependent inhibition by azadiradione (22.2–133.1 μM) and gedunin (20.7–124.3 μM) was observed against HPA activity. As shown in ΔF represents the decrease in fluorescence intensity relative to the fluorescence intensity of the free enzyme on ligand binding. The unbound HPA (16.6 μg) with activity of 0.25 U ml-1exhibited a fluorescence intensity of 193 a.u. at λmax emission of 350 nm. Titration of this enzyme with azadiradione (3.9–118 μM) resulted in quenching of the intrinsic fluorescence to 128 a.u. with maximal quenching of 33% (Kd and Ka) were performed by linear as well as non-linear regression analysis [2 values of 0.95 and 0.975, respectively.Ligand binding studies with fluorescence and CD spectroscopy of azadiradione and gedunin were performed to gain an insight in the mechanism of ligand interaction with the protein. A concentration dependent decrease in fluorescence intensity at 350 nm on binding of azadiradione and gedunin to HPA was observed . ΔF reprg of 33%  and thatg of 33% . No emisanalysis , 41. FigThe ligand binding studies with CD spectroscopy showed the effect of azadiradione and gedunin binding on secondary and tertiary structure of HPA . The farKinetic studies suggested that azadiradione and gedunin exhibited a mixed mode of inhibition, which led us to dock these ligands on to the enzyme-substrate complex. As mentioned in methods, the PPA-oligosaccharide structural X-ray map obtained from a PPA crystal soaked (and flash-frozen) with a maltopentaose substrate, was taken for docking the limonoids. The PPA-oligosaccharide complex showed an electron density pattern consistent with the binding of maltotriose, the cryotrapped reaction intermediate, from subsites −3 to −1. Another maltotriose, interacting with the K 261 of PPA, protrudes into the solvent indicating the site to be a part of the starch-binding site while two other surface sites were involved in binding of maltose and glucopyranose .-1, respectively. The interactions with azadiradione include alkyl-alkyl interaction between Ala 307-C19, Leu 237-C19, Leu 237-C30, Leu 237-aromatic centre of C ring with bond lengths of 4.34, 4.89, 4.41 and 4.76 Å, respectively. Conventional water interactions with a bond length of 3.48 and 3.67 Å was observed between C23 of the furan ring with H2O 690 and H2O 789, respectively. Gedunin forms pi-alkyl interactions between, Tyr 151-C28 of A ring and Leu 237-aromatic ring of furan with a bond length of 4.55 and 4.83 Å, respectively. Alkyl-alkyl interactions are observed between Leu 237-C18 and Leu 237-aromatic centre of C ring with a bond distance of 4.71 and 5.39 Å, respectively. Water conventional interaction was observed H2O 1096-keto oxygen of the furan ring, H2O 842-C23 of furan ring, H2O 1141-C3 keto oxygen of A ring, H2O 1145-C3 keto oxygen of A ring and H2O 871-keto group at C13 position with the bond distances of 3.75, 3.34, 3.24, 3.28 and 3.25 Å, respectively , dissociation constant (Kd) and Gibbs free energy (ΔG°) of binding were calculated for all six temperatures and provided in . The free energy ΔG° values calculated from the binding constants are negative, with a maximum ΔG° of (-21.25 and -21.16 kJ mol-1) for azadiradione and gedunin indicating favorable interactions occurring between HPA and the lead inhibitors. This is in near accordance with the free energy of binding from the docking simulation i.e.  for azadiradione and gedunin respectively. The enthalpy (ΔHvH) and entropy (-ΔS) were determined by linear van’t Hoff plot of Ka as a function of temperature with R2 of 0.9952 (ΔHvH) -41.86 and -139.9 kJ mol-1 while the entropy (-ΔS) 70.09 and 397.84 J mol-1K-1were determined for the temperature (298–333 K) for azadiradione and gedunin, respectively suggesting that binding may be both enthalpically and entropically driven.The association constants , mycaminose (Syzium cumini), kaempferol-3-neohesperidoside (Bauhinia forficate), 2’,4’-dihydroxy-4-methoxydihydrochalcone, 4,5-di-O-caffeoylquinic acid (Artemisia dracunculus L), quercetin 3-, rutin, isoquercitrin , caffeic acid, p-coumaric acid (Ocimum sanctum), curcumin, demethoxycurcumin bisdemethoxycurcumin (Curcuma longa), diosgenin from Dioscorea bulbifera have been isolated and characterized against various antidiabetic targets [A. indica have been used as traditional medicines in ayurveda and day-to-day household remedies such as anti-inflammatory, hypoglycemic, anti-malarial, anti-ulcer, anti-microbial, anti-carcinogenic, antioxidant and so on. However, very few reports are available on the individual secondary metabolites or their mode of action for hypoglycemic property [Natural products have been known to complement current therapies with lesser side effects to treat DM. Various lead metabolites from hypoglycemic plants such as galegine ( targets –47. Inteproperty –53.This 50 72.2 μM) contains a six-membered lactone D-ring with 14,15β epoxy moiety. When this lactone was converted to a five-membered cyclic ketone with an intact epoxy ring, as observed in epoxyazadiradione, the activity was lowered (IC50 100.2 μM). Further replacement of the epoxy ring with unsaturation as in azadiradione, further reduced the inhibitory potency more extensively (IC50 138.4 μM). In absence of 16-keto and 14,15β epoxy group as in azadirone, no activity was observed. Interestingly, insertion of 17β-hydroxyl group as seen in 17β-hydroxyazadiradione resulted in complete loss of activity vis-à-vis azadiradione, which is structurally similar to 17β-hydroxyazadiradione except for the presence of 17β-hydroxyl group. It has also been reported that the tetranortriterpenoid meliacinolin and azadirachtolide from A. indica exhibited an IC50 of 46.74 μgml-1 (91.6 μM) and 55.8 μgml-1 (94.09 μM) with PPA [Careful comparison of α-amylase inhibitory potency of limonoids azadirachtin A, azadirachtin B, salannin, nimbin, azadirone, azadiradione, epoxyazadiradione, gedunin and 17β-hydroxyazadiradione revealed that bioactivity was strictly restricted to the basic limonoid skeleton and no activity was observed with limonoids possessing C-seco type architecture. Variation in activity was observed within the basic limonoid family depending on the presence of skeletal functional groups. These five limonoids contained α, β-unsaturated C3 ketone and 4,4-dimethyl moiety in A-ring as a common feature. They also possess critical structural variations in the D-ring, which may play a pivotal role in α-amylase inhibition. It can be seen that gedunin, showing the highest potency against PPA . This suggests that any substitution in the A ring may interfere in the limonoid binding to HPA. Further support for this can be seen with the limonoids azadirachtin A, azadirachtin B, salannin, nimbin,where the A ring has been substituted with bulky functional groups have no bioactivity. Moreover, the limonoids bind near the active pocket without involvement of active site residues in their binding to free HPA, whereas they bind to a site distant from the active site pocket, when docked to PPA-oligosaccharide complex. In mixed inhibition, as has been observed by kinetic and CD studies for HPA by limonoids, the inhibitor binds to a site other than the active site changing the enzyme conformation leading to a reduction in activity (Vmax). A reduction in HPA activity in presence of limonoids is observed from our experimental data wherein a decrease in Vmax and an increase Km has been shown. Also, CD studies suggests perturbations in the tertiary structure of HPA as seen by changes in environment of Tyr and Trp on binding of limonoids. In a docking study between transchalcone and PPA, Trp 59 and Tyr 62 were reported to be the important aromatic amino acid residues in pi-pi interactions involving the two aromatic rings of ligand and side chain [Protomelas virgatus methanol extract acrylic acid, 11-octadecenoic acid, 9–12 octadecodienoic acid, 6-oactadecynoic acid, hexadecanoic acid and phthalic acid involve interactions between ligand and Trp 59 / Try 62 suggesting these residues to be important residues in binding of inhibitors to PPA [Looking at the interactions between the limonoids and HPA from the docking simulations, it is clear that the A ring C3 keto oxygen and C29 methyl gains importance due to its interaction with Hde chain . Similars to PPA . In the 2 values for the one-site binding fit obtained in ligand binding studies by fluorescence suggest the one-site binding model is in agreement with the stiochiometry of inhibition. A similar stiochiometry has been observed for inactivation of HPA with bisdemethoxycurcumin [Phaseolus vulgaris) has also reported to form a 1:1 stoichiometric complex with PPA at pH 5.4 [Stiochiometry of 1:1 implied binding of one inhibitor molecule to one molecule of HPA. A high Rcurcumin  and PPA curcumin . The amyt pH 5.4 –59.Ki value of (22 μM) for 2-chloro-4-nitrophenyl-α-maltotrioside as substrate [A. indica leaf have been shown previously to exhibit mixed non competitive mode of inhibition with IC50 value of 9.15 mgml-1and 5.0 mgml-1[Phaseolus vulgaris is also reported to exhibit mixed inhibition with maltopentaose as substrate with a relatively high Ki of (5x104 μM) [Azadiradione and gedunin exhibited a mixed mode if inhibition as observed by kinetic experiments. The docking with free enzyme results suggests that both inhibitors likely bind near the active pocket. However, the active site residues were not involved in their binding. Moreover docking of azadiradione and gedunin to the PPA-oligosaccharide complex suggests that binding of the ligands occurs at the site distant from the active pocket. Thus mixed inhibition would be of relevance for the regulated activity of HPA in order to slowly release glucose and thereby control PPHG. With the reduction in the limonoids concentration, the HPA activity would be regenerated due to the non-covalent association as observed in docking simulation and the reversible equilibrium with the enzyme. Acarbose has been reported to exhibit noncompetitive mode of HPA inhibition with a ubstrate .Interest.0 mgml-1. α- Amylx104 μM) .The quenching of tryptophan fluorescence on titration with the ligand and the change in the CD spectrum of a protein in the near-UV spectral region (250–350 nm) on ligand binding are in accordance with the docking simulation results which shows the involvement of Trp 58, 59 and Tyr 62 in azadiradione binding to HPA and Trp 58 in gedunin binding to HPA. The significant change in the environment of tyrosine and phenylalanine in gedunin to HPA is also observed in the docking simulation, where Phe 256 is at a quenching distance of 9.33 Å. Tyr 62 and Tyr 151 present near the active pocket are quenched as the concentration of ligand increases on titration.Thus, the ligand binding studies by fluorescence, CD and docking simulation were found to be well in accordance and supportive to each other.ΔH and ΔS). ΔH <0 and ΔS <0, indicates the involvement of hydrogen bonds between the inhibitors and HPA, which is evidenced in the docking simulations too. The interaction between HPA and the limonoids is a spontaneous process with free energy decreasing.The types of main acting force between pharmaceutical molecule and protein could be judged or assessed according to the relative values change in enthalpy and entropy (A. indica could be justified by HPA inhibition as it could be one of the mechanisms of action. Moreover, this comprehensive study scientifically validates these natural products thereby enabling a better insight with respect to their structure- activity relationship. The study gains importance as these limonoids could be used to design better drug candidates in development of newer inhibitors of HPA for controlling starch digestion in order to reduce post-prandial hyperglycemia.In conclusion, results obtained from this study suggest, azadiradione and gedunin to be lead HPA inhibitory molecules. Thus, the hypoglycemic property exhibited by


Small ubiquitin-like modifier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity.IMPORTANCE Posttranslational modifications with small ubiquitin-like modifier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection. Intrinsic antiviral immunity is the first line of intracellular defense to viral infection. This defense is mediated by constitutively expressed cellular proteins that cooperatively act to restrict the progression of infection  pathway (–During herpes simplex virus 1 (HSV-1) infection, viral gene expression occurs in a tightly regulated temporal cascade consisting of immediate early (IE), early (E), and late (L) gene products. IE proteins play pivotal roles in modulating the intracellular environment in order to facilitate viral replication, including inactivation of host immunity that would otherwise restrict the progression of infection  of proteins with SUMO regulates numerous cellular processes, including multiple aspects associated with virus infection and host immunity , counteracts this host response by inducing the degradation of PML-NB constituent, and other SUMO-conjugated, proteins through a combination of SUMO-dependent and -independent targeting mechanisms  family. These ligases were initially characterized as negative regulators of innate immunity by virtue of their transrepression of key regulators in the interferon (IFN) pathway  supplemented with 10% fetal bovine serum , 100 U/ml of penicillin, and 100 μg/ml of streptomycin . HFt cells are immortalized human foreskin fibroblasts stably transduced with retrovirus that expresses the catalytic subunit of human telomerase, as described previously (Human osteosarcoma (U2OS) cells, human embryonic kidney (HEK-293T) cells, and primary human foreskin fibroblasts (HFs)  were ma2O was used at 50 μM. MG132  prepared at 10 mM in dimethyl sulfoxide  was used at 10 μM. Doxycycline  prepared at 1 mg/ml in Milli-Q H2O was used at 0.1 μg/ml. Cycloheximide  prepared at 5 mg/ml in Milli-Q H2O was used at 10 μg/ml. For transgenic cells, hygromycin , puromycin , or neomycin  was used at 50 μg/ml, 1 μg/ml, or 1 mg/ml, respectively, during selection or 5 μg/ml, 0.5 μg/ml, or 0.5 mg/ml, respectively, for maintenance.Phosphonoacetic acid  prepared at 100 mg/ml in DMEM was used at 400 μg/ml. Acycloguanosine  prepared at 50 mM in Milli-Q Hsyn+, its ICP0-null mutant derivative dl1403 , was replaced with an enhanced yellow fluorescent protein (eYFP) coding sequence, and the resultant eYFP.PIAS4 expression construct was subcloned into the lentiviral vector pLKO.dCMV.TetO/R  via NheILentiviral supernatant stocks were generated and HFt cells were transduced as described previously . Pooled Primary rabbit antibodies included anti-actin , anti-enhanced green fluorescent protein , anti-Daxx , anti-PIAS1 , anti-PIAS2 , anti-PIAS3 , anti-PIAS4 , anti-PML , anti-Sp100 (SpGH ), anti-S5 per well) in 24-well plates were incubated at 37°C in 5% CO2 overnight or at least 4 h prior to infection. Wild-type or ICP0-null mutant HSV-1 was serially diluted in serum-free DMEM. Cells inoculated with sequential viral dilutions were rocked and rotated every 10 min for 1 h and then overlaid with 37°C cell appropriate medium supplemented with 2% human serum (HS). At 24 to 26 h postinfection (hpi), cells were washed twice in phosphate-buffered saline , fixed in 1.8% formaldehyde  and 0.1% NP-40  in PBS for 10 min, and then washed twice in 0.1% Tween in PBS (PBST). Cells were blocked with 5% skim milk powder  in PBST for 30 min before incubation with anti-VP5 diluted in 5% SMP in PBST for 90 min. Cells were washed in PBST three times, incubated with peroxidase-conjugated anti-mouse IgG diluted in 5% SMP in PBST for 60 min, and then washed in PBST three more times. Plaques were visualized with True Blue peroxidase developing solution  according to the manufacturer's instructions.HFt cells . The intensity of protein bands was quantified with Odyssey Image Studio software.HFt cells were washed twice with room temperature PBS and then lysed and collected in SDS-PAGE loading buffer with 4 M urea  and 50 mM dithiothreitol . Whole-cell lysates were immediately used or snap-frozen. Proteins resolved using standard Tris-glycine or Tris-tricine SDS-PAGE systems were electrotransferred onto 0.2-μm nitrocellulose membranes  for 150 (Tris-tricine) or 180 (Tris-glycine) min at 250 mA in Towbin buffer  at room temperature. Membranes were blocked in 5% newborn calf serum (NCS) in PBS at 4°C overnight. Subsequent steps were performed at room temperature on a roller apparatus. Membranes were incubated in primary antibody diluted in 5% NCS in PBST for 2 h, washed in PBST three times for 5 min each, then incubated in secondary antibody diluted 1:10,000 in 5% NCS in PBST for 1 h. Following three 5-min washes in PBST, one 5-min wash in PBS, and one rinse in Milli-Q H5) seeded on 13-mm glass coverslips in 24-well plates were incubated at 37°C in 5% CO2 overnight. For infections, wild-type or ICP0-null mutant HSV-1 was diluted in serum-free DMEM to the multiplicity of infection (MOI) indicated in the corresponding figure legends. Cells were typically infected at a low MOI for 16 to 24 h in order to examine the recruitment of host factors to nuclear sites associated with HSV-1 genome entry or replication compartments in cells at the periphery of a developing plaque , fixed and permeabilized in 1.8% formaldehyde and 0.5% Triton X-100  in CSK for 10 min, and then washed in CSK three times. Coverslips were blocked with 2% HS in PBS for 10 min, incubated with primary antibodies diluted in 2% HS in PBS for 90 min, and then washed in PBS three times. Cells were then incubated with 4′,6-diamidino-2-phenylindole  and secondary antibodies diluted 1:1,000 in 2% HS in PBS for 60 min, washed in PBS three times, and washed twice in Milli-Q H2O. Coverslips were mounted on glass slides using a glycerol-based mounting medium  sealed with nail enamel. Cells were examined using Zeiss LSM 510 or LSM 710 confocal microscopes with 405-nm, 488-nm, 543-nm, and 633-nm laser lines under a 63× Plan-Apochromat oil immersion lens with a numerical aperture of 1.4. For graphing purposes, pixel intensities were acquired using Zen software (Zeiss). Exported images were processed with minimal adjustment using Adobe Photoshop and assembled for presentation using Adobe Illustrator.HFt cells  or TRIzol reagent  according to the manufacturer's instructions. RNA was reverse transcribed using the TaqMan reverse transcription reagent kit  with oligo(dT) primers. Samples were analyzed in triplicate using TaqMan Fast Universal PCR master mix  with the following TaqMan gene-specific primer (6-carboxyfluorescein [FAM]/MGB)-probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), PIAS4 (assay ID Hs00249203_m1), 18S , or glyceraldehyde-3-phosphate dehydrogenase .A total of 1.5 × 10de novo SUMOylation, supporting the concept that dynamic SUMO modification and signaling occur throughout infection  that could mediate these SUMOylation events was analyzed, starting with the PIAS protein family. Consistent with other reports, PIAS proteins in HFt cells were predominantly nuclear, with microspeckled distributions characteristic of localization at matrix-associated regions (MARs) , mock 6–67. Whil–To investigate the roles of PIAS4 during HSV-1 infection, the domains that mediate recruitment into replication compartments were evaluated . While tA panel of lentiviral vectors that encode eYFP fused to PIAS4 with point mutations or deletions in the aforementioned domains was constructed ; Fig. 3AEYFP.PIAS4 protein levels increased substantially with time, without considerable alteration to endogenous PIAS4 expression  and D. AAmino-terminal fusion of eYFP did not adversely affect PIAS4 subnuclear localization . InactivThese data demonstrate that all of the conserved domains of PIAS4 contribute to its characteristic MAR localization in HFt cells. In the absence of stable PIAS4 localization at MARs, the SIM or catalytic activity promotes localization at PML-NBs. The C-terminal AD of PIAS4 is required to stabilize PIAS4 localization at either MARs or PML-NBs and contributes to the predominantly nuclear localization of PIAS4.The subnuclear localization of mutant eYFP.PIAS4 proteins was initially evaluated during ICP0-null mutant HSV-1 infection to avoid potential ICP0-mediated disruption of relevant interactions. EYFP alone did not accumulate in replication compartments, while eYFP.PIAS4 did  and B, dThe mutant eYFP.PIAS4 proteins all accumulated in ICP0-null mutant HSV-1 replication compartments, although they had different localization phenotypes within them  to I. AsMutation of the PIAS4 SIM disrupted localization to PML-containing subdomains within replication compartments  and H, cThese data demonstrate that PIAS4 can colocalize with PML within ICP0-null HSV-1 replication compartments, sometimes in string-like structures. The differential colocalization of PIAS4 with PML in cells that have been infected or not indicates that PIAS4 localization is differentially regulated during infection. While the SIM mediates colocalization with PML, such colocalization is not strictly required for PIAS4 accumulation in ICP0-null mutant HSV-1 replication compartments. This observation is consistent with multiple mechanisms to regulate PIAS4 subnuclear localization during infection.The SIM-mediated colocalization of PIAS4 with PML was predominant, although not necessary for PIAS4 localization within replication compartments. The direct or indirect association of PIAS4 with PML could therefore conceal relevant interactions mediated by the other domains of PIAS4 that contribute to replication compartment localization. To address this possibility, the localization of wild-type and mutant eYFP.PIAS4 proteins was evaluated during wild-type HSV-1 infection, when ICP0 efficiently mediates PML degradation , 71. As The eYFP.PIAS4 C342A, mPINIT, and ΔC34 mutants localized to wild-type HSV-1 replication compartments . MutatioThese data confirm that the localization of PIAS4 in HSV-1 replication compartments is not disrupted by ICP0. Whereas the PINIT motif promotes or stabilizes PIAS4 accumulation within replication compartments, the SAP domain or LxxLL motif considerably enhances it. However, as demonstrated during ICP0-null mutant HSV-1 infection, additional factors are sufficient for PIAS4 localization in replication compartments in the absence of a functional SAP domain or LxxLL motif.de novo SUMOylation events that mediate recruitment of constitutive PML-NB antiviral proteins  of wild-type HSV-1 and increased that of ICP0-null mutant HSV-1, as expected  50). De. De50).  of wild-We show that PIAS4 is upregulated and localizes to nuclear domains that contain viral genomes during HSV-1 infection. The recruitment of PIAS4 to domains that contain infecting HSV-1 genomes is SIM dependent, whereas its accumulation in replication compartments is largely mediated by its SAP domain or LxxLL motif. However, in the absence of ICP0, the SAP domain or LxxLL motif is dispensable for PIAS4 localization in replication compartments, as the SIM mediates colocalization with PML within them. PIAS4 contributes to the restriction of ICP0-null mutant HSV-1 through mechanisms that are synergistic with those of PML. We thus identify PIAS4 as an intrinsic antiviral factor. This unique role for PIAS4 adds to the current understanding of PIAS proteins as immune regulators and warrants future investigation into their novel roles as intrinsic antiviral factors that restrict DNA virus infection.Neither the STUbL-like activities of ICP0 nor the accumulation of HMW SUMO-conjugated proteins in the absence of ICP0 is sufficient to fully deplete the free SUMO pools during infection  and B. Mde novo SUMOylation events, for example, through Ubc9 depletion, significantly impairs intrinsic immunity by hindering the recruitment of individual PML-NB antiviral proteins to domains that contain infecting HSV-1 genomes   , while Ss (MEFs) . Even wi−/− MEFs . In cont−/− MEFs , 92. How−/− MEFs , 93, 94.


However, imaging the atomic displacements in screw dislocations remains challenging. Although advanced electron microscopy techniques have allowed atomic-scale characterization of edge dislocations from the conventional end-on view, for screw dislocations, the atoms are predominantly displaced parallel to the dislocation line, and therefore the screw displacements are parallel to the electron beam and become invisible when viewed end-on. Here we show that screw displacements can be imaged directly with the dislocation lying in a plane transverse to the electron beam by optical sectioning using annular dark field imaging in a scanning transmission electron microscope. Applying this technique to a mixed [ Although screw dislocations impact on the properties of various engineering materials, their investigation on the atomic scale has been challenging. Here, the authors use optical sectioning in a scanning transmission electron microscope to achieve direct imaging of screw displacements around a screw dislocation core in GaN. Such dislocations are conventionally viewed end-on at high resolution in transmission electron microscopes because the tensile and compressive displacements are clearly visible1234789a], [c] and [a+c], respectively. The [a+c] mixed dislocations are frequently found to be dissociated4a]=½[a]+½[a] to form a fault lying in the a-plane). Since the ½[a] shift would give rise to Ga–Ga and N–N bonding and therefore a very high-energy interface, an additional ½[c] shift along [0001] was proposed so that correct Ga–N pairings across the interface are possible. This implies that the screw component is also dissociated according to [c]=½[c]+½[c], and therefore the total dissociation reaction proposed is [a+c]=½[a+c]+½[a+c], and the two partials are bounding a fault with a structure similar to the one proposed by Drum in ref. 5Because of the relatively large lattice mismatch with the substrate, GaN films grown parallel to (0001) generally contain threading dislocations, and during growth the dislocations tend to align themselves close to the [0001] direction. Three types of dislocations are found: edge, screw and mixed dislocations with Burgers vectors [The development of aberration correctors in (S)TEM (transmission electron microscopy) has led to a dramatic improvement in spatial resolution in both STEM1316c-type screw dislocation along [0001] in GaN is used, and atomic positions in single layers parallel to c] .city see , an assue c] see . Far froe c] see . Close te c] see , screw dIn the simulated ADF images , the peaks associated with the atomic columns become double peaks as the focal plane approaches the dislocation depth, and the relative intensities of these peaks change with defocus . For a . The evoa+c] dislocation lying approximately along [0001] in the plane of the sample parallel to c], and therefore the Burgers vector of each screw partial is ½[c], giving a total screw Burgers vector of [c] as expected for GaN. The screw shear is further confirmed by quantifying peak positions from the ADF focal series . A 1-μm-thick GaN film was grown at 1,000 °C on a sapphire substrate by metalorganic vapour phase epitaxy. The TEM specimen was prepared by mechanical grinding and polishing, followed by ion milling using a Gatan Precision Ion Polishing System.The high-angle annular dark field STEM images were obtained using the Nion UltraSTEM100 electron microscope at SuperSTEM, Daresbury, UK. This microscope is equipped with a field emission gun operated at 100 kV, and a Nion fifth-order spherical aberration (Cs) corrector. A probe convergence semi-angle of 30 mrad was used to achieve a 7.28-nm depth of field see . The imaTo simulate the STEM ADF images, we have used the multislice frozen phonon approach implemented in the QSTEM software using the same imaging parameters (accelerating voltage and convergence angle) as those used in the experimental workHow to cite this article: Yang, H. et al. Imaging screw dislocations at atomic resolution by aberration-corrected electron optical sectioning. Nat. Commun. 6:7266 doi: 10.1038/ncomms8266 (2015).Supplementary Figures 1-8, Supplementary Notes 1-5 and Supplementary References


Human essential hypertension is characterized by eutrophic inward remodeling of the resistance arteries with little evidence of hypertrophy. Upregulation of αVβ3 integrin is crucial during this process. In order to investigate the role of focal adhesion kinase (FAK) activation in this process, the level of FAK Y397 autophosphorylation was studied in small blood vessels from young TGR(mRen2)27 animals as blood pressure rose and eutrophic inward remodeling took place. Between weeks 4 and 5, this process was completed and accompanied by a significant increase in FAK phosphorylation compared with normotensive control animals. Phosphorylated (p)FAK Y397 was coimmunoprecipitated with both β1- and β3-integrin-specific antibodies. In contrast, only a fraction (<10-fold) was coprecipitated with the β3 integrin subunit in control vessels. Inhibition of eutrophic remodeling by cRGDfV treatment of TGR(mRen2)27 rats resulted in the development of smooth-muscle-cell hypertrophy and a significant further enhancement of FAK Y397 phosphorylation, but this time with exclusive coassociation of pFAK Y397 with integrin β1. We established that phosphorylation of FAK Y397 with association with β1 and β3 integrins occurs with pressure-induced eutrophic remodeling. Inhibiting this process leads to an adaptive hypertrophic vascular response induced by a distinct β1-mediated FAK phosphorylation pattern. The resistance arteries  are essential for mediating the autoregulation of flow and the stabilization of capillary pressure. An important component of this protective mechanism, called the myogenic response, allows these vessels to change diameter in response to alterations in intraluminar pressure. Besides resulting in enhanced myogenicity of the resistance arteries , sustainThe integrin/extracellular-matrix (ECM) axis transfers tensile forces exerted by blood pressure across the cell membrane. Integrins are cell surface ECM receptors that can heterodimerize in a noncovalent fashion to form 24 different αβ receptors which recognize a wide variety of cellular and ECM components present in the arterial extracellular space including collagen, fibronectin and laminin , while oThere is increasing evidence that FAK is an important mediator for cell attachment and mechanosensing . The SrcWe wished to test the hypothesis that FAK/Src signaling is crucial in mechanotransduction and the eutrophic inward remodeling of resistance arteries as a response to elevated pressure.This study examined the activation levels of FAK/Src in arteries exposed to elevated pressure in vitro and ex vivo, and we report differential FAK Y397 phosphorylation levels present at the integrin β subunits of resistance arteries from hypertensive TGR(mRen2)27 rats as pressure rises and eutrophic inward remodeling occurs. Ex vivo pressurization of arterial segments which exhibit myogenic tone confirmed that FAK Y397 phosphorylation is increased as a response to elevated pressure.The TGR(mRen2)27 rat carries a DBA/2J Ren2 transgene and develops hypertension with vascular structural changes from eutrophic inward remodeling ,16. ThesBlood pressure measurements of TGR(mRen2)27 and SD control animals were performed using tail-cuff plethysmography under light fluothane anesthesia.3, 1.17 mM KH2PO4, 1.17 mM MgSO4, 0.026 mM EDTA, 1.6 mM CaCl2 and 5.5 mM glucose) at 37°C and gassed with 5% CO2 and 95% O2 to maintain a pH of 7.4. Subsequent morphological parameters of live vessels such as the media-to-lumen ratio, growth and remodeling indices were then measured and calculated, as previously described [We performed studies on the small arteries harvested from animals sacrificed at 5 weeks of age, because hypertension-induced eutrophic remodeling takes place from the age of 4 weeks and is completed by week 5, as described previously . SegmentIn order to study the effects of integrin αV antagonism, TGR(mRen2)27 rats were injected intraperitoneally at the age of 4 weeks with cRGDfV or cRADfV peptide  twice daily for 5 days before sacrifice . These aWestern blot analysis was performed according to the methodology described by Laemmli . VesselsAn amount of 5 μg of each antibody was used to precipitate integrin subunits from 250 μg of total resistance artery radioimmunoprecipitation assay extract. The antibodies used for immunoprecipitation were: β1 (AB1952), β3 (AB1932) and α5 (aba5b) integrin antibodies . Agarose IgG/protein A  was used to bind and identify coprecipitated complexes. Subsequent Western blot analysis was performed using FAK Y397  antibody and Src Y418 . Protein A-HRP was used for chemiluminescent detection of coprecipitated proteins .Arterial segments from the proximal region of the cremasteric artery from SD animals were dissected and mounted between two glass microcannulae in physiological salt solution in a pressure myograph . Experiments were carried out at 32°C. PP2 and PP3, i.e. a potent inhibitor of c-Src and an inactive analog (negative control) of PP2, respectively ,24, wereVessels for immunofluorescence were dissected on ice and snap-fixed in ice-cold 4% paraformaldehyde. Paraffin-embedded arteries were sliced at 4 μm, dewaxed and rehydrated. The pFAK Y397 integrin  and a nonimmune control IgG antibody were used separately, followed by donkey anti-rabbit antibody with conjugated TRITC . Diamidino-2-phenylindole  (Jackson ImmunoResearch Laboratories) was used as a blue fluorescent nuclear stain. A Leica DM6000 epifluorescence microscope was used at ×1,000 magnification in conjunction with Leica FW4000 software for image capture and analysis. Superimposing fluorescence and phase-contrast microscopy images allowed the visualization of the VSMC membrane and the internal elastic lamina which indicates the smooth-muscle/endothelium boundary.Statistical analysis was performed using MS Excel. Values are expressed as mean ± standard deviation. Differences between data were tested using the two-tailed unpaired homoscedastic Student t test. p < 0.05 was considered statistically significant.Between 4 and 5 weeks of age, mean systolic blood pressure in TGR(mRen2)27 rats was significantly increased from 121 ± 7.9 to 148 ± 3.0 mm Hg . SD control rats showed no age-dependent increase in blood pressure which ranged from 80.9 ± 8.8 to 93.4 ± 8.1 mm Hg. Pressures in TGR(mRen2)27 rats were significantly higher than those in SD rats at both time points (p < 0.0001).The administration of cRGDfV and cRADfV peptides had no effect on the blood pressure rise seen in TGR(mRen2)27 rats (data not shown).The cross-sectional area of TGR(mRen2)27 rat arteries at 5 weeks was unchanged compared with at 4 weeks of age, with a remodeling index of 97% indicating the development of eutrophic inward remodeling due to the media-to-lumen ratio being significantly increased (p < 0.01). The structural parameters of SD rat arteries did not change over 7 days.cRGDfV treatment reduced the remodeling index to 9% and there was evidence of hypertrophic growth, with a calculated growth index of 17%. Treatment with cRADfV had no effect on remodeling, as predicted (data not shown).Western blot analysis of pFAK Y397 showed significant rises in autophosphorylation levels in resistance arteries from TGR(mRen2)27 animals treated with cRADfV control peptide , compared with vessels from SD normotensive control animals fig. . SurprisPhospho-FAK Y397 immunofluorescent staining of the arteries from normotensive SD animals was faint but present throughout the medial smooth-muscle cells  exhibited increases in FAK Y397 phosphorylation compared with vessels pressurized at 60 mm Hg (n ≥ 5). No passive morphological alterations of vessels to pressure were observed at this stage (data not shown). Phosphorylation of FAK Y397 at 120 mm Hg was abrogated when arteries were coincubated with 1 μM of PP2. In contrast, coincubation of PP3 did not have any effect 27 animals fig. . CoimmunIn conjunction with FAK, c-Src was prominently coprecipitated with integrin α5β1 and, to a lesser extent, with integrin β3 fig. . In contPreviously, we showed that the hypertension-mediated inward eutrophic remodeling of TGR(mRen2)27 rat resistance arteries commenced at 4 weeks of age and was complete at 5 weeks . This prWe established that low basal levels of FAK Y397 phosphorylation in normotensive SD rat mesenteric arteries are predominantly associated with the β3 integrin subunit and that active c-Src associates with integrin α5β1. FAK Y397 phosphorylation was significantly increased in hypertensive TGR(mRen2)27 resistance arteries and recruited to FA sites after blood pressure rose and once eutrophic inward remodeling was complete. In addition, both pFAK Y397 and active c-Src associated with β1 and β3 integrins in the resistance arteries of TGR(mRen2)27 animals. However, the inhibition of remodeling by the administration of cRGDfV peptides resulted in the hypertrophy of VSMCs and a further increase of FAK Y397 phosphorylation. This process was associated with integrins containing the β1 subunit only.We can conclude from these data that FAK phosphorylated at tyrosine position 397 (the autophosphorylation site) is present at both the integrin β1 and β3 cytoplasmic sites in the resistance arteries of hypertensive TGR(mRen2)27 animals, in contrast to SD controls, which exhibit relatively low pFAK Y397 levels only associated with the integrin β3 subunit.We cannot rule out the possible involvement of FAK Y397 phosphorylation in subsequent length/autoregulation processes during the hypertension remodeling processes downstream of the integrin β3 subunit. However, it is now thought that the initial role, in the light of the FAK Y397 phosphorylation observed, is in mechanotransduction, as cRGDfV peptides not only inhibited eutrophic remodeling but also encouraged further FAK Y397 phosphorylation during the onset of hypertension in this model. Therefore, the increase of FAK Y397 phosphorylation observed is more likely to occur as a consequence of increases in wall tension, rather than acting as a direct intermediate of eutrophic inward remodeling. Besides aiding adhesion and migration , β1 and There is the possibility that the increase of FAK Y397 phosphorylation observed in the resistance arteries of the young TGR(mRen2)27 hypertensive animals was induced by enhanced activity of the renin-angiotensin system . HoweveVarious studies have reported the association of FAK with different β integrin subunits; β1, β3 and β5 cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation and these specific regions have been identified . DiffereIt has been established that both β1 and β3 integrin subtypes are required not only in the adhesion of VSMCs to the ECM of the vascular wall but also for pressure-mediated signaling, resulting in vascular myogenic constriction and remodeling in hypertension ,27 summ. In thisRecent data have suggested that the morphological changes in the arterial wall of small blood vessels are associated with cardiovascular prognosis. The normal response to untreated hypertension is eutrophic inward remodeling, i.e. a rearrangement of the vascular wall architecture without any evidence of growth . When hy


Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC PRSS8), also called channel-activating protease, is a type I transmembrane protease that is anchored to airway cell surfaces via glycosylphosphatidyl inositol /[enzyme])). In such plots, activity is 0% when log10 ([inhibitor]/[enzyme]) = 0 for an inhibitor that inactivates a protease with full potency and 1:1 stoichiometry. The normalized IC50 for such an inhibitor would be log10 (0.5) = -0.3. The observed IC50 data are shown in The in vitro, steady state susceptibility of four human airway proteases  to inactivation varied depending on the inhibitor studied. As revealed in 50 of nafamostat’s leaving group, 6-amidino-2-naphthol, was >104-fold higher for matriptase and β-tryptase than the IC50 of nafamostat itself. Although one potential mechanism of high potency of a cleavable inhibitor is generation of a leaving group that is a more potent competitive inhibitor than is the uncleaved parent compound, the very high IC50 of 6-amidino-2-naphthol relative to that of nafamostat itself suggests that this is not the case. Therefore, inhibition by the non-acyl component of hydrolyzed nafamostat does not explain nafamostat’s high potency upon cleavage by these enzymes and, given the very low rates of nafamostat turnover, 6-amidino-2-naphthol would be unlikely to achieve the concentrations needed to reduce matriptase and β-tryptase activity.As shown in At nearly stoichiometric ratios of nafamostat to enzyme, inhibition of tryptase and β-tryptase slowly reverses, as seen in Given the observed nearly 1:1 stoichiometry and slow reversal of inhibition of matriptase and β-tryptase by nafamostat, we sought to detect release of 6-amidino-2-naphthol and to explore the possibility of using nafamostat as a “burst titrant” of active sites in preparations of these enzymes. We did not assess this possibility for prostasin or HAT because inhibition for these proteases did not approach 1:1 stoichiometry. Estimates of active site concentration in our stock preparation of trypsin were similar whether obtained using the colorimetric approach with p-nitrophenyl-p’-guanidinobenzoate or the fluorometric approach with nafamostat . However, the nafamostat method, as implemented using the in-line flow cell of the Jasco FP-2020 Plus Fluorescence Detector, was far more sensitive, requiring dilution of trypsin stock solution ~1,000-fold. The fluorometric nafamostat method, being capable of detecting active sites in the 1-nM range see  using sa7 Absorbance Units410 nm min-1 M-1. Assay of β-tryptase active sites in similar fashion yielded an active site concentration of 2.1 ± 0.1 μM , which is 110% of the value based on the manufacturer’s estimate of protein concentration. Based on the active site estimate, the specific activity of β-tryptase for substrate GPK using our standard assay conditions is 2.5 x 107 Absorbance Units410 nm min-1 M-1. Nafamostat itself has minimal fluorescence at excitation and emission wavelengths optimized for 6-amidino-2-naphthol [Application of the fluorometric nafamostat approach to matriptase and β-tryptase  suggestsnaphthol . The obsThis study focuses on four proteases that share three major attributes: 1) they are trypsin-like, 2) they are found in human airway epithelium and 3) they are proposed as targets for inhibition to treat allergic or infectious airway disorders associated with inflammation and hypersecretion. This first direct comparison of these proteases reveals that each has a distinct profile of susceptibility to the inhibitors shown in In the case of β-tryptase and matriptase, the findings show that nafamostat’s high potency relates in part to actions as a suicide substrate. This results in formation of a covalently bound, inactivating intermediate that is stable for hours in aqueous solution. In this regard, nafamostat’s bifunctionality could influence potency. As shown in It can be noted from the structures in Ki) when tested versus human β-tryptase [Ki calculations, which assume a competitive and reversible mode of inhibition, are inapplicable to nafamostat inhibition of tryptase, for which the resulting data would depend on enzyme concentration. Similar considerations apply to camostat, which yielded apparent Ki values that were >100-fold higher for prostasin than for matriptase [50, which is a more appropriate comparator for a suicide substrate. The discrepancy probably relates to the need to use concentrations of prostasin in tryptic substrate cleavage assays that are high relative to the concentrations needed of matriptase, which is an intrinsically more efficient enzyme [Nafamostat’s ability to form an inhibitory complex at low concentration via a cleaved intermediate is undoubtedly responsible for the report of a highly favorable (sub-nanomolar) dissociation constant  and type II proteases (matriptase and HAT) may differ from that of soluble forms of the enzymes due to active site blockade or allosteric effects. However, matriptase and prostasin can exist natively in shed, soluble forms, and HAT’s shed form may be the native enzyme’s dominant form [The kinetic comparisons of prostasin, matriptase and HAT with the native, soluble β-tryptase tetramer were conducted with recombinant forms of these enzymes expressed as soluble catalytic domains. This is a matter of necessity since the assays developed to detect the activity of membrane-bound tryptic proteases are ill-suited to ICant form . Furtherant form .Although one of the principal findings of this study is the relative lack of inhibitor specificity towards four of the tryptic enzymes that have been considered as candidates for therapeutic inactivation in airway diseases, our data provide grounds for considering that prostasin is the principal source of apical surface tryptic activity in the human airway epithelial cell monolayers generating the data in


This allows for the use of the Voigt average in the wave equation, and velocities are obtained solving the appropriate Christoffel equation. The velocity for vertical propagation, as appropriate to interpret sonic logging measurements, is analysed in more details. Our formulae are shown to be accurate at the 0.5% level and they provide a rationale for previous empirical fits to wave propagation velocities with a quantitative agreement at the 0.07–0.7% level. We conclude that, within the formalism presented here, it is appropriate to use, with confidence, velocity measurements to characterize ice fabrics.The propagation of elastic waves in polycrystals is revisited, with an emphasis on configurations relevant to the study of ice. Randomly oriented hexagonal single crystals are considered with specific, non-uniform, probability distributions for their major axis. Three typical textures or fabrics (i.e. preferred grain orientations) are studied in detail: one cluster fabric and two girdle fabrics, as found in ice recovered from deep ice cores. After computing the averaged elasticity tensor for the considered textures, wave propagation is studied using a wave equation with elastic constants  PolycrStarting with Karal and Keller in the 1960s , stochasc the long axis of the unit cell and by a the short axis on the plane perpendicular to c. In the central part of deep ice sheets, ice is deformed at very low strain rates (10−10 to 10−12 s−1) induced by progressive accumulation of snow layers at the surface. In the simplest case , ice is loaded by uniaxial compression due to gravity, and the fabric evolves from nearly isotropic at the surface towards a cluster fabric, where the c-axes are randomly distributed within a cone with vertical axis. The cone angle decreases with depth or ice age, until a strong single maximum close to the bedrock is reached  (the ± signs correspond to φ=π/2(+) and 3π/2(−)). The problem being two dimensional in , the projection onto  is not needed and the average is defined with θ being considered as a polar angle. For any quantity A, we now haveφ in the average as the plane of rotation of the c-axes has to be defined by a delta-function to fix the φ-values, withH2(θ)=1 for θ in , zero otherwise. Note that we could use θ∈ and φ=π/2 but the calculations shown below are easier using the form above. From equations (c-axes for φ=π/2 and 3π/2 (with projections e2), a is diagonal, witha1 varying between θ0=π/2) and a1=1 (single maximum fabric for θ0=0).This configuration is two dimensional b, and th(i)θ and φ are not adapted to deal with it in a simple way: indeed, as defined, θ∈ and φ∈ describe a thick girdle with VTI symmetry (symmetry by rotation with respect to e3); the thick girdle has HTI symmetry, with a symmetry by rotation with respect to e1. To avoid additional calculations in the forthcoming calculations of the Voigt matrix (see §3), we use the following ‘trick’. (i) We denote  thc-axis is parametrized by e1,e2,e3), it isc-axis is given by equations . As expected, the thick girdles prolongate the range of anisotropy of the partial girdles, with a1 varying from a1=1/2  to a1=1/3 (perfect isotropy for ξ0=π/2).As for the clustered fabrics, two of the eigenvalues are equal. This reflects the equivalence between two of the principal directions of the structure ((c)ai, i=1,2,3, as a function of θ0 for the cluster fabric and as a function of θ0 and ξ0 for the partial and thick girdles, respectively. Inspecting the value of a1 allows us to characterize the degree of anisotropy of a polycrystalline structure in a unified way, that is, independently of the considered fabric.3.cijkl of a single crystal when expressed in an arbitrary coordinate frame, with the c-axis being parametrized by the two spherical angles . The result is further used to derive the elasticity tensors for the cluster and girdle fabrics, describing average macroscopic properties of the polycrystalline materials. We use the elasticity tensor in Voigt's notation +4sΘ2cΘ2(〈C12〉2+2〈C12〉〈C55〉)+〈C55〉2.where we have used that 〈equation ). We ded5.et al. [in situ measurement campaign, the first one being the deep drill hole at Byrd Station, Antarctica, during the 1969–1970 field seasons by Bentley [in situ measurements of the velocities and values of the velocities deduced from the measures of c-axis orientations in thin sections of core samples. The logs as used in in situ measurements provide the time of flight between two receivers located on a vertical logger which can be used to obtain the S- and P-wave velocities. Then, an inverse procedure has to be used to obtain information on the fabric in the interrogated zone. This procedure raises several questions that we will address in this section. They are (i) a comparison of our expressions of the velocities with the semi-empirical determination of the elastic velocities, as proposed by Bennett [In this section, we focus more specifically on the application to ice fabric characterization using a sonic log of the deep boreholes, as tested during January 2011 at Dome C, East Antarctica, by Gusmeroli et al. . To the  Bentley . In betw Bentley ,37 and i Bentley ,39. Exce Bentley , these s Bentley  is the q Bennett  for clus(a)e3 axis to coincide with the vertical direction, a convenient choice to analyse sonic logging measurements of antarctic ice. As a first step, we report below the expression of the velocities for propagation of the wave along the vertical e3 axis. In this case, the expressions from equations . For clquations  and D=⟨CC33, C44 and C55 given by equations , with quations , we getquations , we havea1 (defined in equations (β=δ=γ≃0), and the velocities that correspond to an ice polycrystal with randomly oriented c-axes (equations (a1=1 (single maximum) and a1=1/3 , respectively. It is often tempting to neglect the anisotropy of an ice polycrystal and we report here the relative errors in the velocities when using this approximation—when using single-crystal isotropy (Thomsen's leading order): for the cluster 3.5% and 5.7% (P- and S-waves) and for the girdles 3.8%, 7.7% and 3.7% ; when using polycrystal isotropy: for the cluster 1.9% and 2.5% (P- and S-waves) and for the girdles 1.4%, 2.3% and 3.7% ; thus, polycrystal isotropy is a better approximation.quations , 2.11)  a1 (defiquations ). For coquations  with β=δquations ). As expVmax−Vmin)/(Vmax+Vmin) for each fabricNext, we come back to the range of variation of the velocities with respect to the degree of anisotropy of the fabrics. We report the relative variation 2((b)(i)θ only, that is, the angle between the c-axis and the direction e3 of the wave propagation, and not on the longitude φ. At first sight, this approach appears reasonable. However, there are two pitfalls. (i) The times of flight are calculated for each S- or P-wave separately, assuming that the wave energies originally distributed into S- and P-waves remain unchanged; in other words, the re-repartition of the energy between SH-, SV- and P-waves at each grain boundary by mode conversion is disregarded. (ii) More seriously in our opinion, it is assumed that an ensemble of grains with the same θ but different φ-value behaves as a homogeneous medium, which is clearly not the case. Although this crystallographic arrangement is artificial, it allows us to exemplify the error made when ignoring the φ-dependence. Let us first remark that the structure resulting from such an arrangement has an anisotropy with hexagonal symmetry , thus the two shear velocities are the same for waves propagating along the e3-axis. From appendix A, the velocities for grains with the same θ and different φ-values are equivalent to an HTI structure (with symmetry around e3), and, from equations , equations (VS(θ) but also VP(θ) is false when using equations , the avquations  and VP and VS based on acoustic measurements. The details of the method are given in [e3-axis, Bennett's velocities readX,Y) are the same as used in equations . Bennett's fits are usually reported in the literature with a different form. For convenience, we have rewritten them in the form of equations  and density ρ given by  given above. The agreement is excellent, although less good for the S-wave than for the P-wave, and this will be analysed in more detail in forthcoming work. The resulting agreement on the velocities is 0.07% for the P-wave and 0.7% for the S-wave, without any adjustment  (green (c)cijkl〉 corresponds to anisotropic, or textured, effective media, with orthorhombic or hexagonal symmetry. For ice, δc is a small correction as ice single-crystal anisotropy is weak, as previously commented. Thus, the zeroth order of the Dyson equation, which we have used by solving equation (ϵ2=(δc/c)2 only. It is of importance to get an estimate of ϵ2 as the accuracy of the prediction on the velocities has to be sufficient to resolve the typical variations of the velocities.In our study, 〈equation  instead equation , is justCij parameters , fluctuations occur, with mean values being our 〈C12〉 and 〈C13〉; the dotted line shows the value Ciso  a aCij parquations . Resultsb) the error ϵiso, which defines the accuracy of the results if effective isotropy is assumed,a1=1/3. As expected, the error is significantly higher and it increases with the degree of anisotropy a1 up to about 10%.We also report b the err6.c-axes, that is, in general a system with a low variability in the elasticity tensor of the grains. Motivated by measurements in deep ice cores, we have inspected this small variability case in more detail, revealing an accuracy better than 0.5%. This suggests that velocity measurements can confidently be used to characterize ice fabrics. In a forthcoming publication, we will address the specifics related to a more complete inspection of this problem. More generally, extensions of this study are at least twofold. (i) As few theoretical studies of the effects of texture on backscattering and attenuation have been conducted, it should be interesting to iterate the Dyson equation using the potential defined in equation (We have derived closed forms of the elasticity tensors and of the elastic wave velocities for ice textured polycrystals with cluster and girdle fabrics. The prediction is accurate up to the second order in the local deviation of the elasticity parameters from their average values. This is justified for weak anisotropic single crystals or for fabrics with concentrated equation  to quant