Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH. NDH-2 has been attracting based on its potential medical applications. A series of studies has suggested that Ndi1, one of three NDH-2s from Saccharomyces cerevisiae, would be useful in replacement therapy for mitochondrial diseases caused by complex I dysfunction7. Apoptosis-inducing factor (AIF) and its family member protein AIF-M2 are mammalian members of NDH-2 family9. Although they exert NADH-ubiquinone oxidoreductase activity under certain conditions8, their functional role as a NADH-ubiquinone oxidoreductase in the respiratory chain is dispensable in mammals, as complex I plays a major role. Therefore, the microorganism NDH-2 has emerged as a potential therapeutic target for drugs targeting human pathogenic bacteria13 and parasites16. The quinone binding site of NDH-2 is thought to be an ideal target for chemotherapeutic agents because several compounds showing highly selective toxicity for parasites have been shown to target the quinone binding of respiratory enzymes19. Atovaquone, which has a similar structure to ubiquinone, is used as a therapeutic agent for Plasmodium falciparum malaria. It inhibits the respiration of malaria parasites by inhibiting the binding of ubiquinone to complex III20. The biochemical and structural basis for the function of NDH-2 has been intensively studied for these purposes in recent years16.A monotopic alternative NADH dehydrogenase (Type II NADH dehydrogenase: NDH-2) catalyses the electron transfer from NADH to quinone via FAD or FMN without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondriaMycobacterium tuberculosis and Yarrowia lipolytica23. In this reaction mechanism, two substrates—NADH and ubiquinone—bind to the enzyme sequentially, but not at the same time. However, our previous study could not determine whether the reaction occurred via a one-site or two-site ping-pong mechanism. Recent studies that investigated the structure and biochemistry of NDH-2s proposed a non-classical two-site ping-pong mechanism in M. tuberculosis NDH-224 and a ternary complex mechanism in yeast Ndi125 and NDH-2 from Staphylococcus aureus26. NDH-2 from Caldalkalibacillus thermarum follows either a two-site ping-pong mechanism or a ternary complex mechanism depending on the substrate that is used27. NADPH favoured a two-site ping-pong mechanism, while NADH favoured a ternary complex mechanism27. In a ternary complex mechanism, two substrates—NADH and ubiquinone—simultaneously bind to the enzyme. Thus, these two reaction mechanisms require separate binding sites for NADH and ubiquinone.We reported that the enzymatic reaction of yeast Ndi1 has a ping-pong mechanism, as is the case with NDH-2s from + or ubiquinone and demonstrated that the binding sites of both substrates overlap with each other and that the bound ubiquinone penetrates the plane of the isoalloxazine ring of FAD , both of which were located on the opposite side of the NADH binding site with respect to the plane of the isoalloxazine ring of FAD  quinolone (AC0-12), and compounds with carbon skeletons that differed from quinolone, stigmatellin, myxothiazol, and ascochlorin  and AC0-12 (IC50 = 115 ± 18 nM) exhibited higher potency than 1-hydroxy-2-undecyl-4(1 H) quinolone (AC0-11), the most potent inhibitor of Ndi1 (IC50 = 200 nM) ever identified23.The compounds that have been shown to inhibit quinone binding in several respiratory enzymes  and their derivative compounds were screened to find novel competitive inhibitors for yeast Ndi1. First, the compounds that inhibited the NADH-ubiquinone-1 oxidoreductase activity of Ndi1 by >70% at a concentration of 10 μM were selected. Second, the compounds for which the ICrin Fig. , were neKi and α values obtained by fitting the data to the equation of mixed model inhibition in the Prism6 software program  as a template  are specific for the interaction with myxothiazol. This large overlap reflects the observation that stigmatellin at the STG-1 site and the mixed-type inhibitors overlap near the FAD cofactor Fig. . In contKm and Vmax values for the oxidoreductase reaction were determined using ubiquinone-1 as a substrate with NADH at a fixed concentration of 100 μM , L447N (13%), and M485A (28%) , Q394A (37%), H397A (44%), and L487A (40%) , A393G (83%), and M485E (112%), which increased the Km values  increased the Km or Vmax values. Four mutants of the residue interacting with stigmatellin at the STG-2 site—R479I (0.02%), R479A (30%), R479K (29%), and R479H (0.1%)—reduced the Vmax values. R479A and R479K reduced the Km values.Two mutants of the residue interacting with stigmatellin at the STG-1b site—S484F and S484I—had no effect on either the I site by Feng et al.29. Eight of the ten residues involved in the interaction with ubiquinone at UQI are shared by those involved in the interaction with stigmatellin at STG-1a   Fig. . Similarl., Fig. 29. HowevI . There was a reciprocal relationship between the effect on the Km value and that on the Vmax value: a greater increase in the Km value was associated with a smaller reduction in the Vmax. Conversely, a greater decrease in the Vmax value was associated with a smaller increase in the Km value. M485E and W63F, which caused the greatest increase in the Km value, caused no reduction (M485E)—or rather—an increase (W63F) in the Vmax value. In contrast, P92A, L447N, and M485A, which caused the greatest decrease in the Vmax value, caused no increase in the Km value. These observations suggested that W63 and M485 are essential for ubiquinone binding and that P92, L447, and M485 might contribute to the Ndi1 activity. Since the aromatic head group of stigmatellin overlapped with the binding site of the mixed-type inhibitors, AC0-12 and myxothiazol, the amino acid residues interacting with the aromatic head group appear to be responsible for the mixed-type inhibition of the Ndi1 activity. On the other hand, the mutations of the residues specifically interacting with the aliphatic tail of stigmatellin at the STG-1a site increased the Km value, when the mutation was non-conservative . In contrast, the mutations of the residues specifically interacting with the aliphatic tail of stigmatellin at the STG-1b site had no effect on either the Km or Vmax values, despite the fact that the mutations were non-conservative . These observations suggest that the STG-1a site, especially the site of the aliphatic tail of stigmatellin—but not the STG-1b site—is responsible for the competitive inhibition of Ndi1 by stigmatellin. As a result, the UQI site, which overlaps with the STG-1a site  that specifically interact with the aliphatic tail of STG-1a, the hydrophobicity at I459, but not L444, is conserved among the HDH-2s  are suggested to contribute to the ubiquinone binding, because their non-conservative mutations consistently increased the I site implies that the binding sites for two substrates—ubiquinone and NADH—are distinct. This configuration is apparently consistent with either (two-site) ping-pong or ternary complex mechanisms of the enzymatic reaction of Ndi125. Our previous biochemical studies suggested that the binding sites for ubiquinone and NADH are distinct; thus, a model for the enzymatic reaction that was compatible with either two-site ping-pong or ternary complex mechanisms was proposed25. However, Feng et al., hypothesized that the binding of ubiquinone at the UQI site functioned as a structural component and not as a substrate33. Instead, they proposed that the substrate ubiquinone binds to the UQII site. In the present study, the mutations of the amino acid residue (S484) that specifically interacts with STG-1b, which overlaps with the UQII site, had no effect on the affinity for ubiquinone. The findings of the present study therefore support that the STG-1a/UQI site has a role as a site for substrate ubiquinone binding. STG-1b/UQII may not be functional in the enzymatic reaction of Ndi1. A comparison of the structure of the bacterial NDH-235 to that of the yeast Ndi1 suggests that the side chain of R382 in bacterial NDH-2 may cause steric hindrance of the binding of quinone to the site corresponding to the UQII site in yeast Ndi1. This implies that there is only one quinone binding site in the bacterial NDH-2, which corresponds to the UQI site of yeast Ndi1, and that the site corresponding to the UQII site is non-functional in the bacterial NDH-2.The identification of ubiquinone binding as a substrate at the STG-1a/UQsi-face and the quinone ring on the re-face . The Ultra Yield Flask™ was from Thomson Instrument Company . The HisTrap HP column was from GE Healthcare . The Econo-Pac 10DG column was from Bio-Rad . All of the other chemicals were of reagent grade and were obtained from commercial sources.Ubiquinone-1, stigmatellin, myxothiazol, and ascochlorin were purchased from Sigma-Aldrich . AC0-12 was synthesized according to the method of 1-hydroxy-2-alkyl-4(1 H) quinolone synthesisSaccharomyces cerevisiae Ndi1 enzyme was essentially purified and crystallized according to a previously described method28. Detailed methods of the expression and purification of Ndi1 can be found as Supplementary Methods.The A detailed methods of the amino acid replacements of Ndi1 can be found as Supplementary Methods.23. In brief, the reaction was started by adding 100 µM NADH to the enzyme mixture containing 10 ng/mL Ndi1, 50 mM sodium phosphate (pH 6.0) and 1 mM EDTA, which was pre-incubated with various concentrations of ubiquinone-1 for 1 min. The decrease in 340 nm absorbance was measured as an indication of the oxidation of NADH (ε = 6.22 mM−1 cm−1) with a Shimadzu UV2000 spectrophotometer . The enzymatic activity obtained with 100 µM NADH and 60 µM ubiquinone-1 in the absence of any inhibitors (100% activity) catalyzed the oxidation of 1,890 μmol NADH/min/mg Ndi1. The concentrations of inhibitors required to obtain the half-maximal inhibition (IC50) were estimated by non-linear regression dose-response curves. The inhibition mode of each inhibitor for ubiquinone-1 was determined by the intersection point in double-reciprocal plots , the other for the ternary enzyme-substrate-inhibitor complex (αKi). The value of α ranges from 0 to infinity, and determines the inhibition mechanism. When the constant α shows unity, the inhibition mechanism is considered to be noncompetitive; when α is closer to infinity, the inhibition mechanism is considered to be competitive; when α is closer to 0, the inhibition mechanism is considered to be uncompetitive.This equation provides two dissociation constants, one for the binary enzyme-inhibitor complex  and different concentrations of ubiquinone-1 (2.4–60 µM). Apparent Saccharomyces cerevisiae Ndi1 enzyme was essentially crystallized according to a previously described method28 by the hanging-drop vapor diffusion method. Crystals of the Ndi1-stigmatellin, Ndi1-AC0-12, and Ndi1-myxothiazol complexes were prepared by co-crystallization in the presence of 1 mM inhibitors. The protein solution  glycerol, 0.02% (w/v) DDM, 1 mM inhibitor) was mixed with an equal volume of the reservoir solution  PEG400, 100 mM NaCl, 2% (v/v) ethylene glycol, 5% (v/v) glycerol) and incubated at 20 °C. The crystals reached maximum dimensions of 0.3 × 0.2 × 0.05 mm3 in complex with AC0-12 and in complex with myxothiazol; this size was almost the same as that of the previously reported crystals28. The crystals in complex with stigmatellin were thin triangles with maximum dimensions (one side) of 0.3 mm and a thickness 0.05 mm.The purified 37, as shown in Table 38 using the atomic model of the ligand-free Ndi1 structure (PDB code: 4G9K) as a search model.A crystal was scooped up from a hanging droplets using a cryo-loop and was flash-frozen in liquid nitrogen gas. The X-ray diffraction data of the Ndi1-stigmatellin and Ndi1-AC0-12 complex crystals were collected at −173 °C on beamline BL44XU at SPring-8  and that of the Ndi1-myxothiazol crystal was collected at −173 °C on beamline BL17A at Photon Factory, the High Energy Accelerator Organization . All of the diffraction data sets were processed and scaled with the HKL2000 program package39 at resolutions of 1.85, 3.4 and 3.2 Å, respectively. Further refinement and model rebuilding were performed using Refmac5 and COOT40. The crystal of Ndi1-stigmatellin contains one Ndi1 and two bound stigmatellin molecules in the asymmetric unit. Crystals of Ndi1-AC0-12 and -myxothiazol complexes contain one dimer molecule in each of the asymmetric units. All structures were refined to final Rwork/Rfree values of 0.158/0.192 (Ndi1-stigmatellin), 0.207/0.299 (Ndi1-AC0-12), and 0.220/0.285 (Ndi1-myxothiazol), respectively. The results of the structure determination are summarized in Table http://www.pymol.org/).The model structures of Ndi1-stigmatellin, Ndi1-AC0-12 and Ndi1-myxothiazol were refined using Refmac5 in the CCP4 software programwww.pdb.org .The atomic coordinates and structural factors have been deposited in the Protein Data Bank, Supplementary Information


Berkeleya adeliensis Medlin. Furthermore, the propensity for B. adeliensis to flourish in platelet ice is reflected by an offshore downward gradient in diene II concentration in >100 surface sediments from Antarctic coastal and near-coastal environments. Since platelet ice formation is strongly associated with super-cooled freshwater inflow, we further hypothesize that sedimentary diene II provides a potentially sensitive proxy indicator of landfast sea ice influenced by meltwater discharge from nearby glaciers and ice shelves, and re-examination of some previous diene II downcore records supports this hypothesis. The term IPSO25—Ice Proxy for the Southern Ocean with 25 carbon atoms—is proposed as a proxy name for diene II.The presence of a di-unsaturated highly branched isoprenoid (HBI) lipid biomarker (diene II) in Southern Ocean sediments has previously been proposed as a proxy measure of palaeo Antarctic sea ice. Here we show that a source of diene II is the sympagic diatom  The sedimentary presence of the diatom lipid diene II (hereafter IPSO25) has been proposed as a proxy for palaeo Antarctic sea ice. Here, the authors identify a source of IPSO25 and hypothesize that its sedimentary distribution may provide proxy evidence of landfast ice influenced by freshwater discharge. Sea ice in the Southern Ocean is one of the most seasonal and variable features of the Earth's surface, and has a significant influence on key oceanic and atmospheric processes, which, in turn, have major impacts on global climate825, which has become a well-established proxy for seasonal sea ice in the Arctic1925: Ice Proxy for the Southern Ocean with 25 carbon atoms, by analogy with IP25 for the Arctic25 in certain Arctic sea ice diatoms25  indicative of a sea ice diatom origin1225, for which the source2025. As such, although a number of sea ice reconstructions based on IPSO25 have been reported, spanning recent decades, the Holocene101112131416In recent years, the analysis of an organic geochemical lipid biomarker—a so-called highly branched isoprenoid (HBI) diene  diatom species by manual isolation of individual cells from a bulk sample of mixed sea ice diatom assemblage collected from the West Antarctic Peninsula (WAP) in December 2014, and analysis of its lipid composition by gas chromatography–mass spectrometry (GC–MS). We also determined the abundance distribution of IPSO25 in ca. 150 surface sediments covering different regions of (mainly) West Antarctica, including the Weddell Sea, the Antarctic Peninsula (AP), the Bellingshausen Sea and the Ross Sea, together with its stable isotopic composition (δ13C) in three representative surface sediments from some of these locations. The source of IPSO25 in our sea ice samples, Berkeleya adeliensis MedlinB. adeliensis is particularly well adapted for growth in the relatively open channels of platelet ice, a common feature of landfast ice proximal to coastal Antarctic locations, which probably has important consequences for the interpretation of sedimentary IPSO25.In the current study, we identified IPSOB. adeliensis as the major diatom species (>50%)  consistent with that of other HBI-producing diatomsB. adeliensis.The diatom assemblage in a bulk sea ice sample collected from the WAP in December 2014 consisted of s (>50%) . Further25 could be identified and quantified in 125 out of the 149 samples analysed, with concentrations ranging from 0.22 to 1,830 ng g−1 (−1) were always associated with samples taken from coastal locations, with a general drop-off in abundance for offshore sites  of IPSO25 in surface sediments from Marguerite Bay (WAP), the SE Weddell Sea and the northern AP was found to be −15.00±0.03‰, −13.46±0.02‰ and −14.39±0.07‰, respectively ; however, no HBI lipids (including IPSO25) were identified previously in N. glacieiPleurosigma genus do not biosynthesize HBIs with a 6–17 double bond25   for coastal locations and substantially lower abundances offshore (ca. 0.22–8.51 ng g−1)  were recently shown to be strongly dependent on the nature and length of seasonal ice cover, with highest values for coastal locations experiencing at least partial ice cover extending into the summerThe close association of  ng g−1) . Finally25 in Antarctic sediments are closely related to the ecology of the source diatom, B. adeliensis. If this is the case, the apparently strict association of B. adeliensis to landfast ice might limit the value of IPSO25 as a general Antarctic sea ice proxy. However, instead, the environmental specificity of its source should enable more detailed insights into defining past sea ice conditions to be made with confidence. Thus, since it has been shown previously that B. adeliensis increases in abundance towards the end of the spring bloom and during the onset of ice melt3425 likely signifies the past presence of landfast ice during late spring/early summer. Such an interpretation parallels, to some extent, that for IP25 in the Arctic, the sedimentary presence of which is interpreted as reflecting spring sea ice cover19Collectively, these observations suggest that distributions of IPSO25 have largely been conducted on sediment cores retrieved from near-coastal locations around the Antarctic Peninsula (AP)131411B. adeliensis was not reported in the taxonomic inventories of any of these previous studies, which presumably reflects the susceptibility of this species towards dissolution in the water column and in sediments. Indeed, it has been suggested previously that many sea ice diatoms such as B. adeliensis may be under-represented in Antarctic sediments25 in surface sediments described here, and in previous downcore investigations1011121314151617B. adeliensis from the melting sea ice to underlying sediments, even if subsequent dissolution of its silica frustules is significant. By combining the intracellular concentration of IPSO25 in B. adeliensis determined in the current study with its abundance distribution in surface sediments, we estimate that the corresponding contribution of B. adeliensis falls within the range ca. 102–105 cells per gram, which is lower than typical total diatom cell concentrations in Antarctic sediments (ca. 105–107 cells per gram)494525 likely offers complementary information to taxonomic-based sea ice reconstructions, not least, since a clear distinction can be made between the respective signatures of IPSO25 and commonly employed sea ice diatom taxa in sediments. Thus, IPSO25 is biosynthesized by at least one individual diatom species (B. adeliensis) that resides and blooms within the sea ice matrix itself, and therefore represents a proxy measure of late spring/summer (permanent) sea ice. In contrast, the frequently used diatom taxa for palaeo sea ice reconstruction  abundance changes in IPSO25, even for sediments from proximate locations within each of the AP, the Weddell Sea and Ross Sea. For example, the concentration ranges of IPSO25 are 34–750, 150–1,200, 37–1,100 and 8–1,800 ng g−1 in Marguerite Bay (AP), the NW and SE Weddell Sea, and the Ross Sea, respectively.In previous studies, semi-quantitative interpretations of temporal changes to sea ice conditions have been inferred from the variability of IPSO25 abundances and consideration of the ecology of B. adeliensis may offer particularly useful insights. For example, unlike the source diatoms of IP25 in Arctic sea ice, which have a generally consistent contribution of ca. 1–5% of the total diatom taxaB. adeliensis in Antarctic sea ice diatom communities is highly variable, both spatially and temporally, with seasonal shifts in composition also having been observed in previous studies34B. adeliensis represented the major taxon in our sea ice samples from the WAP, but much lower percentages (and absences) have been reported from the same353625 may be much more strongly influenced by variations in Antarctic sea ice diatom assemblages than is the case for IP25 in the Arctic.We suggest that a number of features other than sea ice concentration and/or duration likely have influence over IPSOB. adeliensis is also known to flourish in platelet ice, where diatom communities, in general, can proliferate to yield significantly elevated biomass compared with consolidated ice or other sea ice forms5556Porosira pseudodenticulata are known to have higher sinking rates compared with solitary pennate species in the AntarcticB. adeliensis from sea ice to the underlying sediments, despite its general fragility towards dissolution may, therefore, reflect its propensity to form elongated strands and mats within platelet ice that have elevated sinking rates. Abundances of B. adeliensis (and thus IPSO25) may thus potentially provide a sensitive proxy indicator of platelet ice, whose occurrence and concentration is commonly associated with the provision of super-cooled low-density sub-surface water derived from nearby ice shelves556061B. adeliensis and IPSO25, with elevated abundances of both for coastal locations, is consistent with ice shelves occupying almost half of the Antarctic coastline and enhanced platelet ice formation within the trajectory of super-cooled surface waters25 by B. adeliensis is not restricted to platelet ice, however, since both have been observed in samples of bottom ice, including those analysed as part of the current study. In fact, production of IPSO25 in both platelet ice and consolidated bottom ice communities may potentially explain some of the differences in one of the frequently cited characteristics of IPSO25 in Antarctic settings, namely, its relatively enriched 13C content (compared with pelagic organic carbon), a feature found previously for other individual lipids and bulk organic matter derived within sea ice57636465662 limitation represents the general consensus view, which effectively partially reverses the photosynthetic preference for 12C assimilation pertinent to CO2-replete, open water environments, resulting in increased (less negative) δ13C values57636465662 concentrations in the brine channels of sea ice13C with greater distance from the ice/water interface2 potentially becomes depleted. It has also been demonstrated that interstitial CO2 drawdown has a significant impact on δ13C for particulate organic carbon (POC)13C and CO2 supply/demand is probably complicated by variable (and inconsistent) nutrient exchange13C for sea ice-derived POC576364656613C enrichment in some cases576313C data for IPSO25 are consistent with significant enrichment of 13C within a relatively enclosed  sea ice matrix, with δ13C=−5.7 to −8.5‰ in sea ice13C=−17.8‰) was found for IPSO25 in surface sediment material from Adélie land, East Antarcticaet al.25 at a time of sea ice melt, when the ice matrix would probably have been more permeable to CO2 and nutrient replenishment. However, given our new findings, we suggest that a more likely explanation for the lighter isotopic composition of IPSO25 in Adélie Land sediment is biosynthesis of this biomarker by B. adeliensis in the relatively open channels of platelet ice, with comparably free supplementation of CO2 from surrounding waters. An alternative suggestion, involving production of IPSO25 in CO2-replete open waters following ice melt seems unlikely given the sensitivity of B. adeliensis towards dissolution in the water column. Consistent with our suggestion, Thomas et al.13C enrichment in POC in the interstitial waters of platelet ice from the eastern Weddell Sea (highest δ13C=−20.9‰) with a mean δ13C value (−24.0‰) only slightly higher than that for the underlying open water (δ13C=−25.6‰). On the other hand, a relative enrichment in 13C for IPSO25 in sediments from Ellis FjordB. adeliensis is known to colonize for this region13C values for IPSO25 in selected surface sediments from the WAP, the northern AP and the SE Weddell Sea, were in the range ca. −13.5 to −15‰ , and thus indicative of a semi-enclosed sea ice host. Interestingly, IPSO25 abundances in these samples were also among the highest across all samples, consistent with the higher biomass generally associated with platelet ice5556The production of IPSOC value −.0‰ only B. adeliensis (and thus of IPSO25) towards nearby glacial and ice shelf water inflow, we provide potentially new insights into the impacts of past changes to such processes by re-inspection of some previous palaeo sea ice reconstructions based on IPSO25 (termed diene II in previous reports). For example, we note that enhanced IPSO25 in sediment core JPC24 from Prydz Bay (East Antarctica) between ca. 10.9 and 10.4 cal. kyr BP  record from the WAP since ca. 1950 AD , during which time, sea ice diatom indicator species  were also relatively abundant, either suggests that a different source may be responsible for IPSO25 biosynthesis in such settings, or that B. adeliensis is also able to colonize sea ice for non-coastal marine settings, especially for those under the influence of nearby glacial meltwater.Finally, although most of the previous Antarctic studies incorporating IPSOediments . The occ25 in the Antarctic sea ice diatom B. adeliensis likely ensures that future interpretations of the sedimentary occurrence of this sea ice proxy can be made with greater confidence and in more detail. Thus, in addition to representing a qualitative measure of the past occurrence of Antarctic landfast ice during late spring/summer, our findings indicate that variability in sedimentary IPSO25 potentially provides further insights into changes to ice shelf and glacial melt processes in long-term records. Further, the determination of the stable isotopic composition (δ13C) may also be particularly enlightening for determining the structural characteristics of the sea ice from which IPSO25 was derived . However, measuring the IPSO25 content in a larger number of sea ice diatoms and in sea ice of different types and from other regions is required before the interpretation of its precise sedimentary signature can be fully deciphered.In conclusion, the identification of the organic geochemical biomarker IPSOTwo landfast sea ice cores were collected from Ryder Bay, situated close to the British Antarctic Survey Rothera Research Station  on 3 December 2014. Bottom sections (ca. 10 cm) of each core were sliced from the main cores and left to melt in the dark at 4 °C. Aliquots of the thawed samples were then filtered , with the remaining material re-frozen and stored at −20 °C until further use. Surface sediment material from the Antarctic Peninsula, the Bellingshausen Sea, the Amundsen Sea and the Weddell Sea was taken from the upper 0–1 cm of archived box cores, multi-cores and gravity cores held at the British Antarctic Survey, the British Ocean Sediment Core Research Facility  and the Alfred Wegener Institute for Polar Marine Research . Additional sediment samples from the AP and the Ross Sea were collected during several R/V ARAON cruises between 2001 and 2015. All surface sediment material was assumed to represent accumulation during the modern era.Berkeleya adeliensis Medlin was identified using scanning electron microscopy (SEM) at the University of Plymouth following the detailed description of MedlinoC) followed by washing of frustules (3 × 4 ml H2O). Cleaned frustules were then dried onto glass and sputter-coated (Cr) prior to observation (JEOL 7001F SEM). Morphometric assessment, including elongated central area, apical and transapical length , number of parallel striae in 10 μm (50) and asymmetric axial area adopting a distinct urn-shape around the simple helictoglossa, support our identification. Individual diatom cells for isolation were identified using a Nikon TS2000 inverted light microscope ( × 10 and × 40 objectives) in phase contrast and isolated manually using a modified Pasteur pipetteThe tube-dwelling B. adeliensis and freeze-dried sediments, using previously published methods2125B. adeliensis, a total hexane extract only was obtained . In each case, the resulting total hexane extract suspensions were filtered through pre-extracted (dichloromethane/methanol) cotton wool to remove cells before being partially dried (N2 stream) and fractionated into non-polar lipids by column chromatography (hexane (3 ml)/SiO2). For sediments, ca. 1 g of freeze-dried sediment material was extracted by sonication  to obtain a total organic extract. Each total organic extract was partially purified to remove polar components, elemental sulphur and saturated non-polar components using silver-ion chromatography25 achieved by comparison of its retention index and mass spectrum with those obtained from a purified standard25 was achieved by integrating individual ion (m/z 348.3) responses in single-ion monitoring mode, and normalizing these to the corresponding peak area of the internal standard and an instrumental response factor derived from a purified standard25 were converted to sedimentary concentrations using the mass of sediment extracted, while a cellular concentration of IPSO25 in B. adeliensis was obtained by dividing the normalized GC–MS response obtained from picked cells by the number of cells extracted.At the University of Plymouth, HBI lipids were extracted from thawed and filtered sea ice samples, picked cells of 13C) of IPSO25 was determined using gas chromatography–isotope ratio mass spectrometry (GC–IRMS) at Isoprime Ltd, Cheadle Hulme, UK. All GC–IRMS measurements were performed using an IsoPrime100 IRMS with GC5 interface and Agilent 7890B GC installed with an Agilent HP-5MS column . All samples were dissolved in hexane (10–150 μl) and injected into splitless mode with the following inlet conditions: 250 °C, purge flow 25 ml min−1, purge time 0.75 min. GC carrier gas (He) flow rate was 1 ml min−1, oven program as follows: 1 min hold at 50 °C, ramp to 310 °C at 10 °C min−1, then 13 min hold. The combustion furnace consisted of a 0.7 mm inner diameter quartz tube packed with CuO pellets, held at 850 °C. GC–IRMS results were calibrated using the certified Indiana alkane standard mix A5  and all results are thus reported in delta notation (δ13C) relative to VPDB. IPSO25 was identified in GC–IRMS chromatograms by retention time comparison with corresponding GC–MS analyses. IonOS software (Isoprime Ltd) was used to process GC–IRMS data; ‘peak mapping' functionality was used to systematically designate specific compound identifications across multiple injections for robust data collation. The A5 alkane mix was analysed between every six sample injections in at least duplicate, with calibrations constructed from at least three interspersed measurements of the A5 mix. Reproducibility of individual alkanes was always ≤0.35‰. Root mean standard error (RMSE) of each of the calibrations was usually≤0.25‰, with an overall RMSE for all calibrations combined of ≤0.21‰, reflecting both the reliability of each calibration, and the long-term stability of the system. Samples containing IPSO25 were run in triplicate.The stable (carbon) isotopic composition (δ25 concentrations and study locations referred to in the text are displayed in Distributions of IPSOThe data that support the findings of this study are available from the corresponding author (S.T.B.) and within the article and its How to cite this article: Belt, S. T. et al. Source identification and distribution reveals the potential of the geochemical Antarctic sea ice proxy IPSO25. Nat. Commun. 7:12655 doi: 10.1038/ncomms12655 (2016).Supplementary Figure 1 and Supplementary Table 1


High consumption of dietary flavonoids might contribute to a reduction of cancer risks. Quercetin and its glycosides have PARP inhibitory effects and can induce selective cytotoxicity in BRCA2-deficient cells by synthetic lethality. We hypothesized that common flavonoids in diet naringenin, hesperetin and their glycosides have a similar structure to quercetin, which might have comparable PARP inhibitory effects, and can induce selective cytotoxicity in BRCA2-deficient cells. We utilized Chinese hamster V79 wild type, V-C8 BRCA2-deficient and its gene-complemented cells. In vitro analysis revealed that both naringenin and hesperetin present a PARP inhibitory effect. This inhibitory effect is less specific than for quercetin. Hesperetin was more cytotoxic to V79 cells than quercetin and naringenin based on colony formation assay. Quercetin and naringenin killed V-C8 cells with lower concentrations, and presented selective cytotoxicity to BRCA2-deficient cells. However, the cytotoxicity of hesperetin was similar among all three cell lines. Glycosyl flavonoids, isoquercetin and rutin as well as naringin showed selective cytotoxicity to BRCA2-deficient cells; hesperidin did not. These results suggest that flavonoids with the PARP inhibitory effect can cause synthetic lethality to BRCA2-deficient cells when other pathways are not the primary cause of death. O-glycoside [O-glycodide, is a natural compound found in lemons and oranges [Flavonoids are naturally occurring chemicals in plants. They consist of 15 carbon atoms of three rings, two of which are benzene rings connected by a chain with three carbons. Flavonoids present in this form are called aglycones, and can be glycosylated. Quercetin is the aglycone form of a number of other flavonoid glycosides, such as isoquercetin (glycosyl form) and rutin (rhamno-glycosyl form), which are found in citrus fruit, buckwheat and onion ,3. Narinlycoside . Hespere oranges ,7,8,9. O oranges ,11.Synthetic lethality arises when the combination of two or more genes leads to cell death, but a mutation in only one of these genes does not, and remains viable by itself. Thereby, a synthetic lethality approach to cancer is currently being explored as a means of developing therapies that reduce off-target effects of chemotherapies. Inhibition of PARP can induce synthetic lethality in homologous recombination-deficient cells . TherefoAlthough the PARP inhibitory effect of quercetin and its glycosyl flavonoids have been identified in in vitro cell culture ,11, this50 = 5 nM) [Quercetin, isoquercetn, rutin, naringenin, naringin, hesperetin, and hesperidin  were pur = 5 nM) , was purHT Universal Colorimetric PARP Assay Kit  was used to assess the capacity of PARP inhibition. Solutions were prepared per the directions provided with the kit from the manufacturer. The histone-coated strip wells were rehydrated using 50 µL of a 1X PARP buffer, and were stored at room temperature. Then, 5 µL of the desired dilutions of the testing compounds and 7.5 µL of PARP enzyme (0.5 unit/well) solution were added to the wells. This was incubated at room temperature for 10 min before the addition of 12.5 µL PARP cocktail, the wells were then left at room temperature for 1 h before washing thoroughly with PBS and PBS with 0.1% Triton X-100. After washing, 50 µL Strep-HRP was added to each well. Again, the wells were incubated at room temperature for 1 h before repeating the washing process. After the wells were washed and dried, by patting on top of a paper towel, 50 µL of TACS-Sapphire was added, and then the wells were placed inside a drawer at room temperature for 15 min before adding 50 µL of 0.2M HCl. The resulting absorbance at 450 nm was read with a Bio-rad benchmark Microplate reader .Chinese hamster V79, BRCA2-deficient mutation V-C8 ,22, humaFive hundred cells were plated in P-60 dishes. Two hours after cell plating, a designated amount of flavonoids were added and kept in the incubator for one week. One week later, cells were fixed with 100% Ethanol and stained with 0.1% Crystal Violet solution. In microscopic observation, colonies containing more than 50 cells were designated as survivors. Survival fraction was calculated by dividing the colony number of drug treated samples by the colony number of control samples for each cell line.p values lower than 0.05 were regarded as statistically significant.All experiments were carried out more than three times, independently. Statistical significance was calculated by GraphPad Prism 6 program  with two-way ANOVA and multiple comparisons with Tukey test. Multiple comparison-adjusted p < 0.0002). As predicted, it was identified that naringenin and hesperetin showed PARP inhibitory effects and significant inhibition at 1000 µM (p < 0.0001). Statistically, quercetin showed the strongest reduction of PARP activity among three aglycone flavonoids at 100 (p < 0.0001), 300 (p < 0.0001), and 1000 (p < 0.001) µM concentrations. Hesperetin and naringenin showed similar PARP inhibition effects (p > 0.58).PARP activity of aglycone flavonoid was analyzed in vitro A. It wasp < 0.0089). Isoquercetin, hesperidin and naringin showed a slight reduction of PARP activity, but this was not statistically significant (p > 0.2) compared to the control.Previous studies showed a low PARP inhibitory effect for glycosylated quercetin . Therefop < 0.05) and 50 µM (p < 0.0001). At 75 µM, gene-complemented cells also showed cellular death (data not shown).50, was approximately 25 µM for V79 cells. There was no significant elevated cytotoxicity for V-C8 cells. On the other hand, naringenin showed a similar cell death pattern compared to quercetin. At 75 µM, V-C8 cells showed statistically significant severe cytotoxicity to naringenin compared to V79 and gene-complemented mutant cells (p < 0.001). At 100 µM, no cell lines formed any colonies. Olaparib, a PARP specific inhibitor, showed clear cytoxocicity in BRCA2-deficient cells.Hesperetin was the most cytotoxic among the tested agents in this study. A 50% cell death value, ICFlavonoids are naturally occurring in plants. Varieties of biological activities were identified and some might explain the potential health benefits from a high intake of flavonoids by one’s daily diet and supplemental intakes. Series of flavonoids have shown anti-cancer effects by anti-proliferation, pro-apoptosis, and anti-inflammatory mechanisms ,7,8. TheIn addition to the previously reported quercetin, isoquercetin and rutin, this study revealed that naringenin and naringin presented elevated cytotoxicity in BRCA2-deficient cells due to the PARP inhibitory effect . Since tThis paper identified that naringenin and naringin, which are rich in orange and grapefruit, also showed similar PARP inhibitory effects and BRCA2 deficiency specific cytotoxicity. However, hesperetin and hesperidin with PARP inhibitory effects did not show BRCA2 deficiency dependent cytotoxicity. Hesperetin was the most toxic chemical among the three aglycone flavonoids tested. Cytotoxicity of hesperetin was approximately three times greater than quercetin and naringenin . In vitrIn conclusion, this paper identified flavonoids other than quercetin and its glycosides to have PARP inhibitory activity and selective cell toxicity in BRCA2-deficient cells through synthetic lethality. In addition to quercetin, other flavonoids showed glycosylation-reduced PARP inhibitory activity and cellular lethality. Further research should be done to investigate in vivo synthetic lethality-induced chemoprevention and the PARP inhibitory mechanisms of these chemicals.


A viable vascular endothelial layer prevents vasomotor dysfunction, thrombosis, inflammation, and intimal hyperplasia. Injury to the endothelium occurs during harvest and “back table” preparation of human saphenous vein prior to implantation as an arterial bypass conduit. A subfailure overstretch model of rat aorta was used to show that subfailure stretch injury of vascular tissue leads to impaired endothelial-dependent relaxation. Stretch-induced impaired relaxation was mitigated by treatment with purinergic P2X7 receptor (P2X7R) inhibitors, brilliant blue FCF (FCF) and A740003, or apyrase, an enzyme that catalyzes the hydrolysis of ATP. Alternatively, treatment of rat aorta with exogenous ATP or 2’(3’)-O-(4-Benzoyl benzoyl)-ATP (BzATP) also impaired endothelial-dependent relaxation. Treatment of human saphenous vein endothelial cells (HSVEC) with exogenous ATP led to reduced nitric oxide production which was associated with increased phosphorylation of the stress activated protein kinase, p38 MAPK. ATP- stimulated p38 MAPK phosphorylation of HSVEC was inhibited by FCF and SB203580. Moreover, ATP inhibition of nitric oxide production in HSVEC was prevented by FCF, SB203580, L-arginine supplementation and arginase inhibition. Finally, L-arginine supplementation and arginase inhibition restored endothelial dependent relaxation after stretch injury of rat aorta. These results suggest that vascular stretch injury leads to ATP release, activation of P2X7R and p38 MAPK resulting in endothelial dysfunction due to arginase activation. Endothelial function can be restored in both ATP treated HSVEC and intact stretch injured rat aorta by P2X7 receptor inhibition with FCF or L-arginine supplementation, implicating straightforward therapeutic options for treatment of surgical vascular injury. Human saphenous vein (HSV) is harvested from the leg and transplanted as a bypass graft into the coronary or peripheral circulation. Vein graft failure rates remain high   for 1 hr at room temperature.RA was manually stretched longitudinally to 200% the  earlier . Stretch2 / 5% CO2 at 37°C for 1 hr. Rings were manually stretched to 3 g of tension, and maintained at a resting tension of 1 g for an additional 1 hr. This produced the maximal force-tension relationship as previously described [5 Newtons (N)/m2] = force (g) x 0.0987/area, where area is equal to the wet weight [(mg)/length ] divided by 1.055, which was calculated using the force (g) generated by the tissue. Percent relaxation was calculated as a change in stress compared to the maximal PE-induced contraction which was set as 100% as described earlier [Rings of RA  were suspended in a muscle bath containing a bicarbonate buffer , equilibrated with 95% Oescribed , 21, 22.escribed . Force m earlier .2 incubator and were passaged at 80% confluence. Cells between passages 2 and 5 were used in the experiments.Human Saphenous Vein Endothelial Cells obtained commercially  were grown in Endothelial Cell Growth Medium , maintained in a 37°C and 5% COG-Nitroarginine methyl ester , ATP (2 mM) plus FCF (100 μM), ATP (2 mM) plus L arginine (2 mM), or ATP (2 mM) plus Nω –hydroxy-nor-Arginine , in growth medium diluted to 50% with basal medium for 1 hr. At the end of the treatment, cells were stimulated with CCH (1 μM) for 10 min and nitric oxide generated was measured as nitrite using the nitric oxide assay kit  per the manufacturer’s protocol. Relative percent of NO generated was calculated. NO generated with CCH stimulation was set as 100%.HSVEC were plated in 60-mm dishes and grew to confluency of >70%. Cells were either left untreated or treated with ATP (2 mM), L-NHSVEC were plated in 60 mm dishes and cells at 70% confluency were treated with ATP , BzATP  or ATP with 10 μM PD 98059 in culture medium diluted to 50% with basal medium for different duration indicated). To examine the effect of inhibitors, cells were pretreated with the inhibitors for 1hr prior to treating with ATP for 30 min. Proteins were extracted using lysis buffer containing 50 mM Tris. Cl pH 7.4, 140 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM EGTA, 0.5% deoxycholic acid with protease and phosphatase inhibitor cocktail. The proteins were separated using SDS-PAGE and transferred onto nitrocellulose membrane, followed by immunoblotting with antibodies against phospho-p38 MAPK Thr180/Tyr182 , p38 MAPK , phospho ERK1/2 . ERK1/2 , phospho SAPK/JNK (T183/Y185) and pan SAPK/JNK . P2X7R expression was determined by extracting proteins using lysis buffer followed by a urea chaps buffer  from untreated HSVEC and human HEK 293 cells using anti P2X7R antibodies . Specificity was determined by probing the membrane with the same antibodies preabsorbed with the immunogen peptide. Protein-antibody complexes were visualized and quantified using the Odyssey Infrared Imaging System. Phosphorylation was calculated as a ratio of the phosphorylated protein to total protein and was then normalized to the ATP-stimulated control with the value set as 100%.P value) of experiments. P value <0.05 was considered statistically significant.Data were reported as standard error of the mean (SEM) responses. Paired t-tests or One-way ANOVA analyses with Tukey’s Multiple Comparison test between columns were conducted in order to determine the significance . eATP dose consistent with intracellular ATP levels  (10 mM) To focus specifically on endothelial cell responses, human saphenous vein endothelial cells (HSVEC) were used. Protein lysates from different passages (2–5) were examined for the expression of P2X7R by immunoblotting. A major band at 75 KDa was identified along with a minor band above 100KDa . CCH stiP2X7R activation in neural injury is associated with activation of p38 MAP kinase (p38 MAPK) –31. To dTo confirm that p38 MAPK activation was due to activation of P2X7R, HSVEC were pretreated with FCF. As a control, HSVEC were also pre-treated with the p38 MAPK inhibitor, SB 203580. The cells were then treated with eATP for 30 min and phosphorylation of p38 MAPK was determined. eATP-induced phosphorylation of p38 MAPK was inhibited by FCF  and by the p38 MAPK inhibitor, SB 203580  and c-JUN NH2-terminal protein kinase  in response to ATP treatment were also examined. HSVEC were treated with 2 mM ATP for different time points and activation of ERK and SAPK/JNK was determined by western blotting. ATP induced a transient increase in the phosphorylation of ERK1/2 at 10 min  or L-arginine, ATP-induced decrease in CCH-stimulated NO production was prevented by NOHA (89 ± 15%) and L-arginine (86 ± 9%) when compared to ATP (50 ± 8%), p<0.05, Since exogenous ATP and P2X7R/p38 MAPK activation lead to endothelial dysfunction by either decreased endothelial NO synthase (eNOS) activity or increased arginase activity, HSVEC were first treated with eATP and eNOS phosphorylation was determined. ATP treatment did not alter eNOS phosphorylation in HSVEC . L-arginTo determine whether impaired endothelial function after stretch injury-induced activation of P2X7R was due to activation of the p38 MAPK pathway and arginase activation in intact tissue, stretch-injured RA tissue was treated with SB 203580, NOHA, or L-arginine. Endothelial-dependent relaxation was restored after treatment with SB 203580 , NOHA , and L-arginine  [−9 to 10−8 M) [Impaired endothelial-dependent relaxation was recapitulated by treatment of RA with exogenous ATP , suggesto10-2 M)  and the  10−8 M) . Thus, t 10−8 M) , 38.We h 10−8 M) .50 of 300–800μM) [ATP stimulation of P2X7R leads to ATP release from P2X7R [P2X7R are activated by ATP (EC0–800μM)  and are om P2X7R , 41. Witom P2X7R . P2X7R aom P2X7R . P2X7R wom P2X7R , 43–47. om P2X7R  suggestiom P2X7R  and it iFinally, P2X7R activation is a potent inflammatory stimulus , 50. ThuATP-mediated inhibition of NO release  was assoin vitro and in animal studies [A growing list of P2X7R antagonists have been used successfully  studies , 56. Ant studies , 50, 57. studies . Two com studies , 60. Tarpathologic injury and not physiologic mechanical forces such as those that occur with arterialization of the graft after implantation. In addition, the subfailure overstretch RA model represents a well characterized, inexpensive, high throughput model and while injury of both HSV and PSV [potentiate the response to injury.Limitations of this study include the use of an arterial tissue model (RA). The RA stretch model is a model of  and PSV , 6, 12 l and PSV , apoptos and PSV  in other and PSV  and incr and PSV , and theTaken together, these data suggest that stretch injury during surgical harvest leads to release of ATP, activation of P2X7R/p38 MAPK, and inhibition of NO production by activation of arginase , which rS1 Fig−7 M) and treated with carbachol (5 x 10−7 M). Representative tracing of control  compared to ATP-treated rings .RA rings were suspended in the muscle bath and the vessels were treated with eATP for 1hr, and were pre-contracted with phenylephrine (5 x 10(TIF)Click here for additional data file.S2 FigA, anti P2X7 R antibodies preabsorbed with peptide (preabsorbed) or B, anti P2X7 R antibodies (P2X7 R). Representative western blot of P2X7 R, N = 4 different passages.HSVECS  or HEK cells were lysed and proteins were separated by SDS PAGE and transferred to nitrocellulose membrane and probed with (TIF)Click here for additional data file.S3 FigHSVEC were either untreated or treated with 2 mM ATP for 2 hr and were stimulated with 1μM carbachol for 10 min at 37°C. Cells were scraped in cold assay buffer and nitric oxide generated was measured as nitrate using the kit (Abcam). * Significant compared to untreated CCH stimulated, n = 4, p<0.05,(paired t-test).(TIF)Click here for additional data file.S4 FigA) Representative western blots of phospho p38MAPK, and p38 MAPK, (B) Cumulative data showing the relative fold phosphorylation of p38 MAPK with respect to zero time point, n = 4–5 passages, * p < 0.05,.(paired t-test).HSVEC were untreated (0), treated with ATP (2 mM) for various time points  and phospho p38 MAPK (P-p38) and total p38 (p38) proteins were quantitated with immunoblotting (adjusted to the loading control GAPDH). ((TIF)Click here for additional data file.S5 FigHSVEC were either untreated (control) or treated with BzATP (0.25 mM and 0.5mM) in 50% growth medium diluted with basal medium for 2 h. Proteins were extracted and separated on 4–20% criterion gels and transferred to nitrocellulose. Phospho p38 and p38 proteins were identified by western blot analysis using antibodies to phospho p38 and total p38 . n = 3, * p<0.05 between control and 0.5 mM BzATP, ns = not significant, (t-test).(TIF)Click here for additional data file.S6 FigHSVEC were untreated (Ctrl), treated with ATP (2 mM) or ATP with PD 98059  20 minutes) and phospho p44/42 MAPK (P-ERK1/2) and total p44/42 (ERK1/2) proteins were quantitated with immunoblotting (adjusted to the loading control GAPDH). Representative western blot, N = 3.(TIF)Click here for additional data file.S7 FigHSVEC were untreated (0) treated with ATP (2 mM) for various time points  and phospho SAPK/JNK (P-p54/p46 SAPK/JNK) and total JNK (SAPK/JNK) proteins were quantitated with immunoblotting (adjusted to the loading control GAPDH). Representative western blot, N = 3.(TIF)Click here for additional data file.S8 FigHSVEC were either untreated (Ctrl), or treated with ATP (1 and 2 mM) for 2hour. Phospho eNOS and total eNOS were identified by western blot analysis using antibodies to phospho eNOS and total eNOS and adjusted to the loading control GAPDH. n = 4 passages, ns, not significant, * p >0.05, for 1 mM and 2 mM, respectively, (t-test).(TIF)Click here for additional data file.


Every spring, many of amphibians are killed by motor vehicles on roads. These road-killed animals can be used as valuable material for non-invasive studies showing the effect of environmental pollution on amphibian populations. The aims of our research were to check whether the phalanges of road-killed toads may be useful as material for histological analysis, and whether various degrees of human impact influence the level in bone abnormalities in the common toad. We also examined whether the sex and age structure of toads can differ significantly depending in the different sites. We chose three toad breeding sites where road-killed individuals had been observed: near the centre of a city, the outskirts of a city, and a rural site. We collected dead individuals during spring migration in 2013. The sex of each individual was determined and the toes were used to determine age using the skeletochronology method. While performing age estimates, we looked for abnormalities in relation to normal bone tissue structure. In urban site, females dominate males (sex ratio 2.6:1), but in populations from rural and semi-urban sites, sex ratio was reverse . However, we did not find any significant differences between age structure of all populations (average age of each population: approximately 4 years). We observed abnormalities in more than 80 % of all toads from the city, compared to approximately 20 % from the rural and semi-urban sites. In particular, we found hypertrophic bone cells, misaligned intercellular substance, and irregular outer edges of bones. We suggest that these malformations are caused by different pollution, e.g. with heavy metals. Current knowledge widely indicates a direct relationship between anthropogenic pressures and the deteriorating environmental state and/or global extinction of amphibian populations  influence the level in bone abnormalities in the common toad. Our hypothesis assumes that toads occurring at an urban site are more prone to the formation of anomalies in bone tissue than those from rural localities. We also examined whether the sex and age structure of toads can differ significantly depending in the different sites.Site 1.—the most polluted urban site was ‘Kajka’, situated near the center of the Poznań city . In the immediate vicinity, there are located an illegal settlement and degraded allotment gardens without sanitary sewer lines. Furthermore, there are two railway lines, and on the east, there is a forest area (approximately 0.5 km from the pond). In ‘Kajka’ site, there is a large proportion of industrial plants in the catchment basin. The pond and surrounding settlement are contaminated by factory halls and storage sites located in the vicinity. Presence of heavy metals and petroleum hydrocarbons have been revealed in sediments which pose a threat for aquatic environment . In the nearest vicinity of the pond, there are farmlands, meadows, fallow lands and small forest. Among the anthropogenic structures, we distinguished railways, roads, a military air base and a few houses—a remnant of the old farms. On the east, the railway line forms a barrier between the surrounding area of toads’ breeding site and the area occupied by residential houses—the developed area. There is no direct discharge of water surface to the pond, but in the whole area, there are drains with the system of wells which causes water seepage. Aquatic vegetation is systematically removed every year. Site 3.—the rural site, natural vegetation (meadows) and a pond located in the forest, are situated near the Owiesno village . The catchment area is similar to the natural environment with the small watercourse flowing in the nearest vicinity of the breeding site. A slight extensive farm and local road with little car traffic is located within a radius of 1000 m is only. Very clean water in the pond promotes the development of freshwater plants.We chose three breeding sites of the common toad that characterised by varied in the degree of urbanisation and pollution. During the site selection, we analysed the use of catchment basin, in particular the ratio between the forest/tree cover with open green area compared to the paved surfaces  with buildings or industrial plants Fig. .. We evat Grabia . Occasiot Grabia . Site 2.As material, we used dead individuals collected from roads situated between their wintering area and breeding ponds (in the immediate vicinity of breeding pond less than 100 m). All toads were picked up during spring migration in March/April 2013. The sex of individuals was determined in the field by the nuptial pad in males and ovaries and eggs in females, as well as by body size and shape. Each toad was put to the single string plastic bag and transported to the laboratory. The age was determined by the skeletochronology method . We also tested frequency of bones abnormalities occurrence between males and females and between years of life using binomial generalised linear model (GLMM) in R. Confidence intervals for prevalence of anomalies (95 % CI) were computed using the Quantitative Parasitology application  the ratio was 1:2.2 and at the rural site (N = 41) 1:1.4 .For histological analysis, we randomly chose 100 individuals from the urban site  and all specimens from the remaining two populations. Five toads from the rural site and four from the semi-urban site were rejected from age estimating due to high concentration of anomalies in entire cross section of bone tissue. The average age of populations was similar: 3.7 years at the urban and rural sites, 3.8 at the semi-urban site Table . The youp < 0.05). Detailed analysis showed differences between urban and the other two sites (p < 0.05) but not between semi-urban and rural sites (p > 0.05). In the rural population, anomalies were found in 19 % of toads; in semi-urban, in 23 %; and in urban, in 82 % (Table p > 0.05) and years of life .Anomalies in bone tissue were observed at all studied sites, but significant differences in the frequency of their occurrence were found  clamitans. Also, Pelophylax ridibundus and Bufotes viridis showed altered sex ratios in anthropogenically polluted biotopes , cadmium (Cd), iron (Fe), copper (Cu), nickel (Ni), zinc (Zn)  in bone tissue. Simon et al.  indicateAdditionally, we found a relatively large proportion of individuals with abnormalities from the semi-urban and rural sites. In other agricultural site in Poland, high concentrations  of sulphur (Si) were found in the shafts of abnormal bones, but a negligible amount (0.3 %) in tissue without malformations (unpublished data). As well, low concentrations of molybdenum (Mo) and aluminium (Al) were found only in the epiphyses of impaired bones. Thus, it seems that other causes may also be responsible for these kinds of degeneration, but this requires further complex research.


Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated . Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca Phospho of cTnI   cascade, could mimic the functional effects of PKA by decreasing myofibrillar Ca2+ sensitivity via phosphorylation of cTnI Ser-23/24 and increasing cross-bridge kinetics by phosphorylation of cMyBP-C at Ser-302 . As Fig. 1A), with about 40% present as the monophosphorylated form (1P) and about 60% as the unphosphorylated form (0P). Unexpectedly, after incubation of the myofibrils with constitutively active PKD, the proportion of cTnI that was monophosphorylated increased to more than 90%, but there was no statistically significant increase in the proportion of the bisphosphorylated (2P) form. Because the cTnI was basally phosphorylated before kinase addition, the effects of PKD incubation on cTnI could not be unambiguously interpreted. We therefore dephosphorylated all subsequent myofibril preparations using λ phosphatase (λ-PP) prior to PKD incubation. This protocol  removed all visible endogenous phosphorylation from cTnI . Subsequent incubation with PKD induced exclusively mono-phosphorylation of cTnI, which was complete after 1 h of incubation. No statistically significant bisphosphorylation of TnI was observed even after 2 h of PKD incubation.We demonstrated previously that PKD phosphorylates cTnI at Ser-23/24 (and cMyBP-C at Ser-302) in isolated myofibrils , 8, 16. C). In the λ-PP–treated myofibrils, the proportions of un-, mono-, and bisphosphorylated forms of cTnI were 88%:10%:2%, respectively. After incubation of the myofibrils in PKD, the pattern changed to 5%:92%:3%, confirming that incubation of the myofibrils in PKD almost exclusively resulted in monophosphorylation of cTnI.As a check of the validity of these results, cTnI phosphorylation was assessed using a different technique: high-resolution MS C. In theA and Fig. S1, PKD induced monophosphorylation of cTnI, whereas PKA induced bisphosphorylation. There was no evidence for a trisphosphorylated state of cTnI, indicating that the preferred target for PKD was one of the “PKA” sites, i.e. Ser-23 or Ser-24. In support of this, an antibody against pSer-23/24 cTnI recognized the PKD-induced cTnI phosphorylation . Furthermore, myofibrils from transgenic cTnI-Ala2 mice  remained unphosphorylated after PKD or PKA incubations, confirming the identity of the phospho-acceptor residue(s) that are selectively targeted by these kinases . We conclude that PKD monophosphorylation occurs at the Ser-23/24 sites in cTnI. It may be noted that the phosphoserine 23/24 cTnI antibody was not selective for the bisphosphorylated form of the protein but detected both the mono- and bisphosphorylated forms (A and B), as reported previously (The above results provide no information about the cTnI amino acid residue(s) targeted by PKD. To examine whether the relevant cTnI site was one (or either) of the Ser-23/24 residues, λ-PP–treated myofibrils were incubated first with PKD and then with PKA. As shown in Fig. 2eviously .C). PKD induced monophosphorylation of cTnI in human myofibrils as in mouse myofibrils. There was substantial labeling of both the mono- and bisphosphorylated forms of human cTnI by the mAb14 antibody. Because there must be full phosphorylation of Ser-24 in the bisphosphorylated samples, the apparent similarity in the Ser-24 labeling intensities in the PKD-treated and PKA-treated myofibrils would suggest that most, or possibly all, of the monophosphorylation of PKD-treated cTnI is at Ser-24.To interrogate whether PKD targets Ser-23 or Ser-24, we used a phosphorylation-specific antibody (mAb14) that has been shown to selectively bind to pSer-24 in human cTnI . This anFig. 232P]ATP was performed to investigate whether other myofibrillar proteins were significantly phosphorylated by PKD (and PKA). The autoradiogram  indicates that cTnI and, to a lesser extent, cMyBP-C were the major myofilament proteins phosphorylated by either kinase.Autoradiography using [To investigate the functional consequences of cTnI monophosphorylation, we used the above dephosphorylation/phosphorylation protocol  to produ2+ sensitivity and cross-bridge kinetics (ktr) were measured in λ-PP–treated skinned trabeculae before and after PKD incubation and then again after incubation in PKA (Table S1). Time-matched controls were included (no kinases) because pilot experiments showed that there was a small but statistically significant decrease in Ca2+ sensitivity over the first hour or so of the experiment (B). The reason for this is unclear, but it may be related to a time-dependent reduction in titin-based passive force, which would decrease myofibrillar Ca2+ sensitivity  falling from 6.00 ± 0.04 to 5.68 ± 0.03 , a mean reduction of 0.32 ± 0.01 (D). In contrast, the subsequent incubation in PKA (performed in five experiments) produced only a small further decrease in pCa50 from 5.70 ± 0.01 to 5.59 ± 0.01 (n = 5), an additional decrease of 0.11 ± 0.01. If we subtract the mean pCa50 changes in the corresponding time-matched controls, then the time-independent fall in pCa50 is 0.24 ± 0.03 for PKD (E) and 0.09 ± 0.01 for PKA alone (after PKD). This suggests that most of the Ca2+ desensitizing action of PKA-mediated bisphosphorylation of cTnI can be mimicked by PKD-mediated monophosphorylation alone. The maximum Ca2+-activated force (Fmax) generated by the trabeculae was unaffected by the incubation in PKD (Table S1). Fmax showed a small but significant decrease with PKD+PKA incubation compared with that in dephosphorylated muscles, but this was not statistically significant compared with its corresponding time-matched control.Myofibrillar Caperiment B. The resitivity . Incubat2 ± 0.01 D. In con for PKD E and 0.02+ sensitivity was mediated by phosphorylation at cTnI Ser-23/24, the experiments were repeated with λ-PP–treated skinned trabeculae from cTnI-Ala2 transgenic mice. Fig. 3C shows that Ca2+ sensitivity showed little or no change after incubation of these muscles with PKD or PKD+PKA. The pCa50 fell by 0.10 ± 0.01 with PKD incubation, but this was not significantly different from the small decrease seen in the time-matched control . We conclude that the Ca2+-desensitizing action of PKD, like PKA, requires the presence of phosphorylatable Ser-23 or Ser-24 in cTnI.To check that the effect of PKD on Caktr to the same extent as produced by trisphosphorylation of cMyBP-C (at Ser-273/282/302) by PKA (ktr) varies with the degree of Ca2+ activation, i.e. with the relative force  by interpolation. The results are shown in Fig. S3 and Table S1. Although incubation in PKD alone tended to increase ktr50, this was not statistically significant . In contrast, subsequent incubation with PKA significantly increased relative ktr50 from 28.2% ± 3.6% (post-PKD) to 37.5% ± 2.1%. Similar results were found with experiments using cTnI-Ala2 mice . There was no change in ktr50 in WT time-matched controls. To investigate why PKD failed to accelerate cross-bridge kinetics significantly in this study, phospho-specific antibodies were used to quantify the magnitude and pattern of cMyBP-C phosphorylation by PKD or PKA . This confirmed our previous finding  of calpain-1 used. Calpain incubation produced two main degradation bands: the first band of a molecular mass of ∼22 kDa appeared with both 10 units and 30 units of calpain and sometimes was seen as a doublet; the second band was of lower molecular mass and became prominent only at the highest activity of calpain. Prior treatment of cTnI with PKD partially protected the full-length protein against subsequent calpain-induced proteolysis, particularly that seen at 30 units of calpain-1 (A and C). Partial protection from proteolysis was also observed when cTnI was first treated with PKD+PKA (B and C); in this case, about 50% of full-length cTnI was preserved after 30-min incubation in 30 units of calpain. The protective effects of PKD or PKD+PKA incubation were not significantly different (p > 0.05).Calpain has been implicated in the proteolysis of cTnI that can occur in cardiac disease. Phosphorylation of the isolated cardiac troponin complex by PKA slows the proteolysis of isolated cTnI by calpain . We invealpain-1 , A and C PKD+PKA , B and CD–F). The results confirmed that prior incubation in PKD or PKD+PKA partially protects cTnI from proteolysis (F). The C terminus antibody recognized the 22-kDa fragment(s) produced by calpain at 10 units or 30 units but not the lower-molecular-weight fragment seen with the polyclonal antibody at 30 units of calpain. This suggests that the first fragment retains the C terminus and thus is likely to be a product of N terminus degradation, whereas the second fragment lacks part or all of the C terminus amino acids 190–196, so is likely to be the result of proteolysis before or within this sequence. To explore the calpain proteolysis sites further, we used the pSer-23/24 antibody that recognizes pSer-23 and/or pSer-24 in the N terminus. This antibody recognizes full-length cTnI when the protein has been phosphorylated by incubation with PKD or PKD+PKA (C and D). The results  confirmed the gradual loss of the phosphorylated, full-length protein with calpain treatment, but the antibody failed to detect any of the degradation products, suggesting that both cTnI degradation products were missing the N-terminal sequence up to at least serines 23/24. Finally, we carried out the calpain experiments using myofibrils from cTnI-Ala2 mice . With these myofibrils, the loss of full-length cTnI and the appearance of one or two degradation bands (depending on the antibody used) were unaffected by prior incubation with PKD+PKA; that is, the protective effect of phosphorylation was absent. Together, these data suggest that calpain leads to N-terminal and then C-terminal proteolysis of cTnI in the myofibrils and that PKD-induced monophosphorylation or PKA-induced bisphosphorylation of cTnI serines 23 and/or 24 can partially protect against this proteolysis.Previous reports have documented evidence for cTnI proteolysis within the C terminus or N terminus or both. To investigate whether the degradation products in our study were the results of N- or C-terminal cleavage of cTnI by calpain, we repeated our experiments using an mAb specific for residues 190–196 in the C terminus of cTnI , D–F. Thteolysis F. The C  PKD+PKA , C and DFig. S7 shows that 30 units of calpain produced near-complete loss of the full-length cMyBP-C and appearance of the low-molecular-weight fragment with a time course similar to the proteolysis of cTnI. Prior treatment of dephosphorylated cardiac myofibrils with PKD alone did not affect the proteolysis of cMyBP-C , but this result is ambiguous because PKD incubation only partially phosphorylated Ser-302 in cMyBP-C . However, prior treatment with PKD+PKA provided a near-complete protective effect against calpain-induced cleavage , showing that PKA-mediated phosphorylation of cMyBP-C, like pseudophosphorylation, strongly protects this sarcomeric protein from calpain-mediated proteolysis.Calpain is also known to cleave cMyBP-C in the N-terminal C0C2 domain, resulting in an N-terminal fragment (∼40 kDa). Pseudophosphorylation of cMyBP-C at serines 273, 282, and 302 in intact hearts protects against cleavage . Fig. S7qPCR/PKD pathway may become increasingly important in heart failure (A). These agonists induced about 80% monophosphorylation of cTnI without any significant bisphosphorylation (B). In contrast, PKA activation by isoproterenol led to cTnI bisphosphorylation exclusively. Activation of PKD by PDBu or ET-1 also led to some phosphorylation of cMyBP-C at Ser-302, although the increase in phosphorylation level at this site was only 20–40% of that seen with activation of PKA . These results showed that, in intact cardiomyocytes, interventions that activate PKD induce monophosphorylation of cTnI, as was the case with isolated myofibrils exposed to exogenous PKD.The experiments above were conducted using constitutively active, recombinant PKD as a tool to induce monophosphorylation. Although PKD activity is low in healthy adult cardiac tissue, the G failure . To inve failure A. These rylation B. In con2+ sensitivity. Here we provide evidence that this is not the case and that phosphorylation of just one site can regulate myofilament Ca2+ sensitivity. This has implications for the regulation of contraction under basal conditions and during inotropic stimulation. We also show that both mono- and bisphosphorylation of cTnI at Ser-23/24 can reduce its susceptibility to degradation by calpain, thereby providing a potential protective role in cardiac pathophysiology.Mammalian cardiac TnI has multiple potential phosphorylation sites that are targeted by a variety of kinases . Conseque.g. about 1.3 mol/mol of cTnI (65% of maximum) (A), so it could be due to a basal activity of intracellular kinases. If functionally active, then this pre-existing phosphorylation would blunt the effects of exogenous kinases added experimentally. Prior attempts to fully dephosphorylate cTnI in the sarcomere using exogenously added phosphatases have been largely unsuccessful , provided that the incubation solution lacks EGTA (see supporting information). λ–PP requires Mn2+ for its activity  , 22. Howmaximum) , 23. Witmaximum) A, so it ccessful . We repoactivity , so the for Ca2+ , so evenfor Mn2+ . Given t by EGTA ) for cat2+ sensitivity , had depressed Ca2+ sensitivity substantially before any kinases were added; this would reduce the effects of kinases added subsequently. This illustrates the need for prior dephosphorylation if the full effects of phosphorylation on myofibrillar function are to be established.By putting the myofibrils into a “ground state” of zero phosphorylation in this way, we removed the confounding factor of pre-existing phosphorylation of the myofibrils. Subsequent incubation with PKD or PKA produced large decreases of myofibrillar Ca2+ sensitivity of skinned cardiac muscles, and we assumed that PKD, like PKA, acts via bisphosphorylation of Ser-23/24. This assumption was based on Western blotting with a pSer-23/24 antibody and on the absence of PKD's desensitizing effect in muscles from cTnI-Ala2 mice. For a more detailed and quantitative measurement of cTnI phosphorylation, in this work, we used Phos-tag gel analysis, which showed that PKD phosphorylates only one of the Ser-23/24 sites in mouse and human cardiac myofibrils . This concept originates from a study by Potter and co-workers (2+ sensitivity was not observed when either Ser-23 or Ser-24 (or both) was mutated to nonphosphorylatable alanine. We cannot explain why our results differ from this, although we note that, as judged by the recovery of force, troponin reconstitution was only 40–70% . The similarity in the intensity of staining for the PKD-treated cTnI and the PKA-treated cTnI (in which the Ser-24 site was fully phosphorylated because there was only a 2P band), suggests that the phosphorylation in the PKD-treated human cTnI was predominantly (or entirely) at Ser-24. This agrees with our previous in vitro work on PKD substrate preference using cTnI Ser→Ala mutants reconstituted into a troponin complex with WT cTnT and cTnC . cTnI monophosphorylation has also been observed in human hearts , basal monophosphorylation in intact myocytes would provide a tonic regulation of basal Ca2+ sensitivity; it would also lessen the magnitude of any changes during subsequent inotropic activation, e.g. of PKA during β1-adrenoreceptor stimulation. We predict that the rate of Ca2+ dissociation from TnC, which determines Ca2+ sensitivity and influences myofilament relaxation rate, is also controlled by cTnI monophosphorylation. Finally, the increased Ca2+ sensitivity in skinned preparations from failing or hypertrophic cardiomyopathy hearts is presumed to be due to the decreased bisphosphorylation of cTnI  because PKD produced near-complete conversion of the 0P to the 1P form but almost no conversion of the 1P to the 2P form . This is probably because only ∼40% phosphorylation of Ser-302 was observed, compared with ∼90% previously  (If the Caonserved ? One pot effect) . Another1–192 that may contribute to stunning by decreasing the force-generating capacity of myofibrils  suggested that the initial product resulted from cTnI N-terminal cleavage at a site after Ser-23; this was then further degraded within the C terminus to form the second product. This sequence is contrary to that found by McDonough et al.  found in intact hearts during simulated microgravity . Trisphosphorylation of cMyBP-C with PKA, like pseudophosphorylation, provided complete protection against this proteolysis, at least under the conditions of our experiment. We were unable to establish whether monophosphorylation of cMyBP-C (at Ser-302) by PKD also has a protective effect because PKD produced only a low level of cMyBP-C Ser-302 phosphorylation under the incubation conditions used.Like cTnI, cMyBP-C is degraded during myocardial ischemia, although in this case, proteolysis occurs in the N terminus, forming a 40-kDa C0C1f fragment that may act as a poison peptide . cMyBP-C2+ sensitivity and the susceptibility of cTnI to calpain-induced proteolysis. These effects are more similar functionally to those conferred by cTnI in its bisphosphorylated form, not in its unphosphorylated form.In summary, our results demonstrate that monophosphorylation of cTnI, primarily at Ser-24, affects both myofibrillar CaMethods section is provided in the supporting information.An expanded Experiments using mouse trabeculae and rat cardiomyocytes were performed in accordance with the Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (UK) and the Directive of the European Parliament (2010/63/EU) and received approval from the King's College London Ethics Review Board. Human samples were obtained after informed consent and with approval of the Human Research Ethics Committee of the University of Sydney . This investigation conformed with the principles in the Declaration of Helsinki (1997).2+ sensitivity and cross-bridge cycling kinetics have been described previously  and clamped to a force transducer and high-speed length controller. Force and cross-bridge cycling kinetics were measured at 18 °C in a series of calcium/EGTA activating solutions containing 0.2–30 μmol/liter free Ca2+ (pCa 6.7–4.5). Cross-bridge cycle kinetics were assessed by performing a rapid release–restretch maneuver to forcibly detach the cross-bridges; the ktr when cross-bridges reattach and generate force after the restretch was used as an index of cross-bridge kinetics (The methods for preparing mouse skinned trabeculae and for measuring their Caeviously . In brieeviously ) and werkinetics , 37.supporting information), the detergent-treated muscles or isolated myofibrils nevertheless exhibited substantial endogenous phosphorylation of cTnI (A). To remove this endogenous phosphorylation, we first incubated the skinned trabeculae in λ-PP. However, in pilot experiments we found that λ-PP was inhibited in relaxing solution, probably because of binding of its Mn2+ cofactor by EGTA (see “Discussion”). On the other hand, EGTA is normally required in relaxing solution to avoid a large and potentially damaging Ca2+-activated contracture caused by contaminant Ca2+. We therefore devised a novel dephosphorylation procedure in which the trabeculae were first put into a low-force rigor contracture by removing ATP in the presence of EGTA, and then EGTA was removed and λ-PP applied. Upon return to relaxing solution, the muscle and its sarcomeres showed no visible damage. This sequence of solutions, but performed instead with skinned myocyte fragments and myofibrils (see supporting information), led to almost complete dephosphorylation of cTnI (B and C) and cMyBP-C . The same protocol was used to prepare dephosphorylated myofibrils from left ventricular samples from human (unused donor) hearts.Although the mice were treated with a β-adrenoreceptor antagonist before euthanasia  after incubation in PKA catalytic subunit. The phosphatase inhibitor calyculin A was included in all solutions following λ-PP incubation. With this protocol, each trabecula served as its own control, allowing paired comparison of pre- and post-kinase data. Time-matched controls were included, in which λ-PP-treated muscles were subjected to the identical incubation protocols except that PKD and PKA were omitted.The overall experimental protocol consisted of first measuring the CaTM phosphate affinity SDS-PAGE. For one set of PKD-treated myofibrils, we assessed the phosphorylation of cTnI using high-resolution MS  by homogenization of ventricular tissue. Myofibrils were treated with the same dephosphorylation/phosphorylation protocols and solutions as used for the trabeculae. The extent and pattern of phosphorylation of cTnI and cMyBP-C in the myofibrils was measured by immunoblot analysis after either standard SDS-PAGE or Phos-tagution MS .AVRMs were isolated and infected with a PKD1WT-EGFP adenovirus or EGFP adenovirus control . After 4Cell Signaling Technology supplied the phosphospecific antibody against pSer-23/24 cTnI 4004) and the rabbit anti-TnI polyclonal antibody (4002) that was used to label total TnI. The phosphospecific antibody against pSer-24 of human cTnI was a kind gift from Steven Marston. A mouse anti-cTnI mAb, with an epitope of residues 190–196 of cTnI, was from Hytest (MF4). Phosphospecific antibodies against pSer-273, pSer-282, and pSer-302 of cMyBP-C (04 and thA recombinant adenovirus (AdV:PKDWT/EGFP) encoding full-length WT mouse PKD1 (PKDWT) and enhanced GFP (EGFP) downstream of separate cytomegalovirus promoters was as described previously .supporting data.PKA catalytic subunit  was from Calbiochem/Merck-Millipore, and λ phosphatase was from New England Biolabs . PKD was a recombinant PKD1 catalytic domain expressed in Sf21 insect cells; this is constitutively active in the absence of the autoinhibitory N-terminal domain . Calpaint test, as appropriate, when comparing data between two groups or by analysis of variance followed by the Bonferroni test when comparing data between multiple groups. p < 0.05 was considered statistically significant.Graphical statistics are shown as individual symbols and mean ± S.D.; some numerical data are listed as mean ± S.E. Statistical comparisons were by paired or unpaired Student's A. M.-G., M. A., and J. C. K. conceptualization; A. M.-G., Y. S., C. G. d. R., S. A.-G., W. C., Y. G., M. A., and J. C. K. data curation; A. M.-G., Y. S., C. G. d. R., S. A.-G., and J. C. K. formal analysis; A. M.-G., Y. S., C. G. d. R., S. A.-G., W. C., Y. G., M. A., and J. C. K. validation; A. M.-G., B. J. B., H. E. S., Y. S., C. G. d. R., S. A.-G., W. C., and M. A. investigation; A. M.-G., Y. S., C. G. d. R., S. A.-G., W. C., Y. G., and J. C. K. visualization; A. M.-G., B. J. B., H. E. S., Y. S., C. G. d. R., S. A.-G., W. C., M. A., and J. C. K. methodology; A. M.-G., B. J. B., H. E. S., Y. S., C. G. d. R., S. A.-G., Y. G., M. A., and J. C. K. writing-review and editing; B. J. B., H. E. S., Y. S., C. G. d. R., W. C., Y. G., M. A., and J. C. K. resources; B. J. B., C. G. d. R., Y. G., M. A., and J. C. K. funding acquisition; B. J. B., Y. G., M. A., and J. C. K. project administration; W. C., Y. G., M. A., and J. C. K. software; Y. G., M. A., and J. C. K. supervision; J. C. K. writing-original draft.


GenIce is an efficient and user‐friendly tool to generate hydrogen‐disordered ice structures. It makes ice and clathrate hydrate structures in various file formats. More than 100 kinds of structures are preset. Users can install their own crystal structures, guest molecules, and file formats as plugins. The algorithm certifies that the generated structures are completely randomized hydrogen‐disordered networks obeying the ice rule with zero net polarization. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. Water has a complex phase diagram as a single component small molecule. At least 17 ice polymorphs have been discovered so far.All the ice phases obey the ice rule of Bernal–FowlerA hydrogen bond network can be regarded as a directed graph. In graph theory terminology, an ice hydrogen bond network is a 2‐regular directed graph (each vertex has two incoming edges and two outgoing edges) if it obeys the ice rule.2,1,0.Water molecules also form clathrate hydrates and filled ices. They are solid solutions of water and hydrophobic small molecules.It is necessary to prepare a large number of properly hydrogen‐disordered arrangements of ice to calculate its properties in computer simulations. In this article, we introduce an open source software, GenIce, which enables one to generate homogeneous and sufficiently randomized hydrogen‐disordered ice structures at high speed. GenIce is designed to be extended easily by installing plugins for lattices, molecules, and output formats. See Appendix how to obtain the software.Stillinger first introduced an algorithm for making hydrogen‐disordered ice structures.3O+ ion and an OH– ion are generated by moving a hydrogen atom along the hydrogen bond. Next, a hydrogen atom of the H3O+ ion is moved along the hydrogen bond to transfer the protonic defect by the Grotthuss mechanism. The random proton transfer is continued until the protonic defect reaches the OH‐ ion generated initially. Since a long distance transfer of H3O+ is required to annihilate the defect pair, this algorithm is called the long‐loop algorithm.Barkema proposed a different method.Both of these algorithms start from a hydrogen‐ordered structure and invert the orientation of the hydrogen bonds randomly to obtain a hydrogen‐disordered structure. These methods may yield a nonisotropic and nonuniform structure due to improper selections of the initial configuration and insufficient randomization. Therefore, separate tests are necessary to certify that the generated structure is indeed sufficiently disordered.Unlike the conventional methods, GenIce uses totally randomized structures as initial states.Push all vertices that do not have two incoming bonds, which correspond to defects in ice, into a FIFO  queue.iN.Pop a vertex from the queue and count the number of incoming bonds for the vertex, iN is less than 2, choose an outgoing edge of the vertex randomly. If iN is greater than 2, choose an incoming edge of the vertex randomly. Otherwise, go to 7.If Invert the chosen edge.If the original vertex is still a defect, push it back to the queue.If the neighbor vertex becomes a defect by the inversion, push it to the queue.Go to 2 unless the queue is empty.We first generate a four‐regular undirected graph based on the hydrogen bond network topology of the ice phase, and make a directed graph from the undirected graph by replacing undirected edges with randomly oriented directed edges. Since the obtained randomly directed graph does not satisfy the ice rule, defects are purged by the following algorithm.Essentially, this algorithm is similar to the procedure proposed by Buch.a, b, or c, is always an integral multiple of a certain constant value for the hydrogen bond network that obeys the ice rule under periodic boundary conditions, and the polarization can be removed only by inverting homodromic cycles of hydrogen bonds spanning the cell. Random inversion of homodromic cycles is a common method to introduce randomness to hydrogen bond networks so as not to violate the ice rule. However, a randomly generated homodromic cycle hardly spans the cell neither by the short‐ nor by the long‐loop algorithm when the system is large. Therefore, more efficient algorithm is necessary to find cell‐spanning homodromic cycles.The procedure in step 1 certifies the uniformity of the obtained hydrogen bond network. However, usually, there remains a nonzero net polarization in the resultant hydrogen bond network. A component of the net dipole vector parallel to the direction of one of the primitive translational vectors, c axis as an example.i, randomly.Choose a vertex of the network, say a,b,c), the c‐antipode of the given point is . Find the vertex, say j, that is closest to the c‐antipode of i.When the fractional coordinate of a given point in the cell is written as .The procedure is applied to the genice 4 ‐‐water tip4p ‐‐rep 3 3 3 > ice4.groTo create a 3 × 3 × 3 unit cell replica of hydrogen‐disordered ice IV (4) in the Gromacs format with the TIP4P water model, enter the following command:See Table genice 4 ‐s 1234 ‐‐water tip4p ‐‐rep 3 3 3 > ice4.groDifferent hydrogen order can be obtained by specifying the random seed with the ‐s option.‐‐nodep avoids the depolarization process.By default, the hydrogen bonds are appropriately arranged to make the polarization be zero. The option ‐‐dens x option where x is the density of water in ice given in g cm−3 (the mass of guest molecules is not included for clathrate hydrates and filled ices).The density of ice is set automatically according to the shortest distance between water molecules. It can be specified by the ‐‐format option. Several output formats are available and they are listed in Table .gro format. GenIce can output .scad files for the OpenSCAD software which can generate input files for 3D printers. Figure The output style is specified with the genice T ‐‐rep 1 1 1 ‐‐format o > T.scad2 molecules in all “12” cages (512) and 50% of “14” cages (51262) of CS1 hydrate, add the “‐g” options as follows:genice CS1 ‐g 12=co2 ‐g 14=co2*0.5 > cs1.groOne can also prepare clathrate hydrates filled with guest molecules. For example, to stuff CO‐g option.genice ‐g 16=me*0.5+co2*0.3 ‐g 12=me* 0.9 CS2 > cs2.groThe option also accepts compositions for mixed hydrates. The syntax is (cage type)=(guest name)*(fill ratio)+(guest name)*(fill ratio)+…, for the There are only a few types of guest molecules prepared by default. They are listed in Table Designing semiclathrate hydrates is complicated. Refer to the manual.GenIce offers extensions by plugins. There are three types of plugins: molecular plugin, lattice plugin, and format plugin. Users can install their own plugins to serve new kind of guest and water molecules, crystal lattices, and output formats for specific applications.2 zeolite frameworks. Such structures can be candidates for stable ice phases under negative pressure because of the low density.https://github.com/vitroid/cif2ice). This tool obtains CIF files of zeolites from the zeolite database web siteIt is possible to make ice structures from porous SiOWe introduce a tool for generating hydrogen‐disordered ice structures, GenIce. The algorithm employed in GenIce certifies the uniformity of hydrogen‐disorder and the zero net polarization. Various ice and clathrate hydrate structures are preset. Users can install new host (water) molecules, guest molecules, lattice structures, and output formats as plugins.The uniformity of hydrogen‐disorder may affect the quality and reliability of computer simulations of ices and clathrate hydrates. Nowadays, the system size of computer simulations is getting larger and larger, and there is a great need for preparing many hydrogen bond networks of different orderliness efficiently. GenIce has already been used in our researches,