The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane-bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique, and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring.  •The myristoyl group of c-Src interacts with the SH3 domain•The Unique Lipid Binding Region modulates myristoyl-related interactions•Mutations in the Unique and SH3 domains affect membrane anchoring•A fuzzy intramolecular complex is retained in the lipid-anchored form of USH3 Src Structural Biology; Protein Structure Aspects; Biophysics Its oncogenic potential was brought to light already in 1970 . Since tImportantly, membrane binding is essential for the transforming activity of v-Src and for the activation of c-Src by a membrane-bound phosphatase . All SFKThe UD is intrinsically disordered and the most divergent region in the SFK. Its role remains poorly understood. Recently, the interplay between the UD and SH3 domain has been deciphered, in which the SH3 domain acts as a scaffold of a fuzzy complex that includes the UD and SH4 domain . MoreoveOn the other hand, the subcellular location of c-Src critically affects its function , and c-SHere we used NMR, very well suited for the study of proteins containing disordered domains in solution, and surface plasmon resonance (SPR) to characterize the myristoylated N-terminal region of c-Src, including the SH4 domain, UD, and SH3 domain, in solution and its binding to liposomes. We found that the N-terminal myristoyl group binds to the SH3 domain  in free c-Src. This provides an additional stabilizing element to the previously described intramolecular fuzzy complex in which the folded SH3 domain acts as a scaffold for the intrinsically disordered regions. In the presence of liposomes or supported lipid bilayers (SLBs) the myristoyl group is released to allow anchoring to the lipid bilayer, but the interaction of the SH4 and SH3 domains and the fuzzy intramolecular complex is retained. Mutations in the UD and SH3 domain affect lipid binding by the myristoylated SH4 domain suggesting a competitive model, in which the availability or exposure of the myristoyl group is modulated by interactions involving these domains.E. coli by co-expression with yeast N-myristoyltransferase following a previously described protocol, ensuring full myristoylation and the absence of spurious lauroylation of USH3  formed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-1′-rac-glycerol (DOPG) in a DOPC:DOPG ratio of 3:1. The protein concentration was 75 μM, and the total lipid concentration was 5 mM. We used the polyunsaturated lipids to ensure that lipid bilayers were in the liquid crystalline state in which proteins and lipids can freely diffuse, even at 278 K.In the presence of liposomes, the RT loop residues displayed chemical shifts typical of free SH3, indicating that the interaction of the disordered regions of c-Src with the RT loop was lost C left. TThese chemical shifts of RT loop residues are compatible with the release of the myristoyl group from the RT loop region of the SH3 domain upon insertion into the lipid membrane. The changes in indole NH signals are also compatible with the RT loop losing its interactions when the myristoyl group inserts into the lipid bilayer, as the side chain of tryptophan 121 is pointing toward the RT loop see .2H-NMR in Myr-Src(2–19) (The insertion of the myristoyl group in lipid membranes had been previously demonstrated using rc(2–19) . Importarc(2–19) B and 3A.As expected, the SH4 domain (positively charged) is strongly perturbed by the interaction of MyrUSH3 with negatively charged LUVs, and most signals are broadened beyond detection A. This cInterestingly, the ULBR, which was named as such because of the interaction with lipid bicelles observed in non-myristoylated USH3, showed no chemical shift changes, in spite of the fact that the myristoylated SH4 domain of the same molecule was anchored to DOPC:DOPG 3:1 liposomes in MyrUSH3 A.If the ULBR does not provide an additional interaction with the membrane lipids, the question that arises is what is its natural “lipid” target. To answer this question, we compared the effect of myristoylation of WT USH3 with that of USH3 AAA, in which the ULBR is inactive.13C, 15N uniformly labeled myrUSH3 AAA sample using the targeted acquisition strategy . The pair of oppositely charged residues was replaced by neutral glutamine, whereas the central residue was mutated to alanine. We used SPR to measure the reversible binding kinetics and the affinities to electrically neutral DOPC, negatively charged DOPC:DOPG (3:1), or DOPC:DOPG (2:1) SLBs obtained from liposome immobilization on phytosphingosine-derivatized sensor chips (XanTec) (To assess the effect of the UD and SH3 domain we compared the binding of MyrUSH3 to SLBs with that of the isolated myristoylated SH4 domain (MyrGSNKSKPKDASQRRR noted MyrSH4). The role of ULBR in the UD was tested using MyrUSH3 AAA. The importance of the RT loop in the SH3 domain was tested using a mutant USH3 domain with key residues in the RT loop (XanTec) .MyrSH4 showed two orders of magnitude higher affinity than the USH3 construct toward neutral SLBs . The AAAThese results are consistent with the NMR data indicating that the myristoyl group is interacting with the RT loop of the SH3 domain assisted by the ULBR in the UD.With negatively charged lipids all the affinities increased owing to the additional electrostatic interaction with the positively charged SH4 domain, but the relative differences were reduced. A possible explanation is that the dissociation of the myristoyl group from the SH3 domain is enhanced by the electrostatic interaction of the strongly charged SH4 domain when it is close to the negatively charged membrane.Protein myristoylation contributes to c-Src membrane anchoring together with a cluster of basic residues that interact with acidic phospholipids. Binding of c-Src to the membrane is mostly reversible, although evidence for clustering and irreversible binding of a small population of c-Src molecules has been reported . This isOur results show that there is a myristoyl-binding site in the SH3 domain. The interaction of the myristoyl group with the SH3 domain restricts the availability of the fatty acid chain to bind to lipids and could similarly prevent its interaction with the kinase domain of another c-Src molecule.Recently, N-terminal-bound myristoyl group has been suggested to mediate c-Src dimerization by interaction with the kinase domain . Mutatiocis, i.e., in the same molecule containing the myristoyl group for myristoyl-SH1-mediated dimerization to occur.Thus the myristoyl group can interact with the same c-Src molecule to which it is bound (through the SH3 domain) or the kinase domain of a second c-Src protein. The two binding events may be linked. In fact, Spassov et al. showed that Y419 phosphorylation of the kinase domain, which changes its interaction with the SH3 domain, was required in The AAA mutation prevents lipid binding by the UD in non-myristoylated constructs. However, this mutation results in an increase in the association rate of the myristoylated constructs with lipids. Thus the effect of the AAA mutation cannot be explained by changes in the direct lipid-binding capacity of the UD, but probably involves an indirect effect of the ULBR on the interaction of the myristoylated SH4 domain with the SH3 domain.The AAA mutation in full-length c-Src has a dramatic effect on the invasive capacity of c-Src-dependent colorectal cancer cells. At the molecular level, myristoylation resulted in duplicated NMR signals only in the presence of the native ULBR sequence, suggesting that the native ULBR contributes to the interaction between the myristoyl group and the SH3 domain. A functional role of the ULBR is also suggested by the very different dynamics leading to distinct broadening of SH4 NMR signals of myristoylated constructs containing either the native ULBR sequence or the AAA mutation in the presence of liposomes.Although our data do not provide any precise structural model, we can speculatively suggest that the ULBR contributes to modulating the balance between the lipid-bound and free forms of c-Src or the way by which MyrSH4 is anchored to the membrane while preserving the fuzzy intramolecular complex. This role of the UD would be in line with the notion that this intrinsically disordered region, exquisitely sensitive to the cellular environment and tunable, for example, by post-translational modifications or alternative splicing , could mOur results have uncovered a myristoyl-binding site in the SH3 domain of c-Src and have shown that the fuzzy complex previously characterized in non-myristoylated proteins is retained in the myristoylated forms, either free or bound to lipid bilayers. From the structural point of view, it implies that the structured part of c-Src, including the SH3 domain, is located closer to the membrane surface than implied with earlier models in which the UD was considered a long spacer.in vitro. The interaction sites contributing to the fuzzy complex have been shown to be compatible with the known X-ray structure of full-length c-Src. As the myristoylated SH4 and SH3 domains are known to independently participate in other regulatory interactions, the known effects of preventing myristoylation, changing the acyl group, or mutating the SH3 domain do not provide additional insight. The disordered UD is not observed in X-ray structures. Direct observation of full-length myristoylated c-Src by NMR, in vitro or in vivo, is not yet technically possible but is the goal of our future research.The interaction between the myristoyl group and the SH3 domain has been demonstrated All methods can be found in the accompanying


Sorting of activated epidermal growth factor receptor (EGFR) into intraluminal vesicles (ILVs) within the multivesicular body (MVB) is an essential step during the down-regulation of the receptor. The machinery that drives EGFR sorting attaches to the cytoplasmic face of the endosome and generates vesicles that bud into the endosome lumen, but somehow escapes encapsulation itself. This machinery is termed the ESCRT  pathway, a series of multi-protein complexes and accessory factors first identified in yeast. Here, we review the yeast ESCRT pathway and describe the corresponding components in mammalian cells that sort EGFR. One of these is His domain protein tyrosine phosphatase (HD-PTP/PTPN23), and we review the interactions involving HD-PTP and ESCRTs. Finally, we describe a working model for how this ESCRT pathway might overcome the intrinsic topographical problem of EGFR sorting to the MVB lumen. Ligand-activated epidermal growth factor receptor EGFR/ErbB1) is ubiquitinated by Cbl ubiquitin ligase  is ubiqu–6. Here,Saccharomyces cerevisiae  pathway, a series of multi-protein complexes first identified in the yeast revisiae . DeletioThe yeast ESCRTs act sequentially see  10–12].–12.10–12Aside from ESCRTs, other components are important for MVB formation. These include the accessory protein Vps31/Bro1 (Bck1-like resistance to osmotic shock 1) , which bThese ESCRT pathway constituents are broadly conserved. However, multiple variants of several components exist in mammalian cells . For exaBoth Alix and HD-PTP share with yeast Bro1 an N-terminal Bro1 domain that harbours a conserved binding pocket for the C-terminus of CHMP4B (charged membrane protein/chromatin modifying protein 4B)  the mos, a coile++-dependent regulator of Alix [++-regulated EGFR sorting pathway.Alix supports multiple ESCRT-mediated processes . While s of Alix . VPS37B  of Alix . Hence, 2L motif [Alix exists in the cytosol in an inactive conformation, in which the PRR is folded across the V domain and forms an auto-inhibitory interaction with the Bro1 domain . This ‘c2L motif ,36 withi2L motif . Several2L motif ; phospho2L motif ; alloste2L motif ,52; dime2L motif .Figure In contrast with the widespread cellular roles of Alix, HD-PTP acts selectively at the endosome and is essential for the forward movement of EGFR from the early endosome towards the lysosome. Its depletion causes a striking accumulation of tubulo-vesicular endosomal compartments that contain a build-up of ubiquitin–protein conjugates and reduces sorting of EGFR to the endosomal lumen ,54. Cons2L motif within the coiled-coil domain of HD-PTP , we envisage that ILV formation might be driven by an endoskeleton that is triggered to disassemble prior to the neck closing (In summary, while budding of transport vesicles into the cytoplasm requires an  closing . Though


Chlorophyllum rhacodes, typically regarded as a rich grassland or open forest “mushroom” species, was found fruiting abundantly on nests of Formica lugubris, occurring in a Pinus silvestris plantation. Fruiting was absent from the rest of the woodland.C. rhacodes mycelium, insect cadavers, fruitbody initiation, and roots of adjacent trees.Research focussed on the activities in the nests that could explain this. Within nests, there was a spatial relationship between C. rhacodes was not mycorrhizal with P. silvestris, but that it had qualities which rendered it suitable for colonization of the rhizosphere in the conditions of the nest mound and for further niche development.In vitro experiments found that Implications of the unusual presence of fruit‐bodies and the distribution of associated hyphae are discussed in relation to the nutritional biology  of the fungus. This includes reference to the relevant physiology of insects and to the accepted evolution of mutualistic symbioses between fungi and the Attini and Termitidae.An argument is presented that the situation observed in vivo provides evidence of a degree of facultative association and what could be tangible support for the theory for the developmental origin of mutualistic fungus cultivation by insects. It is presented as a context for continued experimental research. Chlorophyllum rhacodes, an open forest 'mushroom' species, is occasionally found fruiting on nests of Formica lugubris. Within less mobile thatch of such nests, there was a spatial relationship between C. rhacodes mycelium, insect cadavers, fruitbody initiation, and roots of adjacent trees. The situation provides evidence of a degree of facultative association in the rhizosphere. A discussion is presented for a potential developmental mechanism in support of the accepted theory for the developmental origin of mutualistic fungus cultivation by insects. The discussions present a view on the development of:1.2Chlorophyllum rhacodes (Vittadini) Vellinga (formerly Macrolepiota rhacodes (Vittadini) Singer, a.k.a. shaggy or reddening parasol mushroom) is an open woodland litter saprotroph (associated more with conifer than broadleaved woodlands) and is possibly found in grasslands , in Highlow Wood, a coniferous plantation in the Peak District National Park, UK. No fruit‐bodies of this fungus were seen in surrounding soils or any other substratum in the woodland. Popular guides have noted it in the United States  with some Larix decidua Mill. (European larch). Within the Peak District National Park, UK, the site is situated in a sheltered valley, orientated approximately east–west, with Highlow Brook, a narrow tributary of the River Derwent running through it. Location: centered on O.S. grid ref: SK224799; Latitude: 53°18′54″N, Longitude: 1°39′53″W; elevation: 180 m AOD.Highlow Wood (compartment H8b) was a plantation of (predominantly) fifty‐year‐old 22.1F. lugubris were mapped, and fruiting of C. rhacodes was noted for position on nest mounds and duration. Nest conditions  were mapped against the presence of C. rhacodes fruit‐bodies. Nest mounds were dissected to follow mycelial cords through the nest structure.Field observations were carried out over a period of two years in Highlow Wood. Nest mounds of 2.2C. rhacodes to form a mycorrhiza. The first method used was developed by R. Bradley and D.J. Read (pers. com.) with medium pH amended to match average nest conditions (pH 4.7), and subsequently with potted seedlings in previously sterilized compost, inoculated with mycelial mats from the base of C. rhacodes fruit‐bodies. Roots were observed macroscopically for the characteristic shape of ectomycorrhizal development. Microscopic observations were carried out on roots sectioned and stained with methyl blue to highlight fungal mycelium.Laboratory tests were carried out for the ability of C. rhacodes to utilize ants or their (imago) prey cadavers was investigated. Specifically, lipolytic and protease activities were tested, as was the ability to degrade chitin. C. rhacodes was grown on the indicator medium for lipolytic activity  were examined under scanning electron microscope (SEM) for any recognizable adaptations for mycophagy. Ant fecal pellets were examined for presence of passaged spores.Freeze‐dried specimens of 33.1Chlorophyllum rhacodes fruiting on the nests began in May and continued well into December. Within a season, there were normally multiple flushes recorded on the nests .In this study, temperatures recorded throughout the nests were generally uniform; slightly higher than still ambient air, and as much as 22°C higher than surrounding soil. This could allow a longer season of vigorous mycelial growth and may explain the protracted fruiting season.Chlorophyllum rhacodes fruit‐bodies reached maturity, in an unmolested state, only on active ant nest mounds. On recently abandoned ant nests, fruiting was initiated, but the success of fruit‐body maturation was always zero, with primordia always being fatally damaged by fungivorous activity. This suggests that, in this area, fruiting success appeared to be linked, spatially and temporally, to ant nest activity.Of the 12 nests present at the start of the study, only six produced fruit‐bodies every season. All of the latter were inhabited throughout this period and all were built at the bases of living pine trees.C. rhacodes fruitbody stipes. In that, the cords were never longer than 5 cm, the fruit‐bodies were initiated close to the roots. This close physical association leads to the distribution of fruit‐bodies  where the maximum length of stipe limits how far up the nest the fruit‐body appears  area of the nest mound. Hyphal cords associated with the roots extended to the fruit‐body stipes. They were also associated with cadavers, on which they split into individual hyphae. There was a noticeable hyphal load in and on these cadavers.In nests with a crust distribution of roots, the rhizosphere could be very close to what appeared to be galleries containing complete (nonant) insect cadavers. Food store galleries in 3.23.2.1C. rhacodes was tested for its ability to form mycorrhizal association with P. silvestris, no mycorrhizal roots were developed. Indeed many roots sustained cortical damage, which led to root die‐back. The fungus did not thrive in the axenic, moist environment, so the investigation was attempted in plant pots. A strong ectomycorrhiza was not developed, but there were several instances of hyphae penetrating between healthy root cortical cells.The propinquity of mycelium and roots suggests an association. Stubby, dichotomous root branching suggested an ectomycorrhiza. Microscopy revealed evidence of a Hartig net on roots, but when 3.2.2Temperatures recorded throughout the nests were generally uniform; slightly higher than still ambient air, and up to 22°C higher than soil surrounding the nests. This could allow a longer season of vigorous mycelial growth and may explain the protracted fruiting season. Nests are sited most frequently where they benefit from passive solar gain , therefore, held nutrient resources in the nests until the ant activity ceased on abandonment of the nests. Thus, the same argument suggests that in the area of the nest consolidated by tree roots and hyphae, degradation processes are not being prevented and release of nutrients is happening. The rhizosphere area of the nest mound probably experiences more release of saprophyte‐suitable nutrients than other parts of the nest mound. Root exudates contain sugars that stimulate microbial growth, which, in turn will enhance the decomposition of the immediately surrounding litter material of which the nest mound is built. This will lead to a release of ammonium (soluble) nitrogen.Nests are composed of litter and the frequent redistribution of the material maintains an environment suited to the ants and avoids rapid decomposition. The mixture of tree roots, nest material, and hyphae presented a combined mass that might be less likely to be moved by ants in their general nest material rotation. Kilpeläinen's et al.  study ofTo assay total ammonium nitrogen of different regions, nest crowns and adjacent soils were divided into approximately 20 × 10 × 10 cm portions. Preparation involved thorough mixing of each sample and removal of all recognizable, living plant material.The highest nitrogen levels were found in the nests on which fruit‐bodies were observed. Within these nests, the highest nitrogen levels were recorded in the rhizosphere areas, where fruit‐bodies were initiated Figure a–c.C. rhacodes) normally utilize fast‐decomposing litter, utilizing cellulose, hemicellulose, and lignins  and, therefore, present a source of fatty acids and proteins. To investigate if  Sierra,  and on mLipolytic activity would be recognized by the appearance of crystalline precipitate of insoluble fatty acids. No such evidence was found.Chlorophyllum rhacodes was found to produce exogenous proteases, which cleared milk agars .Sagara  noted a C. rhacodes fruiting in the conditions provided by long‐term F. lugubris nests, which are invaded by tree roots, suggests a possible connection between C. rhacodes and established woodland communities. While nest mound material is frequently cycled throughout the mound structure, the material in the highly branched root network is effectively trapped. This physical stability allows for growth of C. rhacodes among the litter, which is probably enriched by root exudates. Some unknown factors in the association between the fungus, roots, and ants lead to the extensive bifurcation of the tree roots .The facilitation of 4.1et al. 2007 cited in Žižka & Gabriel, C. rhacodes) are rarely strong wood decomposers (outside the tropics) and normally utilize fast‐decomposing litter , quite different from the PDH found in numerous white‐rot, saprotrophic fungi . In the sessile oak woodlands of South Yorkshire, nest mounds commonly contain a substantial proportion of primordial acorns still attached to peduncle and short twig (author's observation). In the pine plantations, such as Highlow Wood, the nest mounds are largely pine needles. As per usual, nests are sited most frequently where they benefit from easterly or southerly sunshine  is thought to be controlled by a bacterium of the genus Streptomyces (Actinomyces)  where fruit‐bodies possess free gills and basidiospores with germ pores. DNA analyses show that C. rhacodes is more closely related to the Leucoagaricus clade than the species type of its former genus, Macrolepiota, hence, the reclassification as Chlorophyllum rhacodes  ants utilize their many chemical means of protection. A comprehensive range of fungistats and antigerminants are produced—the reason why so few ants are pollinators Beatie,  and rela4.5Apis mellifera), there are three distinct food preparations: worker jelly, young worker jelly, and royal jelly. These preparations are different mixtures of the same mandibular gland secretions, hypopharageal gland secretions, and crop contents. After only three days of development, the change in balance of these preparations in a larva's diet will determine its development to sexual maturity or suppression and to physical and behavioral distinctions. While some of the hormones involved are derived from the nurse workers' metabolism, some of the active chemicals are from crop contents   appeared to stimulate fruiting of C. rhacodes fruiting in the conditions provided by long‐established nests, suggests a possible connection between C. rhacodes and woodland communities. A link between C. rhacodes distribution and historic woodland pasture is worth investigating.The facilitation of Chlorophyllum rhacodes is theoretically capable of utilizing degraded insect exoskeletons as a resource and was found present in the nest stores of insect cadavers. In terms of chitin metabolism, C. rhacodes presents no predatory threat to living insects, but is able to utilize chitin and some proteins, released by decomposition of cadavers.Formica lugubris is likely to consume quantities of C. rhacodes mycelium along with their stored food supplies.C. rhacodes will release sugars from pine‐needle derived cellulose and if consumed by F. lugubris, will enhance its nutrition, hinting at a facultative association.The saprotrophic nature of C. rhacodes thereby developing into a more obvious mutualism, by the mechanism postulated over one hundred years ago (op. cit.).As insects are prone to large‐scale morphological and behavioral alterations as a result of different diets, an increase in specific mycelium in their food presents such an opportunity. Among all the possible outcomes, one might be a tendency to select for behavior that maintains or further encourages the presence of Inherent in all these observations is the possibility of the evolution of an obligate ant‐fungus mutualism. Although the most strongly developed fungus‐farming relationships are only known in tropical biomes, the warming temperate climate presents opportunities for changing distributions and interactions of potential symbionts.Douglas Fraser is the sole author of this work. Where results, theories or concepts have been drawn from other authors, these have been properly acknowledged and referenced.


Large-scale urbanization has brought about severe ground subsidence in Kunming (China), threatening the stability of urban infrastructure. Mapping of the spatiotemporal variations of ground deformation is urgently needed, along with summarization of the causes of the subsidence over Kunming with the purpose of disaster prevention and mitigation. In this study, for the first time, a multi-temporal interferometric synthetic aperture radar (InSAR) technique with L-band Advanced Land Observation Satellite (ALOS-1) and X-band Constellation of Small Satellites for Mediterranean basin Observation (COSMO-SkyMed) data was applied to Kunming to derive the time series deformation from 2007 to 2016. The annual deformation velocity revealed two severe subsiding regions in Kunming, with a maximum subsidence of 35 mm/y. The comparison of the deformation between InSAR and leveling showed root-mean-square error (RMSE) values of about 4.5 mm for the L-band and 3.7 mm for the X-band, indicating that our results were reliable. We also found that the L-band illustrated a larger amount of subsidence than the X-band in the tested regions. This difference was mainly caused by the different synthetic aperture radar (SAR)-acquired times and imaging geometries between the L- and X-band SAR images. The vertical time series deformation over two severe subsiding regions presented an approximate linear variation with time, where the cumulative subsidence reached 209 mm during the period of 2007–2016. In view of relevant analyses, we found that the subsidence in Kunming was the result of soft soil consolidation, building load, and groundwater extraction. Our results may provide scientific evidence regarding the sound management of urban construction to mitigate potential damage to infrastructure and the environment. Kunming, the capital and largest city of Yunnan province in Southwest China, is located at a longitude of 102°10′–103°40′ east and a latitude of 24°23′–26°22′ north. Situated in the central Yunnan–Guizhou plateau in Southwest China, Kunming is surrounded by the Dian Lake to the south and by mountains on the other three sides [Early studies initiated a few years ago using ground-based techniques  documented the extent and causes of subsidence in Kunming ,6. AccorTo better understand the spatiotemporal evolution of ground deformation in Kunming, a multi-temporal InSAR technique was utilized for the first time in this study to obtain a decade of time series deformation of Kunming. For this, 20 L-band Advanced Land Observation Satellite (ALOS-1) and 40 X-band Constellation of Small Satellites for Mediterranean basin Observation (COSMO-SkyMed) images were acquired in ascending and descending orbits, respectively. The annual deformation velocity was estimated to characterize the spatial pattern of subsidence in Kunming. Subsequently, comparisons were conducted to validate our results and the time series deformation was interpreted in detail. The causes of subsidence are discussed through correlation analysis.The study area is located at Kunming, which is the capital of Yunnan province in southwest China, as shown in 150), Sheshan fault (F152), Pudu river fault (F54) and Puji-Hanjia village fault (F55) [The study area is a late Cenozoic graben basin controlled by a number of quaternary active faults, e.g., the Heilongtan-Guandu fault (F fault F5 and Pujilt (F55) . These sA total of 20 L-band ALOS-1 images with stripe mode and 40 X-band COSMO-SkyMed (CSK) images were collected to derive the time series deformation over Kunming. Using the collected 20 L-band ALOS-1 and 40 X-band CSK images, time series of deformation was derived from the multi-temporal InSAR technique with GAMMA Remote Sensing and Consulting AG (GAMMA) SAR software. This process included three steps.Firstly, interferometric pairs were generated by setting small temporal and spatial baselines. This depressed noise effects and maintained a high level of coherence . On the Secondly, after selection of the point targets, removal of the topographic and orbital phases, filtering of the interferograms, and retrieval of the absolute phase, unwrapped interferograms were produced. The process of point target selection was based on the method in . The topThirdly, the time series deformation was estimated from the collected L- and X-band images. After the second step, the unwrapped interferometric phase may consist of residual phase errors, such as topographic phase error, orbital error, or atmospheric delay error. Prior to the time series deformation estimation, these residual phase errors should be removed from the unwrapped interferometric phase. In this case, the topographic phase error was safely neglected since high-precision LIDAR DEM was used to simulate the topographic phase. A quadratic polynomial phase model was fitted to reduce the orbital phase error  and the The annual deformation velocity maps in the vertical direction were derived from 59 L-band interferograms and 88 X-band interferograms, as shown in Careful inspection of In order to validate our results, the annual deformation velocities between leveling and InSAR were compared, as shown in Thirty-one additional Sentinel-1A images with ascending orbit  were proTo further validate the CSK-derived deformation, 60 C-band Sentinel-1A images  with desAs shown in To investigate the long-term ground deformation time series, both L-band ALOS-1 and X-band CSK deformation in LOS direction were projected to the vertical direction. After that, the cumulative vertical deformation from the L-band was added to the X-band to connect them. Like Guandu, Xishan is another region with severe subsidence. The time series deformation of Xishan from 8 August 2011 to 6 January 2016 was observed from the X-band CSK images, as shown in Kunming was a typical lacustrine sediment basin that was filled by a large area of soft soil . Figure The relevant materials show that the magnitude of subsidence may be related to the quaternary sediment thickness . Thus, t149 and F150 faults. The Xishan subsiding region presented in a long rectangle shape and extended to the southwest, which didn’t follow the general trends of the surrounding F151, F152, and F55 faults. However, careful inspection suggests that Xishan subsiding region might be bounded by these three faults: the east was bounded by F152, the west and south were bounded by F55, and F152 acted as barrier to impede the horizontal propagation of deformation on both sides. Based on this analysis, there was a certain degree of correlation between ground deformation and quaternary active faults over the area.For further investigation of the relationship between ground deformation and geological setting, the quaternary active faults were superimposed on the L-band ALOS-derived deformation map, as shown in As introduced in To further investigate the relationship between subsidence and building load, the building densities over 35 sampling points were extracted and analyzed over Kunming. Building density is the floor area of the building divided by the total area of the site . It shou3 prior to 2010 [Groundwater is the main water supply in Kunming, where the average daily groundwater exploitation was more than 170,000 m to 2010 . Figure  to 2010 . It was  to 2010 ,46. TherWith the development of urbanization, ground subsidence has become one of the most prominent geological hazards in Kunming and has caused substantial damage to homes, roads, canals, pipelines, and other types of infrastructure. In this study, a decade of time series deformation in Kunming was derived using a multi-temporal InSAR technique to provide scientific evidence regarding the sound management of urbanization. Based on this study, the following conclusions were made.(1) Two severe subsiding regions were observed using L- and X-band SAR data. Using 20 L-band ALOS-1 images and 40 X-band COSMO-SkyMed images, the annual deformation velocity and time series deformation maps of Kunming were retrieved through multi-temporal InSAR processing. The results showed that most areas of Kunming are in a state of subsidence and severely subsiding regions are concentrated in Guandu and Xishan, where the cumulative subsidence reached 210 mm and 98 mm during the period of 2007–2016, respectively. Comparisons of ground deformation between InSAR and leveling observations indicate that RMSE values are about 4.5 mm for the L-band and 3.7 mm for the X-band, demonstrating the reliability of our observations.(2) A correlation analysis was conducted to investigate the causes of the observed subsidence. The results show that the severe subsidence in Kunming might be the result of soft soil consolidation, building load, and groundwater extraction. Among these factors, soft soil was the foundation for the subsidence, and the building load and groundwater extraction factors accelerated the subsidence process.This study contributes to the understanding of the spatial-temporal evolution of ground deformation in Kunming. However, it is difficult to clearly describe the mechanism of land subsidence due to a lack of related materials, such as continuous ground-based deformation monitoring data. Therefore, sufficient in situ measurements will be collected in the future to analyze the mechanism of ground deformation in Kunming.


S1 domain, a structural variant of one of the “oldest” OB-folds (oligonucleotide/oligosaccharide-binding fold), is widespread in various proteins in three domains of life: Bacteria, Eukaryotes, and Archaea. In this study, it was shown that S1 domains of bacterial, eukaryotic, and archaeal proteins have a low percentage of identity, which indicates the uniqueness of the scaffold and is associated with protein functions. Assessment of the predisposition of tertiary flexibility of S1 domains using computational and statistical tools showed similar structural features and revealed functional flexible regions that are potentially involved in the interaction of natural binding partners. In addition, we analyzed the relative number and distribution of S1 domains in all domains of life and established specific features based on sequences and structures associated with molecular functions. The results correlate with the presence of repeats of the S1 domain in proteins containing the S1 domain in the range from one  to 15 (eukaryotic) and, apparently, are associated with the need for individual proteins to increase the affinity and specificity of protein binding to ligands. As is known, many natural proteins fold amino acid chains into compact units, defining structural domains . These dS1 domain is one of the structural versions of the OB-fold, which is considered one of the “oldest” protein folds that are mutation resistant and able to adapt to the binding of a large number of ligands. S1 domain is a β-barrel with an additional α-helix between the third and fourth β-strands; the main function of this domain is RNA-binding . A shortThe S1 domain is widespread in various proteins in three domains of life. Recently, we showed that the S1 domain is mainly identified in bacterial proteins (on average about 83%), then 14% in eukaryotic proteins, and 2.8% in archaeal proteins . One of In this study, we examined the features and flexibility of the predisposition of individual regions in the tertiary structures of typical S1 domains from bacterial, eukaryotic, and archaeal proteins (24 proteins), representing all the major protein families, to investigate the relationship between the S1 structure and its molecular functions.https://www.rcsb.org) and PDBe bases (https://www.ebi.ac.uk/pdbe/). The total number of defined 3D protein structures containing the S1 domain is 216 (RCSB PDB) and 225 (PDBe) , Data on the number of the S1 domain and its taxonomy distribution between the three main domains of living organisms can be found in the RCSB PDB  to further examine the relationship between the S1 domain and its functions originated from the tertiary structure. Our data set consists of S1 domains, which represent all the major protein families. Selected proteins were obtained by X-ray crystallography, electron microscopy with different resolutions, and NMR methods.The most common bacterial proteins containing the S1 domain are polynucleotide phosphorylase (PNPase), ribonuclease , transcription factor NusA, transcription accessory protein Tex, general stress protein 13 (GSP13), and the S1 ribosomal protein S1 family . PNPase In general, for all S1 domains in bacterial proteins, participation in RNA binding processes of was confirmed experimentally.Escherichia coli made it possible to establish the functions of individual protein domains and parts. The correlation between the functions of individual domains and protein structures for this protein family will be discussed in more detail below , which is involved in RNA processing. The catalyst component in the exosome of the eukaryotic protein Rrp44 (Dis3p) binds RNA and orients it in the orthogonal direction to the exosome core for processing long RNA molecules . These pDefined functions of the S1 domain in the considered eukaryotic proteins are given in The participation of S1 domains in processes associated with RNA interaction has been experimentally confirmed for some of the considered proteins. These interactions were established at the level of cellular systems in which these proteins are included.Non-catalytic components of the archaeal exosome Rrp4 and Csl4 form the upper part of the central channel of the circular exosome structure. Exosomes become the central mechanisms of 3`-5` processing and degradation of RNA in archaea. The trimer of Csl4 or Rrp4 subunits forms the surface of multi-domain macromolecular interaction on the RNase-PH domain ring with central S1 domains and peripheral domains of KH and zinc-ribbon . The hetDefined functions of the S1 domain in the considered archaeal proteins are given in Note that in bacterial proteins, the S1 domain is the main in the process of interaction with RNA; in higher organisms (eukaryotes and archaea), it almost always, in cooperation with other domains, forms a central channel for RNA movement. Interestingly, the domains involved in the formation of the RNA entry pore are homologous to the S1 domain, for example, the KH- , PH- 3232, YqgF-Analysis of consensus sequence of S1 domains from Bacteria, Eukaryotes, and Archaea will provide information on the position of critical amino acids and their structural and functional disturbance in various proteins containing S1 domains. As mentioned above (Section Introduction), the S1 domain is a small structural domain of 70 amino acid residues that folds into a five-stranded antiparallel β-barrel bounded by a single α-helix .In one example of the S1 domain, it was shown that the conserved residues F19, F22, H34, D64, and R68 (positions are given for PNPase) are located on the surface of the domain and form a specific RNA-binding site ,21. The F23, F34, H36, D64, and R67  are responsible for oligonucleotide binding and are highly conserved during evolution among bacterial proteins a. The foFor S1 domains from archaea and eukaryotes, only three conserved residues were revealed (of the five residues mentioned): F28, D66 and R71 for archaea, F25, D68 and R71 for eukaryotes b,c. ThisB.subtilis, PDB code: 2k4k) and the S1 domain of PNAse of E.coli (PDB code: 1sro) (exclude PNAse from C. vibrioides and E.coli). This percentage of identity is the highest value among all S1 domains alignments. For eukaryotic proteins, the highest identity (46%) belongs to the S1 domains from the subunit of RNA polymerase II from K. phaffii (PDB code: 6ir9) and the subunit RPB7 of DNA-directed RNA polymerase II from S. cerevisiae (PDB code: 4a3g) (exclude RNA polymerase II subunit G from H. sapiens and K. phaffii). Among archaeal proteins, the highest percentage of identity (43%) belongs to the S1 domain from the component of the exosome complex Rrp4 from  and the S1 domain from the component of the exosome complex Csl4 . The minimum percentage of identity is 5%, which is found between the component of the eukaryotic complex RRP40 from H. sapiens (PDB code: 2nn6) and exosome complex exonuclease RRP44 from H. sapiens (PDB code: 6h25).Alignments of sequences of S1 domains allowed us to calculate the average identity of S1 domains in each domain of life and between them . As you 3D model alignments between protein structures can suggests not only evolutionary and functional relationships, but also allows one to reveal differences that may not be distinguishable when comparing sequences . The strAs mentioned above, the S1 domain is one of the “oldest” protein domains. Thus, the evolutionary analysis of S1 domains offers an example of how, through evolution, the ancient protein domain diverged in many different organisms and potentially plays a diverse role, primarily depending on the conserved mode of functioning. In addition, the occurrence of S1 domains in eukaryotes, archaea, and bacteria suggests their presence before the divergence between the three domains of life. For our data set, phylogenetic trees were constructed using the POSA  Figure  and SALIAn analysis of the dendrograms obtained by the POSA and SALIGN servers  reveals To analyze the structural flexibility of S1 domains, we used the FlexPred program, designed to predict the absolute per-residue fluctuations in a query protein from its 3D structure . FlexPreThe results of the analysis of some S1 structures for representative members of bacterial, eukaryotic, and archaeal proteins are shown in According to the FlexPred program, the S1 domain from the exosome complex component Rrp4 is the most flexible S1 domain in archaea, 25% . For Bacteria, the highest flexible S1 domain is identified in polynucleotide phosphorylase, 60% . For eukaryotic proteins, the most flexible S1 domain according to the FlexPred program is the S1 domain of the G subunit of RNA polymerase II .Using the B-factor gives other results. So, the most flexible S1 domain  is indicated in the exosome complex exonuclease DIS3 from eukaryotes, PDB code: 2wp8. For archaeal proteins, the most flexible S1 domain was found in the exosome complex component Csl4 . For bacterial proteins, the highest flexible S1 domain is identified in the antitermination protein NusA .p-value is 0.49798 for the B-factor; 0.49073 for FlexPred). So, in general, according to the profiles  with the average value of 12Å.A comparison of the radius of gyration for S1 domains as an indicator of the compactness of the domain structure  of bacteAmino acids can be classified as ‘order-promoting’  and ‘disorder-promoting’  residues according to their abundance in ordered proteins and disordered proteins ,44. AnalAs we have recently shown, the S1 domain repeats presence in proteins containing the S1-domain : in the range from one to six for bacterial proteins, almost always in one copy in archaeal proteins, and up to 15 for eukaryotic proteins . ApparenAs we mentioned above, all archaeal proteins contain one copy of the S1 domain , PROSITE ID: PS50051), while the number of repeats in the eukaryotic proteins varies from 1 to 15. In bacterial proteins, the number of repeats is not more than 6, regardless of the protein length [First, the bacterial structure of Qβ replicase, which is responsible for the replication and transcription of Qβ viral RNA, consists of virus-encoded RNA-dependent RNA polymerase (β-subunit), EF-Tu, EF-Ts and the N-terminal part of the ribosomal protein S1. In S1, which is capable of initiating Qβ RNA replication, two N-terminal S1 domains are defined (PDB code: 4q7j) ,46. StruRecently, 70S ribosome was visualized by ensemble cryo-electron microscopy . The cryE.coli obtained by NMR [The multi-functional ribosomal protein S1 is part of the 30S ribosomal subunit. Proteins of the 30S ribosomal protein family interact with mRNAs, participate in the initiation and translation of mRNAs in vivo, and interact with the mRNA-like part of the tmRNA molecule. Similar to some other ribosomal proteins, ribosomal protein S1 is an autogenous repressor of its own synthesis. From the study of the structure of S1 on the 70S ribosome, it was shown that S1 interacts with other ribosomal proteins  forming a dynamic network near the output and input channels of the mRNA in order to modulate the binding, folding, and movement of mRNA. In addition, the Protein Data Bank contains 3D structures of separate domains of ribosomal S1 from d by NMR ,49.As can be seen from In addition, we have shown that the unique S1 protein family differs in the classical sense from a protein with tandem repeats, such as the ANK family, leucine-rich-repeat proteins, etc. , which uhttps://www.ebi.ac.uk/pdbe/), European resource for the collection, organization, and dissemination of 3D structural data (from PDB and EMDB) on biological macromolecules and their complexes according to S1 domain profiles in the Pfam database (PF00575) and PROSITE (PS50126).3D structures of proteins containing S1 domains were selected from the PDBe . Per-residue structural flexibility profiles for chosen S1 domain were obtained using the FlexPred software available at http://kiharalab.org/flexPred/ [http://posa.godziklab.org), using multiple flexible structure alignment with partial order graphs [https://www.python.org/) as implemented in the PyCharm community edition 2017 (https://www.jetbrains.com/pycharm/) development environment Matplotlib Python plotting library and NumPy numerical mathematics extension.A global pairwise sequence alignment  using a dynamic programming algorithm was used. The Multiple Sequence Alignment was implemented by the Clustal Omega service (lexPred/ . Structur graphs . This prhttps://weblogo.berkeley.edu) [WebLogo (ley.edu)  was usedp > 0.05.The Kruskal-Wallis test  was usedhttp://posa.godziklab.org) [https://modbase.compbio.ucsf.edu/salign/) [The extracted protein sequences from PDB structures  were uselab.org)  and SALIsalign/) .The radius of gyration is defined as the root mean square distance of the collection of atoms from their common center of mass, calculated from PDB files using the module from the Pymol ScrIpt COllection library (PSICO).As you know, the S1 domain is one of the “oldest” protein domains. It is presented in different combinations in different proteins, and such combinations are a direct result of the evolution of organisms. A study of the available tertiary structures of the S1 domain showed that this domain is mainly identified in eukaryotes. In this work, we studied a representative data set consisting of 24 S1 domains of different proteins to reveal their structural features necessary for the specificity of ligand binding. The main function of the bacterial S1 domain is RNA-binding through conservative amino acids on the surface of the domain. However, the functions of the bacterial ribosomal proteins S1 are not clearly defined, since the number of domains in S1 varies and is a distinctive characteristic for different phylogenetic phyla. For eukaryotes and archaea, the S1 domain almost always interacts with other domains, forming a central channel for RNA movement. Interestingly, the domains involved in the formation of pores of RNA entry, are homologous and, apparently, have an evolutionary relationship with the S1 domain. We calculated and characterized the predisposition of tertiary flexibility of S1 domains  from bacterial, eukaryotic, and archaeal proteins and showed that S1 domains have similar structural features. The most flexible loop region (40–50 a.a.) in the S1 domain, apparently, can be potentially involved in the interaction of natural binding ligands. Our results show that the number of possible functions for eukaryotic proteins increases due to an increase in the number of structural domains and flexible linkers between domains, but not due to a change in the characteristics of an individual structural unit.


A complete picture of the deformation characteristics (distribution and evolution) of the geotechnical infrastructures serves as superior information for understanding their potential instability mechanism. How to monitor more completely and accurately the deformation of these infrastructures  in the field expediently and roundly remains a scientific topic. The conventional deformation monitoring methods are mostly carried out at a limited number of discrete points and cannot acquire the deformation data of the whole structure. In this paper, a new monitoring methodology of dam deformation and associated results interpretation is presented by taking the advantages of the terrestrial laser scanning (TLS), which, in contrast with most of the conventional methods, is capable of capturing the geometric information at a huge amount of points over an object in a relatively fast manner. By employing the non-uniform rational B-splines (NURBS) technology, the high spatial resolution models of the monitored geotechnical objects can be created with sufficient accuracy based on these point cloud data obtained from application of the TLS. Finally, the characteristics of deformation, to which the geotechnical infrastructures have been subjected, are interpreted more completely according to the models created based on a series of consecutive monitoring exercises at different times. The present methodology is applied to the Changheba earth-rock dam, which allows the visualization of deformation over the entire dam during different periods. Results from analysis of the surface deformation distribution show that the surface deformations in the middle are generally larger than those on both sides near the bank, and the deformations increase with the increase of the elevations. The results from the present application highlight that the adhibition of the TLS and NURBS technology permits a better understanding of deformation behavior of geotechnical objects of large size in the field. The deformation distribution and evolution are present as important indications of the instability of large artificial and natural structures such as tunnels, bridges, and landslide ,2,3,4. CIn contrast, the terrestrial laser scanning has a definite advantage of providing point clouds composed of millions of points with high accuracy and high spatial resolution, and the point clouds can be used to detect vertical and horizontal deformation through relatively simple process. Nevertheless, the point cloud data acquired from the laser scanner cannot be processed in the same way as that for the data from the traditional methods, since the laser pulse emitted from the machine is not necessarily aimed at the same place of the target object in different scanning operations . AdditioHence, how to extract information of the dam deformation from the terrestrial laser scanning (TLS) data is a challenging and important task, which is the focus of many recent works carried out on the deformation detection by the application of TSL ,14,15,16In this paper, a detailed deformation measurement method, based on combination of TLS and NURBS technologies, is presented. The method is established on the basis of the quadrangular surface domain parameters for deformation monitoring. By specifying the quadrangular surface domain parameters, the four corner points of the NURBS fitting surface are exactly positioned at the four vertexes of the control mesh. Thus, the precision of the NURBS fitting surface reaches up to a couple of millimeters and is higher than the traditional NURBS surface. The performance of this present method in the field is illustrated by an application to a selected hydraulic structure, the Changheba Dam, located in Southwest China. To acquire the detailed and accurate 3D data rapidly and efficiently, the TLS technology is first applied to take inventories. To fully take advantage of the point cloud data, the digital surface model of the Changheba Dam with high accuracy and high spatial resolution is established by using the NURBS surface modeling technology. Through the comparison between multi-temporal models, the deformation characteristics of the Changheba Dam in different stages are analyzed. The results presented in this study illustrate the applicability of the present methodology to the precise deformation monitoring over large regions. 2  technology, the TLS is able to record dense point-clouds over an extremely short period of time. The modern laser scanner provided by the manufacturer shows a significant higher speed of data acquisition, compared to the conventional surveying instruments like total station . Moreovec is the speed of light; TOF is the time of flight of the laser pulse; S is the distance from the scanner to the reflecting surface; θ is the angle between the line OP and the XY plane; and α is the angle between X axis and the orthogonal projection of the OP onto the plane XY.Let the origin of the Cartesian (rectangular) coordinate system coincide with the center of the laser scanner, where X and Y are two axes perpendicular to each other lying in the horizontal plane, and the Z axis is oriented upwards and perpendicular to the horizontal plane . Then, tIn this paper, a pulse-based scanner, Riegl VZ-400 , is applied to survey the study area. The scanner is capable of measuring the distance ranging from 1.5 m to 600 m. Due to the echo digitization and online waveform processing technique, the highest angular resolution that this instrument can achieve is 0.0005°, and its efficient measurement rate is up to 120,000 points per second. Besides, it offers a wide field of view up to 100° vertical and 360° horizontal. As such, this equipment is suitable for the data acquisition in the vested condition.Measurement campaigns were performed after the filling of the dam. Due to the great size of the dam, it is impossible to acquire the point cloud data of the entire dam surface with only one scan. In addition, a relatively larger number of scans can increase the density of target point clouds, making sure that the overlap between different scans is enough for the alignment of different data sets.Here, both the particular geologic of this case and the requirement of data processing, nine scans were performed at nine different stations distributed in the surrounding of the dam for the first time in October 2016, when the project began to impound. For each scan, the scanner was placed on the observation station. Then the coordinates of the scanner were measured based on the geodetic network. In this way, it was possible to get more precise coordinates of the scanner and reduce alignment error. It took approximately nine minutes to complete each 360° scan. The whole data set acquired in the first measurement campaign comprises one hundred and thirty-six million points in total, which is treated as the reference point cloud. The second measurement campaign was performed in April 2017 in a similar way as that in the first campaign, and both measurements were referred to the geodetic coordinate system.These two sets of TLS data were preprocessed using the RISCAN PRO software. First, the geodetic coordinates of each scanner were extracted from the total station based on the geodetic network. By simply adding the instrument height, the geodetic coordinates of the scanner center were acquired. Then the geodetic coordinate data was input into the RISCAN PRO software for the raw scans by the backsighting orientation. In this way, the location of each scan center was determined. After the input of the coordinates, the orientation of each scan remained random. Therefore, a manual modification of the orientations was carried out to make the orientations in space correct. The second process was multi station adjustment. This process was performed using the iterative closet point (ICP) algorithm proposed by Besl and McKay . In the After alignment of all nine scans, there were some unneeded objects such as dust and vegetation in the point cloud. Via automatic and manual operation, the data was run through terrain filter to remove such points. Eventually, a single point cloud colored with RGB information obtained from the calibrated camera on top of the terrestrial laser scanner came into being, whose partial view is shown in After the alignment of the point cloud, it comes to the stage of the triangulation. The basic input data that can be processed for NURBS surface reconstruction is the triangulated irregular network (TIN) rather than the point cloud. Hence, the TIN should be constructed based on the point cloud first, i.e., triangulation. The triangulation of points is also called “tessellation”. By using the Delaunay triangulation algorithm, each set of three closest points in the point cloud are connected to form a triangle, resulting in a non-overlapping triangulation as a whole. However, the initial triangulation generated automatically by the Delaunay triangulation algorithm usually has deficiencies, like acute angled triangles and holes. With automatic analysis, inconsistencies are detected and then repaired through automatic or manual operation. The final operation carried out to refine the TIN is the smoothing. The purpose of smoothing operation is to reduce the noise and therefore to ensure both the quality and accuracy of NURBS surface that will be constructed later. The downstream face of the Changheba dam is made of masonry; the dam surface is flat macroscopically. Thus, the smoothing operation is of central significance to the surface reconstruction.The point clouds for the Changheba dam are converted into a polygon object with 3.48 billion triangles in total . Only poThe previously-constructed TIN is exported and then processed into a NURBS fitting surface model of the Changheba dam. With high accuracy and high spatial resolution, this NURBS surface model can be applied to detect the deformation of the dam surface for further study.NURBS is the abbreviation of non-uniform rational B-splines, in which, the non-uniform means that the spacing of the knots is uneven, the Rational implies that the control point can be weighted, and the B-spline represents that B-spline is used as basis function. NURBS is a piecewise rational vector polynomial function advocated by Versprille in 1975 . It can P = {x, y, z} is a piecewise rational surface defined by Equation (3):i,jd  are the control points representing a topological mesh, i,jw is the so-called weights, and the i,kN(u), j,lN(v) are the normalized B-spline basis functions defined on the non-periodic knot. For instance, the mathematical expression of the i,kN(u) can be defined recursively as follow,A NURBS surface i,jd, weight factors i,jw and the knot vectors U and V must be determined. On the grounds of the NURBS theory, the dam surface is constructed from the TIN following the four procedures: panel demarcation, surface patch insertion, grid generation, and NURBS surface construction. The four procedures are executed in sequence to construct the NURBS surface, as explained in the following.To generate a NURBS surface, three groups of parameters, control points Data segmentation must be carried out to precisely construct the NURBS model of dam surface primarily. In this step, the characteristic lines are extracted from the TIN model. The surface curvature detection technique is adhibited for data segmentation. After setting the proper curvature level, the curvature detecting is performed and the contour lines are placed in the areas of curvature. In the meantime, Markers are highlighted on the contour lines. There are two different kinds of markers. A red marker indicates a corner, while a yellow marker represents a non-corner point of inflection. The contour lines and the boundary lines are used as panel demarcation lines dividing the TIN model into forty-three panels a. If a hOn the basis of the result of the panel demarcation, the patch boundary structure is generated, which means that four-sided patches are inserted into each panel b. A surfAfter the surface patches are inserted, a further process is grid generation. A grid is a quadrangular mesh constructed in every patch. The mesh is made up of a forty-by-forty set of rectangles, which means that each panel consists of one thousand and six hundred grids. The quadrangular mesh density can be specified by the user. Needless to say, a finer grid produces greater precision in the eventual NURBS surface. The grid generation process creates an ordered u-v grid in every patch on the model c.For the free-form surface of the dam, the exact NURBS surface can be ultimately generated on the TIN using the surface fitting technique. The NURBS surface of the dam is shown in i T in the test model, the algorithm searches for its nearest corresponding point j T in the reference model and computes the SD vector, iV, that starts in point i and ends in point j (Equation (6)) [Deformation can be detected by making geometrical comparison between multi-temporal surface models. In this study, the shortest distance algorithm is applied to the deformation measurement. It is noted that the normal directions of the two surfaces should be generally consistent before the comparison. The algorithm can still work even when the surface normal vector is biased. For each point ion (6)) .(6)Vi=(For the NURBS fitting surface, the central points of grid are used to calculate the shortest distance. The calculated SD vectors do not necessarily represent the real displacements of the object. They are the shortest distances from the point in the test model to the nearest neighbor point in the reference model mathematically . Yet, thTo analyze the deformation of the structure, it is necessary to choose the proper coordinate system . Herein,It is well known that the earth-rock dams are usually subjected to deformation over a relatively long time after the filling. Obviously, the deformation distribution is an essential indicator of the potential instability of the dam. Thus, periodic monitoring of the dam deformation during operation period is of great importance. The deformation of the surface is always larger than the interior due to the effect of displacement cumulation. Therefore, monitoring the surface deformation is an effective way of determining the serviceability of the dam. By introducing the TLS and NURBS technology, making comparison between different multi-temporal scans provides the plentiful and vital information for analysis of the deformation distribution of the dam.For the Changheba dam studied herein, the NURBS model in October 2016 was treated as reference model, while the NURBS model in April 2017 was regarded as the test model. The reference model was subtracted from the test model. In this way, more than eight hundred thousand central points of grid in the test model were calculated and color-coded mappings of the differences were generated, showing the deformation distribution over the monitored time interval during the operation period.The deformation distribution of the Changheba dam from October 2016 to April 2017 is shown in There are three main regions with positive changes in It is worth mentioning that the deformations in cross section A-A are much smaller compared to those in cross section B-B. One of the reasons is that the lower part of the dam has been subjected to settlement over a relatively longer time than the upper part of the dam. The lower part of dam started to deform right after its filling, at this time the upper part has not been filled. Another reason is that, after filling of the upper part, additional settlement of the lower part will be induced due to the addition of gravity load on the top. Thus, the relative smaller deformation of lower part is the consequence of combination of a longer period of consolidation and higher stress conditions.In this paper, a new deformation monitoring method for large structure is formulated. The field experiments are carried out to test its feasibility. In practical works, reducing error is of vital importance for deformation monitoring. According to the different stages of the methodology, the possible errors can be divided into three parts: instrument error, alignment error and modeling error. Many researches have been done on the instrument error and alignment error for the past decades ,35. HereThe deformation distribution has a significant influence on the stability and safety of large artificial and natural structures. By taking full advantage of the TLS and NURBS technology, a new methodology of deformation monitoring and analysis is presented. Using the earth-rock dam as an example, the point cloud gained by TLS allows the detection of small deformation for its high accuracy and capability of target acquisition. The NURBS model based on the point cloud is characterized by high precision and high spatial resolution. Eventually, the holistic deformation distribution of the downstream face is shown in the cloud chart. The deformation monitoring achieved great success with the proposed methodology. The TLS has proved to be a valid solution for the deformation acquisition in three-dimensional space. The NURBS modeling technology is capable of dealing with a huge number of points and making use of them. In comparison with the traditional monitoring, the methodology that integrates TLS and NURBS technologies permits a better grasp of the deformation distribution of the large structures.The millimeter level measurement requires that the data acquisition is performed with great patience and plenty of time. In the future works, data acquisition in the field should be optimized to reduce the field working time. Besides, the data processing is also time-consuming and complicated. Several procedures require personal experience and expertise. These are several potential areas in terms of improving the performance of the presented methodology, in which further research will be conducted.


Microstructures are observed before and after superplastic deformation. The deformation mechanism maps incorporating the density of dislocations inside grains at temperatures of 973 and 1123 K are drawn respectively. By applying the elevated temperature deformation mechanism maps based on Burgers vector compensated grain size and modulus compensated stress, the dislocation quantities and low-temperature superplastic deformation mechanisms of Ti-6Al-4V alloy at different temperatures within appropriate processing regime are elucidated.The low-temperature superplastic tensile behavior and the deformation mechanisms of Ti-6Al-4V alloy are investigated in this paper. Through the experiments carried out, elongation to failure ( The two-phase α-β Ti-6Al-4V alloy has been widely used for aerospace applications because of an attractive combination of properties such as high specific strength, relatively excellent fracture toughness, and strong heat and corrosion resistance ,2,3. HowThe superplasticity deformation mechanisms under given conditions can be identified according to some workability theories, especially a deformation mechanism map based on dislocation kinetics and metallographic theory. The deformation mechanism map has been successfully applied to stainless steel, aluminum and aluminum composites, magnesium alloy, nickel and its alloy, and titanium etc. ,12,13,14Recently, studies and reports on deformation mechanism diagrams have shown that aluminum base composite, nickel base superalloy GTD-111, industrial titanium alloy CP-Ti, and AZ61 magnesium alloy can also be mapped ,16,17. IA deformation mechanism map proposes a new method in discovering quantitatively the essence of a rate controlling process. The dislocation creep mechanism map of a solid solution alloy was ascertained by two regimes; i.e., dislocation viscous glide mechanism regime and dislocation climb mechanism regime. The dislocation breakaway solute atmosphere mechanism regime in the Langdon model is not included in the above deformation mechanism map. Therefore, it is necessary to construct a new type of deformation mechanism map for solid solution alloys.In this study, therefore, the low-temperature superplasticity and deformation behavior of Ti-6Al-4V alloy was investigated on the basis of the flow curves measured during tensile tests at different tensile temperatures with different strain rates. A new deformation mechanism map containing the dislocation quantity was constructed for the two-phase titanium alloy to predict its low-temperature deformation mechanism, and to analyze quantitatively the dislocation quantity during deformation. The results obtained in the present study can provide a theoretical basis for the application of low-temperature superplasticity of Ti-6Al-4V alloy.−4 and 5 × 10−3 s−1.The material used for the superplastic tensile tests was as a cold-rolled Ti-6Al-4V alloy sheet with a thickness of 2 mm. The gauge part of the superplastic tensile specimens was 6 mm in width, 15 mm in length, and 2 mm in thickness, as shown in 3, and 100 mL H2O, and then were observed using an optical microscope . The mean grain size and volume fraction of the β phase were examined using an image-analyzer .The specimens for the metallographic examination were mechanically polished and etched in a mixed solution of 20 mL HF, 40 mL HNO−4, 1 × 10−3, and 5 × 10−3 s−1 under different deformation temperatures. The changes of peak stress as a function of the deformation temperature and strain rate are shown in −4 exhibits no obvious softening behavior in the strain range of 0.3 to 2.0 σ=Kεnm value, is substituted into Equation (4) to get the activation energy, as shown in The data obtained from tensile tests are processed to plot When the deformation temperature is low and the strain rate is high, many cavities near the fracture surface appear. These cavities are small in volume, many in quantity, and distributed along the tensile direction, as shown in σ/G, against the homologous temperature, T/mT. For the present experiments, it is more convenient to use alternative forms of deformation mechanism maps in which the normalized grain size, d/b, is plotted against the normalized stress, σ/G, at a constant temperature or the normalized stress, σ/G, is plotted against the reciprocal of the homologous temperature, mT/T, at a constant grain size. Very little information is available at present on the construction of deformation mechanism maps for incorporating the dislocation quantity inside the grain metals processed using Low-temperature superplasticity.Deformation mechanism maps are used extensively in the field of high temperature creep to provide simple visual displays of the dominant creep mechanisms over a selected range of experimental conditions. The concept of mapping the flow processes may be traced to an early proposal for the construction of a creep diagram and the subsequent quantification of this proposal in the form of deformation mechanism maps plotting the normalized stress, In the present work, a new deformation mechanism map containing the dislocation density has been proposed for the Ti-6Al-4V alloy to predict its low-temperature deformation mechanism, and to analyze quantitatively the dislocation quantity during its deformation.iS is the state variable that describes the current microstructural state, and jP is the material properties, such as lattice parameter, atomic volume, binding energy, modulus, and rate controlling diffusion coefficient, etc. According to the different deformation mechanisms, iS, jP can be different values and algebraic expressions.Taking the Burgers vector compensated grain size as the Y-axis and taking the modulus compensated flow stress as the X-axis, authors construct the deformation mechanism maps at different temperature controlled strain rates. Each deformation mechanism can be expressed by a rate controlling equation as follows ,26:(5)γin, P are material constants, depending on the deformation mechanism; E is the Young’s modulus; b is the Burgers vector; D is the diffusion coefficient that equals to the lattice diffusion coefficient LD, or the dislocation pipe diffusion coefficient PD, or the grain boundary diffusion coefficient gbD.Specifically, a general deformation mechanism can be expressed by the following constitutive equation ,26:(6)ε˙Data corresponding to different deformation mechanisms are substituted into Equation (6), and three kinds of constitutive equations including diffusion flow mechanism, grain boundary sliding, and slip can be obtained. These equations were used to calculate the boundaries and nodal points of the different mechanism regimes. Computer software was also applied to plot the RWS deformation mechanism map.ν is Poisson’s ratio, The calculation model of the dislocation quantity in a single grain is as follows :(7)ni=2[ follows :The deformation mechanism maps of two-phase titanium alloy at 973 and 1123 K were plotted according to the above method, as shown in According to the flow curves at deformation temperatures of 973 and 1123 K in rographs . The box13) (3.03 × 107), indicating that the stress exponent is 7, and the corresponding deformation mechanism is the dislocation slip. Therefore, the deformation mechanism at 973 K gradually changes from the dislocation pipe grain boundary sliding to the dislocation slip with increasing strain rate. It can be seen from 4) (23), indicating that the stress exponent is 2, and the corresponding deformation mechanism is a grain boundary sliding controlled by lattice diffusion.It can be also seen from −4 are shown in TEM micrographs of superplastic deformation for Ti-6Al-4V alloy at 973 and 1123 K with an initial strain rate of 5 × 10Ti-6Al-4V titanium alloy consists of α and β phases in which the proportion of β phase is 38%. α phase is a hcp crystal structure with three slip systems in single cell; β phase is a bcc crystal structure with twelve slip systems in single cell. During superplastic deformation, the slip systems in β phase firstly operate; β phase can accommodate effectively the stress concentration, eliminate partial deformation resistance, and hence restrain the propagation of the crack source at a stress concentration during the deformation. In the meantime, the coexistence of a dual phase will restrain the deformation-induced grain growth. It is noted in −4–10−3 s−1, where the strain rate sensitivity exponent (m) varies from 0.23 to 0.52 and the deformation activation energy (Q) falls in the range of 106.70–402.61 kJ/mol. A maximum elongation to failure rate of 768% has been achieved with m reaching its peak value at 0.52 and Q nearing the grain boundary self-diffusion activation energy. Q decreases gradually when raising the deformation temperature and it becomes higher than the grain boundary self-diffusion activation energy when the temperature is between 973 and 1023 K. This shows that Ti-6Al-4V alloy has good plastic forming capability. In the deformation mechanism maps incorporating the dislocations inside the grains created, they illustrate the quantitative relationships among flow stress, grain size, and dislocation quantity, and accurately predict the superplastic deformation mechanism of Ti-6Al-4V alloy in a low-temperature range. The low-temperature superplastic tensile deformation mechanisms of Ti-6Al-4V alloy are a grain boundary slip induced by dislocation movement. At different strain rates, the dislocation sources in different directions of the Burgers vector in the grain start to form the grain boundary slip under different stress indices.The results demonstrate that Ti-6Al-4V alloy shows excellent superplasticity in the temperature ranging from 973 to 1123 K over a strain rate 5 × 10


This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIbα.The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib  The A2 domain, situated between A1 and A3, contains a proteolytic site that is exposed under shear and cleaved by the metalloprotease ADAMTS13 [The multimeric plasma protein von Willebrand factor (VWF) plays a key role in haemostasis in particular in the presence of rapidly flowing blood like in arteries and arterioles , 2. SheaADAMTS13 . Thus, tHyperactivaton of VWF for example due to arterial stenosis can cause pathological thrombus formation. For this reason, VWF has been the target of anti-thrombotic therapies. Importantly, recent experimental evidence indicates that inflammatory conditions are likely to increase the thrombogenic activity of VWF , 6. Thisβ sheet surrounded by α helices  calculations to study the effect of methionine oxidation on the kinetic and thermodynamic stability of the A2 domain. Since three of the in total five methionine residues in the A2 domain are located in the vicinity of the C-terminal helix it is plausible that their oxidation might destabilize the fold of the A2 domain by facilitating undocking of the C-terminal helix, similarly to a previous study . It is iThe crystallographic structure of the A2 domain with PDB code 3GXB  was used-8. Snapshots were saved every 10 ps for trajectory analysis.The MD simulations were performed with the program NAMD  using th-1, while the pressure was held constant at 1 atm by applying a pressure piston [Before production runs, harmonic constraints were applied to the positions of all heavy atoms of the protein to equilibrate the system at 300 K during a time length of 0.2 ns. To equilibrate the position of atoms around a methionine sulfoxide side chain, harmonic constraints were kept on all heavy atoms except those of the methionine sulfoxide residue and the neighboring amino acids, and equilibration was continued for another 2 ns. After this equilibration phase, the harmonic constraints were released. In order to analyse changes in the native state of the A2 domain upon oxidation, simulations were performed for in total 40 ns each where no external forces were applied . The fire piston .α atom of the N-terminus. The Cα atom of the C-terminus was attached through a virtual spring with a stiffness constant of 2 kcal/mol to a dummy atom that was pulled at a constant velocity of 5 Å/ns. The initial direction of pull was parallel to the axis through the fixed N-terminal Cα atom and the pulled atom. As the dummy atom is pulled, the spring extends. Using Hook’s law, the resulting applied tensile force is defined as F = Δx * k, where Δx is the extension and k the stiffness constant of the spring. The tensile force can then be plotted in function of time in order to monitor rupture events. It needs to be noted that from a physical point of view the direction of force is not relevant, because the protein would rotate as a rigid body until the axis through the pulled atoms is aligned parallel to the direction of pull, if this was not the case at the start of the simulation. Similarly, it is generally assumed that if the pulling is performed gently enough the force will propagate through the protein and it will not matter which atom is fixed and which one is pulled. Each pulling simulation was run for 15 ns in order to sample the key events in the unfolding pathway that were identified in a previous study with the A2 domain [The simulations with applied tensile force were started from snapshots sampled during the runs with no tensile force described above see also . The pro2 domain .The change in the free energy of folding due to the oxidation of a methionine residue was estimated by making use of alchemical transformations  in combi1494, Met1521, Met1528 and Met1606, to methionine sulfoxide ” is used here to denote the oxidized state of methionine). The goal is to study any structural and energetic modifications resulting from methionine oxidation on the fold of the A2 domain. It is important to note that methionine residues need to be accessible to solvent in order to be oxidized. Experimental studies show that shear stress increases the likelihood of finding methionine residues of A2 in the oxidized state including those that are buried when the protein is folded [Three MD simulations were performed with the A2 domain after replacing all five methionine residues with methionine sulfoxide . In addiulfoxide . The sin1545 has a significant portion of its side chain solvent exposed in both the unoxidized and the oxidized state 1545 (1606 forms hydrogen bonds with water molecules (α4-less loop) and a neighboring helix 1521  shows that as expected Meted state . On the t(O)1545 . All otht(O)1545 . Howeverolecules  althougholecules . Visual in [1606 . Interesne state . Smallert(O)1521 .1495 and Met(O)1545 have a large electrostatic interaction energy due to their accessibility to solvent molecules 1528 interact mainly with polar groups in the interior of the protein , antiphospholipid syndrome and sepsis . Thus, iA recent single molecule force spectroscopy study indicated that the A2 domain can exert its inhibitory function only when the disulfide bond between two C-terminal vicinal cysteine residues is reduced . AlthougOf all three A domains of VWF, the A2 domain is the most sensitive to tensile force because it does not have a disulfide bond linking the terminii, thus facilitating oxidation of methionine residues under shear. However, mass spectrometry measurements show that buried methionine residues in the A1 domain also have an increased rate of oxidation under shear, although not as pronounced as for the A2 domain . Two of 1521 and Met1528, causes a relatively large local destabilization. On the other hand, the free energy of folding appears unaffected when a solvent exposed methionine residue, such as Met1495, is oxidized. This is consistent with an increase of the hydrophilicity of a methionine residue once it gains a sulfinyl group conferring to the otherwise rather hydrophobic methionine side chain the ability to engage in hydrogen bonds. The work presented here is the first report to date where an alchemical free energy method has been applied to an amino acid modification due to environmental factors, like oxidation, rather than an engineered mutation. It would be interesting to apply the method used here to other examples where methionine oxidation is suspected to play a role in protein stability or function. In conclusion, the present study suggests that the experimentally observed activation of VWF under oxidizing conditions [A noteworthy observation from a structural perspective is the correlation between the SASA of a methionine residue and its change in folding free energy upon oxidation. Oxidation of buried methionine residues, such as MetS1 FigThe reported values are averages over three simulations while error bars denote standard errors of the mean.(TIF)Click here for additional data file.S2 Fig(a) SASA of methionine residues in the unoxidized  and the oxidized (Ox) state. (b) Fraction of frames where a methionine sulfoxide forms at least one hydrogen bond with water molecules. (c) Interaction energy between a methionine side chain and (top) the rest of the system or (bottom) the rest of the protein, respectively. Blue circles highlight significant electrostatic interactions between a residue and the solvent or the rest of the protein, respectively. Simulations were performed with the respective methionine residue in the oxidized state Click here for additional data file.S3 FigPresented here is an analysis of a previously published trajectory (run WT_pull_2) .(TIF)Click here for additional data file.S4 FigPresented here is an analysis of a previously published trajectory (run WT_pull_3) .(TIF)Click here for additional data file.S5 Fig2. The blue circles highlight the corresponding peak in force. Running averages over 20 ps are indicated in red for the force and in blue for the SASA, respectively. The time series with the unoxidized with-type are taken from a previously published study (WT_pull_2) [The vertical cyan line indicates the time point where the SASA exceeds 50 Å_pull_2) .(TIF)Click here for additional data file.S6 Fig2. The blue circles highlight the corresponding peak in force. Running averages over 20 ps are indicated in red for the force and in blue for the SASA, respectively. The time series with the unoxidized with-type are taken from a previously published study (WT_pull_3) [The vertical cyan line indicates the time point where the SASA exceeds 50 Å_pull_3) .(TIF)Click here for additional data file.S7 Fig2. The blue circles highlight the corresponding peak in force. Running averages over 20 ps are indicated in red for the force and in blue for the SASA, respectively. The time series with the unoxidized with-type are taken from a previously published study (WT_pull_1) [The vertical cyan line indicates the time point where the SASA exceeds 50 Å_pull_1) .(TIF)Click here for additional data file.