Ice retreat in West Antarctica and Antarctic Peninsula has led to important changes in seafloor communities and gains in benthic blue carbon. In most of the Antarctic, however, sea ice increased between the 1970s and 2014, but its effects on the benthos remain largely unexplored. Here, we provide a 1988–2014 record of macro- and megafauna from the north-eastern Weddell Sea shelf, where benthic biomass decreased by two thirds and composition shifted from suspension feeders to deposit feeders. Concomitant increases in sea-ice cover suggest a reduced flux of primary production to the benthos. As benthic communities are major repositories for Antarctic biodiversity and play an important role in biogeochemical cycling, the observed changes have far-reaching consequences for the Antarctic ecosystem and its feedback to the climate system. The findings underscore the importance of long-term ecological monitoring in a region vulnerable to warming and ice-shelf collapse. Sea-ice cover in Antarctica has increased over the last decades and reached a maximum in 2014. Here, the authors report strong declines in zoobenthic biomass and abundance and changes in community composition on the NE Weddell Sea shelf over 26 years, with implications for blue carbon and biochemistry in a globally important marine region. Antarctic benthos evolved in cold isolation, leading to life-forms characterized by low temperature adaptation, stenothermia, long generation time, late maturity age and brooding as prevalent reproductive strategy9. Extreme seasonal and interannual variations in food availability have favoured the capacity to maintain metabolic activity during periods of food scarcity11. Icebergs scouring the seafloor are a notorious source of disturbance creating large variations in abundance, biomass and species numbers on small spatial scales13.Antarctica harbours a unique benthic fauna displaying unusually high levels of biodiversity, endemism and biomass14, spatio-temporal heterogeneities in sampling effort, inconsistencies in sampling equipment and methods and scarcity of repeated measurements make it difficult to detect temporal changes in Antarctic benthic communities. As a result, only few studies conducted around the Southern Ocean have shown climate-related changes in benthic communities and key benthic groups. Most studies on the Antarctic benthos in a changing climate are in response to glacier, sea ice, anchor ice retreat, increased iceberg scouring and ice-shelf collapse19, establishing a link between pelagic productivity and carbon stored by benthos (blue carbon)21. As opposed to the Arctic, where sea-ice has been plummeting over the last decades22, two thirds of the Antarctic including the Weddell Sea, the East Antarctic shelf and the Ross Sea have seen increasing trends of sea-ice cover over the satellite instrumental period from 1979 up to a maximum in 201424, but the response of the benthos to this larger whitening region of the Antarctic is so far unexplored. Long-term studies from the shallow part of McMurdo Sound in the Ross Sea, showed decadal changes in the benthic community25 and in the growth and recruitment of sponges27, likely in response to changes in local productivity due to grounded icebergs. The only available data for the eastern Weddell Sea continental shelf shows a positive relationship between bryozoan growth and open water extent (and hence productivity) in an area of increasing sea-ice cover18. Given the absence of systematic repeated samplings with standardized methods, quantitative community scale time series data on the benthos from the Antarctica’s whitening deep continental shelves are so far lacking.The extreme patchiness, slow growth of most organisms30. KNA is considered a gate of entry for icebergs into the Weddell Sea31 and a hotspot of iceberg grounding20. As sea-ice cover is inversely related to productivity32 and organic material reaching the seafloor33, while icebergs act antagonistically reducing benthos by scouring34 and consolidating sea-ice between grounded icebergs25, our data set provides an opportunity to assess benthic biological responses to a whitening Antarctica.Here, we present a 26-year record of macrobenthic biomass and abundance off Kapp Norvegia/Auståsen (KNA) in the north-eastern Weddell Sea, and its associated changes with increasing summer sea-ice cover during the last decades36. Here we show temporal changes in benthic macrofauna abundance and biomass, megabenthic 3D complexity, and benthic community composition. Additionally, we relate the faunal changes to concomitant changes in sea-ice related productivity, and variation of iceberg area during our study period.KNA represents the intersection of eight ship-based expeditions between 1988 and 2014  and an increase in the proportion of young successional community stages Fig. . Large Aass Fig.  in addit28, a lower areal extent and duration of productive polynya areas and a lower vernal supply of pelagic food to the benthos32. Model-based studies suggest a delayed response of sea-ice cover to SAM variation38. Based on regression analyses, we were able to confirm SAM variations to significantly affect sea-ice cover changes with a delay of one year . The negative correspondence between SAM and productivity suggests that ice-free days and ice-free area dominate over other factors governing pelagic productivity in Antarctica 32.A positive phase of the SAM is associated with higher summer sea-ice concentrations in the eastern Weddell Seap < 0.05, permutations = 9999; green areas in Fig. t0; p < 0.05, permutations = 9999), commensurate with their direct dependence on fresh organic matter. The instantaneous response of the benthos supports earlier findings of rapid sponge growth26 overturning the long-held paradigm of slow growth in polar waters9. The observed coupling of macrobenthic biomass and productivity also supports the hypothesis that Antarctic benthos is food limited39. Deposit feeders and higher trophic levels (scavengers and predators), by contrast, showed a delayed biomass response  region Fig. , as deliPolarstern52 cruises ANT-VI/3. VII/4, XIII/3, XV/3, XVII/3, XXI/2, XXVII and PS82 (see https://www.pangaea.de/expeditions/cr.php/Polarstern for cruise reports), which represent the years 1988, 1989, 1996, 1998, 2000, 2004, 2011 and 2014, respectively. Only shelf stations were selected, i.e. all those stations located within 100–700 m depth, based on the definition of Antarctic shelves33. We excluded stations that were artificially disturbed in the long-term “BEnthic Disturbance EXperiment” (BENDEX)53, leaving a total of 59 multibox corer54 stations with a total 307 cores for the present study 55, which was excluded from the analyses and categorized separately (see below). Sediments were sieved through 0.5 mm mesh and the organisms retained on the sieve were fixed in 5% borax-buffered formaline and stored until microscopic enumeration and biomass analysis, generally within a few months after collection2. The taxonomic units as defined by Pineda-Metz et al.43 were used to calculate abundance and biomass of four major feeding guilds: deposit feeders, suspension feeders, scavengers and predators. Separation of feeding guilds was done following the classification scheme shown in Supplementary Table Benthic total abundance and biomass, and abundance and biomass data of 35 benthic taxa correspond to stations of the R/V To assess changes in the structural complexity of a benthic community from a flat  to a three-dimensional mature apex community dominated by megafauna , we analysed video- and photo-transects (i.e. imagery transects) from all expeditions, with the exception of 1989 for which no image data are available. Transects representing the years 1988, 1996, 1998, 2000, 2004 and one transect for 2014 (Supplementary Table 34). Categories 1–5 represent later stages of colonization by Antarctic shelf megafaunal assemblages sensu Gili et al.56, of increasing three-dimensionality . The frequency of all 3D configurations was assessed for all imagery transects, and was represented by five categories (from 0 to 4) to facilitate analysis. Each category represents the following proportions: 0 = absent/rare, 1 = uncommon, 2 = common, 3 = mainly, 4 = dominant. This first analysis gave a transect × 3D category table which was used with the prep.fuzzy function of the ade4 package for R57 to transform categorical values to percentage. These calculated percentages were used to estimate the average proportion of each 3D configuration category at each year.Megafauna was described on a categorical basis using an ordinal scale ranging from 0 to 5. Category 0 represented a putative scour mark in its earliest stage of recolonization, characterized by a lack of megafauna and an abrupt change in benthic cover and size along the transect , the OceanColor web data repository62 (Chl a and particulate organic carbon), Solar Geometry Calculator of the National Oceanic and Atmospheric Administration63 (NOAA), ALTIBERG Iceberg data base65 , and the “The Climate Data Guide: Marshall Southern Annular Mode (SAM) Index (Station-based)66.Environmental data were obtained from the National Sea Ice Data Center data repository66 for all summers of the years 1986–2014. We considered as summer the period November to March, which represents the months when the local polynya is open67. Based on this, we calculated the summer SAM index as the average value considering the period November to March.Monthly data of the Southern Annular Mode (SAM) index were obtained58. The daily sea-ice cover data were used to calculate ice-free days in summer for each cell within the KNA polygon. Additionally, data from the Solar Geometry Calculator of the NOAA63 were used to calculate the proportion of a summer day with sun light during summers of the period 1986–2014.Daily sea-ice cover from the Sea ice Index provided by the National Snow and Ice Data Center was extracted for the summer months of the period 1986–201432 found polynya productivity to be directly related to mean daily photo-synthetically usable radiation, number of ice-free days, ice-free area, sea surface temperature, continental shelf width, and basal melt rate. Based on this, we calculated the productivity for the 1979–2014 period as the product of ice-free days and proportion of a summer day with sun light for all cells located in the KNA polygon .Sea-ice and solar data were used to calculate a summer productivity index, referred in the main text as productivity. Arrigo et al.a and particulate organic carbon concentration, we correlated productivity with summer Chl a and particulate organic carbon data obtained from the NASA Ocean Color web62 for our study area, and Chl a data for the whole Weddell Sea67. As all correlations were significant  we extrapolated losses recorded in KNA to other Antarctic whitening shelves. We estimated the area of Antarctic whitening shelves to be 1.85 × 10Spearman correlation coefficients were calculated with 9999 permutations to test the association between productivity and iceberg area with benthic total abundance and biomass, for the total community and the feeding guilds. The same approach was used for 3D complexity. Negative coefficients imply a reduction (reduced), positive coefficients an increase of benthic abundance and biomass (increased) in response to the respective environmental driver investigated.57 and ArcGIS 10.x (ESRI) softwares. R packages included for treatment and analysis of data, as well as to design figures were: raster, car, rcompanion, ggplot2, corrplot, grid, dplyr, ade4, wPerm and extrafont.Statistical tests and figures were done using the RFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Sorghum bicolor L. Moench) is a promising source of lignocellulosic biomass for the production of renewable fuels and chemicals, as well as for forage. Understanding secondary cell wall architecture is key to understanding recalcitrance i.e. identifying features which prevent the efficient conversion of complex biomass to simple carbon units. Here, we use multi-dimensional magic angle spinning solid-state NMR to characterize the sorghum secondary cell wall. We show that xylan is mainly in a three-fold screw conformation due to dense arabinosyl substitutions, with close proximity to cellulose. We also show that sorghum secondary cell walls present a high ratio of amorphous to crystalline cellulose as compared to dicots. We propose a model of sorghum cell wall architecture which is dominated by interactions between three-fold screw xylan and amorphous cellulose. This work will aid the design of low-recalcitrance biomass crops, a requirement for a sustainable bioeconomy.Sorghum ( Sorghum is a source of lignocellulosic biomass for the production of renewable fuels. Here the authors characterise the sorghum secondary cell wall using multi-dimensional magic angle spinning solid-state NMR and present a model dominated by interactions between three-fold screw xylan and amorphous cellulose. These biorefineries are required to address the twin global demands of increased energy availability4 and reduced emissions5. Grasses, such as sorghum (Sorghum bicolor L. Moench), Miscanthus × giganteus, and switchgrass (Panicum virgatum L.), are promising as dedicated bioenergy crops, due to their high photosynthetic efficiency and ability to grow in a range of environmental conditions6. However, plant cell walls, which comprise most of lignocellulosic biomass, have evolved to provide many crucial functions to the plant, including structural support, and protection from herbivory and pathogens7. The resulting highly recalcitrant cell wall architecture is an impediment to the efficient deconstruction of plant cell walls into simple monomers9. Improved understanding of the architecture of intact grass plant cell walls at a molecular level will provide key insights into this recalcitrance, and support the predictable design of both the biomass deconstruction process, as well as the development of engineered bioenergy crops.Lignocellulosic biomass, a renewable source of organic carbon, will be a major feedstock for sustainable biorefineries producing bio-based fuels, chemicals, and materials10. It consists of flat-ribbons of α-1,4-linked glucan chains which self-assemble into a crystalline fibrillar structure via hydrogen bonding. The resulting fibril has surfaces with distinct hydrophilicity that allow potential interactions with other cell wall components. However, cellulose crystallites are not perfect, and there is a significant fraction of cellulose that is less ordered, referred to as amorphous cellulose. Hence, two major domains of cellulose can be distinguished: a crystalline cellulose domain and an amorphous cellulose domain. Xylan is the major hemicellulose, which makes up 20–35% of the dry weight of grass secondary cell walls13. Xylan has a α-1,4-linked xylosyl unit backbone. However, unlike in dicot plants, grass xylan is highly substituted by α-L-arabinofuranosyl units at the O-2 and O-3 positions on the xylosyl units of the backbone and comparatively low density of substitution with α-1,2-glucuronosyl or α-1,2-(4-O-methyl)-glucuronosyl units14. This xylan structure is known as glucuronoarabinoxylan (GAX), though the exact substitution pattern is dependent on tissue and species. In addition, grass xylan is also substituted with acetyl groups on the backbone xylosyl units at the O-2 and O-3 positions, and some arabinosyl units can also carry acetyls at the O-2 position15. Grass xylan is acetylated to a lesser extent compared to dicot plants15. Xylans are believed to be present in two major conformations, two-/three-fold screw conformations, and the conformation is highly dependent on the pattern of substitution. For instance, the even pattern of acetyls and glucuronosyl/4-O-methyl-glucuronosyl units on Arabidopsis thaliana (Arabidopsis) allows these substitutions to lie on one side of the xylan backbone, where every two xylosyl units have a 360° turn (two-fold screw) forming a flat-ribbon shape. Without this even pattern, the xylan will have a 360° turn every three xylosyl units (three-fold screw) forming a helical shape due to steric hindrance16. Additionally, another characteristic feature of grass secondary cell wall is the presence of hydroxycinnamic acids , which are esterified to the O-5 of the α-1,3-arabinosyl substitutions on grass xylan backbone and can be esterified and/or etherified to lignin18. Ferulate units on grass xylan facilitate the covalent cross-link of xylan-xylan and xylan-lignin by formation of diferulates through dehydrodimerization and participation in lignification via radical coupling to bridge the lignin and polysaccharides, and stiffen the cell wall and maintain structural integrity23.All plant cells are surrounded by a thin, extensible, primary cell wall. Thickened secondary cell walls are only deposited in certain cell types at the cessation of cell expansion, but form the majority of biomass. Secondary cell walls are composed of polysaccharides  and a complex polyphenolic network of lignin. Cellulose makes up 35–45% of the dry weight of grass secondary cell walls29. More recently, the architecture of secondary plant cell walls was also investigated via ssNMR. Simmons et al. found direct evidence for the presence of xylan in both two- and three-fold screw conformations in the dicot secondary cell wall. In a cross-polarizing experiment, which emphasizes the immobile components, the xylan-cellulose interaction was dominated by xylan in a two-fold screw conformation, with only a minor proportion of three-fold xylan detected30. Terrett et al. reported similar xylan-cellulose interactions in the secondary plant cell walls of softwoods due to the even pattern of substitution of softwood GAX. Additionally, galactoglucomannan, the other major hemicellulose in softwoods, binds the same cellulose microfibrils. Both cellulose-bound xylan and galactoglucomannan are also associated with lignin31. Kang et al. employed ssNMR to investigate the lignin-polysaccharide interactions in the secondary plant cell walls of maize (Zea mays). Results showed a conformation-dependent bridge behavior of xylan in the secondary plant cell walls, linking the cellulose fibrils and lignin, in which two-fold screw xylan coats the cellulose microfibril hydrophilic surface and the three-fold screw xylan connects the lignin nanodomain via electrostatic interactions32.Although the chemical structures of individual cell wall components are relatively well defined, the interaction between the cell wall components that form the three-dimensional network remains poorly understood for grasses. Methods to access the molecular information of intact plant cell walls were limited by the need for additional chemical extraction and pre-treatments, until a groundbreaking effort by Hong and colleagues which employed multi-dimensional solid-state Nuclear Magnetic Resonance (ssNMR) to reveal the architecture of native primary cell walls in Arabidopsis16. Evenly patterned decorations on the xylan backbone allow the substitutions to orient along one side of the molecule, leaving the undecorated side of the resultant flat-ribbon shape free to hydrogen bond with the hydrophilic surface of the cellulose microfibril34. Although the substitution pattern of the xylan backbone in grasses is not known, the large amounts of arabinosyl units on grass xylan suggests that it may not follow the dicot and softwood pattern, which may in turn alter its interaction with cellulose17.Using Arabidopsis with genetically engineered xylan structures, Grantham et al. determined that an even pattern of xylan substitution is critical for the formation of the two-fold screw conformation and its interaction with celluloseHere, to explore xylan-cellulose interactions in grasses, we perform multi-dimensional ssNMR analysis on sorghum to reveal the native architecture of its secondary cell wall. The results suggest a lack of two-fold screw xylan, with the majority of the xylan showing a three-fold screw conformation. However, this three-fold screw xylan shows relatively high rigidity as compared to Arabidopsis and softwood reported earlier, and close proximity with less ordered amorphous cellulose. We propose that sorghum xylan-cellulose interactions are dominated by xylan in the three-fold screw conformation and amorphous cellulose, in contrast to the interactions between xylan in the two-fold screw conformation and crystalline cellulose in softwoods and dicot plants. We also show that the fraction of amorphous cellulose in the sorghum secondary cell wall is approximately three-fold higher than that in Arabidopsis, a model dicot plant. These findings provide molecular level understanding of the grass cell wall structure. Accurate cell wall models will enable a predictive understanding of biomass deconstruction by identifying the most recalcitrant aspects of the architecture. These models will also aid the identification of molecular targets for developing bioenergy crops with improved biomass properties.13CO2-containing atmosphere in our in-house 13C-growth chamber, as previously described35. Upon harvest, all tissue was immediately frozen in liquid nitrogen, and sliced into 1–2 mm pieces to enable packing into the rotor, preventing them from thawing. No further processing of the tissue was performed, to allow us to investigate a cell wall architecture as close to native as possible. Stem internode tissue  from the 3rd internode was divided into upper, middle, and lower . Leaf and root tissue  was also retained and used for comparison.Sorghum plants were grown hydroponically in a 13C enrichment was confirmed by following the procedure described in35, and all tissue used in this study had a 13C incorporation rate of over 90% , and used for compositional analysis. 13C NMR experiments, which detects all the 13C carbons present in the sample, were performed with a long (30 s) recycle delay  one-dimensional (1D) 13C-13C correlation experiments using the refocused INADEQUATE sequence with cross polarization (CP)36. This measures the chemical shifts of directly bonded 13C nuclei from relatively immobile components of the cell wall  we represent these different environments as follows: superscript [C] or [A] represents the crystalline or amorphous cellulose domain respectively, and superscript3 represents the three distinguished environments of cellulose in each domain. The major chemical shift differences between the two domains of cellulose are present in the cellulose carbon-4 (C4) at ~89 ppm (1–3CC4) and ~84 ppm (1–3AC4), and C6 at ~65 ppm (1–3CC6) and ~62 ppm (1–3AC6). The signals from xylosyl units of three-fold screw xylan backbone were identified , but there were a lack of signals from xylosyl units of two-fold screw xylan (2fXn1–5)  and three-fold  screw xylan backbone, the immobile three-fold screw xylan was the dominant conformation. This could be due to the large quantity of arabinosyl substitutions on the sorghum xylan backbone. Dupree et al. previously demonstrated that the spacing between the substitutions is critical for the formation of two-fold screw xylan due to the steric hindrance effect34. For instance, xylan in dicots has little or no arabinosyl substitution, and acetylations and glucuronic acid substitutions are evenly spaced on a major fraction of the xylan backbone, which allows the formation of two-fold screw conformation. Similarly, although softwood xylan is substituted by both glucuronic acid and arabinose, substitutions are evenly spaced on every six and two xylosyl units respectively on the xylan backbone. Hence, we speculate that a large number of closely spaced substitutions on sorghum xylan would likely disrupt any pattern of regular spacing on a major fraction of the xylan. The result of this would be more xylan in the three-fold screw conformation in sorghum secondary cell walls. This spacing pattern has yet to be confirmed, due to a lack of a glycosyl hydrolase with the required specificity for cleaving at an arabinosyl substitution, akin to the glucuronic acid-dependent xylanase XynC used to determine the spacing pattern in Arabidopsis41. However, since CP experiments emphasize the signals from relatively immobile components of the cell wall, the large amount of three-fold screw xylan detected suggests that this confirmation could be important for xylan-cellulose interactions in sorghum.Two-fold screw xylan with a flat-ribbon shape has been reported as the dominant conformation detected by CP in the secondary cell walls of dicot plants42. We hypothesized that this could be due to the additional lyophilization step they performed on the plant samples prior to analysis, which may alter the native wall structure. To test this, we lyophilized the sorghum stem tissue and rehydrated the sample according to the procedure that Kang et al. described in32. A CP-INADEQUATE experiment was conducted on the rehydrated sample and compared with our previously collected CP-INADEQUATE spectrum on the untreated sample . We found that the previously strong signals from arabinosyl units were lost in the spectrum of lyophilized-rehydrated sample  experiments were performed with CP with three different mixing times . Due to the CP transfer used in the experiment, these measurements report on the immobile fraction of xylan in the sample characterized by limited larger-scale molecular motions about an average structure with motional timescales on the order of µs to ms. This is in contrast to the mobile fraction of xylan, which is characterized by much faster molecular reorientations with motional timescales on the order of ns to µs. PDSD experiments provide information on carbons in close spatial proximity. The longer the mixing time, the longer the distance observed on the spectra.Xylan in the intact plant cell wall can populate both mobile and immobile statesrms Fig. . To inve3fXn4-2AC4 , 3fXn3-2AC4 , 3fXn3-1AC6 , 3fXn3-3AC6 , 3f,AXn3-2AC4 , and 3f,AXn3-3AC6 , all indicate that the xylosyl units from xylan in a three-fold screw conformation is closely interacting with amorphous cellulose. Additionally, carbons from the xylosyl units of three-fold screw xylan, 3fXn2 to 3fXn5, show cross peaks with the C1 from cellulose (105.2 ppm), which derives from cellulose in multiple environments. However, since we observed no cross peaks between carbons from three-fold screw xylan and CC2 to CC6, we conclude that the 3fXn2- to 3fXn5-C1 cross peaks were primarily contributed by the C1 from amorphous cellulose. Furthermore, arabinosyl units from xylan show close interactions with the cellulose C1, as indicated by two cross peaks, A2-C1  and A4-C1 . Since a low abundance of two-fold screw xylan was observed  CP-PDSD experiment is dominated by intramolecular peaks, such as carbons from glucan chains with six identified allomorphs in cellulose microfibrils, xylosyl units in the xylan backbone in both two- and three-fold screw conformations, and arabinosyl units from xylan Fig. . In addi1) at various chemical shifts representing different components  and xylosyl (3fXn2 to 3fXn5) units in relatively immobile three-fold screw xylan, ~5 s. T1 measurements of the cell wall components further demonstrate that amorphous cellulose shares similar molecular dynamics with the relatively immobile fraction of the three-fold screw xylan, while crystalline cellulose has significantly reduced molecular motion. This supports our interpretation that amorphous cellulose and three-fold screw xylan are closely interacting with each other.To further explore the interaction between three-fold screw xylan and amorphous cellulose, we measured the spin-lattice relaxation times  from the 31. To enable a direct comparison of sorghum with Arabidopsis , we also produced 13C-enriched Arabidopsis stems and analyzed them using CP-PDSD with a short-mixing time (30 ms). Figure 1-3AC1–1-3AC6  of amorphous cellulose  in Fig. 2fXn4–2fXn5  in Fig. Since the sorghum secondary cell wall is dominated by three-fold screw xylan and amorphous cellulose interactions, we next investigated how sorghum cell wall architecture, in particular the xylan-cellulose interactions, compare to a wall architecture that is dominated by interactions between two-fold screw xylan and highly ordered crystalline cellulose, such as that described for dicots and softwoodsose Fig. . In the 1CC1–1CC6 , 1CC3–1CC4 , 2CC2–2CC4 , 1-2CC1–1-2CC4 , and 1-3CC6–1-3CC4 , were significantly diminished in sorghum compared to Arabidopsis. This is in contrast to the intensities of cross peaks from the intramolecular interactions of amorphous cellulose, which are similar between sorghum and Arabidopsis. To obtain the ratio of crystalline and amorphous cellulose, integrations were performed on cross peaks from the intramolecular interactions of all crystalline cellulose between C6 and C4  and all amorphous cellulose . The ratio determined for Arabidopsis is 0.8:1, which is consistent with previous studies49. However, sorghum has a crystalline to amorphous cellulose ratio of 1:3, which is approximately three folds more amorphous cellulose in sorghum than in Arabidopsis and softwoods49. This is also verified by the relatively more quantitative 1D 13C CP experiments, from which the crystalline and amorphous cellulose ratio were determined based on the integration of deconvoluted crystalline and amorphous cellulose C4 peaks. Similar results to the cross peak analysis were obtained from the 1D experiments . As a corresponding phenomenon to the low ratio of two- and three-fold screw xylan, more less-ordered amorphous cellulose is found in the sorghum secondary cell walls, which would facilitate interactions with the three-fold screw xylan.Additionally, Fig. 46. However, there is a tremendous amount of information still to be elucidated regarding the networks and interactions between cell wall components. This is especially true for the grasses, since digestibility/recalcitrance of the cell wall is a key feature determining their application in bioenergy, animal feed, and human health53. Previous work demonstrated that the presence of two-fold screw xylan, which requires an even pattern of substitution on its backbone, is critical for interactions with the cellulose microfibrils on the hydrophilic surface in plant cell walls34. However, how these xylan-cellulose interactions are formed when such two-fold, evenly substituted xylan is present in low abundance , remains unclear.Recent studies of plant cell wall architecture using multi-dimensional ssNMR techniques have meant that the molecular details of plant cell walls are better understood than ever for various plant species19.We found a lack of interactions between two-fold screw xylan and cellulose, and instead observed a close proximity between three-fold screw xylan and cellulose in the short-mixing time (30 ms) CP-PDSD spectrum. Additionally, the cellulose that shows closest proximity to the three-fold screw xylan is mostly the less ordered amorphous cellulose. Although the interaction with highly ordered crystalline cellulose on hydrophilic surface via hydrogen bonds requires xylan to be also oriented in a flat-ribbon morphology, the high fraction of helical three-fold screw xylan may promote relatively weak interactions with the less ordered amorphous cellulose domain Fig.  via Van Miscanthus (a grass) enhanced the biomass enzymatic digestibility54. The researchers also found that GAX-derived arabinose was released by cellulase digestion that preferentially took place at the amorphous region, but not by xylanase, which suggested that the xylan which is densely substituted with arabinosyl units is closely associated with amorphous cellulose, consistent with our findings. Similar effects were later reported in other grasses, including rice56, sweet sorghum57, wheat56, and switchgrass59. Our findings provide direct evidence for interactions between three-fold screw xylan and amorphous cellulose, which uncovers the functional role of highly substituted xylan in grass in sorghum secondary cell wall architecture. This work provides foundational knowledge to guide future genetic engineering approaches for the design of cell walls in improved bioenergy, forage and food crops.Li et al. have demonstrated that increasing the degree of arabinosyl substitution in GAX from −2 s−1, 14 h light/10 h dark) for 17 days with four leaves. Seedlings were then transferred into a 12 L plastic container (purchased from U. S. Plastic Corp.) containing 11 L of Hoagland’s nutrient solution pH 6.0, and placed in a self-constructed 13C enrichment growth chamber  for another 29 days, as previously described35. Stem tissue was harvested from the three internodes, and then split into three: “upper”, “middle” and “lower”. Root and leaf tissue were also harvested at the same time.Sorghum seeds (Tx430), a kind gift from Professor Jeff Dahlberg, UCANR, were sown on soil (PRO-MIX ‘HP’ with Mycorise PRO) and germinated in a controlled environment room  and grown vertically for 10 days. Seedlings were transferred into a 5 L plastic container with 4 L of Arabidopsis hydroponic solution, as described previously60 and placed into the 13C enrichment chamber  for 29 days.Arabidopsis seeds (ecotype Columbia-0) were surface sterilized and sown on half Murashige and Skoog salts including vitamins , agar (0.7% w/v) and sucrose (1% w/v). Following stratification (48 h at 4 °C in the dark), the plates were transferred into a growth chamber  overnight. Lyophilized samples were then rehydrated by adding 40 wt% deionized water and sonicated for 30 min before packing into a rotor for solid-state NMR analysis32.Lyophilized-rehydrated sorghum sample were prepared by lyophilization of 13C-enriched plant materials61. Sorghum AIR (1 mg) was pretreated with 4 M NaOH (50 µL) for 1 h at room temperature, then neutralized with 1 M HCl. Ammonium acetate buffer  was added to the sample, followed by digestion with endo-1,4-α-xylanase  overnight at room temperature. Arabidopsis AIR (1 mg) was incubated in ammonium formate  with 4 U/mL xyloglucan-specific endo-α-1,4-glucanase  for overnight at 37 °C.AIR was prepared from the 62 and LXXG/GXXXG63, were used to determine the 13C incorporation into the sorghum and Arabidopsis tissue, respectively. The 13C incorporation of sorghum and Arabidopsis used in this study was 92 and 91%, respectively.The supernatants, containing released oligosaccharides, were mixed with 0.5% (w/v) of 2,5-dihydroxybenzoic acid in 50% methanol in water with 0.1% (v/v) TFA, and spotted on an MTP 384 target plate (Bruker). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry  analysis was used to determine the mass and relative abundance of the released oligosaccharides. The oligosaccharides XAXX13C MAS NMR experiments were performed at the Environmental Molecular Sciences Laboratory (EMSL) on an Agilent 850 WB with a direct drive (VNMRS) NMR console. The never-dried 13C-enriched tissue was sliced into ~1–2 mm pieces using a razor blade on a cold surface (−20 °C). Samples (~30 mg) were packed into 3.2 mm zirconia rotors with O-ring seals (Revolution NMR). All 13C NMR spectra were obtained at 20 °C and with a static magnetic field strength of 20.0 T . The NMR probe utilized was an Agilent 3.2 mm HXY MAS probe tuned in double resonance mode, and the MAS frequency was 13 kHz. CP MAS acquisition was accomplished with a standard ramped CP pulse sequence using a 4 µs 1H 90° pulse, a 1 ms contact pulse (ramped 1H RF amplitude64), and a 2 s recycle delay. SPINAL-64 decoupling65 was applied during acquisition at a 1H nutation frequency of 62–80 kHz. 13C chemical shifts are reported relative to tetramethylsilane [(CH3)4Si] by assignment of the methylene peak in adamantane at 38.48 ppm as an external secondary standard. Two-Dimensional (2D) DQ to SQ correlation experiments (through-bond) were performed via a refocused INADEQUATE67 pulse sequence with either CP or DP. Through-space interactions were measured with SQ–SQ 2D PDSD68 experiments with mixing times of 30 ms, 100 ms or 1.5 s. T1 measurements were performed with a CP analogue of an Inversion Recovery experiment where the polarization transfer step is immediately followed by a π/2 13C pulse to invert the carbon signal. The magnetization is then allowed to relax by a varying delay and then read out with another π/2 pulse. Fitting the signal versus the variable delay to a biexponential (see equation in Supplementary Table 1 constants. All ssNMR data on sorghum stem internodes samples and Arabidopsis stem sample were collected by using VNMRJ software (v4.2 A) and analyzed by using MestReNova software (v14.0). T1 measurement data were fitted by using OriginPro 2016 software.For the sorghum stem internodes samples and Arabidopsis stem sample, solid-state 1H 90° pulse of 3.5 µs, a 13C 90° pulse of 4 µs, 1H-13C CP with irradiation at ~70 kHz and 50 kHz on the 1H and 13C channels, respectively, for 1.0 ms with a 1H ramp of 20%, and 70 kHz TPPM 1H decoupling. The t1 time increment was 20.0 µs and 250 States-TPPI points were collected for a 2.5 ms t1 evolution time. The spin echo delay time was 4.2 ms. 128 scans were averaged with a recycle delay of 1.75 s for a total experiment time of 15.6 h. All ssNMR data on sorghum root and leaf samples were collected by using Bruker Topspin software (v2.1) and analyzed by using MestReNova software (v14.0).For the sorghum leaf and root samples, solid-state NMR measurements were also recorded on a Bruker Avance wide-bore 11.7 T NMR spectrometer using a 4 mm triple resonance solenoid 4TR Bruker MAS probe. Samples of leaves and roots were removed from −80 °C storage and kept on dry ice until prior to packing into a 4 mm zirconia rotor and sealed with cyanoacrylate gel and a vespel cap. Experiments were recorded immediately after packing the rotor with the sample. The refocused CP-INADEQUATE spectra were obtained at room temperature with a spinning speed of 10 kHz, a Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Calluna vulgaris, is a major invasive weed in New Zealand, especially on the Central Plateau, where it has spread rapidly since its introduction in 1912, outcompeting native species. However, the chemical behaviour of heather in its invaded ranges is poorly understood. We aimed to explore the natural variation in volatile emissions of heather and the biotic and abiotic factors influencing them on the Central Plateau of New Zealand. To this end, foliar volatiles produced by heather at four different sites were collected and analysed using gas chromatography coupled to mass spectrometry. Soil properties, herbivory and other environmental data were also collected at each site to investigate their effects on VOC emissions using generalised linear models (GLMs). Our results reveal significant differences in VOC emissions between sites and suggest that soil nutrients are the main factor accounting for these differences. Herbivory and temperature had only a minor effect, while soil water content had no impact. Further studies are needed to investigate how these variations in the invasive plant’s foliar volatiles influence native species.Invasive plants pose a threat to natural ecosystems, changing the community composition and ecological dynamics. One aspect that has received little attention is the production and emission of volatile organic compounds (VOCs) by invasive plants. Investigating VOCs is important because they are involved in vital ecological interactions such as pollination, herbivory and plant competition. Heather,  The intentional or accidental introduction of exotic species, including plants, into new regions, poses a threat to biodiversity . Plant iPlant volatile emissions are species-specific  but alsoChionochloa rubra (red tussock) and Poa colensoi (blue tussock) (both grasses), and Leptospermum scoparium (mānuka) and Dracophyllum subulatum (Dracophyllum)  [Calluna vulgaris (heather) is a European native perennial shrub from the family Ericaceae. This alien species was deliberately introduced to the Central Plateau in 1912 [The Central Plateau is a volcanic area covering the central part of the North Island of New Zealand, including the Tongariro Natural Park (TNP), a world heritage site of natural and cultural value. Common native plants in this area include species) . Calluna in 1912 ,21 and iLochmaea suturalis, have been of limited success due to the persistence of seedbank in the soil and poor establishment of the biocontrol agent [On the Central Plateau, heather has invaded the seral tussock grasslands, modifying soil properties, outcompeting native vegetation and disrupting the natural processes of plant regeneration and succession ,23,24,25ol agent .E)-4,8-Dimethyl-1,3,7-nonatriene. The authors investigated seasonal variation and emission responses to six years of climatic manipulations  in a semi-natural setting and found seasonal variation in VOC emission but also a significant effect of the abiotic variables tested. However, to our knowledge, our study is the first to explore the VOC emissions of heather in its invasive range and the factors affecting their emissions under natural conditions .A recent study explored the VOC emissions of heather as a main component of heath ecosystems in its native range (Denmark)  and founβ = −1.32, X2 = 5.66, p = 0.017) and site 2 . The same was true for homoterpenes, sesquiterpenes and fatty acid derivatives  and site 2 . Total sesquiterpenes were also significantly lower at site 4 compared to site 1 , site 2  and site 3 . The proportion of homoterpenes was significantly higher at site 1 , site 2  and site 3  compared to site 4.After collecting samples from the headspace  of heather branches enclosed in nylon cooking bags and analysing them using a Gas Chromatograph-Mass Spectrometer (GC-MS), we identified 33 volatile compounds and grouped them under their respective chemical classes . The mosivatives . Total fE)-4,8-Dimethyl-1,3,7-nonatriene ((E)-DMNT)  to further explore differences in plant volatile emission between sites. The first axis of principal component analysis (PC1) explained about 31% of the total variance in VOC emissions among the four sites and was mostly characterized by fatty acid derivatives and (E)-DMNT) . PC2 wasWe collected soil samples and environmental data  at each site. Soils analyses revealed that all sites were nutrient poor; however, at site 4, most nutrients, except for N, were lower .X2 = 58.275, df = 3, p < 0.001), with site 2 having a significantly lower temperature compared to the other three sites  and site 1 .The soil water content (SWC) also differed significantly between the four study sites  with site 3 having the highest soil temperature, while the lowest soil temperature was recorded at sites 2 and 4 . There was a greater number of Hemiptera on heather at site 4 in comparison to the other sites , with a greater number of mites at site 4. The number of thrips found on heather at site 1 was higher than the other sites, but only marginally significant  with Hemiptera, thrips, spiders and mites being the most dominant groups . The numer sites . SimilarX2 = 2.475, df = 3, p = 0.480) although slightly higher damage was recorded on the plants at site 2 -3-hexenol, (Z)-3-hexenyl acetate, (Z)-β-ocimene, (E)-β-caryophellene, γ-elemene, copaene and humulene) were not significantly affected by the factors tested in this study  to investigate the effect of biotic and abiotic factors on VOC emissions. For this purpose, the 21 volatile compounds with higher contributions to the first six components selected through the PCA were used . The GLMis study .Z)-2-hexenol , (Z)-3-hexenyl 2-methylbutyrate , (Z)-3-hexenyl benzoate , (Z)-3-hexenyl butyrate , (Z)-3-hexenyl hexanoate , (Z)-3-hexenyl isobutyrate  and (Z)-3-hexenyl valerate . In addition, the emissions of terpenoids -α-farnesene , α-gurjunene , germacrene B , germacrene D , (E)-β-farnesene  and (E)-DMNT  were significantly affected by soil nutrients -2-hexenol , (Z)-3-hexenyl benzoate , germacrene D  and (E)-DMNT . The effect of herbivory on VOC emissions by heather was minimal, only significant for hexyl acetate  and germacrene B , while soil water content had no significant impact on the volatile emissions of heather . This species emits large amounts of terpenoids (E)-DMNT -DMNT .The effect of soil nutrients on VOC emission by plants is still poorly documented, but previous reports suggest that nutrient depletion has a negative impact on plant volatile emissions ,30,31. TThe production of constitutive defence compounds and the inducibility of such compounds can also be controlled by nutrient availability, with some plants investing more in their production at the expense of growth under nutrient limited conditions . This exZ)-3-hexenyl acetate, (E)-β-caryophellene and γ-elemene (Z)-3-hexenyl acetate are typically involved in direct and indirect defence by repelling herbivores and attracting their natural enemies [The main compounds with reduced emissions at site 4, which had nutrient-poor soil, were  comprising about 50% of the VOCs released by plants attacked by chewing herbivores ,13,43. IFeeding damage observed on heather was caused by generalist species, and the slightly lower damage at the heather dominant site (site 1) and site 4, where another invasive plant (Scotch broom) is present, suggests that herbivorous arthropod communities are depauperate at sites where invasive plants are dominant. A recent review found that invasive plant species are often associated with an overall reduction in arthropod abundance and taxonomic richness . Howeverde novo synthesized and stored volatiles [Z)-3- hexenyl benzoate, germacrene D and (E)-DMNT by heather were affected by temperature differences between sites. Temperature did not account for the emissions of other volatile compounds, which is likely due to the homogeneous weather during the VOC collection periods, indicating that there must be some stability in VOC emission that can withstand certain levels of environmental variation [Elevated temperature is also known to increase the emission of various volatile compounds ranging from fatty acid derivatives to terpenoids ,49,50, aolatiles . In thisariation .The effect of water stress on VOC emissions is not consistent in the literature and has been proposed to be dependent on plant species, duration and severity of water stress, as seen in isoprene-emitting species ,53. In o2, drought and night-time warming over six years [2. Experimentally induced drought also had a negative impact on monoterpene emissions immediately after treatment application and in the late growing season, while experimental night-time warming increased total emissions, showing the potential impact of climate change on heather VOC emissions [A recent study found significant variation in the VOC emission of heather in response to experimentally induced elevated COix years . The resmissions . In contCalluna vulgaris at four different sites on the Central North Plateau of New Zealand. Our study provides the first evidence suggesting that volatile emissions of C. vulgaris are influenced by different environmental factors, with soil nutrients (K) being a major contributor to the variation in emissions under natural conditions in its invasive range. This work explores the chemical behaviour of the highly invasive environmental weed Cytisus scoparius), whereas the site with highest emissions was dominated by heather. The other two sites had a combination of heather with native species  evidencing differences in plant community composition  in all sites, suggesting a possible effect of interaction of the target plants with microorganisms . Furtherposition .We therefore encourage further research to investigate the impact of plant-microbe interactions and other variables, such as neighbouring plant identity on heather volatile emissions. We also recommend exploring how these changes in a plant’s emissions influence the foraging behaviour of pollinators, soil arthropods, key herbivores and their natural enemies, as well as their impact on native plants. Such studies could provide valuable information on how volatiles contribute to the successful invasion of plants into novel environments.2 per site) were set up: three within the Waiouru Military Training Area (WMTA) to the east of TNP and the fourth site near Erua, a small settlement on the western border of TNP  has been released into this region as a bio control agent [Five plants of the same size and phenology were selected at each of the four sites. The introduced heather beetle  was inserted in the pull tube ,62. Coll11H22O2) (Sigma Aldrich) as an internal standard. The samples were analysed using gas chromatography coupled to mass spectrometry (Shimadzu technologies) with a 30 m × 250 μm × 0.25 μm TG-5MS column and helium as a carrier gas. Operating conditions were as follows: injector temperature 230 °C; split ratio of 10; initial oven temperature at 50 °C, held for 3 min then increased to 95 °C at a rate of 5 °C/min. Tentative identification of compounds was achieved by comparing them with target spectra in the MS library from the National Institute of Standards and Technology (NIST) and, when available, verified by authentic standards (Sigma Aldrich).Collection filters were eluted using 200 μL of 95% hexane with 10 ng/mL nonyl acetate  were collected at random points surrounding each sampled plant, for a total of 20 soil cores per site. The fresh weight of each sample was measured on the day of collection, and then oven-dried (40 °C) to constant weight. Soil water content (SWC) was measured gravimetrically  and exprThe ambient temperatures for experimental sites were obtained by installing temperature data loggers  from mid-November to mid-December 2017. Soil temperatures were taken from five positions covering each site using a soil temperature probe.Arthropods were collected from each sampled plant using the beating tray technique ,66. BeatThe foliage enclosed in the bags from VOCs sampling was used to estimate herbivore damage. Due to the small size of the leaves, visible herbivore damage was assessed using a handheld magnifying glass. The number of damage marks seen on foliage was counted as illustrated in Statistical analyses were performed using RStudio, Version 1.1.456 (RStudio: integrated development for R) . The ShaPrincipal component analysis (PCA) was performed using all the volatile compounds identified from the headspace of heather. PCA and descriptions of variable presentations in respective components were performed using the “FactoMiner” package . A GenerA second GLM was then performed to determine the effect of environmental variables on volatiles emitted by heather. VOCs with higher contributions in PCA were the response variables. Herbivore damage, soil water content (SWC), average daytime temperature and primary macronutrients  were initially selected as potential predictor variables. These predictors were selected based on their importance to plant performance and VOC emissions ,51,72,73


Caldesmon, an actin-binding protein, can inhibit myosin binding to actin and regulate smooth muscle contraction and relaxation. However, caldesmon has recently attracted attention due to its importance in cancer. The upregulation of caldesmon in several solid cancer tissues has been reported. Caldesmon, as well as its two isoforms, is considered as a biomarker for cancer and a potent suppressor of cancer cell invasion by regulating podosome/invadopodium formation. Therefore, caldesmon may be a promising therapeutic target for diseases such as cancer. Here, we review new studies on the gene transcription, isoform structure, expression, and phosphorylation regulation of caldesmon and discuss its clinical implications in cancer. The calFrom a structural perspective, caldesmon contains amino (N)- and carboxy (C)-terminal domains and a middle region . The N-tThe tissue and cell distributions of H-caldesmon and L-caldesmon are different. H-caldesmon is expressed in vascular and visceral smooth muscle and not in myofibroblasts, rhabdomyosarcoma, or tumors derived from myofibroblasts . Therefo2+/calmodulin binding protein capable of regulating actomyosin contraction, actin filament dynamics, and cytoskeleton remodeling in smooth muscle and non-muscle cells , calmodulin activated by Ca2+ forms a complex with caldesmon, and this complex is freed from actin filaments  and radiation treatment than L-caldesmon-negative cell lines . The exp2+/calmodulin and via phosphorylation  Alternative splicing of the caldesmon gene determines its different structures and the expression of its isoforms. (2) H-caldesmon and L-caldesmon conserve the completely identical sequences in the N- and C-terminal domains, and the central repeating sequence of H-caldesmon is deleted in L-caldesmon. (3) Although H-caldesmon and L-caldesmon have similar functional domains, their tissue and cell distributions are different. (4) Caldesmon can be a biomarker for the pathological diagnosis of tumors and the prediction of chemoradiotherapy response. (5) Caldesmon can suppress tumor metastasis by regulating podosome/invadopodium formation and vasculogenesis. Future research aspects may include (1) clinical data about the relationship between expression of the two isoforms in cancers (primary and metastasis) and patient survival; (2) the effects of the expression of upregulated or downregulated isoform in cancers (primary and metastasis) on cell motility and invasive characteristics; and (3) evidence-based clinical studies or animal models on the role of caldesmon in gastrointestinal motility disorders are critically required.Y-BY and C-FX prepared literature research and wrote the manuscript. J-GL conceived the ideas and wrote the manuscript. CW and Y-BY designed project, wrote the manuscript, and prepared figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Screws from each manufacturer were subjected to maximal stress force until they fractured. The fracture points were recorded and compared among all samples. To compare the mean values of fracture torques, the reference values associated with each brand and the sample results were used in t-tests. ANOVA  was used to compare the maximal resistance limit between brands, complemented with Tukey’s multiple-comparison test. The maximal considered level of significance was 5%. The average fracture force for the brands was 40.07 Ncm for Megagen®, 53.39 Ncm for Dentium®, 39.74 Ncm for Biomet 3i®, and 68.84 Ncm for BTI®. BTI® screws showed the most resistance to fracture. According to the protocol that was applied, the implant–abutment connection demonstrated good resistance and a precise fit between these interfaces; therefore, in some cases, the presented values showed a lack of quality control and low fracture resistance.Micromovements of the implant–abutment connection influence peri-implant bone preservation. This study evaluates and quantifies the maximal torque after a cycle of implant prosthetic screws tightening using original components. A total of 40 samples were tested: Megagen The loss of natural teeth has been a problem for human beings throughout their existence. In primitive societies, most tooth losses were due to trauma, whereas at present, tooth loss is associated with diseases of the oral cavity, the most prevalent being dental caries and periodontal disease. The use of dental implants is of great importance in restoring an edentulous patient’s masticatory, aesthetic, and phonetic functions, contributing to improvements in their oral health and quality of life ,2.The success of implant-supported rehabilitation depends on the direct interaction between bone and implant surface without the interposition of connective tissue or signs and symptoms of infection or inflammation . In addiThis complication has been established as comprising 6% of prosthetic complications ,8,9. TheScrew loosening is more frequent in external connections  than in internal connections and in single implants, although it also occurs in multiple prostheses. However, in the case of multiple prostheses, the perception of screw loosening by the professional and the patient is much more complicated and can lead to the appearance of fractures or bone loss around the implants that still have the screws tightened due to overload, in addition to the possibility that the screws of these implants also loosen ,21,22.On the other hand, screw loosening can lead to the appearance of biological problems at the implant–abutment interface, since it allows bacteria to leak through the interface. This bacterial leakage, together with the mechanical problems derived from screw loosening, can lead to the loss of peri-implant bone, as well as the appearance of infiltrate of inflammatory cells. Furthermore, with the presence of inflammatory infiltrate at the level of the alveolar bone, the possibility of mechanical failure and fatigue and the risk of screw loosening increase. Several studies have linked the presence of periodontal pathogens in the peri-implant sulcus with the appearance of damage to the adjacent hard and soft tissues, leading to implant failure ,24. HoweOcclusal load is a force of multidirectional and variable magnitude ,8,9. AltScrew fractures are reduced when using gold alloy screws or passive connection structures without any mechanical stress on the fit ,15. It iThe screw is tightened by applying force torque. The applied torque develops a force on the screw that is called the preload. As the screw is tightened, it stretches, producing tension. The elastic recovery of the screw brings the two components that are tightened together, creating a clamping force. In contrast to the clamping force, there is a separating force between the two parts that are joined by the screw. Screw loosening occurs when the separation force is greater than the clamping force. Excessive force causes the screw turns to slip, with a loss of preload ,20.The preload is determined by factors such as applied torque; the metal alloy, design, and surface of the screw; and the prosthetic parts. The preload is proportional to the applied torque. In other words, insufficient torque allows for a separation between prosthetic part and implant, and too much torque may grind the screw turns. The ideal torque is 75% of the torque that is required to break the screw ,15.2. In addition, the torque indicated by the manufacturer is on average between 20 and 35 Ncm2. There are intraoral separation forces that include off-axis occlusal forces, excursive laterality movements, interproximal contact points, protrusive movements, parafunctional forces, and structures without passive laying [One variable is the amount of torque that can be applied to the screw and how it is applied: manually via a mechanical device, or digitally. On average, manually applied force varies between 10 and 15 Ncme laying ,22.As soon as masticating forces exceed the preload, the seating and connection become unstable, with vibration and micromovements leading to the loosening of the screw, i.e., a loss of its function and a mechanical failure ,22. In aThe purpose of this study was to evaluate and quantify the maximal torque after a cycle of tightening of an implant prosthetic screw using the original components.The study was a descriptive and inferential experiment.®—Daegu, South Korea; Dentium®—Gangnam-gu, Seoul, Korea; BIOMET 3i®—West Palm Beach, FL, USA and BTI®—Álava, Spain. This study was performed by using 10 laboratory analogs of each brand and 10 prosthetic abutments, where 10 original screws from each manufacturer were tested. In each prosthetic screw, a brand-new screwdriver and torque wrench were used.A total of 40 prosthetic screws from the following brands were tested: MegagenThe prosthetic screws characteristics are described in Inclusion: prosthetic screws, direct implant connection, titanium alloys of the same grade.Exclusion: prosthetic screws from nonoriginal brands.(1)® prosthetic screws; 10 Dentium® prosthetic screws; 10 BIOMET 3i® prosthetic screws and 10 BTI® prosthetic screws.All prosthetic screws from the manufacturers were labeled with serial number and control date: 10 Megagen(2)® SZ61—Tokyo, Japan), and a 90× magnifier was used to evaluate any changes in the surfaces.The presence of anomalies and defects was assessed with a stereoscope (Olympus(3)The prosthetic abutment was coupled to the implant analog (of the corresponding brands) with the prosthetic screw  with theA brand-new manual screwdriver was used every time a prosthetic screw was tested.(4)® The fracture points were automatically recorded in the machine and compared between all samples.(6)®  Excel file was stored on a computer for further graphical and value analyses.The CS-Dental Testing Machine(7)The fractured parts were analyzed under an optical microscope to observe microscopic fracture characteristics.A standard laboratory protocol was established and applied at the Institute for Research and Advanced Training in Health Sciences and Technologies (IINFACTS-CESPU) to test all selected samples.® and another in Biomet 3i®. As these extreme values were not explained by the methodological process, an imputation process for missing values was carried out. Those extreme values were replaced by the means of the remaining nine values obtained in the tests, 53.39 and 39.74 Ncm, respectively.Data analysis was carried out in SPSS, version 24. First, exploratory data analysis was performed, which detected two outliers, one in DentiumIn descriptive analysis, the means and standard deviations for the fracture torques, expressed in Ncm, were calculated after symmetric distribution was confirmed by observing the histograms.Normal distribution was assessed using the Shapiro–Wilk test, used for n < 50, confirming the necessity of parametric tests. The four brands’ homogeneous distribution was evaluated with the Levene test, confirming this assumption.t-test. The reference values for each brand were 35 Ncm for BTI® and Megagen®, 30 Ncm for Dentium, and 20 Ncm for BIOMET 3i®.To compare the mean values of fracture torques, the reference values associated with each brand and the sample results were used in the ANOVA was used to compare the maximal resistance limit between brands. This test was complemented with Tukey’s multiple-comparison test.The maximal considered level of significance was 5%.®, Dentium®, BIOMET 3i®, and BTI®  than the maximal resistance limit referenced by the manufacturers. Megagen® did not produce statistically significant results   .(3.36) = 31.20 (p < 0.001), evidenced by Tukey’s multiple-comparison test, with differences in BTI®, which produced the highest mean value; BIOMET 3i®, which produced the lowest mean value when compared with Dentium®, BTI®, and Megagen®; and Megagen®, compared with Dentium®, had the lowest mean value , leading to the belief that some screws are of inferior quality, even within the same brand. According to the literature ,24, it i®, which may have been due to the surface treatment applied to the screws. This was in line with existing studies [Another factor to be considered is that the brand that had the greatest resistance was BTI studies ,33,34 sh® brand, and this phenomenon occurred at a torque that was slightly higher than the manufacturer’s maximal limit. A key fatigue pattern was established, and the resilience of materials, such as the protection and conservation of prosthetic screws, could be a study focus. Although this was not examined in the present study, it opens a line of investigation for the future [Finally, fractures were observed of the three original prosthetic keys in the BTIe future .® and Dentium®, friction-Fit hex—Biomet 3i®, and four-lobed internal—Bti®). Internal connection geometry influences the degree of abutment movement, requiring a significantly greater pull force to separate abutment from the implant, leading to conservation of prosthetic screw and less prosthetic complications [The type of connection between implant and abutment also plays a significant role in the long-term effect of dental implant treatment , the results could be different.(1)®;The greatest resistance, evidenced by the highest load torque before screw fracture, was achieved by BTI(2)The maximal average fatigue loads were within the parameters defined by the manufacturers. However, there were prosthetic screws that did not reflect this reference value; and(3)There seemed to be a correlation between better results for screws and surface treatment.Based on the laboratory experiments of the present study, the applied methodology, and the obtained results, we conclude the following:Manufacturers’ prosthetic screws torque should be respected and a higher preload to torque (more 5–10 Ncm) should be applied to prevent the settling effect.


The southwest wind was the principal reason for the deformation, which made the deformation mainly occur in the horizontal direction from east to west. Moreover, the inner fast ice kept stable due to the protection of outer consolidated ice. The results showed that the deformation trend and characteristics can be better understood by using InSAR technology that was combined with multi-orbits SAR data to resolve and analyze the landfast ice deformation.Detailed mapping of landfast ice deformation can be used to characterize the rheological behavior of landfast ice effectively and to improve sea ice modeling subsequently. In order to analyze the characteristics, trends and causes of deformation comprehensively and accurately, the Sentinel-1A ascending and descending orbits data were used to detect the horizontal and vertical deformation of the fast ice in the Baltic Sea. Firstly, the fast ice edge lines were acquired through feature extraction with interferometric coherence images and SAR amplitude images. Then, the deformation transformed model was constructed according to the geometric relationship of multi-orbits deformation measurements. Finally, the landfast ice deformations were resolved and the horizontal and vertical deformations were obtained. The results showed that the maximum deformation was—44 cm in horizontal direction and 16 cm in vertical direction within the fast ice region of 960 km The change of sea ice, a sensitive indicator to global climate change, is closely related to the regional ecological environment, production and development in polar and high latitude regions. Landfast ice is a kind of stable sea ice that grows along the coast, islands, reefs and grounding ice ridges and is firmly attached to them. The growth, development and deformation of landfast ice not only affect the thermal balance of the marine environment but also affect the temperature and salinity cycle of the ocean. The stability and area of landfast ice are affected by the depth, temperature and salinity of sea water or other comprehensive factors ,2. DurinIt has always been a difficult issue that how to accurately obtain the deformation of fast ice over large area while ensuring the immediate effectiveness and economy of the method . Most ofInSAR (Synthetic Aperture Radar Interferometry) technology has been proved to be able to obtain centimeter-scale deformation of landfast ice ,4,7,8. DBased on the above, there are two main problems in the detection of fast ice deformation. (i) How to obtain the region of fast ice accurately? The fast ice was separated from drift ice by long time baseline in . AlthougIn this paper, the deformations of fast ice were acquired by InSAR technology with combining the ascending and descending orbits data of Sentinel-1A. The boundary line of fast ice was automatically obtained by using the texture features of coherence and amplitude images, and then the deformation in LOS was transformed into the horizontal and vertical displacement according to the geometric relationship of multi-orbits deformation measurements. On this basis, the deformation trend and reasons of fast ice were estimated accurately. In The study area is located in the north part of the Bothnia Bay of Baltic Sea , as shown in Four SLC (Single Look Complex) images in IW mode acquired from Sentinel-1A were selected. The specific information of Sentinel-1A data were shown in The interferometric parameters were shown in Moreover, the ACE2  DEM released by NASA  was applied to InSAR data processing in this study . In thisAs shown in The fundamentals of InSAR technology can be found in reference ,28. FirsThermalγ is the decorrelation caused by thermal noise of the receiver, which can be determined by the signal-to-noise ratio (SNR). Spatialγ is the decorrelation caused by the difference of incidence angles and proportional to the vertical baseline, so it is also known as baseline decorrelation [Temporalγ is the correlation factor associated with incoherent changes in the scattering medium in interval period. Compared with the two decorrelation factors previously mentioned, Temporalγ characterizes with large variation and poor stability, which is the major factor of decorrelation and the principal quantity measured. In the study of fast ice, the numerical variation of Temporalγ is caused by the movement of ice floes or the change of surface scatterer within a resolution cell [The complex coherence γ between two complex SAR images S1 and S2 is defined as :(1)γ=⟨s1ee parts , i.e.,, and θ is the incidence angle of radar pulse signal. Firstly, according to the geometric relationship, the deformation in the north-south and east-west directions were decomposed into the ground distance direction, i.e.,A_LOS, αAd and Aθ represent the deformation in LOS direction, the azimuth angle of ascending orbit and the incidence angle of radar under ascending mode respectively. In the same way, under the descending orbit mode, the deformation in LOS direction, D_LOSd, can be obtained by Equation (5)Then, the deformations in the vertical and ground directions were decomposed into LOS direction by Equation (4)Since the near-polar orbit radar satellite is not sensitive to the displacement of the north-south direction, it is difficult to obtain the deformation in this direction ,36,37. DThe main research results included coherence images, interferograms and deformation maps. The coherence value reflected the stability and correlation of sea ice during the interval time when the interferometric pair images were acquired. The interferograms reflected the features of fast ice deformations. The denser the fringes are, the more serious the deformations are. The deformation maps directly illustrate the displacement of fast ice.The coherence images were shown in The filtered interferograms were displayed in The displacements in LOS direction determined by the descending orbits data was displayed in In the coherence images, the value decreased in the area near shore significantly, due to the drastic change of scattering geometry and the shallow water that made fast ice grounded and ice ridges formed . The val−4 kg/m3/K, so the effect on fast ice deformation could be ignored. In addition, the temperature on 14 February 2018 was about 0 °C for the most of the day. The Bothnia Bay is a low salinity sea area and the salt water will not freeze when the temperature above −0.2 °C [The factors for decorrelation can be roughly attributed to temperature, precipitation and wind. First, the temperature difference between the acquisitions of tow images within image pair A was so large, as shown in  −0.2 °C , so the Some special interferometric fringes were enlarged and shown in As shown in It was the movements of ice floes driven by strong winds that were the main reason for the deformation of fast ice in study area. The continuous southeast winds existed on 9 and 10 February 2018 with speed over 12 m/s, as shown in λ is the radar pulse wavelength [Another factor for deformation was sea level tilt. Although the absolute sea level cannot affect the interferometric fringes, the sea level tilt might have a certain impact on the interferometric phase . Fringesvelength . In this−4/°C [The low salinity ice will expand with increasing temperature at temperatures beneath about −5 °C, and the expansion coefficient is 10−4/°C . In thisThe researches that utilize InSAR technique to investigate the fast ice in Baltic Sea are few. Dammert, P.B.G. et al.  have detBerg, A. et al.  have uti2.This paper was similar to it in study area, experimental data and survey time. The results in this paper showed that the displacement values in LOS obtained by ascending and descending orbits data were 37 cm and 38 cm within the fast ice region of 960 km2 respectively.Marbouti, M. et al.  have firDue to the lack of field measurements, we compared the results with previous works to verify the effectiveness and advantages of the results in this paper. As shown in In this paper, the deformation of fast ice in Bothnia Bay of Baltic Sea has been studied using InSAR technology with the ascending and descending orbits data of Sentinel-1A. A method for automatically obtaining fast ice edge lines by combining interferometric coherence image and SAR amplitude image was proposed. According to the geometric relationship between multi-orbits deformation measurements, a deformation transformed model was established. It was used to successfully obtain the west-east and vertical deformations of fast ice through converting the displacement values in LOS direction.2 in the study area, during the study time from 2 to 16 February 2018. The main reason for the fast ice deformation was the shear stress caused by the drift of ice floes under the strong southwest wind and the major deformation was horizontal direction from east to west. In addition, the inner fast ice kept stable and its deformation was smaller due to the protection of consolidated ice.The results showed that the maximum horizontal deformation was −44 cm and the maximum vertical deformation was 16 cm within the fast ice region of 960 kmOne of the keys of InSAR technology is to obtain radar data with high coherence. In this study, the time baseline of interferograms was 12 days, which can not only effectively avoid the influence of some ice floes, but also effectively detect the developing trend and causes of fast ice deformation. Because of the lack of filed measurements, such as leveling, GPS measurements etc., however, the deformation results were not verified effectively. And the displacement values were accumulated or counteracted for a long time, which cannot accurately reflect the sudden or short-term sea ice changes, such as ice crack and ice drift. Recently, it has been proved that TerraSAR-X/TanDEM-X has considerable application prospects in the study field of sea ice ,40. In a


IntroductionThe pathophysiology of isolated coronary artery ectasia (iCAE) has not been clearly identified, although multiple abnormalities, including arteritis, endothelial dysfunction, and vascular destruction, have been reported. In this study, we aimed to analyze serum cystatin C concentrations in patients with iCAE and controls.MethodsForty-seven patients with iCAE (mean age: 55.9 ± 11.5) and 32 individuals with normal coronary angiography (mean age: 57.8.1 ± 9.6) were included in the study. Plasma cystatin C levels were measured by using the principle of particle-enhanced turbidimetric immunoassay (PETIA).ResultsSerum cystatin C concentrations were significantly lower in patients with iCAE compared with the control group . A significantly positive relationship was found between serum cystatin C levels and creatinine and high-sensitivity C-reactive protein (hs-CRP) levels in both groups . In multivariate logistic regression analysis, serum cystatin C level found to be a significant predictor for the presence of iCAE , p-value = 0.013). Receiver operating characteristic (ROC) analysis determined that a cystatin C value < 1.02 mg/L had a sensitivity of 56% and a specificity of 78% for the prediction of ectasia.ConclusionWe conclude that cystatin C independently can be a useful predictor for the presence of iCAE. Isolated coronary artery ectasia (iCAE) is characterized as a localized or diffuse, non-occlusive dilatation of epicardial coronary arteries in the diameter of a coronary artery segment of 1.5-fold normal size, considering as such the adjacent non-dilated segments .The pathophysiological mechanism of iCAE has become an important research topic because of its similar mortality rate compared to patients with multivessel coronary heart disease .Unfortunately, there is still limited information on the underlying biological process. Previous trials have suggested that several pathways, including those involved in the response to wounding, lipoprotein remodeling, platelet activation, and blood coagulation, may differ between healthy volunteers and patients with iCAE -5.The main underlying pathophysiologic process in iCAE could be associated with enzymatic degradation of the extracellular matrix (ECM) of the media, especially the overexpression of matrix metalloproteinases (MMPs), which could result in excessive expansive arterial remodeling .Cystatin C is an endogenous secretory protein that inactivates the proteolytic enzymes, such as cysteine proteinases. We believe that cysteine proteases, which predominate because of the reduction or inhibition in cystatin C expression, may take a place in the inflammatory and dilating process of iCAE due to its elastolytic and collagenolytic activities -8. ThereThe present analysis aimed to assess the value of cystatin C as a predictor of ICAE and to further our current understanding of the pathogenesis of iCAE.Study designThe protocol was approved with the registration number of 2011-KAEK-25 2015/20-07 by the local ethics committee in accordance with the ethical principles for human investigations, as outlined in the second Declaration of Helsinki, and conducted at a tertiary referral cardiology hospital. All patients provided written informed consent prior to participating.Patient selectionIn total, 79 individuals were included in this cross-sectional study. The study group included 47 patients with iCAE without any stenotic lesions and the control group consisted of 32 age-and gender-matched subjects who proved to have normal coronary angiograms.Exclusion criteriaAortic or cerebral aneurysm;Patients with high troponin due to any causes (including acute coronary syndrome (ACS), acute and chronic heart failure, pulmonary embolism, obstructive coronary artery disease (CAD) who had coronary stenotic lesions of > 20%, etc.;History of cardiovascular disease including previous myocardial infarction, coronary artery surgery, arrhythmias, or moderate/severe valvular disease);Chronic systemic disease Coronary angiography assessmentCoronary angiography was performed with a femoral approach (with the Judkins technique) without the use of nitroglycerin, adenosine, or a calcium channel blocker by using Siemens Axiom Artis dFC system . The results were evaluated by at least two independent interventional cardiologists.Analysis of cystatin CAll blood samples were collected after a fasting period of eight hours from a large antecubital vein without interruption of venous flow and collected in 13 x 100 mm biochemical tubes with a yellow cap containing gel (BD vacutainer) and 2.0 mL purple top tubes coated with K3EDTA (BD vacutainer). For measurement of cystatin C, serum was separated after centrifugation at 1,500 g for 10 minutes and stored at -80℃ until analysis, and all samples were processed simultaneously. Cystatin C levels were measured with an auto-analyzer . The sensitivity of the assay kit was 0.05 ng/mL with a limit of detection of 0.3 ng/mL. The kit also has intra-assay and inter-assay coefficient of variation of < 10% and < 12%, respectively, and used the principle of particle-enhanced turbidimetric immunoassay (PETIA) to assay the concentration of cystatin C. Results were presented as mg/L.Statistical analysisThe Statistical Package for Social Sciences (SPSS) software package, version 26.0  was used to perform all data analyses. The distribution pattern of the variables was analyzed using the Kolmogorov-Smirnov test. Continuous data are presented as mean ± standard deviation, or median and interquartile range, according to the distribution pattern of the variables. The student’s t-test was used to compare parametric continuous variables, and the Mann-Whitney U test was used to compare nonparametric continuous variables. Categorical variables were compared using the Chi-square test, the results of which are presented as percentages. The correlations were assessed by performing Pearson's correlation test. The effects of different variables on iCAE were determined with multivariate analysis. In all statistical analyses, a p-value of < 0.05 was considered significant.Demographic, clinical, and laboratory characteristics of subjectsThe groups did not significantly differ with regard to baseline clinical and laboratory characteristics. The comparison is summarized in Table Correlation analyses of factors relating to coronary ectasia and cystatin CIn correlation analysis, a significantly positive relationship was found between serum cystatin C levels and creatinine and hs-CRP levels in both groups  Figure . There wRegression analysis for assessment of the presence of ectasiaMultivariate logistic regression analysis was performed to investigate independent correlates of iCAE and other parameters. In multivariate logistic regression analysis, the serum cystatin C level was found to be a significant predictor for the presence of iCAE  . A cystatin C value < 1.02 mg/L had a sensitivity of 56% and a specificity of 78% for the prediction of ectasia Figure .The main finding of this present study is that the patients with iCAE had a lower cystatin C level compared to the individuals with normal coronary artery. Furthermore, cystatin C was associated with the presence of coronary ectasia, and cystatin C levels < 1.02 mg/L had a sensitivity of 56% and a specificity of 78% for the prediction of ICAE. The infographical abstract of the study was seen in Figure The exact pathogenesis of ectasia has not been clearly identified. The main hypothesis of this study was that cystatin C deficiency may result in vascular enlargement with an imbalance in favor of MMP. The results of this study support the view of increased MMP activity in iCAE pathophysiology. This has been demonstrated in vivo studies, but not enough clinical trials have been performed yet -13.Cystatin C is known as a secretory protein that inactivates the proteolytic enzymes, such as cysteine proteinases (cathepsin) and serine proteinase . Thus, cystatin C may have a crucial role in protein catabolism during ECM remodeling -15. OverShowing lower cystatin C levels in aortic aneurysmal tissues in immunohistochemical studies caused plasma cystatin C levels to be investigated in patients with dilating vascular disease.Firstly, Sukhova et al. examined the expression of the potent elastases cathepsins S and K in human atheroma . They foSimilarly, Aoki et al. investigated the role of cysteine proteases in cerebral aneurysms with animal models. A cerebral aneurysm was created surgically in mice, and it was demonstrated by immunohistochemistry methods that after three months of follow-up, the levels of cathepsin B, S, and K in the aneurysmal tissues increased and the cystatin C level decreased. After the use of NT-2300, which is a cysteine protease inhibitor, it was observed that cathepsin activity and collagenase 1 and 4 activity in the aneurysm wall decreased while the level of elastin increased .Dogan et al. found higher plasma levels of MMP-3 and MMP-9 in CAE patients compared to CAD patients and those with normal coronary arteries . Luttun There are a few studies investigating the cutoff for the cystatin C level as a prognostic indicator in process of various cardiovascular conditions. Budano et al. found that a cystatin C level > 1.4 mg/L as a predictor for long-term adverse events before coronary angiography had a sensitivity of 59%, a specificity of 83%, and a negative predictive value of 98% . Mao et We encountered a positive relationship between cystatin C levels and hs-CRP, which is a classic indicator of inflammation and creatinine level, which represents a reduced glomerular filtration rate (GFR). Although the correlation between hs-CRP and cystatin C was found to be statistically significant, very little correlation was observed in real terms. This may be because of the patients with high hs-CRP levels who were excluded from the study. Therewith, our results were complementary to previous studies -24.When we compare these results with our own study, we see that our results are compatible with the literature. However, the first question that may come to mind as a criticism at the end of these studies is why the destruction is only in vascular tissues. When we review the literature from this point of view, unfortunately, despite the developments in preclinical and clinical studies, we have not yet found a credible answer to this question. However, another hypothesis is that cystatin C expression in vascular tissues is higher compared to other tissues, and it is likely that there is another protective mechanism against cystatin C-like elastolytic effects in other tissues. In any case, it is very clear that this point will remain the focus of the researchers.It is unclear whether cystatin C deficiency is acquired congenitally or later. It is also not clear at what age the first coronary enlargement first appeared. Therefore, we recommend performing an age-related subgroup analysis in much larger population studies.This study has some strengths and limitations that are worth noting. One of the strengths is that, in other models of cystatin C, a deficiency in aortic and cerebral aneurysm has been demonstrated and the patient group mentioned was excluded in our study to obtain reliable results. Another one is that, in recent years, many studies related to iCAE have been conducted and many cytokines that may be associated with iCAE have been identified. The feature that distinguishes this study from them is that the results of this study may shed light on the pathogenesis of iCAE. The first limitation is that coronary artery specimens are optimal for studies; however, they are difficult to collect from patients. Thus, blood samples were used in the present study. Secondly, the patients enrolled in our study were candidates for coronary angiography. Thus, our findings may provide indirect evidence for the general population. Third, we used a single baseline measurement for cystatin C.According to the results of our study, we can suggest that serum cystatin C can be used as a marker for the presence of coronary ectasia. ICAE, intracranial aneurysm, and abdominal aortic aneurysm, which are different forms of dilated vascular diseases. All these phenomena can be caused by dysregulated expression of proteases and their endogenous inhibitors. We also recommend that a patient with any of these phenomena should be systematically evaluated for other dilated vascular diseases.