Thelohanellus kitauei, is a member of obligate parasitic myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (− 13 KJ/mol) with cathepsin L whereas a higher binding energy (− 8.6 KJ/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-T. kitauei vaccine in carp culture in the future. Thelohanellus kitauei preferentially infects the intestine of host, and causes intestinal giant-cystic disease of farmed carp  in the host by inhibiting the corresponding protease, which prevented the host from presenting the parasite antigen  was amplified by the specific primer pairs . PCR was performed with the following parameters: initiation denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 2 min. The PCR products were confirmed by DNA sequencing.Total RNA of purified https://web.expasy.org/protparam/) was used to predict the theoretical isoelectric point (PI), the molecular mass, and stability coefficient. NetOGlyc 4.0 and NetNGlyc 1.0  was used to predict potential phosphorylation site  method with MEGA 7.0 (Saitou and Nei Hydra vulgaris (XP_012565994), cystatin-B from Oreochromis niloticus (XP_003439236), cystatin-A from Aotus nancymaae (XP_021529657), cystatin-A from Opisthocomus hoazin (XP_009930686), cystatin-B from Peromyscus maniculatus bairdii (XP_006989515), cystatin-A from Pelodiscus sinensis (XP_006124242), and cystatin-A from Chrysemys picta bellii (XP_023966898) were selected for multiple sequence alignment using ClusterW and Espritt 3.0.The homology search of TK-cystatin protein was carried out by BLASTP, and the top seven hits of similar sequences from other species, such as cystatin-A from https://servicesn.mbi.ucla.edu/PROCHECK/)  , and the main interaction bonds were visualized in the PyMOL Molecular Graphics System, Version 2.0 . The recombination product then was transformed into E. coli BL21 (DE3) and positive clones were determined by DNA sequencing using T7 universal primers. A single positive clone from overnight culture was transferred to 200 mL LB medium with kanamycin (100 μg/mL) according to the ratio of 1 : 100. When the OD600 = 0.6-0.8, induced with various concentrations of IPTG  at 16 °C for overnight. The culture was centrifuged at 12,000×g at 4 °C for 10 min, the bacterial cells were harvested. TK-cystatin protein was purified from recombinant cells using Ni2+-NTA affinity method as described by Huo et al. (4HCO3 (pH = 7.5) based on previous research /pET28aR (5′-TCAGTGGTGGTGGTGGTGGTGTTGCGGATGTTTTGTATCAAG-3′) and TK-cystatinF (5′-CACCACCACCACCACCACTG-3′)/TK-cystatinR (5′-CATGGTATATCTCCTTCTTAAAGTTAAA-3′) were used for amplifying the vector fragment and the target gene fragment, respectively. The PCR-amplified DNA fragments were purified and assembled into recombinant pET28a_TK-cystatin plasmid by NEB/NEBuildero et al. , and wasl-cysteine hydrochloride, 0.005% tritonx-100, pH  5.5) were activated by pre-incubation in 96 well black microplate at 37 °C for 30 min. After that, another two reactants, 10 μmol/L substrates and an increasing-concentration gradient TK-cystatin, were added to each well of this microplate to a final volume of 200 μL. Subsequently, fluorescent products were measured with excitation at 360 nm and emission at 460 nm using fluorescence spectrometer (Bio Tek) every 30 s for 10-min reaction. Each reaction was performed in triplicate.To investigate the potential immune inhibitory function of TK-cystatin, the inhibitory activity of recombinant TK-cystatin protein was measured against three physiologically relevant peptidases including endopeptidases (papain from Carica papaya and cathepsin L from human) and exopeptidase (cathepsin B from human). All the three proteases were purchased from Sigma-Aldrich . It based on real-time mean velocities and determined by nonlinear regression analysis. All statistical analyses were performed in GraphPad Prism Version 6 software .The activity of TK-cystatin protein was expressed as half maximal inhibitory concentration . Combined with the morphological observations, the parasite was identified as T. kitauei.Dissection of infected ace Fig. a. The siace Fig. b, and itpI of 6.97 predicted by the ProtParam , one Casein kinase II phosphorylation site (S42FKD45) and three protein kinase C phosphorylation site (S42FK44、S52FK 54、T91AR93) in TK-cystatin were predicted by NetNGlyc 1.0 and PROSITE  critical to the biological function of cystatins . After induction with 0.1 mmol/L IPTG  at 16 °C overnight, TK-cystatin was purified from the recombinant bacteria cell lysates by Ni2+-NTA affinity chromatography with 200 mmol/L imidazole elution buffer. The purified protein migrated to a predicted ~ 10 kDa band in 12% SDS-PAGE, was confirmed by western blot analysis with anti-His-tag mouse monoclonal antibody  for papain, cathepsin L, cathepsin B inhibition by curve fitting were 0.8 μmol/L, 0.175 μmol/L, and 2.1 μmol/L, respectively. pH and thermal stabilities of TK-cystatin were investigated. TK-cystatin had lower activity stability near pH 7.0, higher stability at alkaline or acidic conditions, with highest stability at pH 11.0 . TK-cystatin, however, lacks of disulfide bond, the SND/S conserved motif relating to the inhibition of legumain-like proteases  and cystatins (family 2) and kininogens (family 3). TK-cystatin has some important features of family-2 cystatins, such as secretory, small molecular weight, rich in phosphorylation sites, and conserved inhibitory regions  and exopeptidase (cathepsin B), but the inhibitory activity of it against cathepsin L is 12 times higher than that against cathepsin B. This result is consistent with the conclusion of the previous studies. Because compared to cathepsin L, cathepsin B contains an additional “occluding loop”structure in the S′ region, which usually restricts the inhibitor from entering the active site (Turk et al.


Pleuragramma antarctica] and crystal krill[Euphausia chrystallorophias]) responses to predation associated with the marginal ice zone (MIZ) of the McMurdo Sound, Antarctica, polynya. Prey abundance and habitat was sampled across a 30 × 15 km area by remotely-operated vehicle, and included locations that were accessible (ice edge) or inaccessible (solid fast ice) to air-breathing predators. Prey and habitat sampling coincided with bio-logging of Adélie penguins and observations of other air-breathing predators , all of which were competing for the same prey. Adélie penguins dived deeper, and more frequently, near the ice edge. Lowered abundance of krill at the ice edge indicated they were depleted or were responding to increased predation and/or higher light levels along the ice edge. Penguin diet shifted increasingly to silverfish from krill during sampling, and was correlated with the arrival of krill-eating whales. Behaviorally-mediated, high trophic transfer characterizes the McMurdo Sound MIZ, and likely other MIZs, warranting more specific consideration in food web models and conservation efforts.Predators impact preyscapes (3-D distribution of forage species) by consuming prey according to their abilities or by altering prey behavior as they avoid being consumed. We elucidate prey (Antarctic silverfish[ The MIZ is defined by this melt water lens, extending out on either side of the ice edge. The physical processes determine phytoplankton abundance and species composition, forming the base of the MIZ food web and providing bottom-up influence toward higher trophic levels8. The zooplankton and micronekton grazers (e.g. krill Euphausia spp.)10 can be abundant in the MIZ, even though their elevated biomass is often maximal beneath the ice cover back from the ice edge12. This indicates that the MIZ may not just be facilitating increased grazer abundance, but may also provide a refuge from air breathing predators under heavier ice cover further into the MIZ. Concurrently, in the outer, open-water portion of the MIZ, predators may have depleted the grazers (see below). In this way, a mid-trophic organism’s location in the MIZ is part of a game of risk, where seeking food must be balanced by predator avoidance14. Part of this game of being consumed or being a consumer is evident at the smaller scale in the patchiness of grazer aggregations and their predators within MIZs19. Important in that aspect of the game are inter- and intra-specific competition between predators, i.e. a top-down influence. This interaction can negatively affect prey availability where predators overlap their foraging21, if predators are abundant enough to have a measurable impact13.In the marginal ice zone (MIZ) of the Southern Ocean, where sea ice-covered waters transition to open ocean, a combination of both biotic and abiotic habitat features and processes lead to elevated abundance in biological communities relative to adjacent habitat. Melting ice floes create a freshwater lens that increases stratification, reduces surface mixing, and increases light reaching the water column due to decreasing ice cover, all of which encourages blooms of diatoms9, grazers12, and upper trophic levels23, investigated simultaneously. Within that large-scale band of sea ice, however, occur pools of persistent open water or less concentrated ice, called polynyas. These mesoscale features are found especially along the continental margin and coast24. Around each polynya is also an MIZ that exhibits enhanced productivity and high concentrations of grazer organisms. The trophic importance of polynyas and their MIZs can be further judged by the high densities of top- and mesopredators found there, including seabirds26, seals29, and whales30. Another important supporting indicator is the significant association of penguin colonies with polynyas32.Overall, MIZs constitute a significant Southern Ocean habitat. Antarctica is entirely encircled by a band of sea ice several hundred kilometers wide, its outer circumference constituting the longest MIZ on the planet. This larger-scale MIZ has received appreciable multidisciplinary research attention, with phytoplankton33. In part, this is a result of logistical difficulties required to make simultaneous observations of predators and the preyscape (3-D distribution of middle trophic levels), along with their immediate environment, especially in the somewhat remote Antarctic. However, recent studies utilizing both established and new technology have directly examined predator foraging in relation to prey availability in comparable areas of the Southern Ocean where sea ice is at least seasonally present35. These studies have shown, by inference, the impact of predator abundances and behaviors on small-scale occurrence patterns of prey36; however the influence of predation and top-down trophic impacts in polynya habitats has not been systematically examined5.The Southern Ocean MIZs of coastal polynyas have received less of the integrated, multidisciplinary research needed to establish ecological linkages and food web dynamics. Apparent ecological linkages have primarily been inferred, based on coincident enhanced productivity and abundance24, and large numbers of predators including penguins37, seals39, and whales41  animal behaviors, specifically predator avoidance behaviors by the prey, and predation pressure, through resource or interference competition among predators; and by B) physical characteristics of the environment. This MIZ exhibits trophic qualities observed in larger-scale MIZs, including high seasonal primary productivity associated with the polynya and fast ice edgePygoscelis adeliae). While measuring the preyscape, we also measured seasonal change in diet, foraging location and behaviors, and population and breeding dynamics, all of which should contribute importantly to the foraging pressure upon two main prey species, crystal krill  and Antarctic silverfish (Pleuragramma antarctica)45. Adélie penguins nesting at Cape Royds, located where the fast ice edge meets Ross Island . Typically, Adélie penguins at Cape Royds and elsewhere on Ross Island switch from feeding primarily on crystal krill in the early season (~100%) to a diet containing more silverfish later in the season , a baleen species that also feeds on crystal krill50. Prior to the arrival of minke whales, Weddell seals (Leptonychotes weddellii) and emperor penguins (Aptenodytes forsteri) predominate along the fast ice edge, where they prey heavily on silverfish, 80% and 20–100% of diet, respectively50.Using what is essentially a natural experiment, we compare the preyscape unavailable to air breathing predators (under the interior fast ice) to the preyscape available to predators in immediately adjacent waters (fast ice edge). The project was organized around a focal predator, the well-studied Adélie penguin  to penguins in the McMurdo Sound MIZ is driven by physical processes , and also by foraging pressure through trophic competition . Specifically, we hypothesize that predation pressure will alter the preyscape, the predators affecting the availability of their prey by means of their predation . Prey should be depleted where accessible to predators, or prey should occur deeper in the water column, compared to where predators have no access under the ice. In the process, we address the question of why the Adélie penguin diet shifts from one almost entirely of krill to one in which silverfish are prominent, co-incident with the arrival of cetaceans. We provide a 3-D examination of the McMurdo Sound preyscape as it evolved during the spring of 2014–2015.13; (2) krill and fish are more prevalent higher in the under-ice water column in the fast ice interior compared to the ice edge, where predators abound12; (3) Adélie penguins forage where there is reduced light in the MIZ, from shading of sea ice cover53; (4) Adélie penguins from Cape Royds, responding to changing prey distributions, change their foraging strategy (distance from colony and depth of foraging) as the chick-provisioning period progresses48; and (5) the diet of Adélie penguins changes to one with more fish due to competition upon the arrival of minke whales, i.e. whales are capable of foraging deeper than the penguins and cause a disappearance of krill at shallow depths21.(1) Krill and fish depth distribution corresponds with seasonal phytoplankton distribution within the bloom, i.e. shallow in the water column, to enhance foraginga m−3 over much of the top 60 m of the water column  foraged in the leads, following the southward-receding fast ice edge .Potential air-breathing competitors to Adélie penguins observed during the study included whales, Weddell seals, and emperor penguins. The first fish-eating, type-C killer whales were seen from Cape Royds on 20 December, and were observed sporadically thereafter Fig. , their p37.Helicopter surveys along the fast ice edge indicated 211 fish-eating emperor penguins were present during the first flight in mid-November Fig. . That nu46, at the start of the 2014 penguin chick rearing period (mid-December), crystal krill dominated penguin diet, but over the next two weeks, 14 to 30 December, the proportional composition of the diet included more Antarctic silverfish . As a proxy for water clarity, which could potentially influence diet by affecting the penguins’ ability to locate and pursue prey, we included mean depths of 1% light level calculated from fluorescence profiles using a model of light transmission . Either case constitutes a behavioral modification of prey availability. In a mini-natural experiment evident in the 2019–20 summer, the Cape Royds penguins had exhibited their shift from krill to fish with arrival of whales . Then, when a 2 km wide band of fast ice  suddenly broke away, penguin diet switched immediately to 69% krill , followed by a switch back to fish  with arrival of more minke whales . As demonstrated in the present project results, it appears that the krill had been avoiding being too close or had become depleted near the fast ice edge.Reduced krill abundance in the near surface layers (10–40 m depth) at the fast ice edge corresponds to where penguins were foraging, overlapping the average recorded dive profiles of penguins (average maximum depths on foraging dives ranged 47–54 m). Simultaneously, minke whales were foraging persistently and on average to 72 m (range 19–130 m)E. superba) growth (length) compared to environmental Chl-a levels show half-saturation constants of just 0.33–0.5 µg Chl-a L−1 57; insofar as crystal krill  growth saturation rates are not appreciably faster, Chl-a concentrations should have been saturating for crystal krill over at least the top 100 m of the water column during our study  while concentrating foraging at the McMurdo Sound fast ice edge, similar to penguins in the Ross Sea that apparently encounter more prey during darker dives62. Typically snow-covered, McMurdo Sound fast ice transmits <1% of incoming solar irradiance64 and underneath the fast ice, light-avoiding nekton appear to shoal52. Any shallow prey that migrate away from the cover of sea ice into the brighter lightscape of the MIZ may experience an increased risk of predation and consequently provide increased foraging opportunity for visual predators, such as fish65 and air-breathing predators.Following from the risk-reward theory, if foraging opportunities for krill were not restricting, then other factors besides risky prey behavior made the McMurdo MIZ a hotspot for penguin and whale foraging during our study period. Heavy pack ice cover associated with the fast ice edge, a stark boundary of both light availability and predator exclusion, may lead to greater prey prevalence nearer the surface, given the light-avoidance behavior exhibited by both krill and silverfish to avoid visual predation21. Instead, the average maximum foraging depth remained at ~50 m. This is the same depth at which Crozier penguins began foraging, with increasingly deeper dives as the chick feeding period progressed. Foraging consistently within 20 km of the colony, instrumented penguins at Royds did not need to travel far to find food. Hypothesis 4, that penguins would change foraging range and diving behavior with changing prey resources, was not supported as their behaviors indicate penguins were prey-replete, despite reduced availability of shallow krill at the ice edge. Prey species were detected at 50% of sampling stations before 20 December and at 100% of the stations afterward. In waters of the MIZ of the Ross Sea Polynya , where the preyscape was quantified in 2012–13, penguins foraged to ~50 m depth early, and increased to 80 m later5. Note that Adélie penguins have reached neutral buoyancy near 50 m, at and below which energy expenditure would be minimized66. At Cape Crozier, three orders of magnitude more penguins, plus competition from minke whales21, apparently depleted krill in the upper water column near to the colony, necessitating that penguins dive deeper and travel farther later in the season; with deeper dives they brought back more energy-rich fish5. The predation from minke whales and the relatively small number of penguins at Cape Royds did not result in inter-seasonal shifts in penguin foraging depth or distance.Cape Royds penguins during 2014–15 did not dive progressively deeper over the chick-feeding season, which is counter to the pattern observed at the nearby, but much larger, Cape Crozier penguin colony21. The penguin diet at Cape Royds similarly shifted to include more fish over time, however the relative abundance of crystal krill increased and silverfish aggregations, insofar as we could measure their availability, decreased, opposite to the trend in diet. However, the correlation between penguin diet and minke whale presence, similar to that observed at other nearby penguin colonies21, may indicate a different form of competition, interference competition, from the minke whales that can consume entire, small krill aggregations in one multi-lunge dive67.Perhaps related to the apparent prey saturation, the prediction of Hypothesis 5, that prey availability related to competition would be a determinant in penguin diet, was not clear for Cape Royds penguins during this study. This hypothesis was derived from the larger Cape Crozier colony, where both very abundant penguins as well as minke whales foraged resulting in local prey depletion and apparent exploitive inter- and intraspecific competition68, which along with emperor penguins are abundant initially at the ice edge  but which then moved to the fast ice interior39 with the arrival of killer whales . Overall, the abundance of silverfish then became lower beneath the fast ice interior, where the seals had moved (emperor penguins exited the area to molt). It is also possible that since our acoustic detection was limited to aggregations, silverfish may have dispersed, and even ascended, to prey upon the increasing abundance of krill, and thereby became more available to penguins.The overall decrease in silverfish aggregation abundance during the study period confounds the foraging picture, and more study will be needed to establish the seasonal patterns of silverfish in the McMurdo Sound food web. The decrease of silverfish detections could be due to predation pressure by Weddell seals48. Adélie penguins, as an easily-studied indicator species of food web status69, foraged preferentially along or within several kilometers of the fast ice edge  and their mid-trophic prey (high levels of phytoplankton), the distribution of krill at the fast ice edge agrees with the expected response to predation pressure, and is indicative of behaviorally-mediated trophic structuring in the McMurdo Sound Polynya MIZ. Such top-down controls could limit trophic transfer in a situation where prey are not saturated21, and could cause diet shifts among predators competing for the same prey. Understanding these dynamics can inform food web models that currently lack competition71, and allow management decisions that more appropriately consider ecosystem function in addition to species’ life histories and abundance. Though a termination time span exists, for now, the recently designated Ross Sea Region Marine Protected Area includes within its boundaries the MIZs of the McMurdo Sound, Terra Nova Bay, Ross Sea, and Ross Passage/Pennel Bank polynyas. As demonstrated herein , these are likely the hottest biological hotspots in the region, and confirm that the Antarctic treaty powers made a wise decision72.Overall, results indicated that the McMurdo Sound Polynya MIZ constituted a trophic hotspot, where high-quality prey patches resulted in concentrated predationge Figs. , 3, and All penguin survey, capture and handling methods performed during data collection for this study were approved under Antarctic Conservation Act (ACA) permits , issued by the U.S. National Science Foundation and the U.S. Antarctic Program; and Institutional Animal Care and Use Committee permits issued by Oregon State University’s . Studies did not involve endangered species. All data collection activity and field study was performed in accordance with these permits and additional relevant ACA guidelines and regulations.2 in area and >700 m deep ; as well as daily records kept at our camp at Cape Royds. The distance from foraging locations to the fast ice edge was found as the minimum distance to the ice edge upon a particular date54.  that overlap with penguin biologging and whale abundance surveys. We began to deploy data loggers on the foraging adult penguins after 20 December, as by that time, chicks had hatched and penguins were foraging much more frequently than earlier in the study. Details of biologging are provided below.To assess the temporal and spatial variability of the preyscape, we sampled the grid Fig.  three ti66. SCINI contained cameras and thrusters, and towed a sensor package consisting of a WET Labs fluorometer (ECO-AFL/FL) and a single-beam Biosonics 120 kHz DT-X echosounder74. The echosounder operated at a nominal ping rate of 1 ping s−1; however, this rate was occasionally adjusted if false bottom signals were observed. The general profile of a dive included a surface transect of ~300 m horizontal distance, where the acoustic transducer faced downward, and also a dive to ~120 m if conditions allowed. Our surveys characterized the surface 200 m because the signal-to-noise ratio of the transducer allowed resolving −80 dB targets at ~ 100 m range.Krill and fish were sampled acoustically and visually using the tethered SCINI ROV75. Echogram data were saved to a depth of 500 m, and background noise was removed76. All acoustic aggregations >4 pings wide were manually delineated, and acoustic energy of the aggregations was integrated into bins of 6 s wide by 1 m in depth. To ground truth acoustic signals, coincident visual targets were identified to the lowest taxonomic classification possible. Where visual targets were not identifiable, acoustics were verified by comparing aggregation target strength, shape, density, and texture and depth to a set of aggregations with positive visual classification.Raw acoustic data were analyzed using Echoview software−1). We did not attempt to transform integrated acoustic energy into biomass, since there was not enough size class information to construct acoustic models of identified organisms. Therefore, with the assumption that krill and silverfish body size distributions did not change substantially over the 8-week sampling period, the acoustic values reported are comparable within the classified groups of krill or silverfish, but not between these two groups.Acoustic returns are presented as integrated acoustic energy , and gray, chunky paste was silverfish (100%)47. Previous work also showed that chick diet represented quite well that of the adults/parents43. Diet observations began on 14 December 2014 (when approximately 5% of chicks had hatched) and continued until 22 January 2015. To obtain adequate sample size, diet observations were grouped into 5–6 d periods, centered to coincide with the start of the second pass of prey sampling .We assessed the overall diet being consumed by penguins in the Cape Royds colony by observing the color and consistency of food as it passed from parents to chicks during feeds observed on a daily basis; this was easily seen by using binoculars for a side view of feeding events2 . We attempted to retrieve the tags after one or two foraging trips  and attach them, using tape, to the next set of individuals78. Only one parent was instrumented from a particular nest. Up to three penguins were instrumented at a given time; penguins recorded from 1–7 foraging trips per deployment.Once sufficient numbers of chicks hatched thus to make their parents easily available (20 December), we equipped randomly 18 selected parents with SPLASH tags (wildlifecomputers.com). Parents with chicks forage intensely, and are also easily located upon return to the colony. The tags recorded depth, light, and temperature each second, and determined position using ARGOS satellites (see below). The tags, applied to the lower back just forward of the tail, weighed 62 g (1.6% of a 4-kg Adélie penguin) and had a cross-sectional area of 3.2 cm79. Because of different temporal resolution between dive (1 s) and accepted location data , we used temporal proximity to assign each dive an approximate geographic location, as we have done previously62. We assumed that the follow-on interpolated positions were adjacent in space and time from a temporally known position (TKP). We removed highly interpolated positions (>30 min TKP), which allowed more dives to be included in the analysis, while reducing the locational errors. In total, the data set included information from 18 tagged penguins making 50 foraging trips.The tags were set to transmit locations every 45 s for the first eight successive transmissions and then switched to once every 90 s, with up to 1440 transmissions allowed per day. They turned off if they were dry for 6 h (penguin out of water) in order to conserve batteries. Diving data were downloaded from the tags after retrieval. We used data from all trips for which both dive data and location data were available, except for cases in which locations indicated errors79. Foraging dives contain undulations in the bottom phase, with undulations considered to represent prey capture attempts; exploratory dives were just up and down with no time spent at depth and “other” were shallow, traveling dives80. Divesum also summarized light recorded during foraging dives into means of light levels at ~ 5 m depth (indicating surface light availability) and light levels during the dive bottom phase (indicating light levels during foraging). Light levels were log-transformed before performing statistical tests81.Dive data were initially processed using the program divesum . This program computed several dive parameters, such as maximum dive depth, time spent at the bottom phase of each dive, and number of undulations . Divesum also classified dives into three types: foraging, exploratory, and other41. We obtained an index to whale presence by visual observations (telescope and binoculars) once or twice per day, weather permitting, from a coastal hill at Cape Royds 30 m above sea level21. Observation periods lasted for an hour. We could effectively scan along the ice edge out to about 3 km, as well as adjacent open or pack-ice-covered waters. Observations were made between 20 November and 26 January, with gaps during 22–26 December, 29 December-9 January, and 19–25 January, in part related to poor visibility.To provide more ecological context, we investigated the prevalence of various potential penguin competitors. Due to inability to assess presence of these species in the open waters of the polynyas, we assessed abundance along the ice edge as an index to prevalence. The occurrence of cetaceans in McMurdo Sound for several years, including the study year, has been detailed in publications elsewhere54).To ground-truth the abundance of whales, and assess abundance of seals and emperor penguins along the entire cross-Sound ice edge, we counted these species by helicopter at several-day intervals, mid-November to mid-January, weather permitting. Flights were made 20 November, 2, 8 and 27 December, and 10 January. The flights followed the fast ice edge in both directions between Cape Royds as well as a few kilometers north, i.e. both arms of the fast ice “L” 83. To test for relationships or correlations among penguin dive parameters, mixed-effect models were evaluated with the distance to the fast ice edge as the single fixed-effect, and individual penguin as a random effect. To test for sinusoidal variability in penguin dive parameters, similar models were constructed predicting dive parameters (treatment) using a cosine function with wavelength of one day and phase shift of pi/6 (i.e. the peak of the cosine function set to 14:00 daily) as the fixed effect and individual penguin as a random effect. Individual tests were performed for each fixed-effect. Light-related variables and the distance of foraging penguins to the fast ice edge were log-transformed to normalize their distributions.Penguin dive data were tested for a relationship to the fast ice edge using regression analysis in the R computing environment48, and diet observations were randomized as opportunistic encounters.Kendall rank correlations were calculated between penguin diet fractions and preyscape variables, with multiple tests accounted for with Holm-Bonferroni corrections . We assumed that penguin diet observations and dive data were representative of the colony population as a whole. This approximation is acceptable given that individual penguins did not forage in completely independent areas41; see Fig. To test for difference in prey distribution at the ice edge, volume backscattering values were pooled into edge  we expect that xj < X for prey species, which would be indicative of lower prey concentrations at edge sites.Thus, we performed a permutation test85 geographic information system software. Data used for coastlines and glacial ice are derived from the SCAR Antarctic Digital Database86 available through the QAntarctica87 package, and bathymetry data is from Antarctica New Zealand and GNS Science88. Figures 89 graphical package, with Fig. 90 package. Figures Figures


In the digested biscuits, the input to ACE inhibitory activity was provided by p-coumaric, sinapic, syringic, vanillic, and protocatechuic acids as well as by kaempherol, quercetin, apigenin, and orientin. Therefore, it can be concluded that cumulative action of those phenolic acids and flavonoids released after digestion is responsible, in part, for the bioaccessible ACE inhibitory activity of buckwheat biscuits.The angiotensin converting enzyme (ACE) inhibitory activity and phenolics profile of fermented flours and of baked and digested buckwheat biscuits was studied. The fermentation of buckwheat flour by select lactic acid bacteria (LAB) caused a decrease in ACE inhibitory activity as compared to the non-fermented flour. The baking process significantly reduced the ACE inhibitory activity of biscuits obtained from fermented flours, whereas digestion significantly increased these properties. In non-fermented and fermented flours and buckwheat biscuits before and after in vitro digestion samples, ten phenolic acids and eight flavonoids were found. Highly significant correlations were found between sample concentration of 50% inhibition of ACE (IC Angiotensin-I converting enzyme ACE, EC 3.4.15.1) plays a main physiological role in the control of blood pressure . Elevate.4.15.1 pRecently, there has been growing interest in using bioactive compounds, mainly peptides, as antihypertensive functional food products to lower high blood pressure . Many AC50 = 6.25 μg protein/mL [It has been suggested that that consumption of buckwheat products may be associated with hypotensive activity ,17. Buckotein/mL . Buckwheotein/mL ,21,22. Totein/mL  found hiotein/mL .Lactobacillus genus [Cereals have been used as the substrates for fermentation by a number of species of the us genus ,25. CompIn vitro digestion models have been widely applied and offer an alternative tool to animal studies to predict the bioaccessibility of ACE inhibitory activity and phenolic compounds due to their simplicity and speed ,27.At present, no information is available regarding the ACE inhibitory properties of fermented buckwheat flours or biscuits based on these fermented flours. Also the bioaccessible ACE inhibitory properties of digested buckwheat biscuits were not investigated yet. Therefore, in this study, we determined (1) the effect of fermentation of common buckwheat flour by selected lactic acid bacteria (LAB) on the ACE inhibitory activity; (2) the effect of baking of water biscuits formulated from fermented buckwheat flours on the ACE inhibitory activity; (3) the effect of digestion in vitro on the potential bioaccessible ACE inhibitory activity from water biscuits; and (4) the correlation between total phenolic compounds, phenolic acids, flavonoids, and ACE inhibitory activity of fermented buckwheat flours and biscuits before and after in vitro digestion.o-aminobenzoylglycyl-p-nitorphenylalanylproline (Abz-Gly-Phe(NO2)-Pro) was obtained from BACHEM . Reagents in mass spectroscopy grade, including acetonitrile, methanol, water, and formic acid, were purchased from Sigma-Aldrich . The diethyl ether (Et2O), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were obtained from Avantor Performance Materials Poland S.A. . The Folin and Ciocalteu’s phenol reagent was obtained from Sigma-Aldrich . Standards of phenolic acids and flavonoids were obtained from Sigma-Aldrich  and Extrasynthese  and were used for IC50 calculation. Water was purified using the Mili-Q-system .Captopril was obtained from Sigma-Aldrich . Angiotensin-converting enzymes (ACE) from porcine kidneys (EC 3.4.15.1) was purchased from Sigma-Aldrich . The substrate Fagapyrum esculentum Moench) was purchased from a local industry plant . The fermentation process and preparation of buckwheat biscuits were carried out as described by Wronkowska et al. [L. rhamnosus GG, which was purchased from ATCC®. Before the fermentation, buckwheat flour was suspended in distilled water (50 g per 950 mL) and then heated at 90 °C for 45 min, autoclaved (121 °C/15 min), and cooled to 37 °C. Buckwheat flour suspension after pretreatment was inoculated with the selected bacteria and fermented at 37 °C for 24 h, and then, the samples were freeze-dried. Water biscuits were prepared according the methods of Hidalgo and Brandolini [Commercially available flour obtained from Polish common buckwheat GD is the ACE inhibitory activity after simulated gastrointestinal digestion (GD), (IC50)BB is the ACE inhibitory activity of buckwheat biscuits (BB), a BI value > 1 indicates high bioaccessibility, and a BI value < 1 indicates low bioaccessibility.For better evaluation of the bioaccessible ACE inhibitory activity in vitro, the bioaccessibility index (BI) was used for indication of the potential bioaccessibility of ACE inhibitory activity of water biscuits formulated from fermented buckwheat flours: 2CO3 solution, and water and left for 25 min at room temperature, and after centrifugation, the absorbance was measured at 725 nm . The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry matter sample.The sample extraction and determination of total phenolic content (TPC) were made according Zieliński et al. . BrieflyThe analysis of polyphenols (phenolic acids and flavonoids) was conducted according to the modified method of Wiczkowski et al. . In the 50, each sample was diluted to various concentrations using deionized water.About 300 mg of analysed samples was 5 times extracted with 70% MeOH. Each extract was evaporated to dryness under nitrogen and then dissolved in deionized water before each assay. The supernatants obtained after in vitro digestion of buckwheat water biscuits were used directly for ACE inhibitory activity. In order to evaluate the ACE inhibitory activities, expressed as ICo-aminobenzoylglycyl-p-nitorphenylalanylproline (Abz-Gly-Phe(NO2)-Pro) by the reaction of ACE. The fluorescence generated by the liberation of the product (the o-aminobenzoylglycine group) was measured immediately after mixing (0 min) and then after 30 min of reaction using multiscan microplate fluorometer. The following equation was used to calculate the % inhibition of ACE:at time 30−RFUat time 0.An angiotensin converting enzyme (ACE) activity inhibitory assay was performed according to the method of Sentandreu and Toldra . The pro50 value indicating the sample concentration of 50% inhibition of ACE activity was determined for varied extracts by using linear regression analysis of logarithmic plots. At least 3 replicates for each extract were conducted. Results were expressed as IC50 values.The IC50 or total phenolic content in fermented flours and in water biscuits before and after digestion were evaluated using a Student’s t-test for less numerous groups (p < 0.05). The differences in the IC50 or total phenolic between all analysed samples—fermented flours, water biscuits, and the sample after digestion—were determined by a one-way analysis of variance (ANOVA) with Fisher’s Least Significant Difference test (p < 0.05). The Pearson correlation coefficient was calculated for correlation analysis (p < 0.05) .The results of the analyses are illustrated as mean values and the standard deviation of three independent measurements. In relations to the control sample, the differences in the ICThe lactic fermentation of cereals such as barley, maize, millet, oats, rice, rye, sorghum, and wheat improves food quality through the development of flavour, lower anti-nutritional effects, and higher bioavailability of minerals .L. delbrucki subsp. bulgaricus 151 and L. acidophilus 145, the IC50 values were comparable to the control buckwheat flour. Generally, it could be said that fermentation of buckwheat flour by LAB caused decrease ACE inhibitory activity. Rui et al. [Monascus fermented soybean, Lactobacillus plantarum fermented lentil, and Bacillus spp. fermented black soybeans, with IC50 of 0.29, 0.20, and 1.81–2.35 mg/mL, respectively. These data indicate that ACE inhibitory effect may depend on the material used and on the applied microorganisms.The results obtained for ACE inhibition of fermented buckwheat flours is shown in i et al.  and Xiaoi et al.  providedi et al. , Torino i et al. , and Juai et al.  showed t50  compared to the flours, control and fermented , L. acidophilus , and L. rhamnosus   . General50 values  compared to flours. For eight biscuits after digestion, no significant differences in the analysed parameter was found compared to the control sample. The statistically significant (p < 0.05) increase of ACE inhibitory activity was found only for biscuits obtained from flour fermented by L. rhamnosus K .The protocol for in vitro digestion included the following steps: first with saliva at pH 7.0, second with gastric acid at pH 2.0, and last with simulated intestinal digestion at pH 7.5. The results obtained for ACE inhibitory activity of the supernatant obtained after digestion of biscuits are shown in The term “bioaccessibility” is a key concept to ascertain the efficiency of food and food formula developed with the aim of improving human health. In this study, the definition of bioaccessibility is the part of ACE inhibitory activity which is released from the food matrix in the gastrointestinal lumen and is used for intestinal absorption . MeasureIn our study, the ACE inhibitory activity of the digested biscuits were significantly higher than those noted in the biscuits before digestion .L. plantarum W42 and L. acidophilus V (21.0) followed by Streptococcus thermophilus MK-10 (20.3) and L. rhamnosus K (19.2). The type of applied bacteria, the physical structure of biscuits, and the enzymes used for in vitro digestion are important factors affecting the potential bioaccessibility of ACE inhibitory activity. Li, et al. [L. plantarum W42, Streptococcus thermophilus MK-10, and L. acidophilus V for fermentation seems to be the most appropriate for enhancing the potential bioaccessibility of ACE inhibitory activity from buckwheat biscuits.The BI values  ranged fus MK-10 .3 and L., et al.  showed a, et al. . In summThe total phenolic compounds (TPC) content in fermented buckwheat flour and water biscuits prepared from them (before and after in vitro digestion) is presented in The ANOVA analysis of the differences in TPC content between all analysed samples  showed that analysed parameters were significantly highest in all samples after digestion compared to the others. Baking generally caused a significant decrease in TPC of biscuits compared to the flour and digested samples. Burton-Freeman  in theirp-coumaric, sinapic, ferulic, t-cinnamic, syringic, vanillic, isovanillic, protocatechuic, and caffeic acids. The average concentration of detected phenolic acids is shown in p-coumaric, sinapic, protocatechuic, and caffeic acids was increased, whereas the concentration of vanillic acid was reduced. Baking caused a reduction in the concentration of phenolic acids with the exception of vanillic and protocatechuic acids: their concentration was ten and twofold increased. These results mostly highlight the influence of thermal treatment on the profile and content of phenolic acids [In non-fermented and fermented flours and buckwheat biscuits before and after in vitro digestion, ten phenolic acids and eight flavonoids were found . Phenoliic acids . After iIn this study flavonoids in non-fermented and fermented buckwheat flours and biscuits before and after in vitro digestion included luteolin, kaempherol, quercetin, rutin, vitexin, apigenin, orientin, and epicatechin. The average concentration of detected flavonoids is shown in 50 and TPC in fermented buckwheat flours (r = 0.44), biscuits (r = −0.25), and the supernatant obtained from biscuits after in vitro digestion (r = 0.29). On the contrary, highly significant correlations were found between IC50 and TPC of fermented flour, water biscuits, and the sample after digestion for each applied bacterium (r = −0.65 (L. delbrucki subsp. bulgaricus 151) to r = −0.93 (L. plantarum IB and L. acidophilus 145) for all samples obtained from fermented buckwheat flour. These evidences clearly indicated the specific LAB-dependent contribution of phenolic compounds in ACE inhibitory activity of the investigated materials.In this study, no correlations were found between ICacterium . The cor50 and TPC of fermented flour, water biscuit, and the sample after digestion for each applied LAB, thus clearly indicating the contribution of phenolic compounds in ACE inhibitory activity. Therefore, it can be concluded that bioaccessible ACE inhibitory activity of buckwheat biscuits is formed by the cumulative action of those phenolic acids and flavonoids for which concentration was increased after digestion.The obtained results showed that fermentation, baking, and digestion significantly affected the ACE inhibitory activity of buckwheat fermented flours and biscuits before and after fermentation. The LAB-dependent variation in ACE inhibitory activity of fermented buckwheat flours was noted. Generally, the fermentation of buckwheat flours caused a decrease of ACE inhibitory activity. Baking significantly reduced the ACE inhibitory activity of buckwheat biscuits prepared from fermented flours. The potential bioaccessible ACE inhibitory activity from digested buckwheat biscuits made of fermented flours was very high and ranged from 5.6 to 21.0. Highly significant correlations were found between IC


This study investigated in vivo the three-dimensional distribution of CT attenuation in the lumbar spine pedicle wall measured in Hounsfield Unit (HU). Seventy-five volunteers underwent clinical lumbar spine CT scans. Data was analyzed with custom-written software to determine the regional variation in pedicle wall attenuation values. A cylindrical coordinate system oriented along the pedicle’s long axis was used to calculate the pedicular wall attenuation distribution three-dimensionally and the highest attenuation value was identified. The pedicular cross-section was divided into four quadrants: lateral, medial, cranial, and caudal. The mean HU value for each quadrant was calculated for all lumbar spine levels (L1–5). The pedicle wall attenuation was analyzed by gender, age, spinal levels and anatomical quadrant. The mean HU values of the pedicle wall at L1 and L5 were significantly lower than the values between L2–4 in both genders and in both age groups. Furthermore, the medial quadrant showed higher HU values than the lateral quadrant at all levels and the caudal quadrant showed higher HU values at L1–3 and lower HU values at L4–5 than the cranial quadrant. These findings may explain why there is a higher incidence of pedicle screw breach in the pedicle lateral wall. The new technique of cortical bone trajectory screw fixation has been proposed to increase the fixation strength in osteoporotic spines, by increasing the contact surface between the screw and pedicle cortical bone, which is stronger than the trabecular bone and less influenced by osteoporotic changes6.Pedicle screw fixation has become a gold-standard method of stabilization for a variety of spinal disorders. However, fixation-screw loosening or failure may occur, particularly in osteoporotic patients7. Unlike long bone diaphysis, the pedicle has a trabecular internal structure. Previous studies on pedicle screw pullout strength have mainly focused on the trabecular computed tomography (CT) attenuation values expressed in Hounsfield Units (HU) values in the vertebral body and the pedicle9. Recent studies using advanced imaging techniques indicated that the local HU values of the trabecular bone surrounding the pedicle screw was a reliable predictor of the pedicle screw pullout strength11. Although no cortical purchase of the lumbar pedicle screw has been reported in a laboratory setting using cadaveric spines, some degree of misalignment can occur during pedicle screw insertion during surgery, which causes redirection of the pedicle screw and breach of the pedicle wall14. It has been reported that cortical purchase of the pedicle screw provides higher fixation strength both with traditional pedicle fixation16 and cortical bone trajectory fixation18.The pedicle is a tubular bony structure with osteons orienting mainly in a longitudinal direction along the pedicle length, similar to long bones20. Regional variation of the pedicle isthmus thickness has been previously investigated and a thicker medial cortex compared with the lateral cortex was reported23. Because bone quality of the cortex is important especially for the cortical bone trajectory screw fixation, recent research on cortical bone trajectory aims to establish noninvasive methods to predict bone quality along the cortical bone trajectory screw pathway25. Bone mineral density has been reported as a reliable predictor of bone material properties, measurable using clinical imaging modalities27. However, to the best of our knowledge, there is no study in the literature describing the three-dimensional (3D) distribution CT attenuation values through the lumbar spine pedicle wall using clinical computed tomography (CT) in vivo. Therefore, the objectives of this study were to investigate the 3D HU values distribution in the lumbar spine pedicle wall in vivo using clinical CT and to analyze variations of the pedicle wall attenuation values by gender, age, spinal level and anatomical site.The pedicular cortex structural properties have been studied mainly with regards to cortical thicknessA total of 84 volunteers participated in this IRB-approved study  including all subjects signing an approved informed consent form. All research was carried out in compliance with relevant guidelines and regulations. From this initial recruitment group, nine of them were excluded for the following reasons: one volunteer had spondylolisthesis, seven volunteers had transitional lumbosacral vertebrae and the last one had a combination of spondylolisthesis and transitional lumbosacral vertebrae. In consequence, a total of 75 subjects were used for the analyses.Each subject underwent CT imaging of the lumbar spine  in a supine position. Axial slice raw image data were exported in the DICOM format.28. These ROIs were acquired on all the axial CT slices of the L1–L5 lumbar levels. Osseous abnormalities and voids such as vascular channels were excluded from the ROIs.Attenuation in the vertebral body was measured using the software ImageJ  to evaluate osteoporotic changes of the vertebral bodies. For each of vertebral bodies the largest possible elliptical ROI was drawn for the vertebral body HU measurementApproximate center points in both the posterior (point a) and anterior (point b) ends of each pedicle were manually determined in the DICOM image(s) of the pedicle level using commercially available software   shown in Fig. To obtain the 3D distribution of HU values in the space adjacent to the pedicle axis, a cylindrical discrete coordinate system centered along the pedicle long axis was defined as follows: 30, the longest axis at the isthmus was calculated and defined as the craniocaudal axis for each pedicle. The four topographic quadrants were defined by 90° intervals as follows: cranial: ± 45°; medial: 45°–135°; caudal: 135°–225°; and lateral: 225°–315° from the cranial axis of each pedicle.Four topographic quadrants: (a) cranial, (b) medial, (c) caudal, and (d) lateral were determined in 3D space, as described next. Since cross-sectional axes decline from the anatomical axes of each vertebrat-test. A gender effect was analyzed by an unpaired Student’s t-test. Age was stratified into two groups: 20 s/30 s and 40 s/50 s. Parameters from these cohorts were then compared via an unpaired Student’s t-test. Regional and level effects of the pedicle cortex were analyzed using repeated-measures ANOVA with Tukey’s post hoc test to probe for statistical differences. Correlations between the mean pedicle wall HU values and the vertebral body HU values at each spinal level were analyzed via Pearson’s correlation coefficients. Results were presented as mean ± SD. Significance was set at p < 0.05.Right and left pedicles in the same vertebral level were compared via a paired Student’s 2 [range 18.9—53.5]) were included in this study  from 75 volunteers    , however the HU values in the cranial region were higher than those in the caudal region at L4 and L5   Fig. , Table 3 Fig. , T07) Fig. , Table 3ly) Fig. , Table 323 and the attenuation values of the whole pedicle or trabecular bone inside the pedicle34, the present study is the first to evaluate in vivo the regional attenuation distribution of the lumbar spine pedicle wall in all lumbar levels using clinical CT data. This report showed that there was an increase in HU values of the pedicle wall from L1 to L2, followed by a consistent plateau of data between L2 and L4 and a decrease at L5 in both gender groups and both age groups. Our regional distribution of attenuation showed that the lateral region had lower HU values than the medial region at all spinal levels. We also showed that HU values in the caudal region were higher than in the cranial region from L1 to L3, but lower at L4 and L5.Although previous studies reported on the pedicle cortical bone thickness32. This finding may be explained by remarkable changes in pedicle dimensions between L4 and L5. Sugisaki et al. reported a significant increase in longitudinal axis of the pedicle isthmus from L4 to L5 by 25% and 27% in females and males, respectively30. Assuming pedicle cross-sectional geometry has an elliptical shape, these increases in the longitudinal axis would increase area moment of inertia to resist a bending moment in females and males by 95% and 105%, respectively. Therefore, the reduced HU values in L5 may be of limited influence on pedicular structural properties, leading to consider that a reduction of pedicular HU values in L5 may be a result of functional adaptation of bone tissue under reduced stress caused by the increased area moment of inertia.The increase seen in the average HU values from L1 to L2–4 may be attributed to a corresponding gradual rise in loading at the lower levels. However, the average HU values decreased between L4 and L5, which is consistent with results of previous research on the whole lumbar pedicle HU values, including the pedicle cortex and interior trabecular bone35 Previous studies looking at regional variation of the cortical thickness in the lumbar pedicle found that the cortical thickness was thinnest in the lateral region23, which has been thought to be a reason for the higher incidence of the pedicle penetration in the lateral pedicle wall. Crawford et al. investigated the trajectory of self-centering lumbar pedicle screws inserted in trajectories starting 0°, 10°, 20° or 30° from the optimal trajectory, either medially or laterally misdirected. The authors found that lateral misalignment as small as 10° was likely to lead to cortical wall violation (3 of 7 violations) whereas medial misalignment usually resulted in safe screw insertion 12. This study indicates that the lateral wall has less strength to resist pedicle screw penetration as compared with the medial wall. Although multiple factors would be involved in the higher incidence of pedicle screw breach in the lateral side, lower attenuation in the lateral pedicle wall shown in the present study should be considered as an important factor favoring pedicle screw breach due to the strong correlation shown by previous efforts between HU values and cortical bone material properties36.The lateral region showed lower HU values than the medial region at all spinal levels. It has been reported that lateral wall breaches are the most common pedicle-screw related failures in thoracolumbar indications37. The architecture of the trabecular bone tract trajectories connecting the articular processes and the lumbar vertebral body38 and radiographically dense trabecular tracts extending from the pedicles15 were also reported. Given that these trabecular tracts connecting the articular processes and the vertebral body bear load transmission between the anterior and posterior columns, the pedicle cortex in the cranial and caudal regions may also play a part in load transmission and resist bending moments borne by the pedicle. A full explanation of why are there higher HU values in the caudal region at L1–3 and in the cranial region at L4–5 remains elusive. We speculate that load transmission patterns between the anterior and posterior column through the pedicle, especially bending in the sagittal plane, may be different between the upper and lower lumbar spines, where spinal curvature and posture may play a role. Future biomechanical studies on the stress/strain distribution in the pedicle and their correlation with the HU values distribution will be required to explain the regional differences in pedicle wall HU values.Significant differences in the HU values between the cranial and caudal regions of the pedicle were found in the present study. While the HU values in the caudal region were higher at L1–3, the opposite case was seen at L4–5. This is clinically important in that the inferior breach of the pedicle screw at L4 and L5 is relatively common and can result in neurological complications. The surgeon should be mindful of the cortical walls with lower HU values, which are lateral in all lumbar levels and inferior at L4 and L5. Trabecular tracts running obliquely from the superior process downward to the inferior endplate through adjacent to the cranial cortex of the pedicle and from the inferior process upward to the superior endplate through adjacent to the caudal cortex of the pedicle were reported as early as 1925 by Gallois and JapoitIn the present study, we provided a new cylindrical HU dataset for each pedicle. HU values of new points in the cylindrical dataset were calculated by trilinear interpolation of the eight adjacent HUs in the original CT voxels Fig. C. In the31. Future studies including an osteoporotic population will be required. Second, we analyzed regional variations in pedicle wall attenuation by quadrant regions along the pedicle axis even though the entire 3D HU values distribution data were available. Analyses using other zoning systems and/or pattern analyses of the HU values distribution considering cortical bone trajectory would be of interest. Finally, the voxel size of the original CT data used in the present study, approximately 0.4 × 0.4 × 1.0 mm, may not be of high-enough resolution when compared with the pedicle cortical thickness if the intent was to evaluate absolute peak HU values at individual points due to partial volume effects.Our study is not without some limitations. First, since this study cohort included subjects from 20 to 50 s, who are relatively active and still young, only two subjects met the criteria of osteoporosis defined by the vertebral HU values at L1In summary, this paper presented a novel 3D pedicle model based on peak HU values located radially away from the pedicle axis and investigated regional variation in lumbar pedicle wall attenuation. The results of present study demonstrated that the mean attenuation value at L5 was significantly lower than the values in L2–4 regardless of gender and age and the HU values in the lateral region was lower than in the medical region at all spinal levels. Since the 3D HU values distribution in the pedicle wall can be measured directly from patient-specific DICOM datasets without creating 3D models, the presented method could be used as a preoperative planning for transpedicular fixation using clinical CT.


AnpIBP was recently isolated from a cold-adapted fungus, Antarctomyces psychrotrophicus. The present study identified an orbital illumination (prism ring) on a globular single ice crystal when soaked in a solution of fluorescent AnpIBP, suggesting that AnpIBP binds to specific water molecules located in the ice prism planes. In order to examine this unique ice-binding mechanism, we carried out X-ray structural analysis and mutational experiments. It appeared that AnpIBP is made of 6-ladder β-helices with a triangular cross section that accompanies an “ice-like” water network on the ice-binding site. The network, however, does not exist in a defective mutant. AnpIBP has a row of four unique hollows on the IBS, where the distance between the hollows (14.7 Å) is complementary to the oxygen atom spacing of the prism ring. These results suggest the structure of AnpIBP is fine-tuned to merge with the ice–water interface of an ice crystal through its polygonal water network and is then bound to a specific set of water molecules constructing the prism ring to effectively halt the growth of ice. Many microbes that survive in cold environments are known to secrete ice-binding proteins (IBPs). The structure–function relationship of these proteins remains unclear. A microbial IBP denoted  Proteins bind to their ligands to perform specific functions. Hydration water molecules in a protein play a significant role in ligand binding and also have roles in dissolving proteins in water and maintaining their tertiary structures ,2. Some a-axes (a1–a3) perpendicular to the c-axis [h) has six primary prism planes and six secondary prism planes along the c-axis. These prism planes lie parallel and perpendicular to the a-axes, respectively. The basal plane lies perpendicular to the c-axis and parallel to the a-axis. Other inclined planes between the basal and prism planes are defined as pyramidal planes. It has been demonstrated that the growth speed of the prism planes toward the a-axis is approximately 100 times faster than that of the basal planes toward the c-axis [a- and c-axes of this golf-ball-size single ice crystal using a polarizer, where its earth-axis corresponds to the c-axis of Ih. When such an ice hemisphere is mounted on a frosty probe with the desired orientation, and soaked in a solution of fluorescence-labeled IBP, a pattern appears on the globular ice surface under ultraviolet (UV) light. This is called the pattern of fluorescence-based ice plane affinity (FIPA) of an IBP species, as presented in [The IBPs isolated from cold-adapted organisms such as fish, insects, plants, and microorganisms  are knowe c-axis . The hexe c-axis . A snowfe c-axis , in whicented in . Variatiented in . From thTm and Tf) of a single ice crystal prepared in an IBP solution; TH = |Tm − Tf| [Tf and slightly raises Tm to generate the TH, which can be evaluated with a photomicroscope system as described earlier [In addition to the FIPA pattern, IBP’s ice-binding ability can be detected by the observation of morphological change in an ice crystal and thermal hysteresis (TH) activity. Both observations require a photomicroscope system equipped with a cooling stage, whose temperature needs to be lowered to deep freezing temperatures, such as −40 °C. IBP changes the photomicroscope image of a single ice crystal into a hexagonal bipyramid, a hexagonal trapezohedron ,15, or aTm − Tf| ,16,17,18 earlier . IBP froMarinomonas primoryensis (MpIBP) has pentagonally arranged water molecules on its IBS [3 groups of Thr side chains and are stabilized on the IBS through hydrogen bonding to the main-chain nitrogen and side-chain hydroxyl groups of Thr, as well as to the side-chain oxygen atom of Asp. The ice-like water molecules are thought to play a significant role in facilitating IBP binding to ice crystals and are known as anchored clathrate water (ACW). Polygonally arranged water molecules have been observed for moderately active type II and III IBPs [Tenebrio molitor (TmIBP) and Dendroides canadensis (DIBP), which consists of a 12-residue consensus sequence CTxSxxCxxAxT repeated tandemly, where C, T, S, and A are Cys, Thr, Ser, and Ala, respectively, and x represents any residue [AnpIBP does not have any tandem repeat sequences [IBPs have been used to elucidate the relationship between hydration water molecules and the ice-binding function. IBPs have one or two ice-binding sites (IBSs), which consist of many hydrophobic residues making a flat surface in which several polar residues are inserted at regular intervals or dispersively . For sev its IBS . These mIII IBPs ,21, as wIII IBPs ,23, but III IBPs ,6,7,8,9. residue . AnpIBP equences , and it AnpIBP. IBP from the Antarctic bacterium Colwellia sp. SLW05 (ColIBP) and that from Arctic yeast (LeIBP) are both contain the DUF3494 domain. The former exhibits high TH activity (~4 °C) at 140 µM [LeIBP is much lower (0.35 °C), even at 370 µM [AnpIBP exhibited TH activity (~0.7 °C) at 300 µM, which was the same level as that of the moderately active fish IBP. An ability to modify ice crystals into a lemon shape is regarded as a typical sign of high TH activity. AnpIBP was reported to generate such a lemon-shaped crystal, although it is only moderately active [AnpIBP and a mutant version for which an extraordinary ice-binding property has been suggested. Here we present new structural data for an IBP identified from a cold-tolerant fungus, Antarctomyces psychrotrophicus (AnpIBP). AnpIBP showed a unique ability to bind to the equator region of a single ice crystal when we prepared it in a globular form, a phenomenon that we called a “prism ring”. IBPs from microorganisms including bacteria , fungi , diatomst 140 µM , whereast 370 µM . The Anpy active . The preAnpIBP and a mutant version were prepared as described previously, using Pichia pastoris as the host organism [AnpIBP was inserted downstream of an α-factor signal sequence in the expression vector pPICZα. The vector was transformed into P.pastoris after linearization by a restriction enzyme. The transformant was precultured in buffered glycerol complex medium, followed by 96–120 h incubation in methanol-containing medium, to express AnpIBP. The culture was then centrifuged to collect the medium containing AnpIBP. The protein was purified by a series of ion-exchange chromatography passes  and further purified by size-exclusion chromatography . The mutant proteins were prepared using the same procedure. The purity of these samples was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have previously reported that A.psychrotrophicus secretes glycosylated AnpIBP at the Asn55 position [AnpIBP under investigation here, expressed in P. pastoris, was also thought to be glycosylated, because it showed multiple or smear bands in SDS-PAGE. We found that the substitution of Asn55 with aspartic acid produced a non-glycosylated form of AnpIBP, and the N55D mutant of AnpIBP is defined as AnpIBP hereafter. Further amino acid replacements were made in this AnpIBP. The S153 of AnpIBP was replaced with Tyr, for example, and is called the S153Y mutant in this article. These samples were dissolved in water or 20 mM Tris–HCl at pH 8.0, and concentrated by ultrafiltration (MW cutoff = 3 kDa) . Their final concentration was determined using UV absorbance at 280 nm with a coefficient calculated on the basis of the amino acid sequence.Wild-type organism . Brieflyposition . The AnpAnpIBP and its mutants was measured by observation of a single ice crystal prepared in an IBP solution, using a photomicroscope equipped with a temperature-controlled stage and a Charge Coupled Device (CCD) camera, as described previously [Tm) while increasing the stage temperature. We then prepared another single ice crystal, and lowered the temperature with a cooling rate of 0.1 °C/min. An ice crystal does not start to grow when the temperature is higher than the lower limit of TH, and it shows bursting growth when this limit is exceeded, which is determined to be the freezing point (Tf). The difference between the two temperatures (Tm − Tf) is defined as the TH value [The TH activity of eviously . A 1-µL TH value ,16,17,18c- and a1–a3-axes of this cylindrical ice crystal were checked using polarizers. The crystal was modified into a hemispherical shape (Ø = 30 mm) with a heated stick and was mounted on a chilled copper rod with a desired orientation. The single ice crystal hemisphere mounted on the rod was soaked in a 40-mL solution of fluorescence-labeled IBP so as to face down the curved front of the crystal. The fluorescence dye used was tetra-methyl-rhodamine (5 (6)-TAMRA-X, SE: Thermo Fisher Scientific, Waltham, MA, USA). The single ice hemisphere was held in the solution for 4–5 h at −5 °C. When it became golf ball sized (Ø = 5 cm), it was detached from the copper rod to observe the FIPA pattern under UV light in a cold room (−1 °C).In order to identify the IBP-bound plane of a single ice crystal, the FIPA pattern was observed for our samples using the procedure described in . A singlAnpIBP and its S153Y mutant prepared without His-tag were crystallized using the hanging drop vapor diffusion method [AnpIBP were obtained when they were dissolved with 0.1 M HEPES-NaOH pH 8.6, 0.95 M ammonium sulfate, and 0.1 M lithium sulfate. The S153Y mutant was crystallized with 0.1 M Bis-Tris pH 7.0 and 4.0 M ammonium acetate. The experiments using synchrotron radiation were performed with the approval of the Photon Factory Program Advisory Committee . Diffraction data for AnpIBP and the S153Y mutant were collected at the beam line BL-17A [AnpIBP was determined using the molecular replacement method using Phenix [ColIBP (PDB ID: 3WP9) [ColIBP coordinate was removed, because the corresponding segment does not exist in AnpIBP. The initial model structure of AnpIBP was further refined using the software packages Phenix, Refmac5 [AnpIBP. The statistics for data collection and refinement [AnpIBP and S153Y mutants are shown in AnpIBP and 7BWY for the S153Y mutant.The n method . A 1-µL e BL-17A  at the Pe BL-17A  and the e BL-17A  program g Phenix  with a cD: 3WP9)  as a sea Refmac5 , and Coo Refmac5 . The strfinement  are summAnpIBP, for which illustrated interpretations are presented in c-axis downward  [AnpIBP between 0.01 and 0.15 mg/mL. The FIPA pattern did not change with increasing concentration; AnpIBP binds to the prism ring but not to the basal plane, even at the highest concentration (data not shown). AnpIBP is a member of the microbial IBPs containing the DUF3494 domain. The FIPA pattern reported for the known DUF3494-characterized IBPs, such as ColIBP [TisIBP [SfIBP [FfIBP [AnpIBP is exceptional, and its manner of binding to the prism ring is controlled by a unique structural property.It has been shown that the FIPA pattern is sometimes changed with increases in the concentration of IBP ,41. For s ColIBP , TisIBP  [TisIBP , SfIBP [P [SfIBP , and FfIP [FfIBP , showed AnpIBP was determined at 1.9 Å resolution using a molecular replacement method utilizing ColIBP (PDB: 3WP9) as a structural template [AnpIBP molecules ; β6, A184–Q201 (18); β5, N166–G183 (18); β4, N145–G165 (21); β3, S117–A144 (28); and β2, A93–A116 (23). The lengths of the β2–, β3–, and β4–loops are 3- to 10-residues longer than those of the others . The three surfaces of the triangular β-helical domain are named the A-, B-, and C-faces  at equal intervals, which sandwich several hydrophobic residues. The role of this C-terminal segment is unknown. The overall structural motif of AnpIBP is highly similar to that of other IBPs from bacteria, despite their phylogenetic distance. This finding is in good agreement with the previous indications that Antarctic ascomycetes acquired AnpIBP from bacteria through horizontal gene transfer [The crystal structure shows that D55, whose original residue is N55 modified with a glycan, is located at the C-terminal end of the long α-helix and is far distant from the B-face containing the IBS. The TH activity of N55D is identical to that of the wild-type, confirming the previous indications that the glycan is not involved in ice binding . The C-ttransfer . AnpIBP, we prepared a mutant protein in which the T58 on the A-face (AnpIBP (wild-type) and its four mutant proteins: T58Y, Y131T, S153Y, and T156Y. They showed a typical linear dependence of TH on the square root of concentration. The wild-type had 0.63 °C of maximal TH activity at a concentration of 250 µM. The T58Y that mutated on the A-face exhibited a similar dependence to the wild-type. Mutations in S153Y and T156Y on the B-face resulted in a decrease in activity to 17% of the wild-type. These results suggest that the IBS of AnpIBP is on the B-face. The Tyr131 located on the B-face protrudes a bulky side chain, which may cause steric hindrance when the B-face binds to an ice crystal. Hence, we expected TH to be increased for Y131T, because threonine is a typical ice-binding residue for most IBPs. However, the mutation affected neither TH nor the ice-shaping abilities, indicating that Y131 is located in such a way as to maintain the ice-binding ability. To confirm that there was no significant influence of these mutations on protein folding, we performed circular dichroism (CD) measurements (AnpIBP) exhibited minimum ellipticity at 217 nm and maximum ellipticity at 197 nm on the CD spectra, suggesting a β-helical structure. The same profile was reported for other microbial IBPs [AnpIBP mutants exhibited highly similar CD profiles to the wild-type, suggesting that defective mutations did not affect the overall structure but collapsed a structure needed for ice binding on the B-face. To identify the IBS location of e A-face A was repurements . The wilial IBPs ,42. All AnpIBP is categorized as a moderately active IBP based on its TH value  on the surface of an ice crystal. This mechanism facilitates rapid association of IBP to ice crystals and has been proposed for several other IBPs that accompany the polygonal water networks. It has been shown that hydration water molecules on an IBP are relevant to its ice-binding function. We examined such water molecules located on the crystal structures of ectively . Among tric unit . We founled Maxi ,23. In Mled Maxi ,49, a tot, S153Y C, whose t, S153Y C,D. MoleAnpIBP, as well as the basal plane. The basal plane is the polar area of the spherical ice corresponding to the top surface of the hexagonal ice  [TmIBP, have regularly arrayed hollows, but they do not accompany a polygonally arranged water network [AnpIBP is the first example to both have polygonally arranged water molecules and have hollows to tightly trap the surface water molecules at regular intervals, allowing the binding of this IBP only to water molecules separated by 14.7 Å, which form the prism ring. The fish type III IBP also accompanies the surface-bound waters [AnpIBP binding to the prism planes effectively inhibits this fast growth, which protects the host organism (A. psychrotrophicus) from ice injuries.The  its IBS B, which  mutant) . This IB network . The oth network . Therefod waters , which ad waters . AnpIBP AnpIBP. The AnpIBP uniquely binds to the water molecules that participate in the primary and secondary prism planes, which were detected as a prism ring in the FIPA experiment. Both the ice-like polygonally arranged water molecules and a row of four hollows to trap the surface water molecules exist on the IBS of AnpIBP and perfectly match the oxygen atom spacings (4.6 and 14.7 Å) of the prism ring. The presence of oxygen atoms separated by 14.7 Å should be key to recognizing the prism ring, whereas four trapped water molecules will bind AnpIBP tightly onto the prism ring. These results suggest that AnpIBP is fine-tuned to merge with the ice–water interface of an ice crystal through polygonally arranged water molecules and then bind to specific water molecules constructing the prism ring through the water molecules trapped in the four hollows. To summarize, the present study determined the target ice planes and X-ray crystal structures of the wild-type and a defective mutant of


For pediatric flexible flatfoot, the subtalar extra-articular screw arthroereisis (SESA) and endosinotarsal device are the most popular techniques in current practice. Nevertheless, scarce literature is available comparing the outcomes between these two techniques. Thus, we aimed to provide a meta-analysis for the radiographic and clinical outcomes, respectively. A systemic search for correction of pediatric flexible flatfoot using subtalar arthroereisis was conducted mainly in Pubmed and Scopus, and the search was completed on 31 Dec., 2019. The standardized mean differences (SMD) of postoperative versus preoperative calcaneal pitch and Meary’s angle were defined as the primary outcomes, whereas the preoperative versus posteoperative AOFAS (American Orthopaedic Foot and Ankle Society) as the secondary outcome. The meta-analysis included 12 comparative studies comprising 2063 feet in total. The quantitative analysis showed a marked improvement in Meary’s angle of endosinotarsal cone implant group  than exosinotarsal screw group . But no significant difference was noted between both groups in calcaneal pitch and AOFAS. The exosinotarsal screw and endosinotarsal device are both effective arthroereisis implant for pediatric flexible flatfoot. While considering the correction of Meary’s angle, the endosinotarsal device is better than exosinotarsal screw. Although we further went through the literature focusing on outcomes of exosinotarsal screw and endosinotarsal device for subtalar arthroereisis procedure, the comparison study is still lacking2. Furthermore, there was no multi-center, multi-surgeon study to compare these two techniques in the previous research. Thus, we aimed to provide a meta-analysis for radiographic and clinical outcomes, respectively.For pediatric flexible flatfoot, the subtalar extra-articular screw arthroereisis (SESA) and endosinotarsal device are the most popular techniques in current practice. Nevertheless, we found that there were limited studies to compare the outcomes between these two techniques. Traditionally, exosinotarsal screw is thought as inexpensive, while endosinotarsal implant is thought to preserve more soft tissue. However, none of the study has provided the difference between the two proceduresPubmed, Scopus, Cochrane Collaboration Central Register of Controlled Clinical Trials, Cochrane Systemic Review, and ClinicalTrials.gov were searched for studies concerning the use of extraosseous subtalar arthroereisis for pediatric flexible flatfoot from the earliest record . The bibliography of included trials and related review articles were manually reviewed for relevant reference. Literature not written in English, not available in full text, adult patients, focus on gait analysis, review articles, patients with neuromuscular diseases, case reports, or techniques except exosinotarsal subtalar screw in calcaneus or endosinotarsal cone-shaped implant were excluded. We investigated studies using endosinotarsal tunnel subtalar arthroereisis for pediatric flexible flatfoot treatment.The search strategy comprised the following keywords combined with subtalar arthroereisis: flexible flatfoot and pes planus. Regarding the types of included studies, we enrolled randomized controlled trials (RCTs), comparative experimental trials, or single-armed follow up studies. We excluded case series and case reports. The target population comprised pediatric patients who suffered from painful flexible flatfoot.Two reviewers examined all of the retrieved articles and extracted data using a predetermined form. We recorded the first author, year, sample size, implant choice, combination of soft tissue procedure, radiographic outcome, and clinical outcome. The methodological quality of enrolled studies was independently evaluated by two reviewers using Jadad scoring for the RCTs and the Newcastle–Ottawa Quality Assessment Scale for the comparative experimental trials.3 A study can be scored a maximum of one point for each item in the Selection and Exposure domains and a maximum of 2 points for each item in the Comparability domain. Between-reviewer discrepancies were solved through discussions under the supervision of the corresponding author.The Newcastle–Ottawa Quality Assessment Scale contains 9 items in 3 categories: participant selection (4 items), comparability (4 items), and exposure (3 items)The standardized mean differences (SMDs) of calcaneal pitch and Meary’s angle between the SESA group and endosinotarsal device group comprised the primary outcomes. Data extracted from the radiographic outcome parameters were evaluated during outpatient department follow up postoperatively. A negative SMD value indicated receiving arthroereisis surgery was a worse option. AOFAS (American Orthopaedic Foot and Ankle Society) score was also retracted, and a negative SMD value of AOFAS indicated receiving arthroereisis surgery was a worse option.4.A random effects model was employed to pool individual SMDs. All analyses were performed using CMA software V3. Between-trial heterogeneity was determined by using I2 tests; values > 50% were regarded as considerable heterogeneity5. Statistical significance was defined as p-values < 0.05.Funnel plots and Egger’s test were used to examine potential publication biasWe retrieved 101 non-duplicated citations for reviewing their titles and abstracts and other 4 articles using manual extraction. We finally included 12 articles for meticulous evaluation after eliminating references violating inclusion criteria   and exosinotarsal screw group    than exosinotarsal screw group  Fig. b.The overall AOFAS score improvement was 3.425   and exosinotarsal screw group  , Meary’s SMD (p = 0.05507), and AOFAS SMD (p = 0.41738). The funnel plot is as shown in Fig. The Egger’s test revealed no significant publication bias regarding the overall calcaneal pitch SMD  technique and endosinotarsal cone shaped implant. However, previous studies failed to reveal significant difference between the two techniques. Our study thus pooled 12 articles and collected 2063 feet to compare the radiographic and clinical outcomes between the two surgical techniquesOur result revealed both techniques improved in both radiographic parameters. In Meary’s angle, the group of using endosinotarsal cone shaped implant technique improves more than the group using exosinotarsal metallic screw.2. This appears that the outcomes in statistics of the studies may be mixed with diverse techniques. In addition, many reviewers could have unclear pictures to recognize which one of the techniques was better for rectifying the pediatric flexible flatfoot. In some articles, authors adopted the same term of “arthroereisis” with different types of implants, even data were from surgeons working in different affiliations. Memeo et al. indicated no statistical difference was found between the two techniques for Costa-Bertani angle, heel inclination, Kite angle and talar declination angle in 402 feet.Most of previous studies about subtalar arthroereisis did not specifically indicate if the procedure was performed with SESA or endosinotarsal deviceApparently, the data from Memeo A et al. were collected for 10 years with quite complete records. However, with mono-clinical practical surgeries under one hospital, it seems that if we could take a further examination in the event of different hospitals, different surgeons, the result could be shared differently for its consequence.We assumed the metallic screw had lesser contact area to bone than endosinotarsal implant, which may lead to screw head sinking into bone and thus less corrective effect. The other reason may be due to screw purchasing in cancellous bone with minor fixation effect. Based on above reasons, the results of corrective effect during follow-up did not meet the expectation. Furthermore, the favorite position and screw axis direction performed by surgeons in processing exosinotarsal screw were different. Thus, the screw position was difficult to change after insertion. On the contrary, surgeon can change varieties of endosinotarsal implant in size when the endosinotarsal implant stands at the improper position or appears a flaw of corrective effect. In light of this, the surgeons may change the endosinotarsal implant depending on personal preferences.24. Second, the variable screw purchase to calcaneus may lead to screw loosening after repeated subtalar motion. Last but not least, the optimal position and trajectory for the screw has yet to be determined. Consequently, the stabilizing effect for exosinotarsal screw would be relatively unpredictable. On the contrary, surgeons can modify endosinotarsal implant size according to correcting effects.Our result revealed both techniques were effective regarding radiographic parameters. For Meary’s angle, the group with endosinotarsal cone shaped implant improved more than the group with exosinotarsal metallic screw. We suggest three possible explanations (there may be more). First, the exosinotarsal metallic screw implant had less contact area to bone than endosinotarsal implants, which may lead to bone erosion and consequent less corrective effect30. Bioabsorbable material may be associated with regional inflammatory response30. Higher body mass index was found to be associate with higher complication rates and less deformity correction with endosinotarsal implants21. Careful selection of patients and techniques based on current evidence may be helpful to reduce the complication rate.There are some factors associated with the occurrence of complications. The type of implant chosen may result in different complication profiles. For example, sinus tarsi pain and implant migration were more frequently seen with endosinotarsal implants. Incomplete correction may be associated with the exosinotarsal implants33.Generally speaking, there are four main types of complications: result of inappropriate indication, technical error, patient adaptation or irritation, and biomechanical failure of implant with incidence of 4.8–18.6%. Complications reported in endosinotarsal device included sinus tarsi pain, implant extrusion, local tissue reaction, and mechanical irritation. Complications reported in extrasinotarsal screw included sinus tarsi pain, subjective limitation, screw breakage, incorrected positioned screw, and local fracture of calcaneus or talus after fall/trauma. While the most common complication is unexpected sinus tarsi pain, the majority of symptoms could be adapted by patient or relieved after implant removal. It seemed to be more easily to handle complications in endosinotarsal device compared with exosinotarsal cases. Up to date, there has been no randomized controlled trial to compare advantages and disadvantages between the two techniques in the current literaturesMost articles presented these 2 types techniques based on retrospective studies. The quality was limited due to retrospective articles in its nature. Newcastle score is between 5 and 7 with a limited number of extreme high-quality studies.We approached some authors by emails for obtaining more detailed data of standard deviations and implant choices. However, there was no response till the end of our study.The papers related to the outcomes of these two techniques are quite limited. AOFAS is widely used by most investigators. However, it is scattered on QxFAD-C (Oxford ankle foot questionnaire for children). Therefore, there is only one study on AOFAS for Exosinotarsal group.24, the best time for the surgeries is 9–12 years old. According to Hsieh et al., most of their patients underwent endosinotarsal implants were before the age of 10, suggesting less bone remodeling in older children. More studies to support the best operation age are required in the future.The range of age distribution is relatively wide in both endosinotarsal cone group or exosinotarsal screw group. According to study of Kubo et al.There are several limitations in our study:The endosinotarsal group had a better corrective result in Meary’s angle compared with exosinotarsal group. For clinical AOFAS, exosinotarsal group also shows an improvement. We assumed that once the sample size was adequate enough, this outcome of clinical AOFAS would be better. Hence, investigators need to examine the outcome of AOFAS clinical scores.1, while others applied metallic materials. We take conservative attitude to the rectification effectiveness on foot alignment while selecting different materials of endosinotarsal device. Considering the foot alignment needs to sustain a long period under same situation for different materials, age and body weight, future studies will be required for investigating the effectiveness.For endosinotarsal device, some articles used bio-absorbable deviceOur study provided the evidence of comparing corrective effects of “subtalar arthroereisis” for pediatric flexible flatfoot using different techniques and implants. We suggest that the surgical types, implant selection, sources and costs should be well documented for a more specific statistics in the future.The exosinotarsal screw and endosinotarsal device are both effective arthroereisis implants to treat pediatric flexible flatfoot. However, the endosinotarsal device shows a better improvement in Meary’s angle than exosinotarsal screw.