Cysteine protease inhibitors (cystatins) are molecules that play key protective roles in protein degradation and are involved in the immunomodulation of host responses to parasites. Little is known about the cystatin gene repertoire, evolution, and lineage-specific adaptations of early-emerging metazoans. Using bioinformatics searches, we identified orthologues of cystatins in basal animal lineages including free-living and parasite taxa. We aimed to explore whether their cystatin gene repertoire and evolution follow similar patterns recognized for derived metazoans and whether the modifications are linked to the organism’s life history. We revealed that cysteine protease inhibitors from early-emerging animal groups are highly diverse, with modifications in gene organization and protein architecture. A new subtype of cystatins was discovered in the parasitic cnidarians, the Myxozoa, which has so far been only reported for a group of derived animals: trematode flukes. We set out hypotheses to describe the driving forces for the origins of this unique cystatin subtype and propose evolutionary scenarios elucidating the current existence of cystatins in the Metazoa, especially in their early-emerging lineages. Our research identified molecules for which future functional studies may help to identify their roles in host–parasite interactions and for the parasite itself. The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity. Members of the cystatin superfamily I25), also known as cystatins, are major regulators of the activity of papain-like cysteine proteases and legumain-type proteases. These endogenous protease inhibitors are widely distributed in all domains of life. Based on sequence homology, the cystatin superfamily comprises three main sub-families: types 1 –3. Type 1 cystatins  are low-molecular-weight intracellular single-domain proteins generally lacking disulfide bonds and signal peptides. Within the stefins, an additional subtype, unusually featuring a signal peptide, is recognized exclusively in liver and intestinal flukes (Trematoda: Digenea) 5, also k. Type 2 Cystatins are involved in the regulation of key physiological processes, such as the protection of cells from proteolysis ,6,7. In The diversity and evolution of the cystatin superfamily is well-documented in prokaryotes and certain eukaryotic lineages ,31,32,33Edwardsiella spp. [Polypodium hydriforme [Edwardsiella and Polypodium parasitize their hosts exclusively during the larval stage of their development [Basal metazoans mostly comprise free-living species, but at least eight independent parasite lineages of early-emerging Metazoa are known, with all but one in Cnidaria . Recentllla spp. ,45, Polydriforme ,47, and driforme ,52,53,54elopment , the micelopment . Memberselopment . Myxozoaelopment ,53,58. TTo explore the gene repertoire, gene organization, and structural features of cystatins in phyla at the base of the metazoan tree, we mined 110 genomes and transcriptomes of 58 early-emerging animal species. We conducted phylogenetic analyses using newly obtained and published cystatin sequences of basal and derived metazoans to provide insights into the origin, evolution, and structural diversification of this group of inhibitors at the time when this superfamily emerged and expanded. We investigated the amino acid sequence structure and phylogenies of cystatins from free-living and parasite taxa to identify structural modifications, which potentially represent lineage-specific adaptations related to parasitic lifestyles. Our investigation thus aimed to reveal whether gene repertoire and evolution of cystatins in early-emerging animals follow similar patterns recognized for derived metazoans and whether the modifications are linked to the organism’s life history.Polypodium hydriforme, Edwardsiella lineata, E. carnea, and all available myxozoans (13 species). We used published the cystatin homologues of metazoans from basal animal species [Representatives of basal metazoan lineages  were selected for mining of cystatin homologues from the NGS data available from GenBank, other public databases, or produced in our laboratories . The mined sequences were examined for the presence of typical conserved motifs of the cystatin domain , by seaP/PW; YF ) includiP/PW; YF  against Buddenbrockia plumatellae (Buddenbrockia EST database (Full gene sequences of the cystatin superfamily homologues mined from unpublished genomes and transcriptomes  were vermatellae  as minindatabase  did not database . The posGiardia cystatin (outgroup) resembling the most ancestral eukaryotic cystatin [Polypodium hydriforme, Edwardsiella spp., and myxozoans; 16 taxa) and bilaterians   of both newly identified and published cystatins. The dataset comprised Choanoflagellates (11 taxa), early-emerging animal lineages (58 taxa), more derived metazoans (58 taxa), and cystatin  (Table S39 taxa) . Multi-dWe aligned 329 amino acid cystatin sequences in MAFFT v7.017 implemented in Geneious v8.1.3  using thCystatin diversity on the amino acid sequence level was analyzed in terms of critical motifs, amino acid conservation, presence/absence of a signal peptide and disulfide bridges, and modifications that might potentially affect the cystatin inhibitory activity. Disulfide bonds were predicted using the DISULFIND webserver . Signal In this study, we retrieved 184 homologues of cystatins (104 stefins and 80 type 2 cystatins) from the NGS data of early-emerging metazoans . Of thesWe observed a large variation in the representation of cystatin subclasses in the investigated animal groups . Type 2 Ceratonova shasta and Physalia physalis) only possessed single type 2 cystatin and/or stefin homologue.We found that basal metazoans had a diverse cystatin gene repertoire: most investigated species had multiple cystatin homologues with significant sequence differences, even on the intraspecies level  or as Genbank contig/scaffold identifiers for sequences mined from published data .The clustering of cystatins in both ML and BI analyses did not follow the generally accepted trends of organismal phylogeny, as early-emerging animal lineages did not occupy basal positions in our trees  as commoHydra, Podocoryna, Physalia, Velella, and Porpita spp. clustering into independent type 2 cystatin clades) or (ii) the occurrence of paralogues . Some of the out-paralogous clades additionally contained multiple sequences from single species   . The results of structural analyses aimed at identifying intron number and positions, conserved regions, and the presence/absence of signal peptides and cysteine bonds were combined to create the comparative scheme of cystatin structural motifs .We identified that introns in cystatin nucleotide sequences of early-emerging animal lineages varied in number (0–2) and location in type 2 cystatins while stefins contained two introns with conserved positions . All intIn amino acid sequence data from our alignment of basal animals , we obseWhile the amino acid structure of the first two key activity regions  was conserved among all sequences, several modifications were seen in other regions. All sponge (Porifera) stefins showed a characteristic amino acid replacement of the LP pair to LD (or LS in one case). Substitution of the conserved C-terminal tyrosine residue by non-aromatic valine was observed in two homologues. The conserved histidine residue located near the LP pair was commonly replaced by methionine or, exceptionally, by histidine or tryptophan (both observed once) .Stefins and type 2 cystatins of comb jellies (Ctenophora) had the typical sequence motifs defined for these molecules in other metazoans. In a few taxa, we observed substitutions of the conserved cystatin PW pair by AW and stefin C-terminal tyrosine residue by proline or histidine .Cnidarian cysteine protease inhibitors showed a wide variety of modifications . CubozoaP. hydriforme had the typical conserved motifs, its stefin was highly modified, with the LP pair substituted with LS and the C-terminal conserved D-x-L-x-Y-F motif replaced with a completely different pattern lacking a tyrosine residue. While this stefin retained the histidine found near the LP pair, the conserved lysine located near the C-terminal was replaced by leucine. Myxozoans, as representatives of early-emerging metazoans, had atypical stefins, which completely lacked the D-x-L-x-Y-F region at the C-terminus and had the normally conserved histidine and lysine residues replaced by other residues. Stefins of an evolutionary older myxozoan subgroup, the Malacosporea, retained the original LP pair, but some stefins of the specious and more derived myxozoan group, the Myxosporea, had replaced this motif with LS, LY, LR, FK, or SA  while others presented multiple stefin genes that all carried the signal peptide  or had genes that represented both variations   .As typical for type 2 cystatins, two conserved disulfide bridges were identified in all basal metazoans with the exception of homologues found in ctenophorans, which were predicted to contain only one disulfide bridge. Classical stefins lacked disulfide bridges, as is typical for this cystatin subfamily type, and this pattern was also observed in atypical stefins .E. lineata, E. carnea, and P. hydriforme) possessed both classical stefins and type 2 cystatins, and no atypical stefins were found in these animals. Their stefins formed a distinct lineage that afterwards clustered with other cnidarians (P. hydriforme) or grouped with their respective anthozoan members (E. lineata and E. carnea). Similarly, clustering of “partially parasitic” cnidarians into distinct monotypic lineages (P. hydriforme) or within anthozoan lineages (E. lineata and E. carnea) was observed for type 2 cystatins . Stefinthe host .Structurally, atypical stefins contain the characteristic stefin LP pair, which is modified in certain myxozoan stefins. Importantly, all atypical stefins lack the conserved D-x-L-x-Y-F carboxy-terminal part that is generally found in classical stefins but is absent from type 2 cystatins . As the Thelohanellus kitauei [Additionally, we identified that the atypical stefins of some myxozoans and all trematodes have a signal peptide typical for type 2 cystatins . In some kitauei ,84. Thes kitauei ,88,89,90 kitauei ,20. Simi kitauei ,25,26,27 kitauei ,92.We propose two mechanisms for the origin of atypical stefins: (i) structural modification of ancestral classical stefins by the acquisition of a signal peptide in some homologues and loss of the C-terminal and its tyrosine residue, or (ii) combination of the features of classical stefins (LP pair) and type 2 cystatins  in chimeric genes. Chimeric genes are important in the evolution of genetic novelty and can allow organisms to adapt to novel environments (or hosts) by acquisition of novel functions ,94. BothSphaerospora sensu stricto, and malacosporeans [In our analyses, the reconstruction of evolutionary relationships in the cystatin superfamily had good support for the more recent clades but not for deeper evolutionary nodes, as reported previously for this group of genes ,35. Unstsporeans ) ResolutFrom their comprehensive analysis of prokaryotic and eukaryotic cystatins, Kordiš and Turk  concludeBased on our novel data and published records , we propStefins are considered relatively conserved genes with characteristics closer to the ancestral superfamily type, while type 2 cystatins are considered to have undergone a more complex and dynamic evolution through numerous gene and domain duplications . In this


The supplemental screw technique was introduced for salvage of lateral hinge fracture in medial open-wedge high tibial osteotomy (owHTO). The efficacy of its use in protection of lateral hinge fracture and corresponding biomechanical behaviors remained unclear. The current study was aimed to clarify if a supplemental screw can provide better protection to lateral hinge in biomechanical perspective.An in vitro biomechanical test was conducted. Tibial sawbones, commercial owHTO plates and a cannulated screw were utilized for preparing the intact, owHTO, and owHTO with cannulated screw insertion specimens. A “staircase” dynamic load protocol was adopted for axial compressive test with increasing load levels to determine structural strength and durability by using a material testing system, while a motion capture system was applied for determining the dynamic changes in varus angle and posterior slope of the tibia plateau with various specimen preparation conditions.Type II lateral hinge fracture were the major failure pattern in all specimens prepared with owHTO. The insertion of a supplemental cannulated screw in medial owHTO specimens reinforced structural stability and durability in dynamic cyclic loading tests: the compressive stiffness increased to 58.9–62.2% of an intact specimen, whereas the owHTO specimens provided only 23.7–29.2% of stiffness of an intact specimen. In view of tibial plateau alignment, the insertion of a supplemental screw improved the structural deficiency caused by owHTO, and reduced the posterior slope increase and excessive varus deformity by 81.8% and 83.2%, respectively.The current study revealed that supplemental screw insertion is a simple and effective technique to improve the structural stability and durability in medial owHTO. The concept of lower limb realignment by medial open-wedge high tibial osteotomy (owHTO) has been widely accepted for the treatment of medial compartment osteoarthritis and varus deformity of knee joint –3, especThe risk of alteration of the posterior slope of the tibial plateau has been reported in several clinical studies –13. KeepPrevious studies have confirmed that supplemental screw insertion plays a successful role in salvage of lateral hinge fracture and partially restores biomechanical stability in medial owHTO cases , 20. Howth generation sawbones were utilized in the current study [th generation, composite, medium; A Pacific Research Company, WA, USA) and 6 standard varus  models were used. The 6 varus models were treated by a single experienced surgeon, with medial owHTO referring to the standard TomoFix Osteotomy System Surgical Guide, and corrected by 10 degrees to valgus using a bi-planar osteotomy technique. TomoFix Medial High Tibial Plates  and locking screws were implanted for fixation. Three specimens were randomly selected from the 6 varus-correction models. An 8-mm cannulated screw  and washer was additionally placed in these specimens . Referring to a previous study by Yang et al., the supplemental cannulated screw was inserted from the lateral cortex to the region beneath the medial-lateral tibial plateau, in an orientation of approximately 50° oblique in the coronal plane and 38.5° oblique in the transverse plane [For purpose of standardization, 4nt study , 24. A tse plane . A speciAll specimens were embedded into a bone cement mould at the distal region. Before the embedding procedure, the distal tibia was shortened by 10 cm to remove the medial malleolus region. Using the cannulated shaft axis of the tibial sawbone as reference, the specimen was place into an aluminium cylindrical mould with a central vertical pole. The cannulated tibial shaft was placed on the pole and the mould filled with commercial bone cement to have an 8-cm embedment. All embedded specimens were placed in room temperature for 48 hours for fully cement polymerization.An Instron 8801 Material Testing System  was used for the mechanical tests. The test specimen was fixed at the base of testing machine, with the tibial shaft axis vertical to the ground. Compressive load was exerted onto both medial and lateral condyles of tibial plateau, through a hinged load distributor to permit a balanced resultant force to be located on the tibial plateau with a 62% lateral offset as described in previous in vitro studies , 25, simAccording to in vitro test protocol for medial owHTO by Agneskirchner et al. , a preloThe reference coordinate system was defined by the tibial shaft axis , anteroposterior axis , and the mediolateral axis (X axis). The load-displacement data for all specimens were recorded by the built-in program of the Material Testing System, representing the structural performance at the Z axis. The compressive stiffness of the specimen was calculated by the cyclic load over the axial displacement , averaged for each load level. After labeling the optical makers in the program provided by MaxTRAQ, the tibial plateau slope changes were defined by the relative angular movement between the superior and inferior fragments in coronal plane (the X-Z plane) and sagittal plane (the Y-Z plane), calculated from the videos recorded by the CCD-cameras, averaged for each load level as well. Positive values of the slope changes represent increased varus  or increased posterior slope  as signs of reduction loss. Three testing groups  were compared.Due to difficulty in determining the exact failure cycle during the test, the durability of the test specimen was represented in a 100-cycle interval . Among aQuantified structural compressive stiffness is represented in th load level (endpoint of the group 1), the increased varus angle for intact, group 1, and group 2 were respectively 0.16, 1.41±0.12 (range 1.3–1.53), and 0.37±0.01 (range 0.36–0.39) degrees. At the 6th load level (endpoint of specimens 1 and 2 for group 2), the increase of varus angle were 0.26 and 0.56±0.03 (range 0.53–0.59) degrees. The 3rd specimen of the group 2 showed an increased varus angle to 0.69 at the endpoint of the 7th load level.th load level the increase of posterior slope for intact, group 1, and group 2 groups were respectively 0.17, 4.34±0.38 (range 4.1–4.77), and 0.93±0.09 (range 0.85–0.91) degrees. At the 6th load level, the increase of posterior slope for intact group and group 2 were 0.263 and 1.42±0.23 (range 1.23–1.68) degrees. The 3rd specimen of group 2 showed an increased posterior slope to 1.68 degrees at the 7th load level (endpoint), while the slope of the intact specimen was 0.32 degrees.As a strategy for postponing the necessity of total knee arthroplasty for patients with varus deformity or medial compartment osteoarthritis, the medial owHTO must take the issue of joint stability into serious concern. In the concept of enhanced recovery after surgery (ERAS), it will be very beneficial to bone healing that patients can return to the original activity level and weight bearing. Early full-weight bearing with adequate interfragmental stability may improve the early clinical outcome for patients who receive owHTO surgery . If the th to 7th load level until the occurrence of type II hinge fracture. The group 1 showed comparatively lower durability to 5th load level. A stress-based biomechanical study has discussed about the strategy in avoidance of hinge fracture during gap expansion by pre-drilling a hole at the end of open wedge [Biomechanical methods are commonly applied in studying the in vitro stability and durability of osteotomy models. For simplifying the test for determining structural stability and durability under different loading magnitudes, Agneskirchner et al. have firstly introduced a “staircase” dynamic loading protocol to represent the biomechanical behavior of owHTO structures with various designs of owHTO implants. A linear displacement transducer was applied to measure the gap motion at the osteotomy site . In theien wedge . Howeverth load level, the group 1 further increased the posterior slope by 4.17 degrees in average compared to the intact group, which is apparently larger than the group 2 (0.76 degrees in average). Taking the full loss of posterior slope by 100% when only the bone plate was applied in the group 1, the group 2 preserved the posterior slope by 81.8% (i.e. only 18.2% loss of reduction). When considering varus correction loss, the supplemental screw insertion also restored the stability to 83.2%. The evidence provided in the current study helped to confirm that the strong support by a supplemental screw can maintain the structural stability without jeopardizing the safety of implanted components in medial owHTO surgery.The posterior slope of the tibial plateau is influential to knee joint function and joint contact force. High varus correction and lateral hinge fracture are risk factors for loss of reduction (increased posterior slope) after owHTO surgery, validated by Jacobi et al. in a cadaveric model . This isSome limitations of the current study were noted:Due to limited research resources, the sample size was not very large. In our current study, the intergroup differences were not highly significant. The specimens were carefully prepared to reduce the possible influence from human factors. However, some other important physiological loading conditions such as torsion and bending moment were not simulated in the current study. A further investigation may be necessary to comprehensively realize the biomechanical performance of the supplemental screw in medial owHTO model.Although the sawbone specimen has been validated for its mechanical behaviors compared to cadaveric bones, the property of sawbone is more like a plastic material lacking flexibility. When performing the opening of the osteotomy site for varus correction, it is very difficult and technically demanding to preserve an intact lateral hinge. Some of the studies created the medial owHTO by removing the wedge from a normal tibial sawbone instead of expanding the gap for varus reduction , 32. In The failure observation was confirmed visually and acoustically. Therefore, the current study did not provide the exact number of failure cycles. Because the lowest frequency for visual confirmation was within 100 cycles, so the interval of 100 cycles were represented in the current study for describing the failure cycles.The test specimen was a stand-alone tibial sawbone without the lateral support by the fibula. The fibula shares around one-sixth of the load from the knee joint. The reduced lateral support may also influence the resistance to possible valgus moments.The pre-tension provided by the supplemental screw insertion was difficult to quantify. It is expected that a fully threaded screw may firmly stabilize the gap between the superior and the inferior bone fragments, while a partially threaded screw (ex. lag screw) may tend to close the gap and offers a secondary compression to the lateral hinge in medial owHTO surgery. Surgeons would decide the tightening level for the partially threaded screw referring to personal experience or preference. However, the artificial sawbone cannot provide similar feedback torque for surgeon. Further study for optimal strategy for supplemental screw insertion technique will be required.The current in-vitro biomechanical study encouraged the usage of supplemental screw in medial owHTO structure with improved mechanical strength, reduced loss of reduction, and better durability to dynamic cyclic load compared to the regular treatment without the supplemental screw. Further studies for more comprehensive biomechanical verifications, and optimizing surgical technique and for confirming clinical efficacies should be conducted in the future.S1 Fig(XLSX)Click here for additional data file.S2 Fig(XLSX)Click here for additional data file.S3 Fig(XLSX)Click here for additional data file.


Chrysochromulina/ Prymnesium) that produced 5.7 g C m−2 new production. This estimate represents about half the annual pelagic production, occurring below sea ice with a large contribution from the mixotrophic algae bloom. The freshwater-influenced, nutrient-dilute and low light environment combined with mixotrophic community dominance implies that phagotrophy played a critical role in the under-ice bloom. We argue that such blooms dominated by potentially toxic mixotrophic algae might become more common and widespread in the future Arctic Ocean.The pelagic spring bloom is essential for Arctic marine food webs, and a crucial driver of carbon transport to the ocean depths. A critical challenge is understanding its timing and magnitude, to predict its changes in coming decades. Spring bloom onset is typically light-limited, beginning when irradiance increases or during ice breakup. Here we report an acute 9-day under-ice algal bloom in nutrient-poor, freshwater-influenced water under 1-m thick sea ice. It was dominated by mixotrophic brackish water haptophytes ( The contribution of phytoplankton productivity beneath ice-covered oceans to annual pelagic net primary production was until recently thought to be negligible, primarily because of the strong light attenuation by snow and sea ice2. However, this view has recently been challenged by observations of extensive diatom-dominated under-ice blooms beneath thick melting sea ice or under refrozen leads5. In these studies, diatom- and Phaeocystis-dominated under-ice blooms were triggered by increased under-ice irradiance due to melting sea ice, refrozen leads and/or melt pond formation (i.e. increased transmittance) and were fuelled by an excess of nutrients in the under-ice waters7. However, an improved understanding of the role, extent and frequency of these extensive under-ice blooms is needed to describe and model future changes in annual pelagic net primary productivity in the Arctic Ocean.Satellite-based remote sensing data suggest that annual pelagic net primary production in shelf areas of the Arctic Ocean has increased by 20% from 1998 to 2009, mainly due to the longer seasonal duration of the ice-free period, and thus a longer pelagic growth season8 and has a pronounced summer stratification that impedes nutrient supply from deeper waters10. The combination of light limitation by sea ice and terrestrial run-off and low nutrient supply in Young Sound is responsible for its low annual pelagic net primary productivity (10.3 g C m−2 year−1)11, which is low compared to the more productive Godthåbsfjord on the southwest coast of Greenland (between 84.6 and 139.1 g C m−2 year−1)12. The future annual pelagic net primary production in Young Sound and across the Arctic Ocean is likely to increase overall as the ice-free season lengthens. Counteracting the effects of a longer pelagic growing season is, however, the recent increase in freshwater fluxes from Arctic glaciers and the general freshening of the Young Sound fjord, adjacent fjords14 and the Arctic Ocean16. This freshening may lead to an intensified stratification and therefore a weakened vertical supply of nutrients17.The Young Sound fjord in NE Greenland is covered by sea ice for most of the year (8–10 months)Prymnesium and Chrysochromulina) can be highly toxic to fish and cause severe damage to the aquaculture industry, e.g. in Norway19. Harmful blooms of Chrysochromulina spp. have been observed in the Skagerrak/Kattegat20, the Baltic Sea21, and in Resolute Bay, Canada22, but this is, to our knowledge, the first observation of a high Arctic under-ice bloom driven by mixotrophic haptophytes.In this study, we demonstrate for the first time an under-ice bloom driven by mixotrophic brackish-water haptophytes beneath 1-m thick sea ice in a nutritionally dilute environment. These haptophyte genera  was similar to those cell concentrations found during the peak of harmful Chrysochromulina blooms in the Skagerrak in southern Norway19. The indication of a mixotrophic-based bloom suggests that mixotrophic algae may play an important role in driving the Arctic spring bloom and thus the ecosystem and carbon dynamics in this area. Some haptophytes tend to be more abundant in less saline waters, as exemplified by records of Chrysochromulina spp. in the low saline  Baltic Sea and in the Kattegat 21. This further implies that mixotrophic-driven harmful algae blooms might become more common and widespread in a more freshwater-influenced future Arctic Ocean.Species of In this study, we document that a mixotrophic-driven under-ice bloom can be an important seasonal feature of Arctic fjords. An improved insight into the extent and frequency of these extensive blooms driven by potentially mixotrophic haptophyte species underneath thick sea ice is fundamental for understanding and modelling future changes in Arctic Ocean pelagic net primary productivity. If blooms of potentially toxic algae become more common in the Arctic, this might have a large ecological and socio-economic impact.−2 s−1 before melt pond formation  and sea ice thaw (from 0.95 to 0.75 m thickness) resulted in a nearly full desalination of the sea ice towards 12 July and a concomitant decrease in under-ice salinity on 9 July   than the under-ice total algal abundance on 8 July (up to 6.2 × 105 cells l−1)  in the sea ice, which indicates that heterotrophic activity may have played an important role in nutrient re-cycling  of the sea ice from 0.21 ± 0.07 on 9 July to 0.11 ± 0.04 on 12 July  entire ice cores, and support the hypothesis that the complete brine drainage terminated the sea ice photosynthetic activity. Nevertheless, we observed a dramatic increase in under-ice water maximum quantum yield (Fv/Fm), biomass specific productivity and total algal abundance from 0.12 ± 0.02, 5.80 mg C mg Chl 0 cm enti30. The under-ice algae were clearly acclimated to low light conditions given the relatively low under-ice Ek values (mean of 75 µmol photons m−2 s−1) derived from 14C incubations  rounded cells with two long flagella and in many cases a long and coiled haptonema typical of e.g., the toxic mixotrophic species Prymnesium polylepis and Chrysochromulina leadbeateri. The under-ice bloom provided up to 740 mg C m−2 day−1 of new production  to have been deficient both in sea ice and seawater  in Table The proposed link between high under-ice productivity and mixotrophy is further supported by the low bulk NOhttps://data.g-e-m.dk/). Sea ice cores were collected on four occasions  using a MARK II coring system (Kovacs Enterprises). All samples were collected within a 10 m2 (3.2 m × 3.2 m) quadrat. Triplicate cores were collected for physical and chemical samples during each sampling and cores for biological parameters were sampled in duplicate. The under-ice seawater samples were collected on four occasions  using a Niskin  water sampler at three depths: 1 m, 15 m and 30 m. The sea ice cover broke up on 15 July after which the ice floes were transported out of the fjord by winds and tides. Open seawater samples were thereafter collected twice (16 and 17 July 2017) also at three depths: 1 m, 15 m and 30 m. Vertical profiles of temperature, salinity, irradiance  and fluorescence in the under-ice and open seawater column were measured using a CTD profiler (Seabird SBE19plus) equipped with additional sensors for Photosynthetic Active Radiation .Sampling was conducted during the sea ice melting season in Young Sound, NE Greenland  in 2017. The under-ice PAR measurements were obtained from a new and calibrated Odyssey PAR censor (Dataflow Systems) mounted on a cable and placed 2.4 m below the ice between 1 June and 7 July 2017. Under-ice PAR time-series were extended to 15 July by applying the transmittance of the ice (0.09), determined as ratio between measured CTD and surface PAR from the nearby Zackenberg Research station  in sea ice, three sea ice cores were cut into 10 cm sections and placed immediately in gas-tight plastic bags33 and transported back to the laboratory in thermally insulated boxes. TCO2 and O2 were determined using standard methods34. Average values of triplicate samples are reported.The air temperature was measured 2 m above the melt pond, and vertical profiles of temperature within the ice were measured using a thermometer (Testo). Light attenuation  was determined using standard methods35. Average values of triplicate samples are reported.In the laboratory, sea ice sections were weighed for later determination of density and then melted in the dark over a 2-day period at 3 ± 1 °C. Conductivity of melted sea ice was measured (Thermo Orion-star with an Orion 013610MD conductivity cell) and converted to bulk salinity233 and O234 analysis following the standard analysis procedure.Unfiltered under-ice and open seawater was transferred by gastight Tygon tubing to tubes (12 ml Exetainer) for TCO43−, NO2− + NO3− (NOx) and NH4+), Chlorophyll a (Chl a) and photosynthetic activity. Duplicate samples were collected for primary production and algal abundance. Melting of sea ice was carried out following the protocols of Lund-Hansen et al.17 to avoid osmotic shock during ice melt, and photosynthetic and photobiological parameters are therefore maximum values.Triplicate melted sea ice cores (10 cm sections) and under-ice and open seawater samples  were analysed for inorganic nutrients (PO−2 s−1) using the H14CO3− incubation technique36, and following the primary production procedure37. The potential primary production (µg C l−1 h−1) measured in the laboratory for different sea ice depths was plotted against the eleven laboratory light intensities and fitted to the function38 . Average values of duplicate samples are reported.Potential primary production was determined in melted sea ice cores cut into three sections , and under-ice and open seawater at three depths  incubated for 3.15–6.49 h at 1 ± 1 °C at eleven laboratory light intensities  before and after addition of 200 µl of a 1 M HCl solution. The fluorometer was calibrated against a pure Chl a standard (Turner Designs). Average values of triplicate samples are reported.For determining Chl 43−, NOx and NH4. The concentrations were measured on a Seal QuAAtro autoanalyzer using standard colorimetric methods. Average values of triplicate samples are reported.For determining inorganic nutrient concentrations, triplicate samples of melted sea ice, under-ice water and seawater without ice were filtered through 0.45 µm filters (Q-Max GPF syringe filters) into 6.5 ml scintillation vials and frozen (− 19 °C) for further analysis of PO40 and the curve-fitting algorithm41. Average values of triplicate samples of maximum quantum yield (Fv/Fm) are reported. Temperature in a snow bath with measuring unit was controlled by an US-T Temperature Control Unit and kept close to 1 °C. Temperature of the samples was measured by a thermometer .Pulse amplitude modulated (PAM) fluorometry  was applied to measure algae photoacclimation and photosynthetic activity in triplicate samples from melted sea ice core sections (10 cm), under-ice, and at depths (1 m) in open seawater with no ice. All PAM measurements were conducted in the dark to keep algae dark-incubated and the photosynthetic parameters were derived by means of the rapid light curve techniqueFor algal abundance measurements, samples of melted sea ice and under-ice and open seawater were fixed with acidic Lugol’s solution  and stored dark and cold (3 ± 1 °C). Algal cells were identified and counted from light-microscopy analyses using the Utermöhl sedimentation technique. Algae cells were analysed within 2 months of collection, average values of duplicate samples are reported.Chrysochromulina polylepis, which is a typical haptophyte with a long and coiling haptonema, has been transferred to the genus Prymnesium.Using light microscopy, it is not possible to identify small naked flagellates such as the haptophytes to species level. The taxonomy of the haptophytes is currently under revision, and the bloom forming mixotrophic species formerly known as Supplementary Figure S1.


Stephanella hina is a little studied freshwater bryozoan belonging to Phylactolaemata. It is currently the only representative of the family Stephanellidae, which in most reconstructions is early branching, sometimes even sister group to the remaining phylactolaemate families. The morphological and histological details of this species are entirely unknown. Consequently, the main aim of this study was to conduct a detailed morphological analysis of S. hina using histological serial sections, 3D reconstruction, immunocytochemical staining and confocal laser scanning microscopy techniques. The general morphology is reminiscent of other phylactolaemates; however, there are several, probably apomorphic, details characteristic of S. hina. The most evident difference lies in the lophophoral base, where the ganglionic horns/extensions do not follow the traverse of the lophophoral arms but bend medially inwards towards the mouth opening. Likewise, the paired forked canal does not fuse medially in the lophophoral concavity as found in all other phylactolaemates. Additional smaller differences are also found in the neuro-muscular system: the rooting of the tentacle muscle is less complex than in other phylactolaemates, the funiculus lacks longitudinal muscles, the caecum has smooth muscle fibres, latero-abfrontal tentacle nerves are not detected and the medio-frontal nerves mostly emerge directly from the circum-oral nerve ring. In the apertural area, several neurite bundles extend into the vestibular wall and probably innervate neurosecretory cells surrounding the orifice. These morphological characteristics support the distinct placement of this species in a separate family. Whether these characteristics are apomorphic or possibly shared with other phylactolaemates will require the study of the early branching Lophopodidae, which remains one of the least studied taxa to date. Phylactolaemate bryozoans are, from an evolutionary perspective, very interesting, as they comprise the sister group to the two remaining taxa, Stenolaemata and Gymnolaemata , 2. TheyStephanella, the sole genus of the Stephanellidae, represents an early branch within phylactolaemate diversification, a notion also supported by more recent molecular analyses ).s, e.g., , 23–25).Stephanella hina is the lack of a distinct, continuous forked canal. In all other phylactolaemates, the latter commences on the lateral, proximal sides of the cerebral ganglion as ciliated openings that lead into two ducts that medially fuse in the distal direction. This medial fusion of the ciliated ducts arches directly above the epistomial coelomic extension located above the cerebral ganglion .Excretion is a little studied process in bryozoans and often seems to be associated with coelomocytes . The secS. hina is similar to that of other phylactolaemates [S. hina and the remaining, particularly vacuolated epithelium as the esophagus until the cardiac valve. However, several studies (including the present study) frequently label the upper vacuolated area the pharynx . This mynx . In the the gut , but theAdditional studies should comparatively analyse all six major phylactolaemate families regarding the anatomy and cytological specifications of the foregut. This should aid in redefining the terminology in terms of more criteria than just ciliation and also investigate whether morphological details of the gut might have any systematic or phylogenetic value.Stephanella hina, shows that the main muscular systems are very similar to previous descriptions. Concerning the six abovementioned muscular systems, the following can be summarized. 1) The body wall possesses a regular muscular grid as present in other phylactolaemates [Pectinatella or lophopodids [S. hina, the situation is similar to that of most phylactolaemates except lophopodids where the bands attach at the diaphragmatic sphincter [S. hina. Hence, the differentiation of the diaphragmatic sphincter structures is not as evident as in other species ..StephaneS. hina. This supports the notion that a regular grid of orthogonal musculature is the ancestral condition for phylactolaemates . In addition, the arrangement of the muscle bands is very dense in other phylactolaemates, whereas S. hina shows a rather loose pattern, particularly in the caecum. In contrast to all other analysed phylactolaemates, S. hina does not show longitudinal muscles in the funiculus (see [3) The tentacle sheath muscles comprise more prominent longitudinal and circular muscles. The arrangement thus reflects the general arrangement of the body wall musculature, as also seen in tes see , 30, 31), 31S. hites see , 17. 4) . 4 S. hilatinosa . In contlus (see ).Stephanella hina. The lophophoral base area where these muscle bands insert differs between phylactolaemates and myolaemates, with a series of small muscular elements being present in the latter. In S. hina, these rootlets are negligible since the frontal muscle bands appear more distally and show no rooting of any sort, and the abfrontal muscles only have a thin elongated proximal extension. This differs from the previous description of the other four families, where the frontal tentacle muscles show one to two different rootlets, which on the oral side are even connected to the pharyngeal musculature. Likewise, the abfrontal muscle bands have a characteristic arrangement not found in S. hina, where only a series of a few stacked muscle bundles, intertwined in a zig-zag fashion, are present. Consequently, S. hina entirely differs in its basal root structure of the tentacle musculature from the other four analysed families [5) In all bryozoans, including phylactolaemates, the tentacles are supplied with two longitudinal muscle bands . These afamilies , 31.Hyalinella punctata [S. hina, the second state, involving individual fibres, is present and shared by lophopodids and pectinatellids, whereas the three remaining families, Cristatellidae, Fredericellidae and Plumatellidae, share the first configuration. It thus remains difficult to assess which type of epistome musculature is ancestral, but since lophopodids and stephanellids are early branching, it seems reasonable to assume individual traversing fibres as the ancestral condition.The epistomial musculature can show two different patterns: muscle fibres embedded in the epithelial linings or individual muscle fibres traversing the epistomial coelom/cavitysee , 30, 31), 3130, 3Cristatella mucedo, Pectinatella magnifica and Hyalinella punctata [Stephanella.Muscles associated with the proximal border of the lophophoral ring canal were detected in punctata  and were6) The retractors show no difference from those of any studied phylactolaemates (see references above). They consist of numerous bundles originating from the body wall and attach at several locations on the oral side of the polypide. They are of a smooth fibre type.Stephanella hina. These proceed distally from the lateral sides of the cerebral ganglion and bend medially to extend towards the lateral margins of the epistome. In other phylactolaemates, the ganglionic horns extend in a straight line distally towards the tip of the lophophoral arms [S. hina is the only representative with such an unusual condition, it is concluded that this characteristic  is apomorphic. The traverse of the bent ganglionic horns is reminiscent of the epistomial nerve , but suStephanella hina appears to completely lack latero-abfrontal neurite bundles in each tentacle, which are otherwise found in all other phylactolaemates [S. hina seems to be the first phylactolaemate recently investigated that lacks any sign of these latero-abfrontal neurite bundles. The current data do not support any abfrontal lateral neurite bundles, but to completely verify the presence or absence of these thin neurites, transmission electron microscopy would be necessary.Additional differences in the nervous system are evident in the detailed tentacle innervation that in laemates , 25 and S. hina is reminiscent of the medio-frontal nerves of myolaemate bryozoans that show a similar origin with mostly two roots emerging intertentacularly from the CON [In addition to the apparent lack of latero-abfrontal neurite bundles, the roots of the medio-frontal bundles mostly emerge directly from the circum-oral nerve ring (CON), with only a few emerging from the radial nerves. The latter condition is described for most other phylactolaemates , 25. Hen the CON , 35, 36.Hyalinella punctata [Cristatella mucedo [S. hina, were also encountered in the aforementioned species.Concerning the remaining, more peripheral neuronal elements, the plexus innervating the digestive tract and tentacle sheath is more diffuse and does not show more prominent neurite bundles on either the oral or anal side of the polypide, which was reported for all other species , 32, 37.punctata  and Crisa mucedo . DistincStephanella hina is clearly a phylactolaemate and possesses many morphological characteristics similar if not identical to those of other members of the clade, there are numerous unique characteristics underlining the special position of this little studied species. These are most apparent in the structure of the lophophore, including the arrangement of the forked canals in the inner lophophoral arc and the ganglionic horns. Additional finer details, such as the lack of funicular musculature, latero-abfrontal neurite bundles or smooth caecum musculature, support that S. hina is a distinct family among Phylactolaemata . Althou


Sea ice cover in the Arctic and Antarctic is an important indicator of changes in the climate, with important environmental, economic and security consequences. The complexity of the spatio-temporal dynamics of sea ice makes it difficult to assess the temporal nature of the changes—e.g. linear or exponential—and their precise geographical loci. In this study, Koopman Mode Decomposition (KMD) is applied to satellite data of sea ice concentration for the Northern and Southern hemispheres to gain insight into the temporal and spatial dynamics of the sea ice behavior and to predict future sea ice behavior. We observe spatial modes corresponding to the mean and annual variation of Arctic and Antarctic sea ice concentration and observe decreases in the mean sea ice concentration from early to later periods, as well as corresponding shifts in the locations that undergo significant annual variation in sea ice concentration. We discover exponentially decaying spatial modes in both hemispheres and discuss their precise spatial extent, and also perform predictions of future sea ice concentration. The Koopman operator-based, data-driven decomposition technique gives insight into spatial and temporal dynamics of sea ice concentration not apparent in traditional approaches. The formation and distribution of sea ice plays an important role in the planet’s climate and thus large amounts of data related to quantitative measures of sea ice have been collected, including continuous satellite remote sensing measurements since 1978. The decreasing extent of Arctic sea ice over the last several decades has had negative effects on Arctic wildlife and local communities, while also potentially opening new regions to maritime commerce and natural resources exploration. The future of sea ice behavior is thus of great significance for environmental, economic, and national security reasons. There are several studies that suggest a nonlinear trend in the decline of the sea ice cover3 and, e.g. kernel analog forecasting4). Statistical approaches were reported by the SIPN to be similarly or more successful compared with dynamical approaches for prediction of sea ice distributions 1–3 months in the future3. Of statistical approaches, spatial mode based approaches5 have particular advantages. Compared to regression or trend based approaches, spatial mode based approaches are powerful tools for studying and predicting the geographic and temporal behavior of sea ice because they decompose the time dependent sea ice data into time varying spatial structures of physical significance. However, such approaches have heretofore been restricted to methods that—explicitly or implicitly—assume statistical stationarity of the underlying process. Here we apply Koopman Mode Decomposition (KMD)7—a method capable of capturing nonstationary, growing or decaying trends in data—to sea ice concentration dynamics and prediction.A variety of approaches have been applied to predict future sea ice behavior over short time scales (1–3 months in the future), including both dynamical  and statistical (data-based) (see the reports of the Sea Ice Prediction Network (SIPN)13 is already widely used to analyze data and provide models for complex dynamic processes. Mathematically, the Koopman operator14 is a linear representation on an observables space of an appropriate group action on state space. As it is a linear representation, it is natural that key objects of analysis will be its eigenvalues and eigenfunctions. It turns out that in distributed systems  in addition to the eigenvalues and eigenfunctions, there is a third class of objects of importance: the Koopman modes13 which are projections of fields of observables on eigenfunctions of the Koopman operator7. The eigenvalues of the Koopman operator provide the time scales on which—a potentially exponential—change in sea ice cover is happening, and the modes indicate the spatial extent of the changes. Crucially, the Koopman operator methods do not require a model—observables like sea ice thickness and concentration are sufficient to compute the eigenvalues and the associated modes.KMD is a mathematical tool well suited to analyzing sea ice dynamical behavior because it identifies important spatial structures and their complex time dependence from large data sets such as those available for sea ice. The Koopman operator theory15 for the study of fluid flows where it was conceptualized as an algorithm to decompose the flow field into component fluid structures, called “dynamic modes” or “DMD modes”, that described the evolution of the flow. The DMD modes and their temporal behavior are given by the spectral analysis of the linear operator, which is constructed from data since it is assumed that direct access to it is not available. The book16 provides references and introduction to a variety of DMD-related algorithms. Rowley et al.13 gave the method theoretical underpinnings by connecting it to the spectral analysis of the Koopman operator. The paper17 provides both enhancements and analysis to the DMD method as well as additional theoretical underpinning for its relationship to the Koopman operator.The most popular computational method for Koopman eigenvalues and modes is the Dynamic Mode Decomposition (DMD), that has become a major tool in the data-driven analysis of complex dynamical systems. DMD was first introduced in 2008 by P. Schmid18, due primarily to the long and continuous time period (from November 1978 to the present) and the large geographic regions over which this data is available. KMD analysis was applied to both entire Arctic and Antarctic sea regions. The geographic regions mentioned below for the Arctic were those given by Boisvert and Stroeve19 and the Antarctic regions were those given by the NSIDC18  with a) have norm greater than a.Figures Comparison of the mean modes in Fig. The combination of the mean and annual variation modes shown in Figs. 24, these modes show that the decrease in sea ice coverage is most pronounced in the regions of the Beaufort Sea and Barents Sea. This is also consistent with the changes in the mean and annual variation observed in the five-year window cases above. Figure 26, so this KMD mode is consistent with that observation and the result from the five-year windows above showing decreased mean ice coverage and increased annual variation near West Antarctica. Note that the exponential decay described by the identified modes occurs in the geographic regions indicated by the spatial content of the mode, so the decrease in sea ice concentration on those time scales occurs locally in those regions.Such modes are apparent in analysis of the entire 40 year period of the data set. Figure The identification of an oscillatory period comparable to or greater than half of the time period of the input observations  is believed to have dynamical significance despite this frequency being less than the frequency resolution that would be obtainable from the discrete Fourier transform (DFT) of this time series data. As described in greater detail in the As described in the Methods section below, forecasts of future sea ice concentration can be made by “recomposition” of the Koopman Mode Decomposition, where a subset of the eigenvalues and modes obtained from performing KMD over a given time period of data are used to calculate predicted concentration or anomaly values for times after the time period of the input data. As described in more detail in the Methods section, the goodness of the forecasts is described by a skill calculation, where the KMD-based forecast is compared to one of four different reference models: (1) a climatological model, in which each future calendar month is forecast to be the mean of the data values of that calendar month for the entire period of available sea ice concentration data, (2) a persistence model, in which the anomaly of each future month is forecast to be the same as in a reference month, (3) a climatological-type model in which the forecast for each future month is the mean of that calendar month in the input data , and (4) a linear fit of the data values for each calendar month in the input data.In the following, the skill, root mean square error (RMSE) and anomaly correlation coefficient (ACC) results comparing the KMD-based prediction to the climatological and persistence models are shown first, as those are standard reference models used for skill calculation. Results are then shown for the remaining two reference models to contrast the resulting skill values and their implications for the nature of the dominant system dynamics.17. It is seen in Fig. Figure Figure Figure Figures Turning now to the reference models based on the monthly means taken over the input period and on linear fits to the monthly values in the input period, Figs. The mean skill for the KMD forecast versus the mean input of the data model is seen to be slightly positive for most future months, though a wide spread is evident in the traces for individual four year input periods, and the skill for the linear fit model case is seen to be consistently and increasing positive, as expected for a model whose design will produce an increasing error over time. These results are seen in the RMSE values, where the KMD prediction has comparable error to the input period mean model and increasing outperforms the linear fit model over time. The ACC values have slightly positive mean values, but as for the 30-year KMD input period results shown above they generally fall below the 0.5–0.6 synoptically useful threshold range.28, including long term decreases in sea ice coverage near West Antarctica and in the Arctic marginal seas, but also that a long-term exponential decrease in sea ice concentration exists and that Koopman Mode Decomposition allows a geographic view of where changes occur on annual and multi-year timescales. Such nonlinear trends in dynamics commonly result from positive feedback mechanisms such as those suggested in sea-ice dynamics studies29. The existence of long-term exponentially decaying modes seems to be of potentially substantial physical significance and could warrant further measurement  and investigation.These results show not only the previously-known existence of long term variation in sea ice concentration30 suggests the circumpolar deep water (CDW) flow as a physical mechanism for the decline of land-based ice due to increased glacier flow, where this increased decline is largest in the regions listed above where the decrease in the mean mode and increase in the annual variation mode is most apparent. The undersea topography of these regions is most consistent with the upwelling of relatively warm water by a strengthening CDW, leading to increase melting of ice shelves and, we suggest, reduced sea ice formation.A limitation of the application of KMD is that, as a purely data-driven tool, it does not provide the physical insight into the underlying forcing or other drivers of a system’s dynamical behavior that numerical or theoretical models can provide. In this case, the geographic heterogeneity of the sea ice concentration behavior in Antarctica suggests a possible link with a proposed physical driver of the decrease in the Antarctic ice mass balance. Recent work31 are not predictable from purely data-driven examination of sea ice concentration.The accuracy of the predictions of future sea ice behavior by KMD reconstruction depends on the extent to which the sea ice concentration dynamics are governed by underlying nonlinear continuous processes, rather than stochastic or discontinuous drivers. That is, KMD-based prediction is expected to accurately predict variations in sea ice concentration due to the interactions of both long-term growth or decay and oscillatory behavior on fast or slow time-scales, however, changes due to “tipping points” such as the greater mixing between the Barents Sea and North Atlantic7, the success of climatological prediction in the southern hemisphere can be attributed to it behaving as a linear system with noise. The noise would in the Koopman framework be modeled by the continuous spectrum33 that we do not consider here.Taken in this light, the significantly different success of the KMD predictions for the Northern and Southern hemispheres suggest different sorts of underlying dynamics to the sea ice measurements. While the prediction results over multiple years for the Northern hemisphere do not display as high skill or ACC values as would be desired for future prediction purposes, the at least marginally positive values, particularly for the summer months, suggest that the observed recent sea ice dynamics are at least in large part a consequence of nonlinear long-term trends in the dynamical processes governing sea ice concentration behavior accessible to KMD when applied to the 30-year windows of data used for the KMD forecast and generally poorly represented by climatological models consisting of long-time averages of a non-stationary process. In contrast, the results for the Southern hemisphere indicate the dominance of oscillatory cyclo-stationary processes and thus are well represented, on average, by climatological models. Note that linear systems in the Koopman framework lead to a fixed set of frequencies that do not contain integer multiples . Since the annual phase averaged mode is equal to the Koopman mode at the annual frequencyThe objective of this study was to apply Koopman Mode Decomposition (KMD) and analysis techniques to existing satellite image data of sea ice concentration. Existing KMD algorithms were applied to the satellite images and the resulting Koopman modes and eigenvalues, defined in the following paragraph, showed the temporal and spatial details of the sea ice concentration  dynamics.n is the number of field observations, I the set of time snapshots and 3434. Note that the dependence on the initial state j  of months in the 1980’s, so we chose to interpolate over the missing months to allow use of all data back to 1979, giving 40 full years of data (1979 to 2018).13 and DMD type methods17. The results from the two categories of algorithms were found to be identical, which was taken to be a strong indication that the results are a good representation of the true Koopman eigenvalues and modes.The arrays for each month were then combined into a 2-D data matrix for performing KMD analysis run using KMD algorithms based on both ArnoldiAs described in the text, the calculated Koopman eigenvalues showed the time dependence (oscillatory and growth/decay) of the Koopman modes, which themselves showed the spatial structure of the time dependence of the input data.To capture relatively short time scale dynamics, the analysis was performed on windowed data sets. The windowing consisted of performing KMD on subsets of the sea ice concentration data covering time periods of 5 to 40 years .x and time index t) can be transformed to anomaly values x computed over the entire period of available concentration data  and i(t) is the calendar month at time t. Note that the KMD analysis described herein can be applied to either The sea ice concentration data values Five forecast models are used in this work: the KMD-based prediction and four reference models. Two of the reference models are the commonly used climatological and persistence models, whereas the other two models (the monthly mean over the input data and a linear fit to the monthly input data) were chosen to illustrate the influence of the relative cyclo-stationarity or nonlinear behavior of the system on the success of KMD-based prediction.k is performed using the Koopman eigenvalues N time steps :Reconstruction of the N Koopman eigenvalues and Koopman modes, where each Koopman eigenvalue is a single complex number and each Koopman mode has dimensions 1-by-Here, there are kth time step in some arbitrary input data kth time step, based on the system dynamics deduced from decomposition of earlier observations. For the specific case examined here where For N years of past data by applying KMD separately to the anomaly N years of past anomaly data and prediction of M years into the future, the 12N months of input data were separated into twelve sets, each set containing N months. KMD was applied once to each of the N data sets, producing twelve sets of N modes and N eigenvalues. A number M future years using the KMD reconstruction equation given above, for time index The reported KMD forecast results were produced using The KMD forecast skill results for the 30-year input data period shown above were calculated by sorting the eigenvalue/mode pairs by the value of their residual, and calculating a KMD-based prediction as described above using the No probability distribution is assumed in the KMD process so no statistical methods were applied. The deviation between the KMD reconstruction-based predictions of future sea ice concentrations and the actual values are due to two factors: the finite dimensionality of numerical realizations of KMD algorithms, which for relatively high-dimensional data as used in this study is not expected to be a major source of error, and the stochastic nature of the underlying climatological processes driving sea ice concentration dynamics, which will produce behavior not predictable in a purely dynamical model such as that produced by KMD reconstruction-based predictions.s months in the future (relative to a time t) will equal the average value for that month of the year in the entire period of available data:i(t) is the calendar month number  is the error of the forecast or reference relative to the true value at location x and time t. The error for the persistence forecast is s months in the future, while the error for the climatological forecast is The definition used for prediction skill of the forecast model relative to a reference model is:t indicates perfect forecast quality at that time, zero indicates no correlation between the forecast and the truth, and The (centered) anomaly correlation coefficient (ACC) is:Supplementary file1


As a widely expressed receptor in the mammalian brain, A1 receptors are implicated in the modulation of a variety of neuronal and synaptic activities. In this study, we investigated the role of A1 receptors in the regulation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in the adult rat brain in vivo.The adenosine A1 antagonist DPCPX, rats were sacrificed and several forebrain regions were collected for assessing changes in phosphorylation of AMPA receptors using Western blots.Adult male Wistar rats were used in this study. After a systemic injection of the A1 antagonist DPCPX induced an increase in phosphorylation of AMPA receptor GluA1 subunits at a PKA‐dependent site, serine 845 (S845), in the two subdivisions of the striatum, the caudate putamen, and nucleus accumbens. DPCPX also increased S845 phosphorylation in the medial prefrontal cortex (mPFC) and hippocampus. The DPCPX‐stimulated S845 phosphorylation was a transient and reversible event. Blockade of Gαs/olf‐coupled dopamine D1 receptors with a D1 antagonist SCH23390 abolished the responses of S845 phosphorylation to DPCPX in the striatum, mPFC, and hippocampus. DPCPX had no significant impact on phosphorylation of GluA1 at serine 831 and on expression of total GluA1 proteins in all forebrain regions surveyed.A systemic injection of the A1 receptors maintain an inhibitory tone on GluA1 S845 phosphorylation under normal conditions. Blocking this inhibitory tone leads to the upregulation of GluA1 S845 phosphorylation in the striatum, mPFC, and hippocampus via a D1‐dependent manner.These data demonstrate that adenosine A 1 receptors maintain an inhibitory tone on GluA1 S845 phosphorylation under normal conditions. Blocking this inhibitory tone leads to the upregulation of GluA1 S845 phosphorylation in the striatum, mPFC, and hippocampus via a D1‐dependent manner.Our data demonstrate that adenosine A Blocking dopamine D1 receptors abolished the responses of GluA1 phosphorylation to DPCPX. These data reveal an inhibitory linkage from adenosine A1 receptors to AMPA receptors. Malfunction of this linkage may be linked to various psychiatric and neurological disorders such as drug addiction and anhedonic depression.In this study, we found that an A12+/calmodulin‐dependent protein kinase II, one of four AMPA receptor subunits, GluA1  receptor is widely expressed in the mammalian brain and is actively involved in the regulation of a variety of cellular and synaptic activities  in the striatum, probably via a signaling mechanism involving the cAMP/PKA pathway  on GluA1 phosphorylation at S845 and S831 sites in different brain regions, including the caudate putamen (CPu), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and hippocampus. We then tested the effect of DPCPX on GluA1 phosphorylation in the presence of a dopamine D1 antagonist SCH23390.In this study, we attempted to evaluate the role of A22.1We used male rats in this study. Adult Wistar rats  were purchased from a vendor (Charles River) and were kept at 12‐hr light/12‐hr dark cycle and 23°C with water and food available ad libitum. At 5–6 days after habituation, animals were used for experiments. The protocol of animal use and care was approved by the Institutional Animal Care and Use Committee.2.2α = .05, β = .2; 80% power). There were no differences in sample size between the beginning and end of the experiments. We based the criteria for inclusion/exclusion on the health state of animals. The healthy animals that showed no sign of illness as evaluated by the body weight and visual observations were used in the analysis.All drugs were administered via intraperitoneal (i.p.) injections. We calculated doses of drugs as their salt form. All drugs were injected in a volume of 1 ml/kg. Age‐matched rats were injected with a vehicle solution and served as controls. A series of experiments were conducted with rats randomly divided into different groups using a computer‐generated randomization table . Size of sample was determined by the sample size calculation  were subjected to an i.p. injection of vehicle or DPCPX at either 0.25 or 2.5 mg/kg and were then sacrificed 20 min after DPCPX injection. Changes in GluA1 phosphorylation and expression in four different brain regions, including the CPu, NAc, mPFC, and hippocampus, were subsequently assayed using Western blots. Doses of DPCPX (0.25 and 2.5 mg/kg) were selected because an i.p. injection of DPCPX at 3 mg/kg blocked the anxiolytic‐like response to the A1 selective agonist and positive allosteric modulator  were used at each time point. Finally, the effect of DPCPX was tested in the presence of a dopamine D1 receptor antagonist in four groups of rats (n = 4 per group). The D1 antagonist SCH23390  was administered 10 min prior to DPCPX . Rats were sacrificed 15–20 min after DPCPX injection for immunoblot analysis of changes in GluA1 phosphorylation in different brain regions.We first investigated the effect of DPCPX, an A2.32EDTA, 1 mM EGTA, 1% NP‐40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β‐glycerophosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin , the dissected brain tissue was homogenized. Homogenates were centrifuged at 800 g (10 min). Protein concentration of the supernatant was determined. Samples were then used for Western blot which was performed as described previously  and R(+)‐SCH23390 hydrochloride (Sigma‐Aldrich). We dissolved DPCPX in dimethyl sulfoxide (DMSO) and 0.1 M sodium hydroxide. Stock solutions of DPCPX were diluted in saline. Final concentrations of DMSO and sodium hydroxide were 15% (v/v) and 8% (v/v), respectively  with a multiple comparison post hoc test was conducted to analyze data. The statistical significance was determined by a p value < .05.Data were statistically analyzed following tests for the normality of data. They are expressed as means ± 33.11 receptors on GluA1 phosphorylation, we subjected rats to a single i.p. injection of the A1 antagonist DPCPX. We sacrificed rats 20 min after DPCPX injection to assay changes in GluA1 phosphorylation at S845 and S831 in different brain regions using Western blots. In the CPu, DPCPX at a lower dose (0.25 mg/kg) had no significant effect on GluA1 phosphorylation at S845 and S831 .To determine the impact of blockade of AWe next examined the effect of DPCPX on GluA1 phosphorylation in the mPFC and hippocampus. DPCPX at 2.5 but not at 0.25 mg/kg (20 min prior to tissue collection) elevated the pS845 level in the mPFC  to rats. We then sacrificed rats at different time points  after drug injection and analyzed changes in S845 and S831 phosphorylation in different brain regions. Like the positive effect of DPCPX observed at 20 min from the above studies, DPCPX at 1 hr elevated pS845 levels in the CPu  10 min prior to an i.p. injection of DPCPX at an effective dose of 2.5 mg/kg. Rats were sacrificed 15–20 min after DPCPX administration for immunoblot analysis of changes in GluA1 phosphorylation and expression. In the striatum, SCH23390 alone reduced the basal level of pS845 signals, while DPCPX alone elevated it as expected  32 kDa at T34 via postsynaptic A1 receptors in striatonigral neurons (Yabuuchi et al., 1/A1‐regulated S845 phosphorylation occurs specifically in the phenotype of striatonigral output neurons. In addition to the postsynaptic D1/A1 interaction, blocking presynaptic A1 receptors by DPCPX may facilitate local dopamine release (Borycz et al., 1 receptors in striatonigral neurons to upregulate GluA1 S845 phosphorylation in these neurons.Dopamine D1 receptors are expressed in the mPFC with a high level in pyramidal spiny output neurons (Rivkees, Price, & Zhou, 1 receptors in both the presynaptic terminals and the postsynaptic structures (Ochiishi, Chen, et al., 1 receptors with DPCPX elevated GluA1 S845 phosphorylation in the mPFC. Thus, A1 receptors in mPFC neurons seem to maintain an inhibitory tone on GluA1 S845 phosphorylation. Additionally, the dopamine D1 receptor is one of principal dopamine receptor subtypes expressed in pyramidal neurons throughout mPFC layers (de Almeida, Palacios, & Mengod, 1 and D1 receptors in mPFC neurons like in striatal neurons may converge on the cAMP/PKA pathway to control activity of PKA substrates such as GluA1. Indeed, D1 receptor activity is required for the A1 antagonist‐induced S845 phosphorylation since the D1 antagonist SCH23390 abolished the response of S845 phosphorylation to DPCPX in the mPFC.A1 receptors. In this structure, strong expression of A1 receptors was present on cell bodies and dendrites of pyramidal and granule neurons and mossy fibers, but not on glial cells (Ochiishi, Chen, et al., 1 receptors are distributed pre‐ and postsynaptically (Ochiishi, Chen, et al., 1 receptors in modulating both presynaptic neurotransmitter release and postsynaptic physiology. To determine the role of A1 receptors in regulating GluA1 phosphorylation in hippocampal neurons, we found that the A1 antagonist upregulated S845 phosphorylation in the adult rat hippocampus in vivo. Early studies found that A1 receptors physically interacted with GluA1 subunits in hippocampal neurons (Chen et al., 1 receptor activation with CPA reduced GluA1 phosphorylation at S845 and S831 and induced GluA1 endocytosis and synaptic depression (Chen et al., 1 receptors on S845 phosphorylation exists. Moreover, dopamine D1 receptors are critical for the A1‐mediated inhibition of S845 phosphorylation in this area. Pretreatment with the D1 antagonist blocked the A1 antagonist‐induced S845 phosphorylation in the hippocampus.The hippocampus is among brain regions expressing a high level of A1 receptor‐regulated S845 phosphorylation is believed to be a biochemical step critical for carrying out normal functions of these brain structures, a topic to be investigated in‐depth in the future.Changes in S845 phosphorylation are thought to have a significant impact on the subcellular expression pattern and function of GluA1‐containing AMPA receptors. Previous studies found that S845 phosphorylation enhanced the surface trafficking of GluA1 (Man, Sekine‐Aizawa, & Huganir, 1‐regulated AMPA receptor phosphorylation have not been reported in striatal neurons to our knowledge, it is important to assess the response of AMPA receptors to adenosine receptor agents in female animals in future studies.Our studies on AMPA receptor phosphorylation were performed in male rats. While sex differences in the AThe authors declare that they have no conflict of interest.


The term vascular anomalies include various vascular tumors and vascular malformations, among them infantile hemangiomas and capillary malformations are the most well-known associated diseases in early ages. Multiple drugs have been introduced for intervention, but susceptibility test in vitro were scarcely reported.To evaluate the inhibition effect of different drugs by adenosine triphosphate sensitivity assay in vitro before the treatment of infantile hemangiomas and capillary malformations.Specimens were selected from 5 cases of infantile hemangiomas and 11 cases of capillary malformations. Propranolol, rapamycin, sildenafil and itraconazole were tested for their growth inhibition effect by using the adenosine triphosphate sensitivity assay.Propranolol demonstrated inhibitory effects on infantile hemangiomas cells. Rapamycin and itraconazole both showed inhibitory effects on infantile hemangiomas cells and capillary malformations cells. Sildenafil has no growth inhibitory effect on infantile hemangiomas cells or capillary malformations cells.Adenosine triphosphate sensitivity assay is a sensitive and useful testing method before the management of vascular anomalies, and individualized medication suggestions for the choice of therapeutic drugs were offered based on the testing result and together with a comprehensive evaluation of each infant. Vascular anomalous entities used to be called in an ambiguous term as “hemangioma” to designate numerous quite different disorders especially in infancy . HoweverInfantile hemangiomas are the most common vascular anomalies in infants , and it There are multiple available treatment prescriptions or approaches for vascular anomalies, including drug therapy , physical therapy . All cases were diagnosed by clinical and color Doppler ultrasonography. Finally, there are 5 cases of severe infantile hemangiomas, 2 males and 3 females, with an average age of 1.5 months, and 11 cases of severe capillary malformations, 5 males and 6 females, with an average age of 2 months, were included. Written informed consent on participation in drug sensitivity assay in vitro was obtained from both parents in all cases.The typical vascular anomalies lesions were selected as sampling sites, and lesions with eczema, scale, erosion, ulcer, crust, necrosis or liquefaction occurred or around were excluded. After a minor excision surgery by trephine, the tissues specimens were immersed in a particular sterile vial containing culture liquid and sent to laboratory within 1 h.Four drugs that most frequently used in treating infantile vascular anomalies namely propranolol, rapamycin (sirolimus), sildenafil and itraconazole were tested for their sensitivity. The corresponding peak plasma concentration  is calculated and determined based on the pharmacokinetic index, drug instructions or manuals and available literature according to their dosage in pediatrics , 19. As This study is only an in vitro laboratory research currently, but according to the previous experimental design, it is planned to be widely used in practice in the future for clinical medication instruction. Therefore, parents are fully encouraged to participated and involved in the formulation of treatment plans and select experimental drugs. The currently available pharmacological mechanism and the administrations dose and pattern of each drug are fully communicated with parents. As fear of or concerned about the adverse reactions of itraconazole, in this study, among 5 cases of infantile hemangioma, three parents chose propranolol, rapamycin, sildenafil and itraconazole as testing drug, but the other two parents only chose propranolol, rapamycin and sildenafil. Among 11 cases of capillary malformations, seven parents chose propranolol, rapamycin, sildenafil and itraconazole and the other four parents only chose propranolol, rapamycin and sildenafil. We fully respect parents’ choices and inclination.This study was carried out by using the adenosine triphosphate-tumor chemo-sensitivity assay to assess the sensitivity of vascular anomalies cells to a range of therapeutic agents. A self-made kit, containing sterile compound digestive enzymes, erythrocyte scavenger, serum-free culture medium, adenosine triphosphate inhibitor reagent, adenosine triphosphate extract reagent, luciferase-fluorescein reagent, etc. is needed during related subsequent laboratory operation.The vascular anomalies tissues specimens were dissected and dissociated adequately, and removal of connective tissue, fibers, fat and red blood cells on the surface of specimens. After soaking, digestion and incubation, the suspension of tissues and cells were prepared like a light and fluffy paste or mush. After equivalent culture medium and different concentration of the four testing drugs were adding to the culture microplate, the suspension of vascular anomalies tissues and cells were cultured into the medium.The suspensions were incubated at 37 °C for 3 days. After that, adenosine triphosphate extract reagents were used to extract adenosine triphosphate from the suspensions, and then, the luciferase-fluorescein reagents were added for fluorescence assay.The fluorescence intensity of these specimens at 560 nm wavelength was measured by fluorescence scanner and illuminometer, which represented and symbolized the levels of adenosine triphosphate and the numbers of viable cell remaining at present. Data were recorded and assessed, and the mean values of three parallel holes were calculated. Combined with MI and M0 and conducted a comprehensive analysis , the actual inhibition rate of the six concentrations of each drug in every specimens was determined and identified. Then, the average inhibition rates of each drug in two groups, infantile hemangiomas group and capillary malformations group, were calculated respectively. Intuitively, inhibition curves could plot and draw when necessary.In traditional adenosine triphosphate-tumor chemo-sensitivity assay, researchers isolate cancer cells from patient neoplasm, culture them and expose them to an array of drugs to assess response. By reference to the M0, these assays typically assess response using cell survival/death rate and a series of related parameters: including IC50, IC90, TGI, SI, AUC, etc. . HoweverDuring the incubated days, the growths of vascular anomalies specimens’ cells were observed daily, to make sure that cells populations were inoculated distributed evenly without aggregation and no bacteria, fungi or mycoplasma contamination was found. Three days later, adenosine triphosphates were extracted from the suspensions and the fluorescence intensity of these specimens at 560 nm wavelength was measured. Then, the actual inhibition rates were calculated and determined Table , Table 2n = 3), the data of itraconazole fluctuated greatly. Except for some isolated data of patient No. 4, the testing result of sildenafil is mostly negative numbers. Just be explained from the experimental results, far from inhibiting the growth of infantile hemangiomas cells, sildenafil even promotes the growth of them on the contrary. Therefore, it can be considered that propranolol, rapamycin and itraconazole can inhibit the growth of infantile hemangiomas cells with different rates, and among them, rapamycin is the most effective. Sildenafil has no growth inhibitory effect on infantile hemangiomas cells.In infantile hemangiomas group Table , no mattIn capillary malformations group Table , the tesThe idea of introducing and carrying out personalized drug sensitivity test in vitro is not new, and researchers have been attempting to develop much higher precision in vitro methods to predict therapeutic response for various diseases such as tumors  or infecAccording to the testing result, in infantile hemangiomas group, though rapamycin or itraconazole appeared to be more effective in inhibiting the growth of hemangiomas cells than propranolol, it is not recommended to prescribe rapamycin or itraconazole in clinical for the consideration of their widely adverse reactions and the anxiety from parents. Infantile hemangiomas are a special type of benign tumour with a distinctive characteristic that can regress or subside spontaneously during their natural history. Therefore, the choices of therapy approaches are required to find a balance between the effect of antitumor and the efforts to reduce side or adverse reactions. Those over-zealous attempts that to perform aggressively treatments only to pursue a high inhibition rate partially should be avoided . On the In capillary malformations group, rapamycin showed a relative smoother inhibition curve, while itraconazole showed a dose-dependent inhibition curve to some extent. Itraconazole is known as a highly efficacious broad spectrum antifungal agent for many years, however, a series of research recently have found that it also has some unknown effects on antineoplastic and immunoregulation by some mechanism such as inhibiting endothelial cells and angiogenesis . This suRapamycin has exhibited similar inhibition effect both in infantile hemangiomas group and in capillary malformations group, while propranolol shows very different manifestations. Propranolol is ineffective for capillary malformations cells, and at present there is no news or report available about the success of using propranolol in treating any vascular malformation . This inIt is worth noting that sildenafil did not show any inhibitory effect in either infantile hemangiomas group or capillary malformations group at least in this study. Although sildenafil has been reported to be effective in multiple vascular anomalies, diverse opinions persisted all through. Sildenafil maybe caused a significant amelioration in the responsiveness or endothelial function of various vascular, but caution is recommended prior to use in clinical .Through ATP-TCA, which drug has a higher inhibition rate on infant subject’s anomalies tissues is much more clear. However, especially in the pediatrics clinical, therapeutic approaches or prescription drugs cannot be selected doctrinaire and avoiding making choices directly completely according to the experimental results without any analysis. It is necessary to evaluate the specific conditions of each infant, combined with the adverse reactions and metabolism pattern of each drug, and then, to give individualized medication suggestions to meet the particular individual needs.Furthermore, more high-quality trials with a large sample and longer follow-up are required, and continuation in-depth study will be carried out both in laboratory and clinical practice: to grope the proper and more suitable conditions for the culture of vascular anomalies cells; to improve, reform and optimize the experimental procedures; to compare the inhibition effect of more drugs such as atenolol, timolol etc., and combine with clinical efficacy and follow-up results; to carry out multi-factor analysis; and so on. After summarizing and concluding these data, to find some general regularity and to provide a basis for the standardized treatment of vascular anomalies in the future.Adenosine triphosphate sensitivity assay is a sensitive assay and suitable for use in clinical settings. Through this experiment, it is clear which drug has a strong inhibition rate on infant subject’s anomalies tissues, thus individualized medication suggestions were offered together with a comprehensive evaluation of each infant before the management of vascular anomalies. Moreover, this assay also has notable advantages for guiding the design of therapy protocols and assessing novel therapeutic drugs.