Hedgehog (HH) signaling is an evolutionarily conserved pathway that is crucial for growth and tissue patterning during embryonic development. It is mostly quiescent in the adult, where it regulates tissue homeostasis and stem cell behavior. Aberrant reactivation of HH signaling has been associated to several types of cancer, including those in the skin, brain, prostate, breast and hematological malignancies. Activation of the canonical HH signaling is triggered by binding of HH ligand to the twelve-transmembrane protein PATCHED. The binding releases the inhibition of the seven-transmembrane protein SMOOTHENED (SMO), leading to its phosphorylation and activation. Hence, SMO activates the transcriptional effectors of the HH signaling, that belong to the GLI family of transcription factors, acting through a not completely elucidated intracellular signaling cascade. Work from the last few years has shown that protein kinases phosphorylate several core components of the HH signaling, including SMO and the three GLI proteins, acting as powerful regulatory mechanisms to fine tune HH signaling activities. In this review, we will focus on the mechanistic influence of protein kinases on HH signaling transduction. We will also discuss the functional consequences of this regulation and the possible implications for cancer therapy. Hedgehog (HH) signaling is a highly conserved pathway playing an essential role in embryonic development, tissue homeostasis and stem cell maintenance. Its misregulation leads to a number of human disorders, including cancer . TherefoDrosophila melanogaster, where it is required for proper embryonic patterning and development [Drosophila has only one Hh and one Gli protein, Cubitus interruptus (Ci), whereas in mammals there are three HH family members, Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog, and three GLI proteins  [Drosophila membrane-mediated Hh pathway, mammalian HH signaling is mainly transduced in the primary cilium (PC) [The Hedgehog pathway has been first identified in elopment . Some ofium (PC) , a solitR and GLI3R) by recruiting the β-transducin repeat containing protein (β-TRCP) E3 ubiquitin ligase [A and GLI3A), which translocate into the nucleus where they begin transcription of HH pathway target genes, including GLI1 (PATCHED (PTCH) is a twelve transmembrane protein that functions as the HH receptor and acts as negative regulator of the HH pathway by inhibiting the GPCR seven-transmembrane protein SMO, the signal transducer. In absence of HH ligand, PTCH1 localizes in the primary cilium, where it inhibits accumulation of SMO . Thus, Gn ligase ,9,10. Biing GLI1 .The GLI proteins are members of the Kruppel-like family of transcription factors (TF). All three GLI TF contain five conserved C2H2 zinc-finger DNA binding domains and a histidine/cysteine linker sequence between the zinc fingers. The GLI TF bind the promoter of target genes at the consensus sequence 5′-GACCACCCA-3′ , with cyPTCH1 gene. Ligand-dependent mechanism is characterized by the presence of HH ligands that activate the pathway and it can be autocrine, paracrine and reverse-paracrine. The autocrine pattern, in which tumor cells secrete and respond to HH ligands, has been reported in several cancer types, including lung, pancreas, gastrointestinal tract, prostate and colon cancers, glioma and melanoma [Several mechanisms of aberrant activation of HH pathway have been described in cancer. Ligand-independent activation occurs mainly in basal cell carcinoma (BCC) and in medulloblastoma. It is due to mutations or amplifications of key components of the HH pathway, which induce its constitutive activation, such as loss-of-function mutations in PTCH1  or SUFU melanoma ,25,26,27melanoma . In the melanoma  and hemamelanoma ,31. Anotmelanoma ,33.Protein kinase A (PKA), glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1) are serine/threonine protein kinases, which work in conjunction in the regulation of HH signaling. PKA is a tetrameric complex whose activity depends on cellular levels of cyclic AMP (cAMP) . The CK1Drosophila, PKA can promote Hh signaling by phosphorylating the C-terminus of Smo at three sites, priming Smo for phosphorylation by CK1. Phosphorylation of Smo promotes its cell surface accumulation and active conformation [Drosophila showed that in presence of Hh, a catalytic subunit of PKA forms a complex with Smo to phosphorylate its C-tail, inducing its conformational change and oligomerization, leading to Hh pathway activation [In ormation . In linetivation . A recentivation .Slimb ubiquitin ligase, leading to Ci degradation [PKA, GSK3β and CK1 have been shown to exert an inhibitory effect on Ci/GLI. Indeed, in absence of Hh, these three kinases phosphorylate Ci sequentially at three clusters of sites, where PKA functions as the priming kinase for subsequently phosphorylation by CK1 and GSK3β ,41. Thesradation ,43. Simiradation ,9,44,45.R formation and blocks GLIA formation, whereas full dephosphorylation of GLI at these clusters produces strong transcriptional activators [PKA is also able to regulate GLI1 protein localization and activity through direct phosphorylation of GLI1 primarily at residue Thr374, which resides adjacent to the nuclear localization signal (NLS) . A recentivators . Moreovetivators . In linetivators .A from degradation by the Cullin 3-based E3 ubiquitin ligase containing the BTB family protein HIB, allowing CiA to accumulate and to induce the expression of Hh target genes. Similarly, CK1 plays a conserved role in the regulation of vertebrate GLIA activity by attenuating its degradation by SPOP, the vertebrate homolog of HIB [CK1 can act as a positive regulator of Ci in presence of Hh. Indeed, CK1-mediated phosphorylation of Ci at multiple Ser/Thr-rich degrons protects Cig of HIB . Howeverg of HIB . It is uGSK3β plays an inhibitory role on the GLI. GSK3β, GLI3 and SUFU can form a trimolecular complex, that stimulates GLI3 phosphorylation by GSK3β and, hence, GLI3 processing. Stimulation of C3H10T1/2 cells with Shh promotes the dissociation of the SUFU/GSK3β complex from GLI3, resulting in the blockade of GLI3 processing . ConsistDrosophila and Zebrafish and very little is known about the role of GRK2 in mammalian. The Drosophila Gprk2 plays a role in the modulation of Hh signaling [The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that belongs to the GRK family. Most of the studies on the regulation of the HH pathway by GRK2 are in ignaling ,57,58, bignaling . Recent ignaling .Drosophila [s, likely at the level of Gαs-coupled GPCRs, such as GPR161, an attenuator of HH pathway [The mechanism of SMO regulation and activation in vertebrates seems to be similar to that described in osophila ,62. In mosophila ,64,65. Uosophila ,67. A re pathway .Drosophila, CK2 has been shown to regulate both Smo and Ci, thus promoting Hh signaling. Indeed, CK2 phosphorylates Smo at multiple sites in its C-terminal cytoplasmic tail, inducing Smo activity. Nevertheless, the effect of CK2 on Smo are not as potent as those of PKA and CK1. CK2 acts also at the level of Ci, by preventing its ubiquitination and thus attenuating its proteosomal-dependent degradation [Casein kinase 2 (CK2) is a serine/threonine kinase that consists of subunits α and β. In radation .The expression level of CK2α is tightly regulated in normal cells, whereas it is upregulated in a number of human cancers . CK2 hasCK2 was recently identified as the main driver of phosphorylation events during proliferation of cerebellar granule cell precursors (GCP), the cells of origin of medulloblastoma, in a phosphoproteome screening performed to discover new candidate drug targets in medulloblastoma. In the same manuscript authors showed that CK2 is required for HH signaling transduction and is critical for the stabilization and activity of GLI2 in medulloblastoma cells .Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are serine, threonine and tyrosine kinases containing a motif called DYRK-homology box. There are five members of mammalian DYRK, subdivided in two classes; DYRK1A and DYRK1B belong to class I, while class II consists of DYRK2, DYRK3 and DYRK4 .Among the five DYRK members, DYRK1A, DYRK1B and DYRK2 are involved in the regulation of HH signaling. DYRK1A exerts an activating function on GLI1, promoting GLI1 nuclear translocation ,77 throuR formation by an unknown mechanism [Other reports suggest that also DYRK1B can play complex and dual roles in the modulation of HH pathway. DYRK1B can inhibit HH signaling, likely blocking GLI2 function and promoting GLI3echanism . On the echanism . Anotherechanism . More reechanism .Finally, a kinome-wide screening identified DYRK2 among 480 kinases as a negative regulator of the HH pathway. The study showed that DYRK2 directly phosphorylates GLI2 at two conserved Serine residues (Ser385 and Ser1011) inducing its proteasome-dependent degradation . In concThe Mitogen-Activated Protein Kinase Extracellular signal-Regulated Kinase 1 and 2 (MAPK-ERK1/2) play pivotal role in controlling several cellular functions, including proliferation and cell cycle. ERK1 and ERK2 are highly similar serine/threonine kinases, which are activated by the upstream MEK1 and MEK2 in the RAS-RAF-MEK1/2-ERK1/2 signaling pathway . SeveralQ61K) and HRAS (HRASV12G) have been shown to activate GLI1 function, enhancing its transcriptional activity and nuclear localization. Mechanistically, both oncogenes counteract GLI1 cytoplasmic retention by SUFU. MEK1/2-ERK1/2 are the possible main effectors of RAS, because MEK1/2 inhibition reverses the effect of oncogenic RAS on GLI1 [A number of reports have shown that the MAPK-ERK1/2 cascade can regulate HH signaling in several types of cancer. For instance, in melanoma cells oncogenic NRAS  is a serine/threonine kinase whose activation is regulated by the level of phosphatidylinositol-3-kinase (PI3K). The PI3K-AKT pathway plays a crucial role in many cellular processes, including cell cycle and apoptosis . Severalis model . Another of GLI1 , suggestp70 ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine kinase that regulates many aspects of cellular biology, by controlling mRNA translation, ribosome biogenesis, cell growth and survival, authophagy, immune suppression and metabolism . The mamThe protein kinase C (PKC) proteins are widely expressed serine/threonine kinases. They consist of three families: calcium-dependent conventional PKC ; calcium-independent novel PKC ; and calcium-independent atypical PKC . All PKC have been shown to modulate HH signaling with different effects.PTCH1 mRNA level [The role of both PKCα and PKCδ in the modulation of the HH pathway is controversial. Regarding PKCα, a report suggests that it negatively regulates GLI1, reducing its transcriptional activity without affecting its nuclear translocation . HoweverNA level . HoweverNA level . The mecPRKCI, the gene encoding for aPKCι, forming a positive GLI-aPKCι feedback loop in BCCs. Direct involvement of aPKC in regulation of HH signaling has been demonstrated also in Drosophila, where aPKC promotes phosphorylation and activation of Smo and Ci [aPKCι/λ has been identified as a GLI regulator in mouse BCC cells. aPKCι/λ directly phosphorylates GLI1 (likely at residues Ser243 and Thr304), resulting in enhanced DNA binding and transcriptional activation . Furthero and Ci .5′-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a serine/threonine kinase consisting of three subunits: α, β and γ. The AMPK activity is fine regulated by the AMP/ATP ratio and by other kinases. It is involved in mitochondrial activity and biogenesis, autophagy and cell proliferation, acting in a context-dependent manner . Xu and AMPK can play also a positive role in HH pathway. AMPK can act downstream of SMO to stimulate metabolic reprogramming towards glycolysis in adipocytes and increase glucose uptake . MoreoveR). On the other hand, in response to SHH, SUFU-ULK3 complex dissociates and ULK3 can act as positive regulator of GLI2 [Unc-51 like kinase 3 (ULK3) is a serine/threonine protein kinase widely expressed, which takes part in several cellular processes, such as autophagy and cell division. ULK3 positively regulates HH signaling by direct phosphorylation of the GLI, enhancing their transcriptional activity and nuclear localization of GLI1 . Interes of GLI2 . Recentl of GLI2 . ULK3 is of GLI2 .GLI1 and GLI2 mRNA levels induced by SHH [The HH signaling has been shown to be regulated also by other PKs. For instance, the serine/threonine kinase PFTK1 (or CDK14), a member of the CDC2-related protein kinase family, positively modulates the protein levels of SMO, PTCH1 and GLI1, thus controlling cell proliferation, invasion and EMT in colon cancer cells . Anotherd by SHH .−/− MEF cells. However, it is still unknown whether the regulation of SUFU by RIOK3 is direct or indirect [The right open reading frame kinase 3 (RIOK3) is a serine/threonine kinase involved in cell proliferation, migration and invasion in various cancers ,120. It indirect . The serindirect ,123. MorThe liver kinase B1  is a serine/threonine kinase that has been found mutated and deregulated in several types of cancers, where it acts as a tumor suppressor ,125. LKBProtein Kinase G (PKG) is a serine/threonine-specific protein kinase that is activated by cyclic guanosine monophosphate (cGMP). In mammalian two PKG genes have been identified, encoding for PGK-I and PGK-II. Upon cGMP stimulation PKG-I has been shown to mediate the response to HH signaling in neural plate cells and in embryoid bodies ,132. TheRecent evidence has shown an involvement of the mitogen-activated protein kinase kinase kinase (MAP3Ks) MEKK1, MEKK2 and MEKK3 in HH regulation. MEKK1 directly binds to and phosphorylates GLI1 on multiple residues at its C-terminal, leading to the inhibition of its transcriptional activity and enhancing its association with the 14-3-3 proteins. This association might explain the inhibitory effects on HH pathway by MEKK1 . MEKK2 aIn the last decade efforts to inhibit the HH pathway have been focused mainly on the development of SMO inhibitors. Several of these SMO antagonists, including vismodegib (GDC-0449), sonidegib (LDE-225), BMS-833923, saridegib (IPI-926), glasdegib (PF-04449913), taladegib (LY2940680), itraconazole and posaconazole, have shown efficacy in reducing growth of mouse xenografts and have been extensively investigated in a number of clinical trials in advanced cancers ,139,140.In this review we have described the multifaceted roles that phosphorylation plays in HH pathway, focusing on cancer. Since the majority of PKs that phosphorylate and regulate components of the HH pathway can be modulated by antagonists/agonists, it is clear that their targeting may have important implications for anti-cancer therapy. Here, we will discuss examples of preclinical and clinical studies focusing on inhibition/activation of the above described HH-related PKs, alone or in combination with HH antagonists , Table 2GLI1 mRNA level in an inducible SMO-mutant BCC mouse model, providing evidence that forskolin inhibits growth of BCCs resistant to SMO inhibitors [Given the role of PKA in promoting Ci/GLI processing, compounds that act by targeting the HH signaling through activation of PKA are currently under clinical investigation. For instance, imiquimod, a synthetic nucleoside analogue of the imidazoquinoline family, was approved for topical treatment of small superficial BCCs. Imiquimod represses HH signaling by negatively modulating GLI activity in BCC and medulloblastoma cells. Mechanistically, imiquimod acts downstream of SMO enhancing PKA activity with consequent GLI2/3 phosphorylation and cleavage into GLI2/3 repressor forms . Consisthibitors .+/−-derived medulloblastoma allografts and to reduce the expression of HH target genes [The anti-pinworm compound pyrvinium is an allosteric activator of CK1α. It acts by enhancing the degradation of the GLI in a CK1α-dependent manner. Pyrvinium is very potent and blocks HH signaling in the nanomolar concentration range, functioning also on vismodegib-resistant SMO and in settings of downstream pathway activation resulting from SUFU depletion. In vivo pyrvinium has been shown to attenuate growth of a Ptchet genes .aPKCι/λ is another potential therapeutic target, that was originally reported as an activator of GLI1 in BCC. Interestingly from a therapeutic point of view, vismodegib-resistant BCCs show high level of aPKCι/λ, and pharmacological inhibition of aPKC with PSI is able to suppress HH signaling and growth of resistant BCC cell lines . A recenA recent phosphoproteomic analysis identified the CK2 as a kinase critical for stabilization and activity of GLI2 and a promising therapeutic target in medulloblastoma. Pharmacological inhibition of CK2 with specific inhibitors decreases proliferation of primary SHH-type MB patient cells in vitro and inhibits growth of murine medulloblastoma that are resistant to SMO inhibitors. In particular, one of these CK2 antagonists (CX-4945) is able to inhibit both wild-type and mutant CK2, suggesting that this drug might prevent or delay acquired resistance . This woS6K1 is another potential therapeutic target that directly phosphorylates GLI1 and promotes its oncogenic activity, suppressing the inhibitory effect of SUFU on GLI1. The report showed that EAC tissues express high levels of S6K1 and inhibition of its upstream activator mTOR with everolimus (RAD-001) enhances the effect of vismodegib in reducing EAC cell line proliferation and mouse xenograft growth . These rGene expression arrays of sonidegib-resistant mouse medulloblastoma have shown upregulation of PI3K, the upstream activator of S6K1. Combination therapy with sonidegib and the PI3K inhibitor NVP-BKM120 markedly delayed tumor resistance in allografted mouse . In glioAnother study reported a novel small molecule DYRK inhibitor able to impair SMO-dependent and SMO-independent oncogenic GLI activity. Authors showed that genetic and pharmacological inhibition of DYRK1B reduces GLI1 expression in a number of cancer types, including human brain and pancreatic cancers, and mouse BCC cells. Interestingly, DYRK1B targeting represses GLI1 expression in both SMO-inhibitor sensitive and resistant cells .Numerous kinases and phosphorylation events that regulate the HH signaling have been described in the last decade. Biochemical and functional analyses of kinases, such as PKA, CK1 and GSK3β, have provided important insights into HH signal transduction mechanisms, and begun to address how SMO and the GLI are regulated in physiological conditions. Despite these achievements, insights into the phosphorylation of several kinases, such as ERK1/2, AKT, PKCα and PKCδ, are still lacking. In addition, the full spectrum of phosphorylation of GLI1 and GLI2, the final effectors of the HH pathway, has only begun to be explored. Although PKA-mediated phosphorylation of the GLI has a predominant inhibitory effect on HH signaling, it is clear that the GLI undergo other important phosphorylation events that modulate their activities. For instance, S6K1 and aPKCι/λ directly phosphorylate and activate GLI1, respectively, in esophageal adenocarcinoma and BCC cells. It will be interesting to determine whether these phosphorylation events play a role in other types of cancer.Given the dual role played by several kinases, such as PKA and CK1α, in the regulation of Smo/SMO and Ci/GLI, it is critical to understand how phosphorylation by these kinases is regulated. In addition, more work is needed to provide a clear picture of the exact role of these kinases in regulating the HH pathway in cancer, especially in those with non-canonical HH pathway activation. This aspect is particularly important in view of future therapeutic approaches targeting these kinases. Thus far, preclinical studies suggest that the most promising therapeutic targets may be S6K1, aPKC, CK2, PI3K and AKT. Targeting these kinases in combination with SMO or GLI antagonists may improve response rates and reduce resistance mechanisms, although more functional and preclinical studies are needed to define these relationships.Finally, several kinases involved in phosphorylation events that modulate HH pathway have been identified, whereas most of the corresponding phosphatase are still unknown. Indeed, very few phosphatases have been described to regulate SMO, the GLI proteins or other HH pathway components; nevertheless, the molecular basis of their functions and, in particular, the specific residues are largely unknown. Therefore, in the near future efforts need to be directed towards the identification of phosphatases that play a role in the regulation of HH pathway.


Mathematical modelling along with velocity and depth estimates of the presumed methane hydrates coincided with values observed from echograms. Modelling results also showed that a methane hydrate fragment with initial radius of 2.5 cm or greater could reach the surface of Lake Baikal given summer water column temperature conditions. Results further show that while methane bubbles released from the deep sedimentary reservoir would dissolve in the Lake Baikal water column, transport in hydrate form is not only viable but may represent a previously overlooked source of surface methane with subsequent emissions to the atmosphere. Methane hydrates captured within the ice cover may also cause the formation of unique ice structures and morphologies observed around Lake Baikal. Sampling of these ice structures detected methane content that exceeded concentrations measured in surrounding ice and from the atmosphere demonstrating a link with the methane transport processes described here.This paper provides a novel report of methane hydrates rising from bottom sediments to the surface of Lake Baikal, validated by photo and video records. The ascent of hydrates in the water column was confirmed by hydroacoustic data showing rising objects with velocities significantly exceeding the typical speeds (18–25 cm s Inland freshwater bodies make significant contributions to the atmospheric methane budget2 . Because lakes are major contributors to the freshwater carbon cycle, their methane production and release dynamics require more precise understanding and quantification3. A common assumption regarding methane in lakes is that it is produced primarily by anoxic sediments and/or in the deep-water column5. Some of this methane can reach the lake surface due to convection during lake turnover, but water column oxidation significantly reduces the dissolved methane concentration such that only a small fraction reaches the atmosphere7. Alternatively, methane bubbles originating from the sediment may ascend to reach the atmosphere, but this transport mechanism depends on the initial size of the bubble and its release depth8. Several recent studies have also suggested that methane production and transport in lakes can be highly variable and may include significant methane production within the oxygen-rich upper water column9. These results demonstrate that methane release from well-oxygenated lakes may not always be negligible10.Methane production in inland freshwater bodies is an intensive area of ongoing research due to the greenhouse potential of methane11. The possible presence of methane hydrates in bottom sediments of Lake Baikal was initially postulated from results collected by a joint expedition of the Limnological Institute and VNIIGAZ (Moscow) in 197812. Seismic profiles13 further indicated the presence of methane hydrates but unambiguous evidence was not obtained until drilling in deep water areas of Lake Baikal in 199611. In recent decades, several expeditions have studied methane hydrates in near-bottom sediments15 as well as gas seeps in both shallow and deeper areas of the lake18.As the world’s deepest lake, Lake Baikal is the only lake known to host solid phase methane (methane hydrates). Formation of methane hydrates is facilitated by high pressure and low temperatures in abyssal areas22. Recently, unique ring structures observed within Lake Baikal ice have been interpreted as resulting from methane hydrate release23. Earthquakes have been shown to influence the formation and decomposition of methane hydrates in near-bottom sediments of Lake Baikal25. The question of whether sediment-formed methane hydrates can reach the lake surface once they break away however remains unresolved. Brewer et al.26 performed experiments on the controlled transit of methane hydrates through the marine water column (Pacific Ocean) using a remotely operated underwater vehicle (ROV). This research showed that hydrate fragments of 8–10 cm diameter rising from 800 m depth can reach the surface and deliver methane to the atmosphere. Zhang and Xu’ll27 performed numerical modeling of a methane-seawater system along a T-P-depth profile similar to that of Blake Ridge (east of the U.S. South Carolina coast). These researchers found that only methane hydrate fragments larger than 9 cm in radius could rise through a 530 m water column and reach the ocean surface. Methane hydrates ascending to the surface of Lake Baikal have not been previously reported or systematically studied.Hydrates embedded within lake bottom sediments can be mobilized by water level variations, earthquakes, or other disturbances in water column pressure29. Historically, kolobovnik has been observed in different pelagic areas of the lake  suggested that they were derived from the littoral zone of the lake, but unfortunately methane analyses were not conducted31.Lake Baikal is covered by ice from January until May. If methane hydrates reach the lake surface, their traces can appear in the form of altered surface ice structure and/or increased methane concentrations in ice. Two distinctive forms of surface ice found around Lake Baikal may record the ascent of methane hydrates and their interaction with the lake’s ice cover. One form of ice is referred to as”kolobovnik”, a local name for ice containing large amounts of lumpy, granular ice with a rough surface with ice-balls from a few centimeters to 0.2–0.5 m in diameter, frozen in the ice cover. Sediment particles may occur within the ice32. Tsurikov described sopki as cone-shaped ice hillocks surrounding a hollow area or an opening that faces toward the lake. The structures are described as resembling a tent in shape32. Sopki may form as isolated units, in groups, or in series (a “mountain range”) with the largest features reaching heights of up to 6 m. Obvious holes present in sopki differentiate them from typical coastal ice mounds formed by water waves and splashing. However, sopki and kolobovnik morphologies and formational mechanisms have not been systematically described in the scientific literature.Another ice form potentially associated with methane hydrates is locally referred to as “sopki’. Sopki appears more frequently on the east side of the lake but sometimes occurs along the northwest shore14.This study utilized visual observations and acoustic echosounding surveys to document the ascent of methane hydrates through the Lake Baikal water column. To estimate the minimum size of hydrate capable of reaching the lake surface, we developed a numerical model and analyzed theoretical methane hydrate transformation during ascent from bottom sediments to the lake surface. We also report novel data demonstrating increased methane concentrations in kolobovnik and sopki ice structures. Data support the interpretation that these ice features could form from methane hydrates ascending to the lake surface. In contrast to bubbles or dissolved methane, methane hydrates are more stable in the water column to transport methane from Lake Baikal sediment to the atmosphere. The ascent of the methane hydrates through the water column thus represents a potentially important pathway linking methane produced in deep lacustrine sediments to the surface water methane3 km3) freshwater lake in the world. Located near the center of the Siberian high-pressure system, the lake is subject to strong cooling in winter, which results in a 3–5 month ice-covered period33. Lake Baikal is the only freshwater lake where methane hydrates are present in deep-bottom sediments11 including subsurface areas4 (beneath the sediment – water interface).Study site and field methods. Lake Baikal (N53°13′00″ E107°45′00″) is the deepest (max. depth 1642 m) and most voluminous (23.6 × 10−1 as the speed of sound in water). The pulse repetition frequency was 0.2 Hz. A hole was excavated in the ice to position the transducers above an area of gas seeps previously detected at the lake bottom. The transducers were mounted on a specially designed rig that ensured their axis remained vertical. A CTD (Conductivity-Temperature-Depth) probe SBE-25 (SeaBird USA) was used to performed temperature and conductivity profiles for later processing of acoustic sounding data and use in modelling . An Infinity EM 2D electromagnetic current meter  was used to measure the current velocity profile . Current speed measurements were averaged for 2 minutes at each depth as determined by rope markers.An acoustic survey was performed in the winter of 2013 with a Furuno FCV-1100 echosounder equipped with two channels (28 and 200 kHz) scanning a 24° and 8° field of view, respectively at up to 3 kW power. The pulse duration was 3 ms . Methane concentrations in the gas were determined as parts per million per mole with 5% precision. Identification of methane was performed by its retention time and quantified using a set of gas standards with a pre-defined methane content of 0, 3.94, 8.37 and 10000 ppm .Methane concentrations in the ice cover were measured from ice cores retrieved using saws. Each ice core was cut into 5–10 cm sections from top to bottom. Sections were weighed and then submerged in saturated brine to melt within a closed vessel. A hose with a clamp and syringe was attached to the vessel to collect the gas from air bubbles included in the ice. After ice melting, the volume, temperature and pressure of gas obtained from each section was determined and then the measured volume was recalculated to standard temperature and pressure conditions (20 °C and 101.325 kPa)p and increasing temperature T. Decomposition and dissociation on the solid surface results in formation of an intermediate layer  saturated with gas, fragments of decomposing hydrate crystals, and possible water41. The model treats the methane hydrate, ice (snow), and water as incompressible. A basic description of the modelling approach is given below while the online Supplementary methods section gives a more detailed description.A numerical model was constructed to describe phase transitions, velocity of ascending methane hydrates, and kinetics of hydrate decomposition. The model assumes a single hydrate fragment rising in an unconfined volume of water at decreasing pressure ah encapsulated within an ice crust of thickness δi = ai – ah, where ai is the equivalent spherical radius of the outside border of the ice crust. The equation for determining the ascending speed w(t) of the spherical body is obtained from the balance of momentum for a solid body with variable mass:mh is the mass of hydrate, mw is the mass of encapsulating water, and fA, fT, fW, fM are buoyancy, gravity, hydrodynamic resistance, and reactive force, respectively. The forces acting on the hydrate fragment (right side of Eq. z(t), rising velocity w(t), and the inner and outer radii of the ice crust denoted as ah(t) and ai(t), respectively. The system can be solved numerically for a given set of initial conditions, namely temperatures and pressures measured from the environment. The model could also inversely estimate the initial radius of a hydrate and thermal properties of the ice crust using experimentally observed ascending speeds of hydrates as inputs.The model assumes an ascending hydrate fragment as a sphere of radius 15. Some methane hydrate occurrences coincide with deep-water gas seeps (flares)17 but direct observations of methane hydrates are still rare. A 2009 expedition of the deep-water submersible MIR41 made a visual detection of methane hydrates at 1350 m water depth in sediments near a mud volcano referred to as St. Petersburg . Estimated depths and velocities for ascending hydrate fragments of different radii agreed well with echogram data  methane hydrate in freshwater containing only methane as the guest molecule occurs at depths of 360–380 m for a seasonal temperature range of 3.40 to 3.65 °CThe porous and inhomogeneous structure of kolobovnik ice serves to diminish thermal conductivity relative to that of clear/normal ice. This effect reduces heat flow from water which makes it possible to differentiate the lower temperature of the kolobovnik ice in infrared satellite imagery. Infrared images of ice cover in 2014 show two fields of kolobovnik as discrete cold regions, one small and another larger region nearby Fig. . Figure −1 of ice  occurred in the upper 10 cm of the ice. These values greatly exceeded atmospheric concentrations (about 85 nmol L−1 or 1.9 ppm). Gas concentrations then decreased to 2000 nmol L−1 (44 ppm) in the layer between 20–30 cm and increased to 8000 nmol L−1 in the layer between 30–40 cm. Below 55 cm, ice became clear and its gas volume did not exceed the 0.2 mL kg−1 detection limit of the analytical method used. Ice samples from areas surrounding the kolobovnik did not show any increased methane concentrations and were close to equilibrium with the atmosphere.Ice cores were collected from the larger field of kolobovnik and surrounding areas (for background conditions) Fig. . Fig. 6bice Fig. . Maximum−1 (4 ppm), a value which exceeds average values observed in the atmosphere by a factor of two. Sopki ice structures were also identified along the western shore of Lake Baikal, near Pereyomnaya River (51°34′51′′N 1105°16525′′E) on 11 January 2018. An ice core was collected from this site on 17 January , which is close to equilibrium with the atmosphere.In 2017, sopki ice structures were observed on a strip of land separating the open water of Lake Baikal from Posol’skiy Sor Bay Fig.  revealed −1 Fig. . The sub −1 Fig. . We also−1. This speed exceeded typical speeds of concurrently registered gas bubbles by a factor of two. McGinnis et al.8 summarized experimental observations to show that ascent velocities for bubbles of different sizes do not exceed 35 cm/s. While velocities over 40 cm/s could be theoretically achieved by large bubbles (more than 35 mm diameter), these structures are unstable and quickly dissociate into smaller bubbles. The rapidly ascending objects may represent hydrate coated bubbles which can be formed in Lake Baikal42, but simple calculations using Stoke’s Law indicate that they would need to be about 6 mm in diameter. However according to McGinnis et al.8, a complete hydrate rim forms around bubbles 2.7–4 mm in diameter and bubbles with larger sizes cannot be completely covered by a hydrate rim, and therefore, cannot reach such speeds. Also, detection of these objects at depths shallower than the gas hydrate stability zone suggests that they should already be dissolved. Velocity and depth estimates provided by mathematical modelling matched values observed in echogram data to support this interpretation. Echograms show the objects in question appearing at depths of 250 m  will began to form around a rising gas hydrate fragment at the depth where the temperature of the hydrate phase transition becomes equal to or less than the freezing point of water. The small bubbles trapped within the ice crust thus becomes a highly efficient reflector. These conditions allow for consistent hydrate detection on echograms. Gas inclusions can also increase hydrate buoyancy and corresponding ascension rate  and especially its usual speed (3–5 m s−1) combined with hydrate ascension rates would not allow features to remain in the echosounder field of view for the necessary detection interval. The probability of observing gas hydrates reaching the surface from an RV . Vessel drift  than previously reported minimum size estimates for hydrates ascending through the marine water column (about 10 cm)44. Data obtained from the water column in the area of the ring structure on the lake ice near cape Krestovskiy in June 2017 showed local decreases in the concentration of dissolved ions and water temperature44 probably due to decomposition of methane hydrates.Detection of methane hydrates ascending to the lake surface also supports our hypothesis that gas released during the decomposition of gas hydrates in the water column may contribute to local upwelling of deep waters and generation of circular anticyclonic currents45 has described the anomalous effect of self-preservation of gas hydrates at atmospheric pressure and temperatures below the freezing point of water (240 K ≤ T ≤ 273 K). This preservation is expressed in the long existence  of the hydrate remnants in metastable form without significant further decomposition. Observations confirm the formation of protective ice coating around hydrates during their dissociation but the mechanism for ice formation remains poorly understood. We hypothesized that spherical ice morphology (kolobovnik) in pelagic areas of the lake forms due to ice encapsulation of hydrate fragments. Ice formation is mediated by heat absorption during hydrate decomposition (phase transition). When the water temperature is close to zero, discrete ice spheres can freeze together and form a field33 or be washed up on the land as fast ice29. Analysis of kolobovnik ice cores detected methane concentrations in upper ice layers that greatly exceeded atmospheric concentrations. The atmosphere is thus not a plausible source of methane observed in ice. Elevated methane concentrations beneath winter ice cover7 (methane accumulation during limited gas exchange with the atmosphere) do not suffice to explain the high concentrations observed in kolobovnik ice. Additionally, concentrations observed in the ice could not be supplied by gas bubbles because the majority of these dissolve into the Lake Baikal water column7 before reaching the surface. Only the much more stable hydrates could deliver the methane volumes observed in ice. The presence of minor soil particles and other detritus within the kolobovnik (Fig. 46.Appearance of methane hydrates at the lake surface during ice cover formation may generate unique structures in the ice. Previous researchnik Fig.  indicateSopki also represents a form of ice likely related to the preservation and decomposition of methane hydrates transported to the lake surface Fig. . We prop7. Methane hydrates ascending to the surface may provide an additional mechanism for methane release into the atmosphere in Lake Baikal and probably in the marine environments, but their contribution requires further investigation. Paull et al.47 have considered the possibility of methane transport by hydrates released by marine slumping and rising through the ocean water column. Kvenvolden48 in turn, interprets these scenarios in terms of geological/climatic risk.This study provides novel evidence that methane hydrates released from sediment at 500–1000 m depths can reach the surface of Lake Baikal. Field observations and model results suggest that hydrates exceeding 2.5 cm in radius can ascend and contribute methane to the atmosphere. Current estimates for Lake Baikal methane release assume only diffusion and ebullition exchange processes, and that most sedimentary methane oxidizes within the water columnin situ detection of the methane hydrates randomly ascending from bottom sediments of a deep lake. Hydroacoustic properties of the hydrate fragments make them difficult to detect in the water column beneath the depth of ice crust formation. At shallower depths, however, they may be obscured behind a large number of accompanying bubbles, since they have comparable target strength. This could be the main reason that no one has observed them before. Specialized long-term and large-scale studies on the frequency of such events in Lake Baikal and in marine environments can further help to quantify the contribution of this methane hydrate transport mechanism.This report provides novel Unique ice morphologies and structures found around Lake Baikal likely form due to methane hydrates ascending from lake sediment. Analysis of gas inclusions from the ice structures revealed elevated methane concentrations relative to atmospheric values and relative to those measured from surrounding ice. While sopki typically appear on an annual basis, kolobovnik occurs only rarely in pelagic areas of the lake. A study of interannual variation in the distribution of these ice forms can help elucidate long term spatial and temporal patterns in methane hydrate transport and release. The unique kolobovnik and sopki morphologies arise from environmental conditions and physical mechanisms that contribute to their formation. Detailed observations can further clarify the processes occurring during the earliest stages of kolobovnik and sopki formation.Supplementary Information


This review elaborates current knowledge on cerebellar inhibitory interneurons , starting from their ontogenesis and moving up to their morphological, physiological and plastic properties, and integrates this knowledge with that on the more renown granule cells and PCs. We will focus on the circuit loops in which these interneurons are involved and on the way they generate feed-forward, feedback and lateral inhibition along with complex spatio-temporal response dynamics. In this perspective, inhibitory interneurons emerge as the real controllers of cerebellar functioning.Neuronal inhibition can be defined as a spatiotemporal restriction or suppression of local microcircuit activity. The importance of inhibition relies in its fundamental role in shaping signal processing in single neurons and neuronal circuits. In this context, the activity of inhibitory interneurons proved the key to endow networks with complex computational and dynamic properties. In the last 50 years, the prevailing view on the functional role of cerebellar cortical inhibitory circuits was that excitatory and inhibitory inputs sum spatially and temporally in order to determine the motor output through Purkinje cells (PCs). Consequently, cerebellar inhibition has traditionally been conceived in terms of restricting or blocking excitation. This assumption has been challenged, in particular in the cerebellar cortex where all neurons except granule cells (and unipolar brush cells in specific lobules) are inhibitory and fire spontaneously at high rates. Recently, a combination of electrophysiological recordings  Both are excitatory and use glutamate as neurotransmitter. The MF input originates from several nuclei in the brain stem and spinal cord. In the GL, MFs make excitatory synapses onto granule cells, whose axons rise vertically to the ML, where they divide to form T-shaped branches called parallel fibers , the Purkinje cell layer (PCL) and the granular layer GL; . The pri CF Ito, . The inh CF Ito, . Basket  CF Ito, . Their a CF Ito, . In addi CF Ito, . Golgi c CF Ito, . Recentl CF Ito, . Golgi c CF Ito, . Lastly, CF Ito,  in the r CF Ito, .Ptf1a is crucial for the initial specification of the GABAergic lineage, ensuring the appropriate cell number production and their subsequent survival  cell precursors , neurogenin-2 (Ngn-2), and Ascl-1 genes  gives origin to GABAergic neurons, whereas the rhombic lip (RL) generates glutamatergic types  with morphological and electrophysiological characterization in order to distinguish several subtypes of GABAergic interneurons  have been described in detail for the cerebellar cortex . Moreover, the modulation of the firing rate of ML interneurons using optogenetics proved able to drive movement kinematics in awake mice. In eyeblink conditioning, a pause in PC firing occurs after training  relationship by decreasing the membrane resistance of granule cells, leading to a reduction of the excitability  plays a general role in locally controlling the excitatory-inhibitory (E/I) balance within a neural circuit . To date, the main forms of plasticity involving cerebellar inhibitory interneurons are the following. A form of LTD has been observed at the connection between PFs and Golgi cells, following high-frequency activation  center-surround organization in the GL; (2) gain and timing regulation in the GL; (3) synchronous low-frequency oscillations in the GL; (4) beam organization in the ML; (5) burst-pause regulation in PCs; and (6) gating of synaptic plasticity. Since the spatiotemporal pattern of cerebellar cortical activity is de facto controlled by cerebellar cortical interneurons, these cannot anymore be considered “subordinates” to excitatory cells  but rather integral parts of diverse microcircuits for multimodal information processing (Casali et al., The last decades have been characterized by considerable progress in understanding the diversity of cerebellar inhibitory interneurons, in terms of embryological and developmental origin, localization and distribution of neurochemical markers, morphological and intrinsic properties. The concept has emerged that neuron properties and network dynamics in the cerebellar inhibitory circuit are more complex than originally thought. Both in the granular and in the ML, cerebellar interneurons are involved in complex inhibitory chains generating feedback, feedforward and lateral inhibition that regulate spatio-temporal dynamics of fundamental importance to determine the processing capabilities of the cerebellar cortex. Interneuron inhibition leads to the emergence of: FP organized and wrote the manuscript. LM prepared the figures and contributed to writing the text. ED’A contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer CH is currently the co-Field Chief Editor of Frontiers in Cellular Neuroscience with one of the authors, ED’A, and confirms absence of any collaboration to the handling editor.


Basophils and mast cells have high affinity IgE receptors (FcεRI) on their plasma membrane and play important roles in FcεRI-associated allergic diseases, such as pollen allergy, food allergy, chronic spontaneous urticarial (CSU), and atopic dermatitis (AD). To date, several reports have revealed that high IgE antibody concentrations activate mast cells—which reside in tissue—in the absence of any antigens . However, IgE antibody-induced activation of basophils—which circulate in blood—has not been reported. Here, we investigated whether IgE antibodies may regulate functions of human peripheral basophils without antigens in vitro. We successfully removed IgE antibodies bound to FcεRI on the surface of human peripheral basophils by treating with 0.1% lactic acid. We also demonstrated that high IgE antibody concentrations (>1 μM) induced histamine release, polarization, and CD203c upregulation of IgE antibody-stripped basophils. Thus, high IgE antibody concentrations directly activate basophils, which express IgE-free FcεRI on the cell surface. This mechanism may contribute to the pathogenesis of patients with AD and CSU who have higher serum IgE concentrations compared to healthy donors. It is well known that basophils circulate in blood and account for less than 1% of peripheral blood leukocytes . BasophiMast cells, resident in tissue, are recognized as similar cells to basophils due to their characters and functions ,6. Mast Therefore, the mechanism of basophils activation induced by antigen-IgE antibody interactions on FcεRIs has been well investigated. However, the mechanism for direct activation of basophils by IgE antibodies themselves has remained unclear. In this study, we investigated if high IgE antibody concentrations directly induce the activation of human peripheral basophils without any antigens.The elevation of IgE antibodies in serum of patients with chronic spontaneous urticaria (CSU) and patients with atopic dermatitis (AD) has been reported by several groups in Europe and the USA ,11. Here−10 M) [Since almost all IgE receptors of basophils isolated from human peripheral blood are already occupied by IgE antibodies circulating in a donor’s blood due to the high affinity of FcεRI for IgE antibodies (Kd around 10−10 M) , we firsWe then investigated the effect of various concentrations of IgE antibodies on the functions of human peripheral basophils of which IgE antibodies on FcεRI were partially removed by the treatment with 0.1% lactic acid saline. Though the low concentrations—50 ng/mL—of IgE antibodies that are usually applied for IgE sensitization for mast cells in vitro could not activate basophils, high IgE antibody concentrations (>1 μg/mL) induced the release of histamines from IgE-stripped basophils at the concentration dependent manner a. High IWe then investigated the effect of high IgE antibody concentrations on the adhesion and polarization of human peripheral basophils. Though IgE antibodies (5 μg/mL) without IL-3 slightly induced polarization of IgE stripped basophils, high IgE antibody concentrations (5 μg/mL) with IL-3 (1 ng/mL) clearly increased the number of IgE stripped basophils with polarity on the fibronectin coated glass-slide. This suggests that IgE antibodies together with IL-3 induce the migration of basophils a. MoreovIn this study, we demonstrated that high IgE antibody concentrations activate human peripheral basophils functions such as histamine release, polarization, and upregulation of surface antigen (CD203c) in the absence of any antigens . Histamine release in response to anti-IgE antibodies and analysis of FITC-IgE and APC-anti-IgE bindings by means of flow cytometry revealed that the stripping of IgE antibodies by treatment with 0.1% lactic acid saline did not remove all IgE antibodies bound to FcεRIs on the surface of basophils a,b. Howe®), in a number of clinical studies [The critical role of IgE antibodies in the development of allergic disorders, such as chronic urticaria and AD, is supported by the effectiveness of anti-IgE antibodies, such as omalizumab . IL-3 was sourced from R&D Systems Inc .Ficoll-Paque Plus was sourced from GE Healthcare Japan Corporation . EasySep™ Human Basophil Enrichment Kit was sourced from STEMCELL Technologies . Reverse-phase HPLC was sourced from Shimadzu . Tetramethylrodamin B isothiocyanate (TRITC)-phalloidin and adenosine were sourced from Sigma-Aldrich Japan . Human IgE Quantitative ELISA kit was sourced from Bethyl Laboratories, Inc., . Human IgE Purified (AG30P) was sourced from MILLIPORE . Human IgE (P50) was sourced from Nordic-MUbio . Human IgE (HE1) was sourced from Bioporto Diagnostics A/S . Fluorescein Labeling Kit-NH2 was sourced from Dojindo laboratories . Anti-IgE receptor-FITC, anti-CD123-PE, anti-CD203c-APC, anti-IgE-APC, and anti-CD203c-FITC were sourced from BioLegend . AllergenicityHuman Peripheral Blood Mononuclear Cells (PBMCs) were isolated from the fresh, heparinized blood of drug-free healthy donors using Ficoll-Paque Plus density gradient separation. Basophils were isolated from PBMCs by magnetic depletion of non-basophils using the EasySep™ Human Basophil Enrichment Kit, as performed in our previous work . Purity Concentrations of IgE antibodies in human serum were measured by ELISA according to the manufacturer’s instructions of Human IgE Quantitative ELISA kit.FITC was bound to IgE antibodies (MILLIPORE) using Fluorescein Labeling Kit-NH2 according to the manufacturer’s instructions.IgE antibodies on the surface of basophils from drug-free healthy donors were removed by the treatment with 0.1% lactic acid (pH 3.9), as described previously . The eff® kit according to the manufacturer’s instructions. Expression levels of FcεRI on the surface of basophils were detected by flow cytometry. PBMCs were labeled with anti-FcεRI-FITC, anti-CD123-PE, and anti-CD203c-APC. For the detection of IgE-free FcεRI on the surface of basophils, PBMCs treated with or without 0.1% lactic acid were stained with FITC-labeled IgE antibodies, anti-CD123-PE, and anti-CD203c-APC. For the detection of IgEs on the surface of basophils, PBMCs treated with or without 0.1% lactic acid were stained with anti-CD203c-FITC, anti-CD123-PE, and anti-IgE-APC. Expression levels of CD203c on the surface of basophils were detected by using AllergenicityHistamine release tests with human basophils were performed as described previously with a goat anti-IgE antibody (670 ng/mL) as positive control . HistamiHuman peripheral basophils were stimulated with 10 μg/mL IgE antibodies with or without IL-3 (1 ng/mL) on the fibronectin-coated glass bottom dishes. After 30 min incubation at 37 °C, cells were washed with PBS and fixed with 4% paraformaldehyde. The cells were then treated with PBS containing TRITC-phalloidin for 20 min. Fluorescence was observed using confocal laser scanning fluorescent microscopy.We here demonstrated that high IgE antibody concentrations directly activate peripheral basophils, which have IgE-free FcεRIs on the surface of cells. This mechanism may contribute to the pathogenesis of allergic disorders such as AD and CSU.


Depletion of basophils before the tick re-infestation abolishes ATR in guinea pigs infested with Amblyomma americanum and mice infested with Haemaphysalis longicornis, demonstrating the crucial role of basophils in the manifestation of ATR. The activation of basophils via IgE and its receptor FcεRI is essential for ATR in mice. Histamine released from activated basophils functions as an important effector molecule in murine ATR, probably through promotion of epidermal hyperplasia which interferes with tick attachment or blood feeding in the skin. Accumulating evidence suggests the following scenario. The 1st tick infestation triggers the production of IgE against tick saliva antigens in the host, and blood-circulating basophils bind such IgE on the cell surface via FcεRI. In the 2nd infestation, IgE-armed basophils are recruited to tick-feeding sites and stimulated by tick saliva antigens to release histamine that promotes epidermal hyperplasia, contributing to ATR. Further studies are needed to clarify whether this scenario in mice can be applied to ATR in other animal species and humans.Ticks are blood-sucking arthropods that can transmit various pathogenic organisms to host animals and humans, causing serious infectious diseases including Lyme disease. Tick feeding induces innate and acquired immune responses in host animals, depending on the combination of different species of animals and ticks. Acquired tick resistance (ATR) can diminish the chance of pathogen transmission from infected ticks to the host. Hence, the elucidation of cellular and molecular mechanism underlying ATR is important for the development of efficient anti-tick vaccines. In this review article, we briefly overview the history of studies on ATR and summarize recent findings, particularly focusing on the role for basophils in the manifestation of ATR. In several animal species, including cattle, guinea pigs, rabbits and mice, basophil accumulation is observed at the tick re-infestation site, even though the frequency of basophils among cellular infiltrates varies in different animal species, ranging from approximately 3% in mice to 70% in guinea pigs. Skin-resident, memory CD4 Ticks, particularly ixodid family members, are blood-sucking ectoparasites of vertebrates and can transmit various pathogens to animals and humans during blood feeding for days, causing serious infectious diseases, including Lyme disease, babesiosis, Rocky Mountain spotted fever, human monocytic ehrlichiosis and severe fever with thrombocytopenia syndrome . BesidesDermacentor variabilis, guinea pigs develop resistance to subsequent tick infestations  mice in that only basophils express green fluorescent protein (GFP), confirmed the basophil accumulation at the 2nd but not 1st tick-feeding site  or anti-CD200R3 (Ba103), just before the 2nd tick infestation completely abolished ATR with no apparent effect on the number of other types of cells, including monocytes/macrophages, neutrophils and eosinophils  have been identified in tick saliva . They shestation , the conH. longicornis, Dermacentor andersoni, and Boophilus microplus larval ticks are highly reactive to histamine in the induction of tick resistance while A. americanum and Ixodes holocyclus ticks are less responsive to histamine (It has been reported that istamine . The foristamine , suggestnd but not 1st infestation. Importantly, the recruitment of basophils can be observed in previously uninfested skin, far from the 1st infestation site, of sensitized animals, implicating that the 1st tick infestation may induce systemic alteration in the skin throughout the body, so that basophils can readily infiltrate the tick re-infestation site anywhere in the body. We have recently demonstrated in mice that skin CD4+ memory T cells play an important role in basophil recruitment to the 2nd tick-feeding site, leading to ATR (+ effector T cells are generated during the 1st tick infestation and distributed to the skin all over the body, and some of them are retained as skin-resident memory T cells (Figure nd tick infestation, tick saliva antigens delivered into the skin stimulate these memory T cells present in the skin to produce IL-3 that is required for basophil recruitment to the 2nd tick-feeding site (Basophils circulate in the peripheral blood under homeostatic conditions, and they infiltrate the skin at the tick-feeding site during the 2g to ATR . Tick aning site . Even thing site , leadingH. longicornis (D. variabilis-infested mice (H. longicornis, histamine derived from basophils but not mast cells is essential for the manifestation of ATR (nd tick-feeding site is comparable between mast cell-sufficient and -deficient mice (nd tick-feeding site are more motile and less-clustered around a tick mouthpart in mast cell-deficient mice than in mast cell-sufficient mice (As described above, mast cells contribute to ATR in mice infested with gicornis , 1990 whted mice . As far n of ATR , even thn of ATR . The defn of ATR , suggestent mice , indicatent mice . Therefost tick infestation triggers the production of IgE against tick saliva antigens. In the 2nd infestation, IgE-armed basophils are recruited to the tick-feeding site and stimulated by tick saliva antigens to release histamine that functions as a key effector in ATR, probably through promotion of the epidermal hyperplasia that in turn interferes with tick attachment or blood feeding in the skin (Figure Recent development of a series of analytical tools in laboratory animals has advanced our understanding of the cellular and molecular mechanism underlying ATR. In several animal species, basophil accumulation is observed at the tick re-infestation site Figure , and basHK has substantially contributed to the literature review and drafted the manuscript. SY contributed to the discussion and drafting, and editing of the manuscript. YT, TO, and TW reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to identify, quantify, and characterize the chemical compounds with the highest degree of inhibitory activity of these enzymes, contained in a Ludwigia octovalvis hydroalcoholic extract. Chemical purification was performed by liquid–liquid separation and column chromatography. Inhibitory activities were measured in vitro, employing acarbose, orlistat, and a Camellia sinensis hydroalcoholic extract as references. For structural elucidation, Nuclear Magnetic Resonance was carried out, and High Performance Liquid Chromatography was used to quantify the compounds. For α–glucosidases, L. octovalvis hydroalcoholic extract and its ethyl acetate fraction showed half–maximal Inhibitory Concentration (IC50) values of 700 and 250 μg/mL, for lipase, 480 and 718 μg/mL, while C. sinensis showed 260 and 587 μg/mL. The most active compounds were identified as ethyl gallate  and gallic acid ; both displayed competitive inhibition of α–glucosidases and isoorientin , which displayed uncompetitive inhibition of lipase. These data could be useful in the development of a novel phytopharmaceutical drug.Current antiobesity and antidiabetic tools have been insufficient to curb these diseases and frequently cause side effects; therefore, new pancreatic lipase and  In the case of the determination of α–glucosidase type of inhibition, the substrate was changed from corn starch to maltodextrin , in order to have greater uniformity in the reaction.The type of inhibition was determined quantifying the activity with and without inhibitor at different substrate concentrations (5–0.35 mg/mL for m) and apparent Km (Kmapp) were obtained analysing the Lineweaver–Burk plots. These values allowed to obtain the inhibition constant (Ki) for competitive inhibitors using  and LoEAF . Samples of these materials and CsHAE were analysed in thepase see .α–glucosidases by 58.9% and the pancreatic lipase by 23.6%, while CsHAE produced an 80.8% inhibition of α–glucosidases and 34.8% of pancreatic lipase. The organic fraction, LoEAF, had more inhibitory activity than LoAqF fraction or LoHAE extract in both assays, with an 82.8% inhibition of α–glucosidases and 31.2% inhibition of pancreatic lipase.LoHAE inhibited the α–glucosidases, while C1F6 was the most active for lipase  that was analysed by HPLC see  and its data see .1H NMR analysis . HPLC, UV spectra  260 μg/mL, while LoHAE produced IC50 700 μg/mL. Ethyl gallate (C1F1) and gallic acid (C2F1) IC50 values were 832 μM and 969 μM, respectively. Luteolin  was used as a naturally occurring reference displaying an IC50 = 1257.7 μM.All graphs corresponding to concentration–response curves in the hown see . CsHAE dm (intersection x-axis) increase, but maximal velocity  remains the same, as expected for a competitive enzymatic inhibition , so it produced uncompetitive enzymatic inhibition of pancreatic lipase [As observed in the graph see , isooriec lipase .α–glucosidase inhibitors are currently the most effective and safest for postprandial glycaemia control as well as intraday and interday glucose fluctuation [β–cell dysfunction [β cells, leading to β–cell exhaustion [According to several studies, postprandial hyperglycaemia periods, even the relative short–lasting ones, contribute to the development of chronic diabetes complications even more than basal hyperglycaemia . Moreovectuation . On the ctuation  and alsofunction . What isfunction , but alshaustion ; this phhaustion , 35.L. octovalvis hydroalcoholic extract has the advantage of displaying both α–glucosidase and pancreatic lipase inhibition activities. This is the first time that these mechanism modes are described for this species. Besides L. octovalvis is an interesting option as antidiabetic because it was described as innocuous according to the OECD [the OECD .α–glucosidase inhibition was also observed in LoAqF, indicating the presence of other polar compounds with high inhibitory activity of these enzymes. Nevertheless, according to HPLC quantitative analysis, the bipartition process successfully increased the concentration of the two α–glucosidase inhibitors in the organic fraction. Therefore, it would be proper to design an extraction or separation method that concentrates these polyhydroxy benzoic acid derivatives. Although gallic acid has been previously described for L. octovalvis [50 ≈ 1257.7 μM) which has been described as good inhibitor of α–glucosidases [In this study, the concentration of low and intermediate polarity compounds contained in LoEAF considerably increased the inhibition of both digestive enzymes, although an increase of tovalvis , this isosidases –38.α–amylase on rat and Saccharomyces sp. α–glucosidases on rat maltase [α–glucosidases [α–amylase [50 values of gallic acid and ethyl gallate in the inhibition of maltase  and sucrase  in rat were considered significantly high values [The inhibitory activity of carbohydrate degrading enzymes by gallic acid and its esters, such as ethyl gallate, has been described with inconsistent results. According to some authors, gallic acid showed very low or no inhibitory activity on porcine and Bacillus sp.  maltase –43. Howe maltase , 44 and osidases  and on p–amylase . Furtherh values .α–glucosidase inhibitors varies according to the origin of the enzymes and the type of substrate used. According to Oki et al. [α–glucosidase inhibitors for clinical use, mammalian enzymes and natural substrates of each type of enzyme should be used. Results of this work strengthen the hypothesis that these phenolic compounds  could be active in the inhibition of human α–glucosidases.This inconsistency of results could be due in part to the diversity of enzymes and substrates used for these tests; it has been shown that the effect of i et al. , to perfi of naturally occurring compounds are described on digestive enzymes [In this study, ethyl gallate and gallic acid displayed a competitive enzymatic inhibition, in which the inhibitor competes directly with the substrate for the binding site in the active site of the enzyme . This is enzymes .In the case of lipase inhibition, the most active compounds were enriched in the organic LoEAF fraction. Further purification by silica chromatography allowed us to obtain a C–glycosylated flavone: isoorientin . This flThese kinds of C–glycosylated flavonoids have shown high inhibition of pancreatic lipase and according to some authors, glycosylation in position C–8 seems to significantly increase this biological activity –44. L. octovalvis extracts with pancreatic lipase inhibitory action.Considering that it is desirable to have reference compounds to standardize a phytopharmaceutical drug, isoorientin could fulfil this purpose in L. octovalvis did not display acute toxicity in mice when it was tested at 5000 mg/kg nor subacute toxicity at 400 mg/kg during 28 days [ L. octovalvis displayed at least two mechanisms of antidiabetic and antiobesity action, which are synergistic and complementary.According to a toxicity analysis of this plant, an alcoholic extract from 28 days , which i L. octovalvis treatments were as potent as the reference drugs, there are reports where in vitro digestive enzyme inhibition of naturally occurring compounds is lower than acarbose or orlistat but when tested on in vivo models, they produced similar pharmacological activities [Although none of thetivities , 50. L. octovalvis hydroalcoholic extract which is bioactive in α–glucosidase and pancreatic lipase inhibition allowed the identification and pharmacological characterization of one flavone (isoorientin) with considerable inhibitory effect of pancreatic lipase and two isolated compounds with high inhibitory effect of the α–glucosidases . These findings bear out one of the possible mechanisms of action by which this medicinal plant could help in the prevention and treatment of type 2 diabetes and obesity; therefore, these data will be useful in the development of a potential novel phytomedicine.The chemical separation of


Ice action on ships and offshore structures is commonly determined by calculating the contact ice pressure. The aim of this paper is to describe the empirical background for determining the ice pressure. This review article describes six different test series where ice edge indentation and contact ice pressure have been investigated. These test series are ice pressure measurements onboard IB Sisu in the Baltic in 1977, pendulum tests carried out at Arctec in Ottawa, Canada, in 1979, laboratory and full scale ice crushing tests at WARC in 1988 and onboard IB Sampo 1989, medium scale indentation tests on Hobson's Choice Ice Island 1990, ice crushing tests at NRC, Ottawa 1992 and the JOIA tests in Hokkaido 1996–1999. These tests were selected as at each series a new phenomenon was observed. The aim of the paper is to introduce the main features for ice–structure contact empirically through the description of tests. The paper is concluded with a short description of the existing models for ice pressure, especially to gain an insight and highlight the main observations in each test series and how the models for ice pressure have developed based on the observations.This article is part of the theme issue ‘Modelling of sea-ice phenomena’. As the contact (loosely) includes an area where the contact exists, instead of the contact force, the contact is described by contact pressure, as this quantity is easier than contact force to relate with ice characteristics (see e.g. ). While The present description of ice edge failure and ice pressure reviews a set of experiments carried out to observe the contact between an indenter  and ice edge, and also to measure the ice pressure. The paper does not present any new results but tries to put measurements already made in the context of understanding ice pressure. The selection of the measurements described is naturally very subjective, and a quick apology is offered to those who feel that their measurements have been neglected. The paper is completed by a short review of the models suggested for describing or determining the ice pressure.Many of the figures are reproduced from the original sources as no other figures or references are available. Thus the quality of these leaves a bit to be desired.2.Ice pressure is defined as the pressure acting on the interface between the structure and ice edge. This definition immediately awakens two main questions. Firstly, why ‘ice edge’? When a level ice sheet drifts against a stationary structure that offers a vertical side against the ice, the meaning of ice edge and its geometry is clear. When a ship proceeds in level ice, the geometry of the edge is less clear. The ice edge arises from the repeated ice failure in bending, which leaves the ship to collide repeatedly on the edge of the newly broken ice.direct contact between ice and the outer shell of the structure. The fact that ice often fails by crushing, i.e. being broken into a granular material containing also very small ice particles, obscures the idea of a direct contact that is defined to occur between intact ice and the structure. There may be a direct contact in the interface and also contact through the crushed ice.The second question is related to the ‘interface’. It sounds clear that there is a nominal contact area. The nominal contact area is the area where the contact would be if the geometries of the structure and the ice feature were extended to the contact without considering deformation or especially cracking. Practically, all the models for ice pressure use the concept of the nominal contact area to define the ice pressure; see, for example, the collection of models in [projected nominal area. This is the nominal area projected on a tangent plane of the contact that is normal to the relative velocity.Local ice edge cracking can also create a contact that is smaller than what is usually described as the odels in . It shouAnom = π/2i·D·h, where D is pile (column) diameter and ih ice thickness. The projected nominal area is simply Aproj = i.D·h In the ship case, the contact areas are much more difficult to determine as the edge geometry is not clear—obscured by the crushed ice even in more clear cases as in When a structure indents an ice edge, the ice edge is described to fail in ‘crushing’. (a) ice pressure is assumed to be uniform over the whole nominal contact area and also on the projected nominal contact area;(b) the local structural response can be elastic or plastic;(c) global structural deformation is (linear) elastic;(d) local ice failure is either described as ‘crushing’ or spalling by cracks created at the contact;et al. [(e) the hydrodynamic reaction force has a static part (due to buoyancy) and a dynamic part (due to accelerating water); studies like Keijdener et al.  show tha(f) the length of the contact  is taken into account only in very simple geometries; and(g) ice bending is modelled as a simple plate on an elastic foundation.In the modelling of structure–ice interaction, all the deformation and motion components should be taken into account especially to determine the relative motion between ice and structure. effect of temperature and indentation speed on the magnitude of ice pressure and failure process;the ratio of the actual contact area, i.e. area where the pressure is not zero, to the nominal contact area; andice pressure distribution and magnitude on both the nominal and actual contact area.The measurements carried out have especially focused on the following items:3.Ice pressure has been and is measured by measuring the deformation caused by ice pressure on a selected area. This may be the deformation of the structural outer shell or a purpose-built panel within the structural shell. In any case, the pressure is obtained based on the force–deformation characteristics of the gauge, i.e. pressure is obtained from the measured force, which is obtained from the measured deformation. Numerical or physical calibration to obtain the force–deformation relationship is done using a certain gauge area. This area in many cases is clear especially if the gauge consists of a separate face against ice. The gauge calibration usually assumes a uniform pressure on the gauge area, an assumption that is more exact the smaller the gauge area is.Mechanical gauges cannot be used to get an idea of the pressure distribution as the gauges cannot cover the whole surfaces without gaps. Two electric gauges have been used that can be used to improve the situation: polyvinylidene fluoride (PVDF) film, which is piezo-electric , and theIn the following, six measurement campaigns are described in which ice pressure was measured and the ice edge failure observed. The presentation follows chronological order. The ice used in these tests has a large influence on ice pressure value. Thus the ice used is described to the extent it is possible based on the original sources. The grain size or direction is not described in most of the tests—only the test series in \S3b and \S3e report it. It is unfortunately not possible to describe all similar campaigns and the selection here serves an aim to develop further the modelling of ice pressure—this is described as the last section of the paper.(a)et al. [Yσ = 235 MPa). The deflection of the icebreaker plating inside the cylinder perimeter was measured relative to the cylinder. The calibration with a uniform pressure yields the deflection versus pressure. The gauges were calibrated using the finite element (FE) method and also with an external hydrostatic pressure. Besides the pressure sensors, gauges to measure the stress on the shell plating in the longitudinal direction  were installed , to find the maximum pressure pmax value  that gives the maximum stress measured. This maximum stress was 70% of the yield, i.e. about 170 MPa. The results of calculations are shown in figure 5b as the maximum pressure versus load patch height using the load patch length as a parameter. As the measured maximum pressure is about 8.5 MPa, acting as an assumption on a stiff area, it can be seen that a relatively small load height (about 20 cm) and a triangular shape fit the data best. It should be noted here that the minimum pressure pm was kept at 2.4 MPa, which was assumed to be the uniaxial horizontal compressive strength of Baltic ice.The stress in the plating was measured in the adjacent frame spacing to the pressure gauges. The measured stresses did not tally with the measured pressure if the pressure is assumed to be uniform. Thus a hypothesis stemming from the theory of stiffened plating resting on an elastic foundation was made, i.e. that the stiffer part of the shell carries more pressure. This was simplified to a triangular pressure distribution as shown in figure 5— the measured ice pressure is clearly higher than the uniaxial compressive strength of ice (which is about 4 MPa), up to 8.5 MPa;— the ice pressure distribution is not uniform; and— high pressures are measured in spring (beginning of May) when ice is melting; this is intriguing as the strength of melting ice is very low .Conclusions from the pressure measurements onboard IB Sisu:All these conclusions were surprising at the time of these measurements but were put into some physical context later, using more detailed measurements (as described below).(b)−1, were achieved using a pendulum by Glen & Comfort [The high ice pressures measured with the Sisu instrumentation stimulated a laboratory test series with high indentation speeds. This resulted in a test series where the high speeds, up to about 5.6 m s Comfort  A large set of ice crushing experiments was carried out in 1988 at the Wärtsilä Arctic Research Centre (WARC) with the cooperation of the Helsinki University of Technology . The intent was to measure the ice pressure with PVDF film (see ) and at −1. The indenter plate was either covered by the PVDF pressure sensors or was transparent, enabling filming through the plate. Apart from the ice pressure, the normal and tangential force were measured and naturally the indentation.The test specimen cross section was always 25 × 25 cm (height 65 cm) while the edge that was crushed was either the edge of a rectangle with an inclined indenting plate or a wedge . The tesc. If the focus is on the beginning of the test, before the first major crack (thus up to an area of about 50 cm2), it is clear that the trend is not linear with the increasing area but rather somewhat below the linear. If a power fit is done to the first part, i.e. a fit as Fn(t) = mCA(t), then the area exponent is between −0.28 and −0.43. This is referred to as a process pressure–area relationship—mainly to distinguish it from a pressure–area relationship where pressure peaks from different tests are plotted .A typical normal force–time history is shown in figure 10The measured pressures were not, however, the main new feature in these indentation tests. This came from the visual observations of the contact area. The contact seems to be transmitted through a small narrow band that is shown black in photographs—black, as the external light disappears in ice at the direct contact. The observation of the narrow high-pressure band induced the question whether this is an artefact from laboratory tests. A test in full scale was decided to be done onboard icebreaker Sampo. IB Sampo is an old Finnish line icebreaker that now acts as a tourist cruise icebreaker at the port of the northern city Kemi. The PVDF plate was attached to the hull and beside the PVDF plate a window was made at the waterline of the icebreaker . The tesIn p = CAn) is made (similar to figure 10c), an area exponent of about −0.5 is obtained. This is somewhat steeper than the process pressure–area relationship—having an area exponent of about −0.3 to −0.4—fitted on the collection of force–time histories. It is, however, important that both the process pressure–area relationship (figure 10c) and the one on maxima et al. [figure 10c—surprisingly as the ice used in these two test series was different. It is noteworthy that the measured maximum average pressure was 22 MPa, and that these maximum average pressures when plotted versus nominal contact area gave rather low area exponents, about −1. This value of the exponent actually suggests a constant force.Indentation tests were carried out on an ice island named Hobson's Choice in 1990 just north of the Canadian Arctic archipelago. The tests are reported in Frederking et al.  and for et al. . The iceet al. [An interesting observation in these tests was that even if the cones (pyramids) were quite flat—angle about 18°—linear high-pressure areas were detected. These are shown in et al. .Figure (e)−1. Ice was fresh water ice and kept in all tests at a temperature of −10°C.An ice edge indentation test series was carried out at the National Research Council of Canada (NRC) cold rooms at the Montreal Road campus in Ottawa in 1992. The aim of the tests was to investigate in detail the effect on the ice crushing process—ice edge indentation—of several factors, like the grain orientation of S2 (columnar grained) ice, the extrusion of the crushed ice and flexibility of the indenter. The tests are reported extensively in Tuhkuri –25). TheThe measured time history shows much variation during the test but, looking a bit more closely, it is noticed that the time history consists of a triangular pattern . The domTests were carried out with a flexible plate . In thisFinally, it should be mentioned that the NRC indentation tests were used to investigate the spalling cracks that were observed. The study is reported in Tuhkuri . The cra(f)et al. [et al. [in situ on sea ice with salinity of about 5 ppt. The test ice was floating and an apparatus was installed to indent the ice edge . This triangular pattern  relates to colder ice temperature; unfortunately, no exact temperature value is available. Sodhi et al. [figure 25a while the lowest indentation speed failure is ‘ductile’. This is interesting as ‘brittle flaking’ is often coupled with triangular force peaks.Time histories of the indentation force at different indentation speeds are shown in figure 25i et al.  described. The observation on local and global pressures induces the question on describing the pressure versus the nominal contact area: Should the indentation speed be included as a parameter?It is also noteworthy that the pressures measured with the tactile sensors  are much higher with higher indentation speeds than with lower ones. This should be contrasted with the fact that the average  ice pressure on the nominal area is higher at the lower indentation speeds. This is demonstrated in figure 25The observations made from the tests described the ice failure at the high indentation speeds as brittle flaking. This flaking created a narrow linear high-pressure area (band) where the individual pressure peaks look like a mountain range. The visual observation by Joensuu & Riska  shows th(g)−1 and 3 mm s−1.— There seems to be a mode change in ice failure from ductile (viscous/plastic) at low indentation speeds to brittle  at high indentation speeds. JOIA tests with relatively warm sea ice suggest that the mode change occurs between the indentation rates of 0.3 mm s— At higher indentation speeds the high pressure is concentrated on a narrow band that aligns with the nominal contact area geometry, preferably along the long geometric axis of the area.— Contact area with ice is much larger in the ductile regime than in the brittle regime. Local ice pressures are, however, higher in the brittle regime whereas average pressure is higher in the ductile regime.— The flow of crushed ice has an influence on contact; with the restricted flow the contact area becomes larger.— In the ductile regime, the contact pressure is (more) uniform whereas in the brittle regime, the contact pressure consists of peaks that are not much correlated. This gives validity to the suggestion that the ice failure is by spalling in the brittle regime and that the spalls formed are essentially three-dimensional.— The triangular pattern was present in most tests where the force–time history could be measured. The reason for this is allocated to a failure mode change from ductile (low indentation speeds) to brittle (higher indentation speeds). Only in some of the tests in the last test series described (JOIA), the triangular pattern was missing from some tests even if ice failure was described as brittle flaking.Results from several test series about ice–structure interaction through ice edge indentation were described above. Some immediate conclusions from the tests can be made, such as the following:4.It may be of interest to look at the various models for ice pressure that have been suggested. It is important to keep the above conclusions in mind when investigating the different ice pressure models. In the following, a brief review of models presented for ice pressure is made.(a)m, value 0.9 to 1), indentation factor based on the multi-dimensional stress state in ice  and contact factor based on the non-simultaneity of the contacts within the nominal contact area . Thus the Korzhavin ice pressure isσc is the compressive strength of ice.The first model for ice pressure assumed pressure to act uniformly on the nominal contact area. If tangential  forces are ignored, then the pressure is uniform also on the projected nominal contact area. The magnitude of pressure was assumed to be proportional to ice compressive strength. The resulting equation is called the Korzhavin equation as it originated from Korzhavin  based onThe Korzhavin equation has been much criticised, as the coefficients are somewhat arbitrary; see, for example, Palmer & Croasdale . The othv is the indentation speed and k an empirical parameter representing both the strength of ice and the viscosity of the crushed ice. The pressure magnitude was obtained using the so-called specific crushing energy that was determined mainly by drop ball tests where the energy consumed to crush a certain volume of ice was calculated. This pressure model was used in developing a theory for ship ice loads by Popov et al. [A formulation that was developed in view of ship ice loads is based on the assumption of a crushed ice layer between the structure and intact ice; see, for example Kurdjumov & Kheisin . Figure v et al. . RussianSimilar criticism as to the Korzhavin equation can be directed to the hydrodynamic ice pressure model, i.e. the description that ice strength is empirical and based on compressive strength. Further, the value of viscosity of crushed ice—and in general the assumption of viscous fluid—is somewhat problematic; see, for example, Sayed & Frederking . Furtheret al. [w, the pressure required to create this is p = kw, where k is the foundation modulus. The situation modelled is shown in The hydrodynamic ice pressure theory does not state anything about the pressure distribution in horizontal  direction. The measurements onboard IB Sisu have been used to develop the theory of the pressure distribution in the longitudinal direction for transversely framed ships. This is based on a stiffened plate resting on a Winkler foundation; see Riska et al. . The cruk is the foundation Winkler coefficient, E steel Young's modulus, ν steel Poisson's ratio, t plate thickness and s frame spacing)pav must be determined by some other means, usually empirically, as for the other formulations above.The shape of the ice pressure is given aset al. [The criticisms presented towards the Winkler foundation model are mostly focused on the foundation model; if the contact is mediated by crushed ice, the foundation model should be different, and if by direct contact, then the foundation model should include contact with elastic solid. The pressure shape has been, however, validated with two test cases; see Riska et al. .(b)et al. [Sanderson  collecteet al. . The areet al.  and Timcet al. .Figure 2.The pressure–area relationship is often called a scale effect as it introduces a geometric scale for ice pressure. As such, the measurements used to derive it do not suggest any pressure distribution on the area and the pressure used in the plots is simply the average pressure on the sensor active area—usually the sensor calibration assumes a uniform pressure distribution. pc and zero on other areas, and that this pressure is measured with sensors having different rectangular active area as shown in et al. [A simple thought experiment shows that the pressure–area relationship is connected with brittle ice failure probably by spalling if it is assumed that the pressure along the direct contact in the test result shown in n et al. .Figure q which essentially represents the averaged pressure over the contact height (q = pc·hc). The pressure–area relationship is converted in this case to a normalized line load versus load length  relationship, which for the rules is assumed to be of form  largest response for each structural element. Offshore regulations, even if using the pressure–area relationships, do not recognize this question of the design load patch being dependent on the particular pressure–area relationship used; see ISO [The application of the pressure–area relationship is not straightforward in design. In principle the local structures, especially steel structures, should be designed so that different areas are used and the response checked with these. The design area (load patch) is the one that gives the largest response. This principle is followed, for example, in the Finnish–Swedish Ice Class Rules  where thund, see )4.4whe see ISO .5.−3 s−1 and decreases then to settle on a constant value at very high strain rates.The description of several ice edge indentation test series with ice pressure measurements has shown the gradual increase of insight into the complexities of describing and modelling ice pressure. The ice failure mode is influenced by the indentation rate (and ice temperature) so that at high indentation rates (or low temperatures) ice failure is brittle. The failure mode influences the pressure magnitude, thus different indentation speed regimes  give presumably different pressures. This indentation speed effect on pressure magnitude is stated to come from ice compressive strength dependence on stress or strain rate; see Michel & Toussaint  and Peytet al. [et al. [Controlled indentation tests in high indentation rates or compressive strength tests at high strain rates are very difficult to conduct. Results from special measurement techniques (Hopkinson split bar) have suggested that the drop in ice strength that is assumed to take place at higher strain rates does not occur; see for example, Shazly et al.  and Orti [et al. .Much ice pressure data come from measurements with ships where the indentation rates are presumably high. These data are, however, somewhat ambiguous, as the indentation rates are high but temperatures are close to melting point. Further, in many ship tests the contact terminates when the relative speed between ice and the ship is zero—this means that the indentation speed varies between the ship speed and zero.Overall, it is not clear if the failure mechanism in compressive tests and in indentation tests is the same. In compressive tests, the test specimen fails at a crack originating from small cracks present on the specimen surface or inside the specimen. The indentation ice failure is, on the other hand, a process where the failure itself is influenced by the process.The introduction of tactile sensors has made the measurement of ice pressure possible on the whole nominal contact area. Much knowledge has been gained from several test series carried out using these tactile sensors. The development of the high-pressure area has received attention but less so the pressure on other areas or pressure in the ductile ice failure regime. This state of knowledge is reflected in the models developed for the ice pressure. The most common model—pressure–area relationship—is empirical. The effect of the failure mode on this relationship is not clear. It would be very interesting if laboratory or other controlled tests were carried out to improve the modelling of the ice pressure.et al. [−1. This would mean that practically all ship–ice interaction occurs in the brittle range. The triangular, pulsing force–time history causes structures to vibrate and is also, for this reason, an important research topic.In most of the indentation tests the force–time history included a triangular pattern. This was, however, missing in some of the level sea-ice indentation tests in Hokkaido. The reason for the pattern is usually placed on brittle ice failure even if the test where this pattern was missing ice failure was described as brittle flaking. The observation of flaking is not very new; see, for example, Matlock et al.  or the iet al. ). Indent