Since the beginning of the twentieth century, the temperature has gradually increased, and it is estimated that by the end of this century the increase in average temperature could be around 4.0°C. In this context of global warming the ice deposits of the Pyrenean caves are undergoing a significant regression. Among this type of caves, that on the Cotiella Massif in the Southern Pyrenees is one of the southernmost studied in Europe. These types of caves house microbial communities which have so far been barely explored, and therefore their study is necessary. In this work, the microbial communities of the Pyrenean ice cave A294 were identified using metabarcoding techniques. In addition, research work was carried out to analyze how the age and composition of the ice affect the composition of the bacterial and microeukaryotic populations. Finally, the  In recent years much research has been done on the microbiology of the cryosphere , especiaThe existence of ice caves in northern Spain has been well known since the pioneering reconnaissance work in the Monte Perdido Massif  in the mid-twentieth century . Since tIn this research work, microorganisms from the A294 cave ice were identified and cultivated at two different temperatures (0 and 4°C), emulating the warming scenario to which these populations could be exposed to. Their metaproteome was studied to understand how they adapt their molecular machinery to increasing temperatures.This work has important implications on the microbiology of the cryosphere and its alteration by climate change, and tries to explain interesting questions such as (i) which microorganisms live in perennial ice caves, (ii) how climate change affects them, (iii) how their molecular machinery adapts to increases in temperature, and (iv) how their metabolism varies in response to temperature rises.2; A294 ice cave  is located within the Armeña cirque in the Cotiella massif  in R  were taken from the ice body in July 2018, using a 9 cm diameter-1-m-long Mark II ice auger (Kovacs) to perform microbiological analyses . The ret.0; clam  with the.0; clam .A summary of the overall experimental strategy is represented in Each ice core was cut into three parts obtaining 3 sampling replicates. Thus, a total of 12 samples: 4 samples  with 3 replicates  were analyzed. Protocols for aseptic sampling and the tracing of potential contaminations were followed. The meltwater samples were either individually used for cultures or filtered through filters with pores of 0.22 μm attached to a vacuum pump in a flow hood, previously sterilized with ethanol. Both filters and meltwater were used for DNA extraction and chemical analysis.4+, NO2–, NO3–, SO42–, soluble reactive phosphorus (SRP), and dissolved organic carbon (DOC) from each filtered meltwater sample were performed by ion chromatography in an 861 Advance Compact IC system . Ions in ice samples were identified and quantified with internal and external standards prepared from Certified Standard Solutions (TraceCERT®) (Merck). Chromatograms were analyzed with the Metrohm IC Net 2.3 SR4 software . Extraction procedures were identical for all samples. DNA concentration was determined using a NanoDrop 2000p. The diversity of uncultured microeukaryotes and bacteria was assessed by Illumina MiSeq 16S and 18S rRNA gene amplicon sequencing. The amplification and sequencing of the V3–V4 regions of the 16S rRNA gene  were performed to identify bacteria. Microeukaryotes were identified by amplification and sequencing of the V4–V5 regions of the 18S rRNA gene .Quality analyses of reads were performed using FastQ Screen software (version 2) . ContigsThis study combines cellular cultures and proteomic approaches, in an attempt to simulate the molecular adaptation of cave microorganisms to warming temperatures. During the summer and part of the fall season, the cave maintains temperatures close to 0°C. The estimated future temperature increase is of 4°C.Both cultures (100 mL) were grown as in 1 via the PRIDE partner repository , rinsed in PBS and stored at −20°C. Afterward, proteins were extracted as explained in previous reports . Proteinpository  with the2). All data were expressed as media ± SD of three sampling replicates. The effects of the chemical composition of glacial ice, as well as the influence of the depth on the microbial community composition, were investigated by a combination of multivariate statistical analysis -Detrended Correspondence Analysis (DCA), Principal Components Analysis (PCA), and Canonical Correspondence Analysis (CCA)- developed with CANOCO 5 software   . The par4+, NO2–, NO3, SO42–, SRP, and DOC in the ice samples were represented in p < 0.05**). These values were also higher in sample M4 (approximately double) than in the results for M1 and M2 , Bacteroidota (21%), and Actinomycetota (16%) were the most abundant groups . The bacteria of the phylum Pseudomonadota were the most abundant in all samples. The Bacteroidota phylum was mainly identified in the most recent samples of the ice body , while the Actinomycetota phylum was found principally in the innermost areas of the ice block . Cyanobacteria were mostly identified in the first layer samples exposed to sunlight. In general, the results obtained in this study were consistent with previous work, in which these same phyla were found in samples from ice caves  were identified. Unexpectedly, thermophilic bacteria such as Thermoanaerobacter and Thermodesulfovibrio were found, especially in M4. These bacteria had already been identified in other ice samples from glacial environments , Nucletmycea (19%), and Chloroplastida (15%)  were obtained, only 142 species were identified, across 13 phyla. Most eukaryotic microorganisms belonged to da (15%) . Most ofs papers . AlthougRhizaria, while the most modern samples  contained microeukaryotes of the phyla Nucletmycea and Chloroplastida. At the genus level, the most abundant in all the samples was the cercozoa Heteromita. This flagellate had already been identified as a very abundant genus in other ice samples  were representatives of the phylum  samples . Its inf samples . Furtherlization . So, theTo determine the distribution of microbial communities in the ice cave samples, several multivariate statistical analyses were carried out . Given tRhodoferax, Sphingobacterium, Oxalobacter, and Luteimonas among bacteria, and by the genus Heteromita among eukaryotes. To find out which factors influence the gradient, various CCA were made using as variables both the age of the samples and the concentrations of the dissolved ions in the ice. Age explained the gradient better in the case of eukaryotes , along with respiration or fermentation, which oxidize organic matter to CO2, stand out. Also relevant in this system is the action of chemolithotrophic organisms, responsible for assimilating the carbon available in the rocks (Streptomyces), is relevant. Additionally, the presence of methylotrophs was detected, which under aerobic conditions catabolize C1 compounds such as CH4 (Methylosinus or Methylotenera). Several proteins that take part in the carbon metabolism, such as the alpha-L-fucosidase (Sphingobacterium), alpha-amylase (Acidipropionibacterium), pyruvate dikinase (Helicobacter), fructose-bisphosphatase (Pseudomonas), acetaldehyde dehydrogenase (Geobacillus), and alcohol dehydrogenase (Moorea) were too identified in the system. Among microeukaryotes, the enzyme orotidine-5′-phosphate decarboxylase from the diatom Thalassospira was found (The identified microorganisms that participate in the carbon cycle obtain this element from either atmospheric COas found .4+ or NO2– to NO3–), denitrification , nitrogen fixation (assimilation of atmospheric N2 so that it can be used by organisms), ammonification (transformation of NH2 from protein groups to NH4+), and anammox (NO2– and NH4+ are converted into gaseous N2). Although most of the nitrifying bacteria are strict aerobes, NH3 can also be oxidized under anoxic conditions in a process known as anammox. In the anammox reaction, ammonia is oxidized with NO2– as the electron acceptor to yield N2. This process is carried out by some members of the Planctomycetota phylum which constitute an unusual group of obligately anaerobic Bacteria. In our study some of these bacteria were identified such as Scalindua brodae . This data and the fact that some parts of the cycle are carried out in abiotic situations, make this cycle more complex. Sulfur can be found naturally in minerals and as gaseous SO2 from volcanic eruptions. Some examples of microorganisms that take part in this cycle could be Thiobacillus, a sulfur chemolithotroph capable of oxidizing H2S or S0 compounds to SO42–; Desulfovibrio or Desulfobacter, which anaerobically reduce SO42– to H2S; and Desulfuromonas, responsible for reducing S0 to H2S. Sulfur assimilation is essential for the synthesis of amino acids with -S- (methionine) and -SH (cysteine) groups.Sulfur is an element with many oxidation states, although only three of them have biological importance  identified the prokaryotic and eukaryotic microbial communities that inhabit a frozen cave in the Pyrenees; (iv) studied the molecular modifications that affect the cellular machinery of these microorganisms; (v) found several proteins and enzymes that show adaptation of cells to a higher temperature; (vi) documented the influence of climate change on biogeochemical cycles; (vii) identified several proteins that are expressed at higher temperatures and could be considered sensors of climate change.This study provides information on the hidden microbial ecosystem in the perennial ice from a Pyrenean ice cave. The results (i) gave important information about a hidden ecosystem that is under imminent risk of disappearing; (ii) simulated the The microorganisms that inhabit remote ecosystems are poorly understood. Further research is needed on the identification of genetic sequences and the unknown and hypothetical proteins they encode. These proteins and enzymes have applications in various industries and are also indicators of the climatic changes to which ecosystems are undergoing.PRJNA663780). The sequences obtained by 18S rRNA sequencing were also deposited in the European Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena/submit/sra/). The assigned ENA accession number was PRJEB32471. The mass spectrometry proteomics data are deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifiers PXD029615, PXD033406, and PXD033535.Sequences obtained by 16S rRNA and 18S rRNA sequencing have been deposited in NCBI Short Read Archive (SRA) (accession number CC and AM designed the study and wrote the manuscript. AM led the scientific project. EG-L, CC, ML, VM-H, and FR-B generated the data and conducted the analysis. CC, MB, and ML participated in the field campaigns. All authors participated in the interpretation of the results and reviewed, commented, and edited the manuscript.


Staphylococcus epidermidis and Escherichia coli in long-time treatments. In a preliminary microdilution test, a MIC value was obtained for thyme EO against S. epidermidis and E. coli. After that, disk diffusion and disk volatilization tests were set up to study the influence of main cultural parameters on EO activity in liquid or vapor phase. Both bacteria were inhibited by thyme and oregano EOs when applied pure (100% v/v) or diluted (75% and 50% v/v): a higher inhibition was observed in a disk diffusion test in which the antimicrobial effect was due to both liquid and vapor phase components. Finally, a comparison with literature data was carried out even if it was not so easy because standard methods are usually modified and adapted to specific case study. For this reason, the results have to be interpreted in relation to the analytical method applied.The resistance to antimicrobials (AMR), especially antibiotics, represents a serious problem and, at the same time, a challenge. In the last decade, a growing interest in the use of essential oils (EOs) as antimicrobial substances was observed. Commercial thyme and oregano EOs are reported to be the main responsible of the oil antimicrobial efficacy against both Gram-positive and Gram-negative pathogenic bacteria. The aim of the present work was to study the efficacy of EOs against  The problem of resistance to antimicrobials (AMR) necessitates the identification of new, safe, and effective substances. In recent years, particular attention was paid to products of natural origin due to their low toxicity, biodegradability, and their broad spectrum of action compared to synthetic antimicrobial substances . In thisEscherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis and are mainly tested with agar diffusion assays [E. coli and S. aureus ; in [S. aureus and S. epidermidis proved to be resistant to the same volume of thyme EO.Essential oils (EOs), secondary metabolites of vegetable origin, are known to be blends of various polar and non-polar chemical molecules such as terpenes, terpenoids, and phenolics . Thanks n assays ,7. As ann assays , 10 µL oThyme EO was described as a natural preservative with antimicrobial properties more evident against Gram-positive bacteria probably due to the absence of the external membrane and a cell wall less resistant to the activity of EOs and their components . It contAs regards the antimicrobial efficacy of oregano EO, it mainly depends on the presence of carvacrol, which can interact with the other oregano constituents to enhance efficacy . As for In a recent review of , methodsAgar methods for the in vitro evaluation of the EO antimicrobial activity are reported in ; among tBased on the previously reported papers, it appears clear that it is very difficult to reliably compare data from the literature because many parameters have an influence on the results due to the strong variance of the used antimicrobial susceptibility testing methods, as reported by .S. epidermidis and E. coli, were used. MIC values were first investigated for commercial thyme and oregano EOs against both bacteria by applying a microdilution test. After that, specific antimicrobial tests were set up to study the influence of main parameters  on EO efficacy in liquid and vapor phases; the obtained values were compared with MIC results. The results obtained with the selected bacteria were compared with available literature data on pathogenic strains to possibly validate them as surrogate microorganisms.In the present work, in order to overcome safety risk during test execution, two Biosafety Level 1 bacteria, Staphylococcus epidermidis and Escherichia coli, and the obtained MIC value was 7.5 µg/mL for both bacteria. On the contrary, for oregano EO, a MIC value was not obtained.The microdilution test was carried out diluting commercial thyme and oregano EOs with 1.5% TEGO; the EO concentrations were in the range 7.5–0.03 µg/mL. At the tested cultural conditions, only thyme EO inhibited the growth of S. epidermidis and E. coli strains. As regard the liquid phase, the agar disk diffusion test was used to examine, at the same time, the EO effect in the liquid and vapor phase. As reported by [In order to distinguish the activity of the EOs in the liquid and in the vapor phase, specific antimicrobial tests were set up against the chosen orted by , the inhorted by ,21; in tS. epidermidis grown at 37 °C is described. The measures of the inhibition halo diameter, checked after 24 and 48 h from the EO addition, are reported in The antimicrobial effect of 100%, 75%, and 50% EOs diluted with 1.5% TEGO on As regards the thyme EO, it was able to influence bacterial growth at all the tested concentrations A. LookinE. coli grown at the optimal temperature of 28 °C. Differently from the results obtained in the presence of S. epidermidis, the haloes did not vary between 24 and 48 h of incubation; for this reason, only the results at 48 h are shown. Moreover, in all the tests performed in the presence of E. coli, a double halo was clearly visible, consisting in an inner portion, where the bacteria are absent, surrounded by a circular crown of “faint” microbial growth .The two EOs were also tested against at 37 °C B, the saS. epidermidis, the incubation was prolonged till 168 h, and the inhibition halo was evaluated each 24 h.The antibacterial properties of thyme and oregano volatile components were detected by means of disk volatilization test carried out in Petri dishes. In this case, the test was conducted with two different EO volumes, namely 3 and 10 µL, and paper disk diameters, namely 5 and 10 mm, respectively. The inhibition zone diameters were always measured at 24 and 48 h; in the tests with the higher EO volume, in the presence of As previously reported for disk diffusion tests, also in this case, between 24 and 48 h of incubation, a progressive reduction of the inhibition zone was observed.S. epidermidis growth in a concentration-dependent way. In the first 24 h, in all the concentrations tested, the behavior of the inhibition halo was similar, with the measured diameters being equal to 26 mm, 20 mm, and 16 mm for thyme and 21.3 mm, 18 mm, and 15.3 mm for oregano EO, respectively. At 48 h, for both EOs, a bacterial growth recovery was observed, and the inhibition halo present at 24 h disappeared. In As it is shown in S. epidermidis with 3 µL of EOs on 5 mm Ø paper disks (0.13 µLEO/mm2) gave a defined inhibition halo only in the first 24 h. To improve the inhibitory effect and to check how long it would be effective, the test was realized with 10 µL of 100, 75, and 50% EO on a 10 mm disk, maintaining a comparable ratio volumeEO/paper disk area (0.15 µLEO/mm2), and the bacterial growth was prolonged for 168 h.The disk volatilization test conducted against As regards the thyme EO, at the two highest concentrations, a progressive halo reduction was measured until the 144th h, and then, stabilization of the inhibition was registered until the 168th hour A. At theConsidering 100 and 75% oregano EO, the curves were overlapped, with an equal slope between 24th and 48th h B. After The antimicrobial efficacy of oregano appears consistently superior to that of thyme at all the tested concentrations.E. coli grown at both 28 °C and 37 °C, utilizing 3 µL of EO on a paper disk of 5 mm Ø. Only 100% oregano EO was effective in the vapor phase; at 28 °C and 37 °C, comparable inhibition haloes  were measured. The results obtained at 37 °C are reported in S. epidermidis.The antibacterial effect of the volatile phase of thyme and oregano EOs was also evaluated against S. epidermidis, the same test was repeated against E. coli at 37 °C. After 48 h of incubation, an inhibition halo was measured for all the tested concentrations. The inhibition zones resulting from 100 and 75% thyme EO were similar, i.e., 38.5 and 35 mm, while that related to 50% was about halved, being equal to 19 mm. 100% Oregano EO allowed to obtain the highest inhibition halo, 43 mm, which is fivefold higher that that associated with 3 µL, while that related to 75% was slightly lower at 37.5 mm. Finally, 50% oregano EO showed an inhibition zone of 33 mm, comparable with those obtained with the highest concentrations of this EO. In Considering the inhibition improvement obtained with a higher EO quantity (10 µL) against E. coli; indeed, the inhibition haloes are always higher than those measured for S. epidermidis  of both EOs: in fact, only the maximum oregano EO concentration was inhibiting. When the higher EOs volume (10 µL) was applied, at all the concentrations, an inhibition was observed, probably in relation to an EO quantity above the threshold value.The disk volatilization test allowed to verify the antibacterial activity of the phenolic EO compounds due to the evaporation at 37 °C . Both thorted by . The antorted by ,28; the orted by . ConcernGenerally, in the literature, Gram-positive bacteria are reported to be more sensitive than Gram-negative ones regardless of the applied EO ; the resFrom the obtained results, it is clear that, with the EO volume being equal and with the paper disk diameter and the bacterial inoculum concentration, the inhibition obtained by both disk diffusion and disk volatilization methods were different. The detected efficacy after 48 h of incubation was always higher in the presence of a direct contact between the microorganism and the EO (the disk diffusion assays) independently of the tested oil and concentration.In the case of the disk diffusion assay, the EO inhibitory effect is due to both the EO liquid phase in direct contact with the bacterial cells and the EO vapors inside the free head space; consequently, the haloes are higher than those obtained with the disk volatilization tests. Diameters measured at 48 h of incubation in the disk volatilization test are always lower than those obtained in the disk diffusion one. This observation could be explained by the fact that, in the last case, the measured inhibition halo was the result of the effect due to volatile compounds and those present in the liquid phase. Considering all the above observations, the application of both agar disk diffusion and disk volatilizations tests is mandatory.B. subtilis, S. aureus, and L. monocytogenes and the Gram-negative E. coli, S. typhimurium, P. aeruginosa, and Campylobacter spp., is high in comparison with applications against non-pathogenic bacteria [The tests applied in the present work are the most frequently found in the literature due to their easy set-up and rapidity of results . They arbacteria . Taking bacteria .5 to 108 CFU/mL [The comparison between results obtained for pathogenic and non-pathogenic strains, although interesting, is not easy to be conducted; in fact, since the tested EO and the bacterial strain were equal, differences in the test set-up are represented by the EO volume, the paper disk diameter, and the CFU/mL of the inoculum. In analyzing the set-up, realized in recent times, from 2019 to 2021, it can be evidenced that microbial inoculum can vary from 108 CFU/mL ,34,35,368 CFU/mL ,32,37,388 CFU/mL ,37,38.Staphylococcus epidermidis ATCC 12228 and Escherichia coli ATCC 8739, came from the Belgian Coordinated Collections of Micro-organisms (BCCM). They were maintained at +4 °C or cultivated at +37 °C on Mueller–Hinton agar  in aerobic conditions.The tested standard strains, E. coli was cultivated at 28 °C, using the same cultural medium. Mueller–Hinton broth  was utilized for the microdilution assays in well plates.As indicated in the following chapters, in some antibacterial tests, Thymus vulgaris EO, OE0970) and oregano  were purchased from Witt Italia Spa Company, Poirino, Italy. They were applied pure (100%) or diluted (75% v/v and 50% v/v) using a 1.5% solution of Polysorbate 20 . This solution was also used for the positive controls to check the potential interaction with bacterial growth.Commercial essential oils (EOs) from thyme  filled with 20 mL of Mueller–Hinton agar, giving a 10 mm high head space corresponding to a free volume of 63.62 cm3  for the control. Plates were sealed off with Parafilm®  and enveloped with PVC film to prevent EO vapor losses.The antimicrobial susceptibility testing for thyme and oregano EOs was carried out by applying the “Agar disk diffusion method”  using a After 24 and 48 h of incubation, the inhibition zones at the two perpendicular diameters, as shown in EO/mm2. An amount of 100 µL of 1.0 × 105 CFU/mL bacterial suspension was spread over the surface of Mueller–Hinton agar. Once set up, Petri dishes sealed with Parafilm®  M and enveloped with PVC film were incubated inverted on top of the lid.The disk volatilization assay was set up to test the antibacterial efficacy of the two EOs in vapor phase. To this purpose, a single filter paper disk was attached to the lid of the Petri dish by means of a bi-adhesive tape. Used disks had a diameter of 5 or 10 mm and were soaked, respectively, with 3 µL or 10 µL of 100, 75, or 50% EO or 1.5% TEGO. The obtained ratio “EO volume/disk area” were similar and, respectively, equal to 0.13 and 0.15 µLAfter 24 and 48 h of incubation, the inhibition zones on the agar surface below the imbibed paper disk were measured at the two perpendicular diameters at the point of complete inhibition as judged by the naked eye, S. epidermidis and E. coli, belonging to Biosafety Level 1, were chosen as indicators of the EO efficacy against a Gram-positive and a Gram-negative bacterium. The effect of commercial thyme and oregano EOs was tested by means of microdilution in broth, agar diffusion, and disk volatilization tests. As regards the first test, an equal MIC value was found against S. epidermidis and E. coli in the presence of thyme EO. Concerning the agar disk diffusion test, both bacteria were inhibited by all the tested EO concentrations. The inhibition obtained in the disk diffusion assays was higher than that in disk volatilization test; in the first case, the antimicrobial effect is due to both the EO components in the liquid and in the vapor phase.In the present work, The comparison of the obtained results with those present in the literature was not so easy; consequently, it can be concluded that when an assessment is needed, it is necessary to interpret the data with a high attention in relation to the applied analytical method used. In fact, even though standard methods are usually applied, numerous modifications are brought to adapt the set-up to the specific case study. Moreover, it is mandatory to evaluate the threshold value of the EOs concentration in the real conditions of application.


ΔΔG = −6.1 ± 0.1 kJ mol−1 for calcium. Site I phosphorylation largely increases both site I and site II affinity for uranyl and uranyl-binding cooperativity. Combining site I phosphorylation and site II Thr7Trp mutation leads to picomolar dissociation constants Kd1 = 1.7 ± 0.3 pM and Kd2 = 196 ± 21 pM at pH 7. A structural model obtained by MD simulations suggests a structural role of site I phosphorylation in the affinity modulation.Uranyl–protein interactions participate in uranyl trafficking or toxicity to cells. In addition to their qualitative identification, thermodynamic data are needed to predict predominant mechanisms that they mediate in vivo. We previously showed that uranyl can substitute calcium at the canonical EF-hand binding motif of calmodulin (CaM) site I. Here, we investigate thermodynamic properties of uranyl interaction with site II and with the whole CaM N-terminal domain by spectrofluorimetry and ITC. Site II has an affinity for uranyl about 10 times lower than site I. Uranyl binding at site I is exothermic with a large enthalpic contribution, while for site II, the enthalpic contribution to the Gibbs free energy of binding is about 10 times lower than the entropic term. For the N–terminal domain, macroscopic binding constants for uranyl are two to three orders of magnitude higher than for calcium. A positive cooperative process driven by entropy increases the second uranyl-binding event as compared with the first one, with ΔΔG = −2.0 ± 0.4 kJ mol Uranium is a radioactive metal, naturally present in the Earth’s crust, which is exploited for the nuclear industry and for military applications. The risk of its distribution in the environment is associated with mining activities, uranium processing, leaching of radioactive wastes or accidental situations in nuclear power plants. Uranium presents radiological and chemical toxicity to living organisms ,2,3 and 22+ cation in biological media, and its coordination geometry with five to six ligands in the equatorial plane is similar to the Ca2+ coordination geometry of seven ligands arranged in pentagonal bipyramidal structures  is the free metal concentration , according to Equation (4) , K1 and K2 were calculated for each titration with a numerical Newton–Raphson method, by taking in account all the complex-formation equilibria, the acid-dissociation constants of IDA and the fluorescence variation (Equation (2) or Equation (4), depending on the peptide), as detailed in Supporting information.In the presence of multiple competing reactions involving M (formation of both uranyl–IDA complexes and uranyl–peptide complexes), the analytical estimation of the free M concentration and, consequently, of KI and KII), are related to macroscopic ones (K1 and K2) according to equations:To evaluate the peptide inter-site cooperativity, it is necessary to characterize the apo- and holo-peptide, as well as its intermediate microstates. In a two-site peptide, four microstates are possible with the population of each depending on the microscopic thermodynamic constants of the system C. MicrosThe inter-site cooperativity was quantified using the parameter ΔΔG, that is, the difference in free energy between uranyl binding to the apo-peptide and uranyl binding to the peptide already loaded with one uranyl ion, as described in . ΔΔG alli is the change in the number of moles of each species in solution, and ∆Hi is the respective molar binding enthalpy.For ITC measurements, we used the same equilibria described for the fluorescence experiments (Equations (1)–(3)). For the one-site peptides, experimental data were fitted to Equation (8):I and ΔHII are the molar enthalpies associated with the binding of the metal ion at site I and site II of the apo-protein, respectively; ∆nMP,I and ∆nMP,II correspond to the change in number of moles of the two possible mononuclear complexes  at each injection; ∆nM2P is the change in number of moles of the dinuclear complex, M2P, at each injection; ΔHc is the molar enthalpy associated with the cooperative effect [I, ΔHII and ΔHc are microscopic parameters. The change in the number of moles, ∆n, between the injection j and the injection j − 1, is related to the concentration of the corresponding species and, consequently, to the equilibrium constants KI, KII or K2: For a cooperative binding in a two-site macromolecule, the heat released or adsorbed at each injection is defined by the equation:e effect ,43. ΔHI,I and KII and the macroscopic constant K2:Using Equations (9)–(12), it is possible to evaluate the heat of reaction as a function of the microscopic constants KII and ΔHII were evaluated from the ITC curves of the one-site peptides, KI, ΔHI and K2 were obtained as parameters of the fit for the ITC curves of the two-site peptides. The macroscopic constant K1 was evaluated from the obtained KI and KII, according to Equation (5).Concentrations of M, P and MP were evaluated using the Newton–Raphson method. KThe entropies for the binding reaction were calculated from measured values of K and ΔH of the corresponding process, by means of Equation (14).I = RTlnKI + ΔHI), the entropy of uranyl binding to site II of the apo-protein (TΔSII = RTlnKII + ΔHII) and the entropy associated with the inter-site cooperativity (TΔSc = RTlnKc + ΔHc). Kc, the cooperativity constant, is an equilibrium constant calculated as KI,II/KI = KII,I/KII [For the two-site peptides, we evaluated the entropy of uranyl binding to site I of the apo-protein . The structure of CaM1 Y I-II P was completed and refined with the molecular dynamics program CHARMM [all36 for proteins [δ atom, aspartate and glutamate residues were kept unprotonated (negative charge) and all arginine and lysine residues were positively charged. Missing hydrogen atom coordinates were built with CHARMM and the structure was energy minimized down to a gradient of 0.1 kcal mol−1 Å−1 with the ABNR algorithm and subject to harmonic restraints on heavy atoms with force constant of 5 kcal mol−1 Å−2. Ten sodium counterions were added to neutralize the system. A 66 × 48 × 48 Å3 box of TIP3P water (5088 molecules), sufficient to accommodate the protein maintaining all atoms 10 Å away from the boundaries, was energy minimized and equilibrated. The CaM structure was then immersed into that water box , energy minimized allowing the box size to vary, and pre-equilibrated with 10 ps Langevin dynamics at a temperature of 300 K with restraints on all backbone atoms of the protein to yield the structure “NonOe”. The initial distances between site I Thr9P P atom and the U1 and U2 atoms were 6.99 Å and 11.67 Å for site I and site II, respectively, and the N-Cα-Cβ-Oγ dihedral angle of Thr9P was −164.7°. Looking for a new conformation where the phosphoryl group would interact with uranyls, NOE-type distance restraints were applied using a bi-harmonic potential with a force constant of 20 kcal mol−1 Å−2 between the site I Thr9P P atom and uranyl U1 atom of site I (above 3.0 and below 5.0 Å) as well as uranyl U2 atom of site II (above 3.0 and below 9.0 Å)The initial model was built from the three-dimensional X-ray structure of m CHARMM . The allproteins  was usedP was −92.5°.The system was energy minimized and subject to 200 ps Langevin dynamics at 300 K to yield the structure “With-nOe”. After the restrained distance dynamics, distances between P and U decreased to 5.18 Å and 9.40 Å, respectively, for site I and site II and the N-Cα-Cβ-Oγ dihedral angle of site I Thr9NonOe and With-nOE systems were translated into Amber format with the ff14SB force field [P. The uranyl parameters used for the polarizable MD simulations are taken from [Pτ = 2.0 ps) dynamics from the minimum energy structure followed by 100 ps NPT dynamics, all with polarization. A time step of 1 fs, a temperature of 300 K using Berendsen control and a cutoff for the non-bonded interactions set to 12 Å were employed. Particle Mesh Ewald was used for electrostatics with Car–Parinello scheme for dipoles, dipole temperature constant dipτ = 9.9 ps and order 5 for B-spline interpolation. Finally, 12 ns NPT MD simulations were run with the same conditions.The Amber program was then used to run production dynamics runs using a polarizable force field . The prece field . A TPO rken from . The AmbP at site I.To study the binding properties of uranyl to CaM site II, the effect of cooperativity between binding sites I and II of the CaM N-ter domain and the joint effect of cooperativity and phosphorylation at site I, eleven mutants were produced . Five “oSite I Tyr7 is an efficient probe of uranyl binding at both sites I and II, since we observed a decrease in its fluorescence emission (302 nm) upon uranyl binding at site II in the CaM Y II and CaM1 Y II peptides and at site I and site II in the two-site peptides. For the peptides having a tryptophan at position 7 of site II, CaM YW II, CaM1 YW II, CaM YW I-II, CaM1 YW I-II and CaM1P YW I-II , the met22+ were satisfactorily fit using binding isotherms, which represent the formation of 1:1 complexes  with UOomplexes . All theous work .1 = KII).For the one-site peptides, since only site II is able to bind the metal ion, the measured macroscopic constants correspond to the microscopic constants of the active binding site (KII = (3.7 ± 0.5) × 106 M−1 and KI = (4.0 ± 0.2) × 107 M−1, respectively . The uranyl-binding affinity of site II is not significantly affected by the site I Thr10Ala and Lys11Ala mutations, with a value of KII = (3.5 ± 0.7) × 106 M−1 for CaM1 Y II (KdII = 286 ± 47 nM).The binding affinity of site II for uranyl is one order of magnitude lower than that of site I with KII = (5.1 ± 0.5) × 106 M−1 for CaM YW II and KII = (5.9 ± 0.6) × 106 M−1 for CaM1 YW II, respectively  measurements of UO1 were in good agreement with those obtained from spectrofluorimetric analysis (−1), and for CaM YW II and CaM1 YW II (−8.9 ± 0.7 and −15.0 ± 2.5 kJ mol−1). The entropic term for each uranyl–peptide interaction was calculated using Equation (14). As previously pointed out for Ca(II) binding to one-site peptides [−1. The values of the macroscopic constant Kanalysis . The entanalysis . On the peptides , in the 22+ binding to two-site peptides were analyzed using a dinuclear model, providing two macroscopic constants, K1 and K2  as the microscopic constants KII in Equations (5) and (6), together with the experimental macroscopic K1 and K2 constants obtained for the corresponding two-site peptides. This strategy is justified by the fact that the substituted residues at site I Ala1 and Ala3, which impair metal binding at site I, were shown not to be involved in the site I–site II interaction [I thus calculated for CaM Y I-II and CaM1 Y I-II (3.1 ± 0.3 × 107 M−1 and 2.7 ± 0.3 × 107 M−1) are consistent with the KI previously measured for the corresponding one-site peptides CaM Y I and CaM1 Y I . 22+ binding to the CaM N-ter domain, with ΔΔG = −2.0 and −1.8 kJ mol−1 for UO22+ binding to CaM Y I-II and CaM YW I-II, respectively. Consequently, the affinity of the binding sites increases when one metal ion is already bound to the peptide . Even if this positive cooperative effect is evident, the ΔΔG obtained for UO22+ binding is three times lower than that obtained for Ca2+ binding to the same peptides  is associated with UOectively ).c  and (13) allowed us to evaluate KI, ΔHI for site I, the macroscopic constants K1 and K2 and, finally, the enthalpic contribution of the inter-site cooperativity, ΔHc . For CaM YW I-II and CaM1 YW I-II, the difference between ΔHI and ΔHII is smaller, because ΔHII is slightly higher, and the enthalpy associated with cooperativity is much smaller, as compared to peptides without the tryptophan with ΔHc = 3.8 ± 0.1 and 8.9 ± 0.3 kJ mol−1 (The obtained macroscopic constants are in good agreement with spectrofluorometric results . ResultskJ mol−1 . AccordiI) ranging between 3.2 kJ mol−1 K−1 and 44.5 kJ mol−1 K−1 were obtained. The calculated values of ΔHI and the enthalpic contribution to the Gibbs free energy are larger than the entropic term, except for the two-site peptide CaM1 YW I-II  and Tyr7 (site I) are part of an inter site β-sheet structure, which can tune the interaction between the main chain carbonyl of both the residues and the metal ion [II were almost equivalent in all the variants, and only slightly lower in the peptides bearing the tryptophan residue  is favorable for all the investigated peptides. This result is consistent with the assumption that CaM mutants undergo conformational changes upon binding the first uranyl ion, which favor the binding of a second ligand, which happens as well for Ca2+ binding to CaM [Entropy values were evaluated from the corresponding ΔH and equilibrium constants, using Equation (14). For uranyl binding at site I, entropic terms  increases the affinity for uranyl of both sites I and II. At pH 6, we obtained macroscopic stability constants for the phosphorylated variants from 5- to 14-fold higher than the constants of the corresponding non-phosphorylated peptides  × 107 M−1 at pH 6  × 106 M−1 (Kd = 270 ± 42 nM). At pH 7, the affinity of CaM1 Y II P for uranyl further increases, with KII = (1.8 ± 0.4) × 108 M−1  × 108 at pH 6 is very close to the experimental value of (2.0 ± 0.4) × 108 previously reported for the CaM1 Y I P peptide [The microscopic constants and ΔΔG for the two-site phosphorylated peptides were evaluated using their macroscopic constants, K peptide .II value was used also for the evaluation of the microscopic constants of CaM1 YW I-II P. As detailed above, the effect of site II Trp7 insertion on uranyl binding (KII 1.4 times higher with site II Trp7) is low compared to the effect of site I phosphorylation (KII circa five times higher). Therefore, we consider that the CaM1 Y II P macroscopic constant could represent a good estimation of KII for CaM1 YW I-II P as well.CaM1 Y II P does not have the site II Thr7Trp mutation, but its K−1, respectively, as compared to (−2 ± 0.4) and (−1.8 ± 0.3) kJ mol−1 for the non-phosphorylated peptides.The ΔΔG values obtained for the two phosphorylated peptides show a significant enhancement of cooperativity in the presence of the phosphoryl group. The ΔΔG values for CaM1 Y I-II P and CaM1 YW I-II P are (−3.4 ± 0.4) and (−4.7 ± 0.4) kJ mol1 two orders of magnitude higher, and K2 almost 10-fold higher, as compared to pH 6  × 1011 and K2 = (5.1 ± 0.6) × 109 correspond to dissociation constants in the picomolar to sub-nanomolar range, with Kd1 = 1.7 ± 0.3 pM and Kd2 = 196 ± 21 pM, respectively. The two phosphorylated two-site variants, CaM1 Y I-II P and CaM1 YW I-II P, thus present a very high affinity for uranyl at pH 7.For CaM1 YW I-II P, similar conclusions can be proposed, although the value of ΔΔG approximately doubles at pH 7. This value depends, however, on the microscopic K2 solution  by performing spectrofluorimetric titrations of CaM1 Y I-II P and CaM1 YW I-II P with a CaCl22+ in water. As detailed in the P to the uranium atoms U1 and U2 of site I and site II, respectively. Large distances were obtained in a structure without constraints. Shorter P-U distances and a different N-Cα-Cβ-Oγ dihedral angle for site I Thr9P were obtained in a structure with NOE-type distance restraints applied between the Thr9P P atom and U1 and U2. Production dynamics were run with the Amber program, starting with the structures without constraints (“No-Restr”) or with constraints on the initial distance P-U1 and P-U2 (“With-Restr”).To better understand the influence of phosphorylation on the affinity of uranyl to both site I and site II and the strong increase in affinity at site I of the phosphorylated two-site peptide at pH 7, as compared to phosphorylated site I only, we have modelled the complex formed by CaM1 Y I-II P with two UOcirca 6 ns and the total energy followed the same evolution 2 system is stable in two different conformations, with different rotamers of site I Thr9P. The structures after 12 ns MD of the “No-Restr.” and “With-Restr.” systems are compared in P is clearly closer to the U atoms in the “With-Restr.” System, corresponding to the second rotamer mentioned above. This structure could explain, at least in part, the experimental results showing an effect of the phosphoryl group on both site I and, to a lesser extent, site II. Interestingly, the inter-site β-strand motif is longer in the “With-Restr.” simulation  = 270 ± 42 nM, which is 200 times lower than the dissociation constant measured for calcium (Kd(site II) = 55 µM [vacuum and indeed, time-resolved laser-fluorescence spectroscopy and extended X-ray-absorption fluorescence spectroscopy strongly indicated the presence of a hydroxyl ligand [We showed that the CaM site II has a 10-times lower affinity for uranyl than site I, with K CaM Y I . This co = 55 µM ). For si = 55 µM . These Ml ligand . The seqI,II > KI and KII,I > KII for all the investigated peptides. To the best of our knowledge, only few studies have been performed on metals’ cooperative binding to calmodulin or calmodulin-derived peptides [A notable improvement of the CaM N-ter domain ability to bind uranyl is observed when both binding sites are activated. Obviously, in this case, each peptide is able to bind two uranyl ions instead of one, but on top of that, a cooperative effect increases the affinity of each binding site, with Kpeptides ,50,51,52−1, while we previously found ΔΔG = −6.1 kJ mol−1 and ΔΔG = −6.9 kJ mol−1, respectively, for calcium binding to these peptides [2+ and Mg2+ binding to a peptide containing sites II and III of CaM showed that the α-helical content of the peptide increased by 12–24% in the presence of Ca2+, while no conformational change was observed for Mg2+ binding [A positive cooperative effect is observed for uranyl binding to the non-phosphorylated peptides, even if it is lower than that observed for calcium binding. The ΔΔG parameter for CaM Y I-II and CaM YW I-II lies between −2.0 and −1.3 kJ molpeptides . The coo binding . The sta binding .The conditional constants recently reported for calcium binding  are at lI and KII, are almost twofold higher for the peptides having the site II Thr7Trp mutation, compared to the corresponding peptides without this mutation. This small affinity improvement for both sites was already observed for calcium binding to the same peptides [A modest contribution to the improvement of uranyl affinity for CaM N-ter domain is given by the insertion of a tryptophan residue at position seven of site II. In fact, our results show that this mutation slightly increases the affinity of both sites of the CaM N-ter domain for uranyl. Both microscopic constants, Kpeptides  and it wI and ΔHII < 0). However, only binding at site II is clearly entropy driven, showing small values of ΔHII compared to TΔSII. The ΔHII values for uranyl binding are similar to those obtained for calcium binding to the same site [Calorimetric measures provided further information on the thermodynamics at the microscopic level of uranyl binding to the calmodulin N-ter domain. Both bindings at site I and site II are exothermic  complex stability is obtained with site I Thr9 phosphorylation. We previously showed, by FTIR and EXAFS analysis of the one-site peptide CaM1 Y I P, that the phosphoryl group is directly involved in uranyl coordination [32- at pH 7. The most significant improvement of both mononuclear (MP) and dinuclear  may extend the inter-site antiparallel β-sheet motif that involves residues at positions seven and eight of the metal-binding loops. The change in uranyl-binding cooperativity may be explained by this structural difference, which could modify the peptide secondary structure upon first metal binding, leading to a more accessible conformation for binding a second metal ion.The insertion of a phosphoryl group can lead to protein conformational changes that improve the affinity at the two sites. Such an effect may also explain the higher inter-site cooperativity ΔΔG for phosphorylated peptides at pH 6 as compared to the non-phosphorylated peptides . By site-directed mutagenesis, we could produce CaM variants with one and two uranyl-binding sites and evaluate both macroscopic and microscopic (site-specific) thermodynamic parameters associated with uranyl binding to site I and site II of the N-ter domain. This strategy allowed us to modulate the affinity of the CaM N-ter domain for uranyl, by identifying structural factors governing this interaction at the molecular level. We showed that the phosphorylation of Thr9 at site I increases the natural inter-site cooperativity of calmodulin N-ter domain, and leads to peptides able to strongly complex uranyl, with picomolar dissociation constants and a high selectivity towards the cognate ion calcium. These findings give further insights into the principles of uranium interaction with proteins, and its possible toxicological effect on biological targets. Our approach could also be a starting point for developing uranyl-selective biological tools, based on mutated peptides, for bioremediation and biodetection purposes.


Ice on aircraft wing changes the aircraft aerodynamic shape, and has negative effects on flight dynamic characteristics, seriously threatening flight safety. Plasma ice shape regulation is a new de-icing method. Plasma actuator produces an apparent thermal effect, which is designed to dissolve the continuous ice into intermittent ice pieces. How to achieve the optimal regulation ice shape to improve the flight dynamics characteristics under icing conditions is a technical problem restricting the application of this method. A simulation ice shape based on previous ice tunnel experiments and a scale model of swept wing were established. The aerodynamic parameters of no ice, full ice, and two regulation ice schemes were obtained based on wind tunnel. Six degrees of freedom flight dynamics model was established, and flight simulation had been carried out. As the analysis of trim characteristics, dynamic stability, and maneuverability, flight dynamics characteristics were better improved when the ratio of ice width to the mean aerodynamic chord was 0.15. The evaluation method of plasma ice shape regulation schemes was proposed. The proposed method, which can compare and optimize the arrangement of plasma actuators, realized the optimal regulation ice shape on the premise of balancing flight safety and energy consumption. In severe cases, it may even lead to losing control of the aircraft.3. In recent years, many serious flight accidents have been caused by icing4. Russian Saratov airliner An-148 got the wrong speed information for pilots due to icing on the airspeed tube, eventually crashed in 2018. General Aviation B-10GD Model Precipitation Enhancer crashed because of propeller icing in 2021 and five crew members were killed. Research showed that UAVs were more sensitive to ice conditions than that larger and faster manned aircraft and aircraft icing was one of the major hazards for airplane operation during takeoff and climbing missions at low temperature regions5,6. The aerodynamic characteristics of the aircraft are greatly affected by the ice accretion problems7. Innovative de-icing techniques are urgently needed.Ice on an aircraft wing changes the aerodynamic shape8. Compared with vibration, heating de-icing system is mostly used for small components such as airspeed tubes, which has a slow response and low thermal efficiency9. Meanwhile, the low reliability of the vibration de-icing system would greatly damage the original aerodynamic shape of the aircraft10, and reduce the aerodynamic performance of the wing11. The de-icing technology of plasma has the advantages of low power consumption12, quick responses, and simple geometry13. Experiments have shown that its total energy efficiency is better under certain conditions14. Meng Xuanshi found that plasma de-icing method can improve the loss of aerodynamic characteristics15. Cem Kolbakir found that actuators in the stream wise laid out had a better de-icing effect than the actuators in the span wise layout. The results showed that plasma actuators in stream wise lay out not only prevented ice accretion near the airfoil leading edge but also allowed plasma-induced surface heating to further downward convection to prevent ice accretion near the airfoil trailing edge18. Rodrigues F. F found that de-icing technology based on plasma excitation had a better effect. The team studied the influence of the thickness of the exposed electrode on the performance of plasma actuators in the de-icing system, which found that the actuators with thicker exposed electrodes can induce higher average surface temperature, compared with the traditional actuator21. Isaac Ball found that the water film formed a virtual cathode on the surface of the plasma actuators, resulting in higher gas temperature and a larger heating area22. The ice shape regulation experiments with simulation ice were carried out. The results were consistent with the ice wind tunnel experiment. Compared with continuous ice, WU Yun found that plasma ice shape regulation can significantly improve aerodynamic performance, which can save about half of electric power and is very beneficial for application23.At present, the two common de-icing techniques are heating and vibration24. However, due to the residual energy limit of the aircraft, ice-tolerant flight still exists25. The application of the plasma ice shape regulation method aims to improve the flight d1ynamic characteristics. The above scholars have studied the mechanism of plasma de-icing technology but did not carry out the flight dynamics characteristics. Obtaining the variation law of flight dynamics characteristics under plasma excitation is the premise of the application of this method. It is also the basis for optimizing the plasma ice shape regulation. Overall, this paper mainly studied: aerodynamic parameters under two kinds of regulation ice shapes based on wind tunnel experiments; six-degree-of-freedom flight dynamics model of the background aircraft is established; stimulation and comparison of the trim characteristic, dynamic stability, and maneuverability.Under certain conditions, the plasma actuator has a significant thermal effect. Its application has become a promising technique in the field of de-icing in the future23. The measurement of aerodynamic parameters of a wing with natural ice accretions is very difficult and expensive26. Icing wind tunnels are a critical engineering tool for simulating natural ice accretion on aircraft surfaces27. Our group has realized the ice shape regulation in the previous plasma de-icing experiment. Based on the ice wind tunnel, a preliminary exploratory experiment was conducted to study the plasma ice shape regulation under two meteorological conditions. As shown in Fig. 28.Plasma ice shape regulation mainly uses the thermal effect of the actuator to cut the continuous ice into intermittent ice. Figure 29, a wind tunnel test with simulation ice based on a typical icing configuration was carried out. The aerodynamic parameters were analyzed under different conditions. In addition, due to the intense coupling relationship between flight parameters, the measurement of de-icing technology needs to be reflected in the flight dynamic characteristics. Aerodynamic parameters provide input for the simulation analysis of flight dynamic characteristics below.How to select the optimal ice shape regulation parameter which achieves the best de-icing effect is a new problem. Due to the high cost of the ice wind tunnel test30, the laws of swept wing icing were obtained by numerical simulation. The results showed icing thickness gradually increased from the wing root to the tip, which provides ice shape model support for the ice shape regulation experiment of the background aircraft wing scaling model. As shown in Fig. 23. As shown in Fig. 2. 23.In referenceAs shown in Fig. Ice shape regulation experimental system includes a low-speed backflow wind tunnel, swept wing scale model, 3D simulation ice shape, and six-component force balance. Figure mentclass2pt{minimAs shown in Fig. The experimental schemes are shown in Table 31. As shown in Table The reproducibility parameters mentclass2pt{minimAs shown in Fig. The drag coefficient The pitching moment coefficient The lift-resistance ratio is This section analyses lift-resistance characteristics and pitching moment characteristics under four states. It can show that the method of ice shape regulation can improve the aerodynamic efficiency under the full ice state and slow down the damage to the aerodynamic characteristics under the ice accretion state. When the scheme of regulation ice A (0.15) is selected, the aerodynamic characteristics are better improved compared with the scheme of regulation ice B (0.2).Figure 32:Taking the longitudinal Dynamics Characteristic as an example, the mathematical model of background aircraft is constructed as shown in Eqs. –5)32:2\32:2\5)32x and z direction. In Eqs. , the elevator angle, and the throttle equivalent. The elevator angle for balancing refers to the angle of the elevator required to maintain the aircraft balance. The AOA for balancing refers to the angle required to maintain the pitch balance. The throttle equivalent for balancing refers to the thrust equivalent provided by the engine during balancing, which represents the position of the throttle rod. This section calculated the AOA, elevator angle, and throttle equivalent for balancing under four states of no ice, full ice, regulation ice A and B. And the trim characteristics were compared and analyzed under four states.In order to ensure the stable level flight of the aircraft, the force and torque acting on the aircraft must be balanced. The process of a stable balance of the aircraft is called balancing. At present, there are two ways to maintain the balancing state of the aircraft in horizontal flight. To make longitudinal torque zero, one is that pilot manipulates the control stick changing the size and direction of the pitching torque. The other is using trim tab, and the pilot operates without a control stick34:35.In order to characterize the icing severity under different regulation ice shapes, the concept of icing factor . In Eq. , the aerFigures Compared with the no ice state, the AOA The results show that the AOA, elevator angle, and throttle equivalent for balancing is reduced by the decrease of dimensionless ice shape value. Especially, the scheme of the regulation ice A (0.15) is selected, which can better improve the longitudinal trim characteristics of the aircraft. The efficiency of balancing is enhanced.The flight height was set to 400 m. The speed was set to 0.21 Ma. Under this state , the AOA Under the disturbance with However, compared with the full ice state, the peak value of the initial AOA Under disturbance, the results show that the oscillation period and amplitude of the short/long period mode parameters are significantly reduced and can converge back to a stable state faster by ice shape regulation. Especially, the scheme of the regulation ice A (0.15) is selected, the longitudinal dynamic stability characteristics are better improved compared with the full ice state.A step signal with width of 5 s and amplitude of 2° was imposed on the elevator. The dynamic response including pitching angle .In the future, schemes of different regulation ice shapes will be studied under different airfoils and different icing conditions. More detailed work will be studied on the laws of ice shape regulation schemes with aerodynamic and flight dynamics characteristics and flight quality. The essay is supposed to offer technical support for plasma ice shape regulation technology and reference for disaster mechanism of icing.


Cyclin-dependent kinases (CDKs) are presumed to control the cell cycle by phosphorylating a large number of proteins involved in S-phase and mitosis, two mechanistically disparate biological processes. While the traditional qualitative model of CDK-mediated cell cycle control relies on differences in inherent substrate specificity between distinct CDK-cyclin complexes, they are largely dispensable according to the opposing quantitative model, which states that changes in the overall CDK activity level promote orderly progression through S-phase and mitosis. However, a mechanistic explanation for how such an activity can simultaneously regulate many distinct proteins is lacking. New evidence suggests that the CDK-dependent phosphorylation of ostensibly very diverse proteins might be achieved due to underlying similarity of phosphorylation sites and of the biochemical effects of their phosphorylation: they are preferentially located within intrinsically disordered regions of proteins that are components of membraneless organelles, and they regulate phase separation. Here, we review this evidence and suggest a mechanism for how a single enzyme’s activity can generate the dynamics required to remodel the cell at mitosis. It is unclear how a single kinase’s activity can phosphorylate the plethora of CDK substrates ,2,3,4 anStudies from the Loog lab suggested that the timing of a substrate’s phosphorylation (and thus ordering of the cell cycle) depends on differential substrate recognition by different CDK/cyclin complexes and is encoded in a substrate docking site in the cyclin, with a built-in mechanism of lower specificity of early CDK/cyclin complexes towards mitotic targets . For mulSpecificity is also dictated by where a particular substrate is present inside the cell and when a given complex is present and active. This was illustrated by the study of Pagliuca and colleagues , which aTo understand this redundancy, it is important to consider the general biochemical and biophysical effects of protein phosphorylation and the structural nature of substrates. Analyses of CDK1 phosphosites in budding yeast ,29 proviThat phosphorylations or other post-translational modifications  preferentially locate to intrinsically disordered regions (IDRs) of proteins is not a new concept . In 2004Several questions emerge from these results. Are CDKs distinct or similar in this respect to other kinases? And what about other cell cycle kinases? Or MAPK kinases, which share with CDKs the proline-directed consensus site, but regulate signalling pathways rather than cell cycle processes? Is the preferential phosphorylation of IDRs by CDKs a conserved phenomenon? Is the observed enrichment of phosphorylations in disordered regions a simple consequence of the compositional bias whereby serines and threonines, as well as prolines, are in general enriched in intrinsically disordered regions? And are the biochemical and biophysical effects of CDK-mediated phosphorylations on diverse substrates similar? Recent work by Valverde, Dubra and colleagues addressed these exact questions . Xenopus laevis, whose early embryonic divisions are naturally synchronous—a system devoid of artifacts due to cell synchronisation approaches. High-resolution mass spectrometry analysis of phosphorylations throughout early divisions of single X. laevis embryos as well as during the DNA replication time course and in mitosis in Xenopus egg extracts provided an unbiased map of dynamic cell cycle phosphorylations. A quarter of proteins with such dynamic sites are homologues of known human CDK targets, while motif analysis revealed that more than half of the dynamic phosphosites comply with at least the minimum CDK consensus motif, underlining the predominance of CDK phosphorylations in the regulation of the cell cycle. Correction for compositional bias established that, indeed, despite a higher number of serines, threonines and tyrosines located to IDRs, CDKs preferentially target sites in disordered versus structured regions, both in yeast and humans. The same was true for Plk1, Aurora kinases, DYRK, NEK and MAPK sites, and thus is not specific to CDKs. However, it emerged that human and yeast CDK targets, as well as Xenopus dynamic cell cycle phosphoproteins are twice as disordered on average as other phosphoproteins  allows cells to organise molecules  into three-dimensional compartments, so-called membrane-less organelles (MLOs), that are found both in the nucleus  and in the cytoplasm (including stress granules and P bodies) and participate in a variety of biological functions, such as RNA metabolism, transcription, nucleocytoplasmic transport, DNA damage, stress responses and others ,51,52. MThis applies to CDKs. At the entry to mitosis, CDK1 phosphorylates many nucleoporins . NUPs fThe examples described above demonstrate that CDKs can regulate the organisation and assembly of specific MLOs. However, given the predominance of CDK-mediated sites among cell cycle phosphorylations, the general high intrinsic disorder of CDK substrates , and theTo test the hypothesis that CDK might act as general MLO dissolvase, Valverde, Dubra and colleagues  assembleThese results are consistent with another recent study , based oThe existing data thus provide evidence that CDKs have a much less constrained choice of targets than previously thought, and they differ from other phosphoproteins in that they are more disordered and a large fraction of them localise to MLOs. CDK-mediated phosphorylation appears to act as a key regulator of homotypic interactions of IDRs, and it can either favour or inhibit phase separation. Consequently, changes in the ratio of the overall CDK to phosphatase activity can provide a general mechanism for initiation of various processes during the cell cycle, with its highest level triggering a switch-like cell reorganisation in mitosis and disassembly of most phase-separated condensates. There is therefore no need to invoke a highly complex code of docking motifs and phosphosites on the substrate, since the overall phosphorylation of multiple sites modifies protein–protein interactions, and the absolute kinetics of each phosphorylation is probably not critical. Furthermore, since there is double negative feedback between the CDK and its opposing phosphatase, a small relative increase in the overall CDK activity can trigger a complete change in substrate phosphorylation. This is consistent with recent data (see above) on the synchrony of mitotic phosphorylation in which no differences in timing of phosphorylation of highly diverse substrates are observed. Our model also provides a mechanistic explanation for how a single CDK-cyclin’s activity can drive a fairly normal cell cycle, and it suggests that CDKs were selected over evolutionary timescales to be rather un-specific kinases that are very efficient at phosphorylating disordered proteins, since much of the reorganisation that occurs in a dividing cell relies on such proteins. Other mitotic kinases  may have evolved to act in a similar manner but targeting IDRs that have more acidic regions around the phosphorylation site, that CDKs cannot readily phosphorylate.


Relapsed/refractory peripheral T‐cell lymphoma (R/R PTCL) has a poor prognosis. Romidepsin (Ro) and brentuximab vedotin (Bv), combined with ifosfamide, carboplatin, and etoposide (ICE) has not been significantly studied in PTCL.We report outcomes of Bv‐ICE in CD30 (+) and Ro‐ICE in CD30 (−) R/R PTCL treated in “Blinded for peer review” Cancer Center.We retrospectively identified R/R PTCL patients treated with BV‐ICE or romidepsin‐ICE from May 2016 to September 2019. Out of 13 R/R PTCL patients, 6 were treated with Bv‐ICE and 7 with Ro‐ICE. Bv‐ICE had an overall response rate (ORR) of 66.7%, with all the patients achieving a complete response. ORR was 71.4% for Ro‐ICE with 57.1% of patients achieving a complete response. Two patients treated with Bv‐ICE and three treated with Ro‐ICE received transplantation.In our experience, treatment with Bv‐ICE and Ro‐ICE based on CD30 positivity is feasible and effective to treat patients with R/R PTCL. Our primary objective was to assess the efficacy in terms of ORRs of both salvage chemotherapy regimens. Secondary objectives included duration of response (DOR), hematopoietic cell transplant rate and toxicities.We queried the medical record database for age, sex, therapy response, date of death/last follow‐up, transplant date as well as laboratory studies including hemoglobin, platelet count, white blood cell count, creatinine, bilirubin, and liver enzymes. We used frequencies and medians to analyze the quantitative variables. Response was assessed by radiologic evidence (CT or PET scan) showing a decrease of tumor burden or resolution of the malignant lesions. Toxicities such as cytopenias, liver and renal function abnormalities were defined per the Common Terminology Criteria for Adverse events version 4.0. The study protocol was reviewed and approved by The Houston Methodist Hospital Institutional Review board.2 (continuous infusion over 24 h on day 2), carboplatin with an area under the curve of 5 (day 2) and etoposide at 100 mg/m2 (days 1–3). Mesna was given concurrently on day 2. Brentuximab vedotin IV was given at 1.5 mg/kg (days 1 and 8) if CD30 positive and romidepsin IV at 8 mg/m2 (days 1 and 4) if CD30 negative. Notably, dose modifications were done per physician's choice. BV‐ICE and romidepsin‐ICE were administered as 21‐day cycles.Chemotherapy with Bv‐ICE and romidepsin‐ICE were given depending on CD30 status. ICE regimen was administered intravenously (IV) using the following doses: Ifosfamide at 5000 mg/m3Thirteen patients with R/R PTCL were identified: Six with relapsed and seven with refractory disease. Bv‐ICE was given to six patients and romidepsin‐ICE to seven. The male to female ratio was 5.5: 1. The median age was 65 years old (range: 22–83). More than 75% had stage IV PTCL. The most common subtype was PTCL, not otherwise specified (46.2%) followed by angioimmunoblastic T‐cell lymphoma (30.8%). Out of the six relapsed PTCL patients, median time from initial chemotherapy to relapse was 10.5 months (3–24 months). Further details in Table 2). ICE was reduced in six patients for ifosfamide (median dose 3750 mg/m2), two for carboplatin (median AUC 5), and six for etoposide (median dose 75 mg/m2). All patients got at least 1 dose of G‐CSF  except for 1 patient in each group; both were not treated with ifosfamide and received less than 2 cycles of chemotherapy.Dose reductions were made for four patients receiving Bv (median dose 1.4 mg/kg) and two receiving romidepsin (median dose 8 mg/mAs detailed in Table Cytopenias (of any grade) occurred in all patients receiving Bv‐ICE and romidepsin‐ICE. Grade 3 and 4 cytopenias were seen more frequently after treatment with romidepsin‐ICE (85.7%) than with Bv‐ICE (66.7%); anemia was the most common cytopenia for both. However, severe neutropenia was seen at a higher rate in patients treated with Bv‐ICE (83.3%) than romidepsin‐ICE (42.9%). Three patients treated with Bv‐ICE had neutropenic fever requiring hospital admission; no patients treated with romidepsin‐ICE were affected.Overall, metabolic abnormalities were seen at a lower rate than cytopenias. An elevated creatinine and abnormal LFTs (elevated AST/ALT and bilirubin) were seen in two patients (33.3%) treated with Bv‐ICE and two patients (28.6%) treated with romidepsin‐ICE. Three of these events were grade 3 or higher (2 in Bv‐ICE and 1 in romidepsin‐ICE). Peripheral neuropathy was seen in a third of patients receiving Bv‐ICE.4Combination with salvage chemotherapies in R/R PTCL has not been well studied. Our approach was to use the tumor's biology to determine the potential best treatment. Therefore, we would choose between two FDA‐approved drugs for R/R PTCL, romidepsin, and brentuximab vedotin, based on CD30 positivity and combine them with salvage chemotherapy (ICE). Romidepsin, a histone deacetylase inhibitor, targets the epigenome via histone modification and has cytotoxic properties; its ORR is below 40% in phase II studies of R/R PTCL.Other combinations of romidepsin with salvage chemotherapy have been less successful. In a phase I study, R/R PTCL patients were treated with GDP and romidepsin. From 10 evaluable patients, an ORR of 60% with no CRs was noted. Toxicity higher than grade 2 was significant for myelosuppression and infections (55%–75%).Brentuximab vedotin is an antibody drug conjugate consisting of an anti‐CD30 monoclonal antibody and a microtubule inhibitor, monomethyl auristatin E.Bv‐ICE has also been effective as a salvage regimen in HL. Two phase I/II studies in relapsed/refractory HL patients showed response rates over 90% with CRs ranging from 69% to 74%; both cohorts had over 40 patients.There have been few studies exploring the combination of brentuximab with other agents for PTCL. A prospective analysis of lenalidomide in combination with BV in 17 patients with relapsed and/or refractory T‐cell lymphoma  showed an ORR of 33% with two CRs and three partial remissions (PR), similar in efficacy to single‐agent BV.Even though grade 3–4 anemia and thrombocytopenia were lower with Bv‐ICE in comparison to romidepsin‐ICE, severe neutropenia and neutropenic fever were more common. Studies using romidepsin and brentuximab as single agents reported less than 20% grade 3–4 cytopenias, except for brentuximab which had a neutropenia rate of 21%.In conclusion, treatment with Bv‐ICE and romidepsin‐ICE can be effective salvage regimens for R/R PTCL. Both could improve remission rates prior to transplant with ORRs and CRs over 50%. In our study, more than a third of patients were able to receive consolidation transplantation. Even though the rate of grade 3 and 4 cytopenias were higher than 50% with the combination of romidepsin or brentuximab vedotin with ICE, most patients tolerated treatment well. In our experience, a treatment strategy based on CD30 positivity is feasible and effective to treat patients with R/R PTCL. Further prospective studies are needed to evaluate the promising role of these regimens in this rare disease.The authors have stated explicitly that there are no conflicts of interest in connection with this article.Cesar Gentille: Conceptualization (lead); data curation ; formal analysis (lead); investigation (lead); methodology (lead); writing – original draft (lead); writing – review and editing (lead). Humaira Sarfraz: Data curation (supporting); investigation (supporting); supervision (supporting); writing – review and editing (supporting). Jitesh Joshi: Supervision ; visualization . Jasleen Randhawa: Supervision ; visualization . Shilpan Shah: Supervision ; validation . Sai Ravi Pingali: Conceptualization (lead); supervision (lead); validation (lead); writing – review and editing (lead).The study protocol was reviewed and approved by The Houston Methodist Hospital Institutional Review board. Need for informed consent in this retrospective study was waived.


Solanum lycopersicu). Metabolites of mature tomato were separated into 55 fractions by liquid chromatography, and then each fraction was examined using the phosphorylation assay of AMP-protein kinase (AMPK) in C2C12 myotubes and in AdipoR-knockdown cells by small interfering RNA (siRNA). Several fractions showed AMPK phosphorylation in C2C12 myotubes and siRNA-mediated abrogation of the effect. Non-targeted metabolite analysis revealed the presence of 721 diverse metabolites in tomato. By integrating the activity of fractions on AMPK phosphorylation and the 721 metabolites based on their retention times of liquid chromatography, we performed a comprehensive screen for metabolites that possess adiponectin-like activity. As the screening suggested that the active fractions contained four carotenoids, we further analyzed β-carotene and lycopene, the major carotenoids of food. They induced AMPK phosphorylation via the AdipoR, Ca2+/calmodulin-dependent protein kinase kinase and Ca2+ influx, in addition to activating glucose uptake via AdipoR in C2C12 myotubes. All these events were characteristic adiponectin actions. These results indicated that the food-derived carotenoids, β-carotene and lycopene, activate the adiponectin signaling pathway, including AMPK phosphorylation.Adiponectin, an adipokine, regulates glucose metabolism and insulin sensitivity through the adiponectin receptor (AdipoR). In this study, we searched for metabolites that activate the adiponectin signaling pathway from tomato ( AdipoRole cells ,10. ThesSolanum lycopersicum) is highly valuable as a staple of the human diet and in terms of scientific significance. Novel techniques, such as whole genome sequencing and CRISPR editing, have been applied to tomato as a model system  glucose (1 μCi/well) for 10 min at 37°C. The reaction was terminated by washing with ice-cold phosphate-buffered saline three times. The cells were lysed in 0.1 N NaOH (400 μL/well) and cell radioactivity was subsequently evaluated by scintillation counting . Glucose uptake values were corrected for non-carrier mediated transport by measuring glucose uptake in the presence of 10 μM cytochalasin B.The uptake of 2-deoxyglucose by C2C12 myotubes was evaluated as follows. The cells . Mice were kept in individual cages in a temperature-controlled room at 23 ± 1°C and maintained under a constant 12 h light/dark cycle. Mice had access to commercial chow diet  and water ad libitum for 3 weeks. Eight-week-old mice were divided into different treatment groups . After fasting for 7.5 hours, all-trans lycopene (200mg/kg body weight) and cis-lycopene (200mg/kg body weight) were orally administered once and blood samples were collected. Plasma glucose level was determined with glucose CII-test Kit . All the animal experiments were approved by the Animal Care Committee of Kyoto University.Five-week-old male t-test or one-way ANOVA and multiple comparison tests (Dunnett’s or Tukey–Kramer). Differences were considered significant at p < 0.05.All data are presented as the mean ± SEM. The data were analyzed by Student’s For preliminary selection of the extract containing metabolites that activate adiponectin signaling pathway, hydrophilic and hydrophobic tomato extracts were prepared. A biological assay was used to evaluate the ability of the two tomato extracts to phosphorylate AMPK in C2C12 myotubes. The hydrophilic extract did not significantly affect AMPK phosphorylation , whereasTo select the primary targets of metabolites that activate adiponectin signaling pathway, we assessed the adiponectin-like activity of HPLC fractions of the hydrophobic tomato extract. The tomato extract was separated into 55 fractions. Functional assays were subsequently performed to determine the ability of these HPLC fractions to enhance the phosphorylation of AMPK, similar to adiponectin. A few fractions enhanced the phosphorylation of AMPK Fig . HencefoTo screen for metabolites that activate adiponectin signaling pathway from tomato, we integrated the biological assay and non-targeted metabolite analysis. A comprehensive estimated metabolites in the hydrophobic tomato extract was annotated using non-targeted metabolite analysis under the same analytical conditions as those used for the HPLC fractionation . The tomato extract contained a wide variety of 721 metabolites, such as carotenoids, lipids, isoprenoids, flavonoids, amino acids and other compounds . IntegraIn the next screening step, we integrated the biological assay and the non-targeted metabolite analysis to investigate 721 metabolites in each structural category. We detected 12 metabolites in the amino acids/sugars/nucleic acids category, one of which (8%) was active and annotated as adenosine . Of the In the next screening step, a detailed analysis of the 60 active carotenoids selected by integrating the biological assay and the non-targeted metabolite analysis was performed in terms of their metabolic pathways and chemical nature. The 60 active carotenoids were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and 16 carotenoids were registered in the KEGG carotenoid biosynthesis pathway  and theiTo identify the compounds with adiponectin-like activity in tomato, authentic samples of the five selected carotenoids were evaluated in their biological activity and chemical properties. The five carotenoids enhanced AMPK phosphorylation, while the carotenoid precursor and degradation product, geranylgeraniol and retinol, did not significantly affect AMPK phosphorylation . To iden2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibition by the selective inhibitor STO-609 suppressed AMPK phosphorylation stimulated by β-carotene or lycopene , p38 and AMPK phosphorylation at the tested times. The ACC and AMPK phosphorylation induced by β-carotene or lycopene peaked at 5–15 min, while p38 phosphorylation persisted for 1–6 h , in which the hypoglycemic effects of adiponectin and AdipoRon have been reported [cis-isomerized lycopene was shown to lower plasma glucose levels more strongly than all-trans lycopene (cis-isomers of these carotenoids (9-cis or 13-cis) [trans configurations in C2C12 myotubes  ,52 were myotubes . This efguration . These oNon-carotenoids that activate adiponectin signaling pathway might be present in tomato. In this study, tomato extract that included a wide variety of metabolites displayed adiponectin-like activity in the phosphorylation of AMPK . By inteThe precise molecular mechanism of the action of identified carotenoids in activating the adiponectin signaling pathway is not fully understood. By integrating 721 tomato-derived metabolites and the activity of HPLC fractions on AdipoR-dependent phosphorylation of AMPK, we screened for metabolites that activate adiponectin signaling pathway from tomato. Whereas this activity-based metabolite profiling efficiently identified β-carotene and lycopene as active metabolites with adiponectin-like activity, the precise molecular mechanism of these carotenoids remains unclear. Recently, the crystal structure of human AdipoR was determined at high resolution, which represents a novel class of receptor structure . A synth2+ influx. These carotenoids activated glucose uptake via AdipoR in C2C12 myotubes. All the observed activities are characteristic of adiponectin. The collective findings demonstrate that the food-derived carotenoids, β-carotene and lycopene, activate the adiponectin signaling pathway, including AMPK phosphorylation.In conclusion, integration of biological assay and non-targeted metabolite analysis allowed the screening for metabolites that activate adiponectin signaling pathway from tomato. The screening suggested that four carotenoids have adiponectin-like activity. We further analyzed β-carotene and lycopene, the tomato major carotenoids. Both induced AMPK phosphorylation through AdipoR, CaMKK, and CaS1 Fig(A) AdipoR-knockdown of C2C12 myotubes by AdipoR1-specific siRNA transfection. The total cell protein was extracted from treated C2C12 myotubes and analyzed by western blotting. Data are presented as mean ± SEM from independent experiments (n = 6 /group). **p < 0.01 vs. control.(TIF)Click here for additional data file.S2 Fig(A) Structure of the major carotenoids found in common dietary sources. (B) Effect of major carotenoids found in dietary sources on AMPK phosphorylation in C2C12 myotubes. C2C12 myotubes were incubated with each carotenoid (1 μM), geranylgeraniol (1 μM) or retinol (1 μM) for 10 min. The total cell protein was extracted from treated C2C12 myotubes and analyzed by western blotting. (C) The carotenoid biosynthesis pathway based on the KEGG database. Metabolites denoted by red font elicit AMPK phosphorylation. Metabolites denoted by green font do not elicit AMPK phosphorylation. Data are presented as mean ± SEM from independent experiments (n = 6/group). **p < 0.01 vs. control. pAMPK, phosphorylated AMPK; AMPK, total AMPK.(TIF)Click here for additional data file.S3 Fig(A) Plasma glucose levels after oral administration of all-trans lycopene (200mg/kg body weight) or cis-lycopene (200mg/kg body weight) in 8-week-old male db/db mice. The isomerized lycopene was used as cis-lycopene in animal experiment. . Data are presented as mean ± SEM (n = 6–7/group). *p < 0.05 vs. control. (B) Structures of the naturally occurring and major cis-isomers of β-carotene and lycopene. (C) Effect of β-carotene and lycopene cis-isomers on AMPK phosphorylation in C2C12 myotubes. C2C12 myotubes were incubated with each carotenoid (1 μM) for 10 min. The total cell protein was extracted from treated C2C12 myotubes and analyzed by western blotting. Data are presented as mean ± SEM from independent experiments (n = 5–7/group). *p < 0.05 vs. control. #p < 0.05 vs. all-trans carotenoid. pAMPK, phosphorylated AMPK; AMPK, total AMPK.(TIF)Click here for additional data file.S4 Fig(A) Effect of β-carotene and lycopene on Glut4 translocation to the plasma membrane in C2C12 myotubes. C2C12 myotubes were incubated with each carotenoid (5 μM) for 1 hr. The plasma membrane fraction was extracted from treated C2C12 myotubes and analyzed by western blotting. (B) Effect of β-carotene and lycopene on LKB1 translocation to the cytoplasm in C2C12 myotubes. C2C12 myotubes were incubated with each carotenoid (5 μM) for 10 min. The cytoplasm fraction was extracted from treated C2C12 myotubes and analyzed by western blotting. Glut4 PM, Glut4 protein in plasma membrane fraction; Glut4 CL, Glut4 protein in cell lysate; LKB1 CY, LKB1 protein in cytoplasm fraction; LKB1 CL, LKB1 protein in cell lysate.Translocation of Glut4 and LKB1 associated with the adiponectin signaling pathway (TIF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Water can form a vast number of topological frameworks owing to its hydrogen-bonding ability, with 19 different forms of ice experimentally confirmed at present. Here, the authors comment on open questions and possible future discoveries, covering negative to ultrahigh pressures. A hydrogen bond can be characterised as O–H⋯O, where the hydrogen atom is linked via a short covalent bond to the donor oxygen and via the longer bond to the acceptor. The one feature that makes ice structures so diverse is that four hydrogen bonds can be formed by a water molecule, although it has only three atoms. Thereby, two atoms may act as H donors, two as H acceptors. In ice, as opposed to water clusters or liquid water, all four hydrogen bonds are developed, resulting in a tetrahedral coordination around each water molecule. This local coordination motif is called the Walrafen pentamer and is found in almost all ice structures1.The hydrogen bond as envisioned by Linus Pauling is key to the structures of H2O breaks down in two stages. First, the H atoms jump back and forth between the two O atoms , currently. Above about 300 GPa, a range of many other post ice X structures is predicted that have yet to be discovered in experiment. Their space groups are given at the high-pressure end in Fig. 15.Figure 6. Nonetheless, two empty clathrate hydrates could be realised in experiments that are now known as ices XVI7 and XVII8. Ice XVI is prepared in vacuo by pumping on a neon-filled clathrate hydrate of cubic structure II over extended periods, thereby emptying it7. A similar strategy was adopted to produce ice XVII starting from hydrogen-filled ice as a precursor8. On thermodynamic grounds, one would expect them to be stable only at negative pressures (see predicted stability range of ice XVI in Fig. 10. These are derived from known guest-filled clathrate hydrates or zeolite type networks (listed at the left in Fig. Turning to the negative pressure regime, ultralow-density forms of ice are encountered that are significantly less dense than common hexagonal ice found on Earth’s surface. These are open structures that have cages large enough to fit even guest molecules. Such structures may be derived from naturally occurring clathrate hydrates, such as methane hydrates. Without guest molecules, such open ice forms are supposed to form from hexagonal ice under tensile stress. Such experiments have not been successfully done, though, mainly because icicles rupture easily when pulling on them. By contrast, experiments on liquid water at negative pressure were done in the past, avoiding cavitation and the transition to the stable vapour11. They have, therefore, been declared as a mineral by the international mineralogic association. At depths of several hundred kilometres, these ices might be of relevance to build friction between cold-subduction slabs, resulting in seismic events one could term “ice quakes.” High-pressure forms of ice are even more abundant in space, where the icy moons are covered in layers of ice up to 1000 km thick so that their own weight suffices to transform the ices to high-pressure polymorphs such as ice III, V, VI, or VII. On Earth pressures within even the deepest glaciers are not high enough to transform hexagonal ice to high-pressure ice forms. The density increase from ice I to ice III to ice V to ice VI and to ice VII (indicated in Fig. 2O networks that are fully interpenetrating.Apart from the ultralow- and ultrahigh-density ices described so far, all other experimental discoveries pertain to the highly versatile intermediate-pressure range, between about 0.1 and 2.5 GPa as depicted in Fig. 12. However, the water dipoles are oriented randomly causing dielectric constants of above 100. Currently, the six hydrogen disorder-order pairs Ih–XI, III–IX, V–XIII, VI–XV, VII–VIII, and XII–XIV are known. The order-disorder transition temperature varies between 72 K for the ice Ih/XI couple and 273 K for the ice VII/VIII couple. To actually make H-ordered ices and to release the frustration in the H-subnetwork kinetics is the key challenge. It has taken until the 1970s before Kawada achieved to make partly H-ordered ice XI from its disordered parent ice Ih. The ordering process requires waiting times of days at <72 K13, but only if extrinsic defects are injected into the hydrogen-bond network through dopants. HCl and KOH have been the most successful dopants, where KOH allows accessing ice XI, and HCl allows accessing the H-ordered high-pressure ices XIII–XV14. As of now, it is still unknown why HCl accelerates ordering dynamics in high-pressure ices, and KOH in low-pressure ice, but not the other way around.The single property that has resulted in the most discoveries of ice phases is hydrogen order. In an ice crystal, both the H atoms and O atoms may occupy lattice positions, resulting in oriented water dipoles (coloured in blue in Fig. 15. In our experiments we recently discovered a second one, which we call ice β–XV16. This represents the 19th polymorph of ice, where elucidation of its crystal structure based on diffraction experiments is ongoing work. While still being contested, this makes the case for the first phase of ice, in which transitions between two differently ordered H-sublattices may be studied—and it is fully open how such a transition progresses. Eighty-five years ago, Pauling considered all H-ordered structures as degenerate, which can be accessed through permutation of H atoms. It is clear now, though, that there are subtle differences in enthalpy, lattice parameters and density. Yet, it is still unclear for all ices known today whether a more stable arrangement of the H atoms exists, and whether this will be realised experimentally someday.One specific and unique case that has received much attention lately is ice VI. Not only one, but in fact 45 different types of H order can be imagined that all obey the Bernal–Fowler ice rules. The antiferroelectric ice XV structure discovered in 2009 is only one of the H-ordered pendants of disordered ice VI18. That is, it is still far from clear how many condensed phases of water can be distinguished. A recent machine learning study on ice phases actually suggests in total 51 ice polymorphs, more than 30 of which have not yet been reported in experiments19. If the stable structures that are predicted in simulations can actually be achieved in experiments, we expect to discover more ice phases in the future than have been discovered up to now. Rather than the candidates indicated between the question marks in Figs. Even at ambient pressure ice still holds surprises, as shown through the realisation of a cubic stacking sequence in ice I without hexagonal stacking faults in 2020