Polymers of ribonucleotides (RNAs) are considered to store genetic information and promote biocatalytic reactions for the proto life on chemical evolution. Abiotic synthesis of ribonucleotide was successful in past experiments; nucleoside synthesis occurred first, followed by phosphorylation. These abiotic syntheses are far from biotic reactions and have difficulties as a prebiotic reaction in reacting chemicals in a specific order and purifying intermediates from other molecules in multi-steps of reactions. Another reaction, ribose phosphorylation followed by nucleobase synthesis or nucleobase addition, is close to the biotic reactions of nucleotide synthesis. However, the synthesis of ribose 5′-phosphate under prebiotically plausible conditions remains unclear. Here, we report a high-yield regioselective one-pot synthesis of ribose 5′-phosphate from an aqueous solution containing ribose, phosphate, urea, and borate by simple thermal evaporation. Of note, phosphorylation of ribose before the nucleoside formation differs from the traditional prebiotic nucleotide syntheses and is also consistent with biological nucleotide synthesis. Phosphorylation occurred to the greatest extent in ribose compared to other aldopentoses, only in the presence of borate. Borate is known to improve the stability of ribose preferentially. Geological evidence suggests the presence of borate-rich settings on the early Earth. Therefore, borate-rich evaporitic environments could have facilitated preferential synthesis of ribonucleotide coupled with enhanced stability of ribose on the early Earth. Abiotic formation of RNA monomers  on the prebiotic Earth is considered as an essential step in the prebiotic RNA synthesis. However, the process of the abiotic formation of ribonucleotides on the Hadean Earth needs further elucidation.Abiotic formation of functional polymers with catalytic capabilities and information-carrying capacities would have been essential for the emergence of primitive life. The discovery of the catalytic RNAs supports that RNA might have served these functions in the primordial life, as proposed by the RNA world hypothesis12. In abiotic nucleoside syntheses, condensation between ribose 1 and nucleobase results in the formation of purine nucleosides in low yields and pyrimidine nucleosides in no detectable yields3. A high yielding nucleoside synthesis needs step-by-step reactions of small reactive molecules with separation and purification of the intermediate chemical compounds6; the geological settings/events that would have permitted such step-wise synthesis remain unclear. On the other hand, phosphorylation of nucleosides occurs by simple drying of nucleoside and phosphate 2 with catalysts such as urea 312. Ribose 1 can form via a simple condensation of formaldehyde, which can, in turn, be formed by a photochemical reaction between CO2 and H2O14. These hint at a possibility of phosphorylation of ribose 1 before the nucleoside formation on Hadean Earth. Further, phosphorylation of sugar before the nucleoside formation is seen in nucleotide biosynthesis in extant life. Phosphorylation of sugar is also an essential reaction related to metabolism in all living cells. Given this, we investigated the phosphorylation of ribose 1 under prebiotically plausible condition.Nucleotides have been chemically synthesized via the formation of nucleosides and subsequent phosphorylation of these molecules2 and ribose 1 with phosphodiester linkages at 5′- and 3′-hydroxyl groups on ribose 1. Simple heating of ribose 1 and phosphate 2 with cyanogen 4 selectively yields ribose 1′-phosphate 515. Further, the reaction of ribose 1 with amidotriphosphate selectively yields ribose 1′,2′-cyclic phosphate16. The synthesis of ribose 1′-phosphate 5 was also demonstrated by artificial electrospray-ionized microdroplets using ribose 1 and phosphate 217. The synthesis of ribose 5′-phosphate 6 has only been achieved by similar artificial electrospray-ionized microdroplets using ribose 1 and pyrophosphate18. Nonetheless, the availability of electrospray-ionized microdroplets on the Hadean Earth is questionable. Thus, the synthesis of biologically relevant ribose-phosphates  under plausible prebiotic conditions remains elusive.The sugar phosphate backbone of RNA consists of phosphate 3, a simple amide molecule, has been used as an efficient abiotic catalyst for the phosphorylation of nucleosides19. In addition, previous research reported that urea 3 could be a nucleobase precursor on the prebiotic Earth21. Urea 3 is formed via hydrolysis of cyanamide that can form from ammonia and cyanide19. Both ammonia and cyanide can be formed by impact-induced reactions22. These reactions would have been common on the Hadean Earth when the impacts of large meteorites and asteroids were more frequent than today23. Thus, it is reasonable to assume urea as a prebiotically available molecule on the early Earth. However, the effects of urea 3 on the phosphorylation of sugars have not been investigated. Therefore, we investigated the phosphorylation reaction of aldopentoses in the presence of urea 3.Urea 24. Previous studies have found that several oxyanions can improve the stability of sugars26. In particular, borate 7 can strongly stabilize ribose 1 compared to other aldopentoses28. This indicates that borate-rich environments could have been advantageous for nucleotide formation as ribose 1 could have accumulated in such environments. Furthermore, borate 7 is known to control the phosphorylation site of nucleosides29. The effects of borate 7 on other steps involved in prebiotically plausible nucleotide synthesis have been reported in many previous studies31. Therefore, we investigated the effects of borate 7 on the phosphorylation of ribose 1 and other aldopentoses under evaporation.Sugars are readily decomposed by heating. In particular, ribose is the least stable aldopentose6 from ribose 1 and dissolved phosphate 2 in the presence of urea 3 and borate 7 under simple drying conditions  was dried by heating at 80 °C for 24 h. The precipitates were hydrolyzed in acidic water (pH ~ 1) at 90 °C for 1 h and analyzed using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS). The formation of significant amount of ribose 5′-phosphate 6  in the reactions containing boric acid was confirmed by its LC retention time, MS/MS fragmentation pattern, and 31P-NMR  was detected  was also detected in the product formed with boric acid before acid hydrolysis. Any ribose phosphate was not formed in the acid condition from ribose and phosphate . Regent grade d-ribose 5′-phosphate disodium salt dihydrate, adenosine 2′-monophosphate, and adenosine 3′-monophosphate were obtained from Sigma-Aldrich and used as standards. All chemicals were used without further purification. LC–MS grade ammonium formate (Sigma-Aldrich) and acetonitrile (Merck) were used for LC–MS/MS analyses. Water was purified using Milli-Q Integral (18.2 MΩ·cm and < 3 ppb TOC).Reagent grade LC–MS/MS standards for ribose 2′-phosphate and ribose 3′-phosphate were prepared by heating 20 mM of 2′-AMP and 3′-AMP, respectively, at 90 °C for 1 h in 2 wt% sulfuric acid solution to hydrolyze N-glycosidic bond between ribose and urea. LC–MS/MS standard of ribosylurea phosphate was prepared by heating 20 mM of ribose 5′-phosphate and 80 mM urea at 60 °C for 24 h.The phosphorylation experiments were conducted in Eppendorf tubes. The 20 µL reaction mixture contained 20 mM ribose, 800 mM urea, 40 mM boric acid, and 160 mM disodium phosphate in water. The starting materials were heated at 80 °C for 24 h with the lids of the tubes kept open. After heating, the residues were resuspended in 200 µL of water. Borate was removed from the product by adding 4 µL sulfuric acid (95%) to the resuspended sample solution before LC–MS/MS analysis. For investigating the acid hydrolyzed product, a sulfuric acid solution containing the experimental residue was heated at 90 °C for 1 h with the lid closed. The hydrolysis would have progressed slowly in the prebiotic ocean, where pH was close to neutral, and the temperature was lower than the acid treatment, although the rate of reaction should be lower.The control experiment in acidic conditions was conducted in Eppendorf tubes. 20 µL of reaction mixture containing 20 mM ribose, 800 mM urea, 40 mM boric acid, and 160 mM disodium phosphate in acidic water adjusted pH ~ 1 with sulfuric acid was dried down at 80 °C. The experimental and analytical procedures were the same as other experiments. For investigating the effect of acid treatment on phosphorylation, the starting material before the experiment was diluted to tenfold with pure water, adjusted at pH ~ 1 with sulfuric acid, and heated at 90˚C for 1 h.The separation and detection of the products were conducted using Shimadzu LCMS 8040  with the hydrophilic interaction chromatography mode using HILICpak VT-50 2D column . The sample was eluted with isocratic elution with an aqueous solution containing 80% 25 mM ammonium formate and 20% acetonitrile at a total flow rate of 0.2 mL/min at 60 °C. Mass spectrometry was conducted in negative mode with desolvation, source, and heat block temperatures set at 250 °C, 120 °C, and 400 °C, respectively.31P-NMR spectra were acquired with Bruker AVANCE III 500 spectrometer. High concentration samples necessary for the NMR analysis were obtained by the following method. The 20 µL aqueous solution containing 200 mM ribose, 4 M urea, 400 mM boric acid, and 1.6 M dipotassium phosphate was heated at 80 °C for 24 h with the lids of the tube kept open. Dipotassium phosphate was used instead of disodium phosphate due to its better solubility. After heating, the residues were resuspended in 200 µL of water, added by 4 µL sulfuric acid (95%), and heated at 90 °C for 1 h. Before the 31P-NMR analysis, pH was adjusted to about 8 with sodium hydroxide solution.Supplementary Figures.


Ice cleats may help prevent ice-related falls in places with icy roads, but there is limited evidence about the association between ice cleat distribution and ice cleat use. Our study examined the association between Swedish municipal distribution programmes and ice cleat use among older adults (65+ years).We combined data on municipal ice cleat distribution programmes (n=63) with repeated cross-sectional self-reports of ice cleat use in Sweden from 2007, 2010, 2014 and 2018. Respondents (n=63 234) were classified as exposed if they lived in a municipality with a programme, belonged to an eligible age group and responded after distribution (n=2507). Dose-response was assessed using distributed ice cleat pairs per capita (mean: 0.38). Linear probability models were used to estimate probability differences in ice cleat use between exposed and unexposed respondents, adjusting for age, sex, country of birth, education, survey wave and municipality. Ineligible age groups living in programme municipalities, who should be unaffected by ice cleat distribution, were used for bias assessment.Exposure to ice cleat distribution programmes was associated with 7.5 percentage points (95% CI 4.2 to 10.9) higher self-reported ice cleat use after confounding adjustment. The association was larger in municipalities that distributed one pair of ice cleats per capita (17.3 percentage points (95% CI 11.2 to 23.4)). No association was found among the ineligible age groups (−2.3 (95% CI −5.5 to 1.0)).Distributing ice cleats to older adults may help increase their use of ice cleats in settings with icy road conditions. Previous research reports that the distribution of a pair of free anti-slip devices (ice cleats) to older adults (65+ years) may decrease ice-related fall injuries. Such distribution programmes are also low cost and likely to require minimal behaviour change to be cost-effective. However, there is currently no direct evidence that distribution influences ice cleat use, which means that the mechanisms through which ice cleat distribution affects ice-related injuries can be questioned.We find an association between ice cleat distribution in Swedish municipalities and increased ice cleat use among older adults targeted by the programmes. The findings also show that greater reach (distributed ice cleats per capita) is associated with greater increases in ice cleat use.Our study suggests that targeted ice cleat distribution by local governments may increase ice cleat use among older adults in settings with icy road conditions. While confirmatory trials are required before drawing strong causal conclusions, our study provides evidence for decision making that may help improve pedestrian safety for older adults.Falls are the most frequent cause of injury among older adults.Many Swedish municipalities have begun distributing ice cleats to older adults to alleviate the increased risks of pedestrian falls associated with icy road conditions.It is well established that ice cleats can reduce ice-related injury risks at the individual level,In this repeated cross-sectional study of the association between ice cleat distribution and ice cleat use, we collected data on municipal ice cleat distribution programmes and combined them with data on self-reported ice cleat use in the Swedish population aged 18–79 years.As part of a previous process evaluation study of ice cleat distribution in Sweden,n respondents=88 676; average response rate across all waves: 52.2%; see We also used data from existing repeated cross-sectional surveys conducted by Statistics Sweden in 2007, 2010, 2014 and 2018 on behalf of the Swedish Civil Contingencies Agency. In each wave, stratified random samples of the Swedish population aged 18–79 years were invited to answer questionnaires about perceptions and behaviours related to safety and security  on their shoes’ during icy road conditions. The available response categories were on ordinal scales in 2007 and 2010  and on a binary scale (yes or no) in the 2014 and 2018 waves. To homogenise the data, we classified those who reported using ice cleats sometimes, often, or always as ice cleat users in the earlier waves (never or seldom were classified as non-users). The exact phrasing of each question and our coding is presented in For our primary exposure variable, respondents were classified as exposed if they lived in a municipality with a programme, belonged to an eligible age group and participated in a survey wave after ice cleat distribution. This definition implies an intention-to-treat perspective: All individuals are considered exposed even if they did not participate in the programme, in line with recommendations for estimating real-world effectiveness.As a secondary exposure, we defined a continuous exposure variable where we assigned each exposed individual a value corresponding to the number of ice cleat pairs distributed per age-eligible citizen in their municipality . This exposure was used to establish a dose–response relationship between programme reach and ice cleat use and to estimate programme efficacyBelow, we refer to results for the primary exposure as the association at average reach and the secondary exposure as the association at perfect reach to highlight the difference in interpretation.Respondents living in municipalities that did not respond to our programme survey were excluded due to unknown exposure status (n=25 442). Individuals with missing responses or who responded ‘don’t know’ to the ice cleat questions were treated as non-users (n=3070). Register data on age, sex, place of birth and municipality of residence were complete in all waves. Individuals with missing data on education (n=719) were assigned to the most common category (no postsecondary education).2 tests and t-tests to assess group differences in categorical and continuous variables, respectively. Associations between the exposure variables and self-reported ice cleat use were then estimated using linear probability models.We first conducted descriptive analyses of the sample characteristics and self-reported ice cleat use by programme exposure status. We used χAll SEs were clustered by municipality, in line with recommendations to cluster at the level at which the intervention is provided.We conducted subgroup analyses by age group, sex, place of birth and educational attainment to assess potential effect measure modificationWe conducted a sensitivity analysis including respondents who reported seldom using ice cleats in the definition of an ice cleat user see . We alsoWe used a negative control group, that is, explored associations among a group that should not be affected by the intervention, to assess the risk of bias.No patients or members of the public were involved in the design of the study.Seventy-three out of the 228 municipalities that responded to our survey responded that they had distributed ice cleats to older adults. We could link programme data from 63 of these programmes to the survey respondents (no one from the remaining ten participated in the surveys after implementation). Of these, 1 had implemented a programme by the 2007 wave, 4 had implemented a programme by 2010, 11 had implemented a programme by 2014 and 63 had implemented a programme by 2018. Regarding eligibility to participate in the programmes, 56 programmes had set the minimum age at 65+ years, 6 had set the minimum age at at 70+ years and 1 had set the minimum age at at 75+years.After excluding the 25 442 respondents from non-responding municipalities, our final analysis sample contained 63 234 survey participants, of whom 2507 (3.9%) were classified as exposed to ice cleat distribution programmes. The average number of ice cleat pairs distributed per age-eligible citizen was 0.38 . In addition, 2192 participants were classified as negative controls ). The participating municipalities and included programme municipalities are listed in We present the characteristics of all respondents and those aged 65 years and older in The age-adjusted ice cleat use was 10.6 (95% CI 5.4 to 15.8) percentage points higher among respondents exposed to the average ice cleat distribution programme than those who were not exposed (The age-adjusted dose-response relationship between ice cleat use and programme reach among older adults (65+ years) showed that greater programme reach was associated with higher ice cleat use . SpecifiThe subgroup-specific associations were too imprecise to establish significant effect measure modifiers, as all 95% CIs overlap . HoweverSensitivity analyses using the alternative definition of ice cleat users and logistic regression showed slightly smaller associations, but the conclusions remain the same (The negative control analyses showed no associations between ice cleat distribution and ice cleat use among younger, ineligible individuals living in programme municipalities (This is the first study to assess the association between ice cleat distribution programmes and ice cleat use. Our results are consistent with the hypothesis that ice cleat distribution increases use: We found an association between programme exposure and usage rates, which was stronger in municipalities that distributed more ice cleats per age-eligible citizen, implying a dose–response relationship. We also found suggestive evidence association may be stronger in groups with lower baseline use (especially men and non-native Swedes; As noted in the introduction, it is relatively well established that using ice cleats can reduce the risk of ice-related injuries at the individual level.Together with this study, our interpretation is that the current research supports the idea that ice cleat distribution may be an effective injury prevention programme in places affected by icy road conditions, especially when considering the cost-effectiveness of these devices. A model-based economic evaluation that combined effect estimates from a randomised trialA weakness of our study is that we could only make ecological connections between individuals and ice cleat distribution programmes.Additionally, our observational approach is prone to confounding bias. To assess the risk of bias, we used younger, ineligible individuals within the same municipality as negative controls.Our exposures may also be susceptible to misclassificationWe used repeated cross-sectional data from national samples, enhancing generalisability to the Swedish population. However, selective participation is always a concern despite using a random sampling design.Distributing ice cleats to older adults in settings affected by icy road conditions may help increase ice cleat use. Confirmatory research with randomised distribution would help further establish a causal relationship between ice cleat distribution and changes in ice cleat use, and empirical research on reductions in ice-related fall injury rates is required to confirm the cost-effectiveness of the existing ice cleat programmes in Sweden.


Mean HR, time spent >85% HRmax (t85HR), as well as the total number of intense accelerations and decelerations were higher for defensemen. However, demands were similar when expressed relative to time on-ice, except that defenders performed more Dec2/min than forwards in all periods, whereas forwards spent more t85HR during the 2nd period (r = 0.46–0.57). Time spent on ice was inversely correlated with the total number of accelerations (Acctot), accelerations >2 m·s-2 per min (Acc2/min), total decelerations per min (Dectot/min), Dec2/min and t85HR (r = -0.63 to -0.18) and positively correlated with mean HR and peak HR (r = 0.20– 0.53). No significant correlations were found between physical fitness and game activity variables scaled by individual time on ice. Absolute acceleration and HR demands of professional ice hockey seem to differ between playing positions, but not in relation to time on ice. Further, no clear signs of fatigue were captured, possibly due to the longer duration of rest intervals in the 3rd period.The aim of this study was to describe the game activity profile of a professional ice hockey team with special emphasis on fatigue development and playing position. Data were collected using a wearable 200-Hz accelerometric system and heart rate (HR) throughout eight official games in a professional ice hockey team . On-ice 10- and 30-m sprint performance, repeated sprint ability and HR responses to the submaximal Yo-Yo Intermittent recovery level 1 test were assessed to determine associations with game performance. Although the 3 Professmponents . However2 . In thiobserved . Additioobserved , 2021. MIrrespective of the playing position, ice hockey players require adequate fitness to cope with the aforementioned demands of the game. Beyond the limited information available about ice hockey athletes’ characteristics using specific on-ice testing approaches  and no iIn summary, little evidence exists regarding the physical and physiological demands of professional ice hockey match-play with special emphasis on position-specific acceleration and deceleration profiles and potential fatigue development over the course of a game . Thus, tAnthropometric characteristics, physical capacities and match activities were collected from 17 professional ice hockey players (6 defenseman and 11 forwards) competing in the highest Danish tier . A detailed description of participant characteristics is reported in Game data were collected using a wearable accelerometric system and HR monitors  throughout a four-week competitive period, between February and March 2020. Pre-game warm-up data were excluded. Only players who spent a minimum of 4 min on the ice were considered as criteria to normalize game data. No input was given about the research design to the technical staff throughout the data collection period. Data were collected in the context of the regular player’ monitoring routine. A total of 122 individual files from 8 competitive professional games  were analysed.SUB) were assessed twice 30 days apart. The best results of these two evaluations (lowest sprint time and lowest HR) were retained as representative of the individual players. To remove the effect of game duration, correlations between physical capacity and game activity variables, as well as between game exposure and accelerometer- and HR-based variables were computed on individual time on ice.Sprint performance (10- and 30-m), RSA and HR during the submaximal Yo-Yo Intermittent recovery ice hockey – level 1 test  and a dual beam setup was used to prevent interference from swinging limbs or the stick accidentally activating the gates prematurely. Verbal encouragement was provided to motivate players to provide maximal effort each time. The mean time of the three sprints was recorded as an indicator of RSA, as previously described . The besSUB with HR measurements , as previously described  and ecological validity  in ice hockey players . HR res players .tot), accelerations above 2 m·s-2 , total decelerations (Dectot) and decelerations below -2 m·s-2 . These were expressed as absolute values and relative to on-ice game exposure.Acceleration and HR data were recorded using a wearable device incorporating a 200-Hz accelerometer and a gyroscope  and a 1-s interval telemetric system, respectively. The device  was placed on the lower sternum using an elastic band. Data were stored in the device and downloaded using the manufacturer’s software . Acceleration variables included the number of total accelerations (Accmean), peak HR (HRpeak) during exercise  and time spent above 85% HRmax (t85HR) based on a recent ice-hockey study . Differences between periods of the match and playing positions were analysed using a mixed model taking into account that different players took part in a different number of matches (r) , 0.1–0.3 , 0.3–0.5 (moderate), 0.5–0.7 (large), 0.7–0.9 (very large) and ≥0.9 .The Kolmogorov-Smirnov test revealed that game data and physical capacity data were normally distributed ( matches . The perches (r) . Within-ches (r) . Additiop ≤ 0.05. Data analyses were performed using Statistical Package for Social Science software .Statistical significance was set at rd period compared to the 1st and the 2nd period  and positively correlated with HRmean and HRpeak . Moreover, Acc2 was moderately to largely correlated to Dectot, Dec2 and t85HR . When expressed as relative values to time on ice, positive correlations were observed between Acctot/min and Dectot/min and between Acc2/min and Dec2/min . A detailed description of the within-subject correlations between game activity variables is reported in In absolute values, Accp > 0.05).No significant correlations were found between individual characteristics and game activity variables scaled by individual time on ice  no clear signs of fatigue appear towards the end of a game; (ii) defensemen appear to experience greater physical and physiological demands than forwards, with similar demands in relation to individual time spent on ice; (iii) an inverse relationship exists between time on-ice and the total number of accelerative efforts; and (iv) anthropometric characteristics and skating-based physical capacity are not associated to the work rate during official professional ice hockey matchplay.tot or Acc2 and Dec2 during the 3rd period, neither when expressed in absolute nor relative terms to on-ice exposure , enabling players to recover between intense efforts and maintain performance. Accordingly, an inverse relationship between total accelerations and decelerations and individual time on ice was observed in the present study, revealing that the most frequently used players performed the lowest amount of changes in speed and direction per minute over the course of the game likely due to pacing strategies in order to avoid exhaustion prematurely or due to shorter rest between each shift throughout the game compromising performance.In contrast to previous studies, we did not observe a significant decrease in the number of Accexposure . HoweverThus, the combination of extended recovery periods due to more interruptions and possible application of pacing strategies for the most frequently used players may have minimized fatigue development toward the end of the game. Also, a decrease in performance cannot be ruled out for specific players, but our sample size did not allow for accurate sub-analyses of match-activities grouped according to playing exposure. Potential fatigue development during a game could be related to fatiguing mechanisms associated with a marked degradation in muscle glycogen (~53%) with a major part of both type I and II fibres being depleted towards the end of an ice hockey game  as well No between-period differences were observed in HR-based variables, which is in accordance with previous findings . However-1) and fast (17–21 km·h-1) skating speed in defensemen during one official game. On the other hand, the same authors reported greater very fast (21–24 km·h-1) and sprint (>24 km·h-1) skating distance covered by forwards in absolute numbers and substantially larger amounts of high-intensity skating relative to on-ice duration . Our results contrast previous findings from duration . Similarmean and HRpeak may indicate an increased aerobic contribution with game exposure . Similarly, our lack of association between HR responses to the Yo-Yo IR1-IHSUB is in contrast to This is the second study to examine game activity variables in relation to individual physical characteristics in male professional ice hockey . In the Despite analyses of multiple matches, our sample size is limited to one team, and thus, the observed differences in physical and physiological demands between defensemen and forwards could be partially explained by game-or-team specific situational factors  or stratIn summary, the present study characterized the physical and physiological demands of ice hockey matches with special emphasis on fatigue patterns and playing position, as well as correlations of match activity variables, using a set of multiple games. This study provides further insights into performance and physiology of ice hockey, being of potential interest for coaches and any professionals involved with ice hockey players.Defensemen experienced greater absolute physical demands than forwards, but similar demands when acceleration and HR data were expressed in relation to time on ice. No clear signs of fatigue were captured as indicated by stable physical performance between game periods. However, players who spent more effective playing time on ice performed less accelerations and decelerations, possibly indicating compromised physical performance due to reduced rest periods on the bench, or possible pacing strategies to manage fatigue. Additionally, individual anthropometric characteristics and skating-based physical capacities seem not to be representative of game-related performance, possibly indicating that well-conditioned players were employed for longer shifts.


Canine atopic dermatitis is a disease of dogs in which the skin becomes permeable. Part of treating canine atopic dermatitis involves restoring the skin barrier function; however, few effective therapeutic options exist. The goal of this study was to evaluate the effect of a spot-on containing fatty acids, ceramides and essential oils on two parameters to evaluate the skin barrier function of dogs. We found that this spot-on had a protective effect on the skin barrier function for both parameters. This study suggests that the investigated product may be useful as an adjunctive treatment for canine atopic dermatitis.® spot-on significantly reduced acute skin barrier impairment in a dog model. The use of this product should be further evaluated as a potential treatment for skin barrier defects such as canine atopic dermatitis.Skin barrier restoration is an important part of atopic dermatitis therapy. We investigated the effect of a spot-on containing plant-based essential fatty acids and essential oils on skin barrier parameters in a dog model of acute skin barrier disruption, using five healthy beagle dogs maintained in a laboratory setting. Four test sites on the dorsum and a control site on the abdomen were defined on each dog. Transepidermal water loss (TEWL) and skin surface hydration (SSH) were measured before and after tape stripping on the first day and then for three consecutive days, over four consecutive weeks. The spot-on was applied at the end of each of the first three weeks. The increase in TEWL after tape stripping was reduced after the spot-on application and reached control values in Weeks 3 and 4. SSH after tape stripping was reduced in Week 4 compared with the baseline. Thus, the ATOP 7 The skin’s primary function is to form a barrier that protects against noxious environmental stress, physical and chemical damage, infections and allergens. The skin barrier is composed of host cells (mainly keratinocytes); proteins, such as junction proteins and moisturizing factors; lipids, organized in structured lamellae containing cholesterol, free fatty acids and ceramides; and the skin’s microbiota ,2.Transepidermal water loss (TEWL) and skin surface hydration (SSH) have been widely used to assess skin barrier function, especially in atopic dermatitis, a frequent dermatosis associated with lipid, protein and cellular abnormalities. Atopic dermatitis can lead to a dysfunctional skin barrier, allowing the penetration of and sensitization to allergens, as well as increased microbial adherence ,4. The rTape stripping is a minimally invasive method that removes layers from the stratum corneum via successive applications of acetate tape on the skin . This me® spot-on, Dermoscent®, Castres, France) has been used as an adjunctive treatment to lokivetmab in a clinical study of dogs with atopic dermatitis [Although the skin barrier has been extensively studied in dogs, few treatments effectively improve the skin barrier function both experimentally and clinically in this species ,13,14,15rmatitis . It cont® spot-on  on TEWL and SSH in a dog model of acute skin barrier disruption. The objective of this study was to evaluate the effect of ATOP 7All procedures were approved by the Institutional Animal Care and Use Committee of our institute (Ethics Committee reference number 1734).The study included five healthy male beagle dogs, aged from two to four years, and without any history of dermatological disease or sign of pruritus.The study was conducted over four consecutive weeks. Each week  was divided into T0 (before any intervention except clipping), D1 (one hour after tape stripping), D2 (24 h after tape stripping), D3 (48 h after tape stripping) and D4 (72 h after tape stripping). The study protocol is synthesized in 2 each  were demarcated on the back of each dog in the paralumbar region. A spot-on application site  was defined at the center of the four test sites, equidistant to the center of each site . The ventral control site (“control”) was the same for the duration of the experiment. Tape stripping was performed on the designated test site on the first day of each week. On Week 1, before any spot-on application, tape stripping was performed on Site 1 until the skin was shiny. The number of tape strips required varied from 16 to 22. The same number of tape strips was repeated for a given dog over the three following weeks. On the last day (D4) of Week 1, Week 2 and Week 3, a spot-on was applied at the “spot-on” site, for a total of three successive weekly applications. Site 1 was therefore assigned as the baseline site, before spot-on applications, whereas Site 2, Site 3 and Site 4 represented test sites after one, two and three spot-on applications, respectively.® CM 825 , with results expressed in arbitrary units as chosen by the manufacturer. Dogs remained in the test room with controlled conditions  for at least 30 min before measurements. Three successive measurements were performed and averaged at each point to reduce intraoperator variability. Each week, TEWL and SSH were measured at both the test site and the control site on Day 1 before tape stripping (“T0”), 1 h after tape stripping (“D1”) and then on Day 2 (“D2”), Day 3 (“D3”) and Day 4 (“D4”).TEWL was measured using an AquaFlux™ AF200 . SSH was measured using a CorneometerAdverse events were noted if present.p-values less than or equal to 0.05 were considered significant. Shapiro–Wilk tests were used for normality testing and paired t-tests were performed to evaluate the difference in means between the control and test sites or between different time points at the same site.Statistical analysis was performed using GraphPad Prism 9.0.0 and p > 0.05).The mean TEWL values of all sites before tape stripping (T0) were not significantly different from those of the control site for any week  and D2 . The elevated TEWL values progressively decreased on D3  and D4 , compared with the control. For Site 2 (Week 2), after the same number of tape strips and one spot-on application, similar results were obtained at the test site and control site, respectively . For Site 3 (Week 3) and Site 4 (Week 4), after the same number of tape strips but two and three spot-on applications respectively, no data points were statistically different from the control  versus the control on D1 . In contrast, the mean TEWL values for Site 3 on D1 (p = 0.009) and Site 4 on D1 (p = 0.0006) were significantly different from Site 1 on D1  were used to compare the maximum increase in TEWL for each site. The mean TEWL values of Site 1 on D1 and Site 2 on D1 were not significantly different , ® spot-on. Two or three successive weekly applications of the spot-on significantly reduced the effect of tape stripping on TEWL and SSH, respectively, as shown by the absence of significantly different values compared with the control site at those time points. These results suggest a skin barrier function improvement and protection after using ATOP 7Skin barrier restoration is one of the therapeutic cornerstones for canine atopic dermatitis, as skin barrier dysfunction seems to be at the root of the pathogenesis of this condition ,19,20. IIt is not known whether this spot-on could reach lesional sites on a dog when applied on a single site. Our study only assessed the effects of this spot-on on locations close to the spot-on site and not on distant sites. These sites were chosen to limit, as much as possible, the differences in the TEWL and SSH between locations on a dog . This mo® spot-on is only partially known. In a model of atopic dermatitis using a reconstructed canine epidermis, the active ingredients of ATOP 7® spot-on seemed to lead to an improvement in the morphology of the epidermis, an increase in filaggrin expression and a decrease in IL-8 secretion [The mechanism of action of ATOP 7ecretion . These mecretion ,36,37, iecretion . Additioecretion ,42,43,44This study has several limitations. The low number of dogs included in this study may have limited the statistical significance of our results, especially for SSH values. Another possible bias was the abdominal location of the control site, whereas the test sites were located on the dorsum. This abdominal location of the control site was deliberately chosen to minimize the impact of the evaluated spot-on treatment on the control site, allowing us to use each dog as its own control. Additionally, no significant differences in TEWL or SSH were detected between the test sites and the control site before tape stripping. Finally, this model does not directly translate to in vivo trials and only serves to experimentally investigate the effects of such products.® spot-on protected the surrounding skin’s barrier function in a tape stripping model of repeated acute barrier disruption in dogs. The increase in TEWL and SSH after tape stripping was significantly lower after the first two consecutive weekly applications of the product. Therefore, ATOP 7® spot-on may be an effective adjunctive treatment for diseases with skin barrier defects, such as canine atopic dermatitis, but further studies are needed to confirm this effect.Weekly applications of ATOP 7


Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2.Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in  Thromboembolic diseases have become a significant global health risk due to the aging global population and drastic lifestyle changes. Despite many years of research, thrombolytic drugs often suffer from low specificity (causing non-specific bleeding) and a short half-life, raising further complications during the treatment of embolism patients ,2,3. ThePichiapastoris is different from that of mammalian glycosylation, which can be immunogenic in vivo and can also lead to accelerated clearance, thus limiting its therapeutic value . According to the standard curve of [pNA], velocities were plotted against the S-2765 concentration and were analyzed using the Michaelis–Menten equation to obtain the kinetic parameters Km, Kcat, and Kcat/Km. Data were presented as means ± SD (n = 3).The assay volume was 0.15 mL and contained 10 nM samples, 100 μg/mL of fibrin/fibrinogen where stated (0.15 U/mL human thrombin in case of fibrin), and 0.01–5 mM of S-2765 in assay buffer  in the range of 30–90 °C for 1 h without substrate, and the activity was then measured by means of S-2765 hydrolysis. The optimal reaction pH was analyzed after the enzymes were incubated at 4 °C for 16 h with 1 M of PBS, pH 3.0–12.0, without substrate, and the activity was then measured in the same way as the means of the optimal reaction temperature. The mean values were given as an average of three replicates (n = 3) and the data were presented as means ± SD (n = 3). The thermostability was detected by a Nano-SPDF analysis, three replicates for each sample.The N-glycosylation predictions were performed using the NetNGlyc v1.0 Server . The amiA homologous model can be used to predict the 3D structure of proteins, which would help us verify the feasibility of these mutations ,20,21. WIn the spatial structure, all of the mutation sites are located near the key functional sites of DSPAα2. Sites N153 and N185 are located in the K1 domain close to the fibrin-sensitive sites and the ligand-binding sites, while sites K368 and N398 are located in the P domain close to the active pocket. We used the PyMOL tool to predict all of the interaction distances of the selected glycosylation sites, and we determined the corresponding functional sites. As can be seen from Ultimately, the experiment identified four mutation sites, and five mutants were constructed .P. pastoris and were purified using a Ni-NTA chromatography. All of the clones were identified by genomic PCR as well as RT-PCR analysis , the in vitro clot dissolution method was used a. The reIn summary, the plasminogen activation of DSPAα2 was highly correlated with the N-glycosylation sites (N368 and N398) close to its active center. Glycosylation mutations around the active pocket greatly reduced plasminogen activation. The introduction of the glycosylation site belonging to uPA did not increase its plasminogen activation as had been expected. We speculated that the structure of its active center was coated or damaged by additional N-glycosylation. Therefore, when introducing this glycosylation site in the future, attention should be paid to its structural distance and relative position to the active pocket. The glycosylation mutation at the N185 and N153 sites had little effect on plasminogen activation.Previous studies have shown that glycosylation affects protein stability . As a poConsidering that the N153 and N185 sites may have affected the fibrin sensitivity of DSPAα2, S-2765 hydrolysis was used for identification. Mutating the two N-glycosylation sites led to changes in the glycan chains of this active recognition region, affecting the ability of DSPAα2 to recognize fibrin and thus decreasing plasminogen activation. Additional fibrin (fibrinogen) was added to the S-2765 hydrolysis as a supporting substrate to verify the fibrin sensitivity of DSPAα2 and its mutants.Kcat/Km ratios of the two proteins containing fibrin showed greater fold changes in comparison to those without any cofactors (~7–11 folds). The smaller the Km, the lower the substrate concentration required for the enzyme to carry out the reaction and the greater the affinity between the enzyme and substrate. The Km values of these two proteins also significantly decreased in the presence of fibrin compared with the absence of cofactors (~100-fold). We failed to observe this phenomenon in QNGlyα2 and QNGlyα2-1, which could be related to the loss of the N-glycan chain at the N185 site.The glycan chain at the N185 site is extremely important for the fibrin sensitivity of DSPAα2. Both rDSPAα2 and QNGlyα2-2 had high fibrin sensitivity. It can be seen from Kcat/Km of the three proteins  that were originally at the N398 site were much higher than those of QNGlyα2 and QNGlyα2-2 (at least 53 mM−1S−1 larger). Kcat is the time it takes for an enzyme to convert a substrate. The larger the Kcat value, the faster the enzyme can convert substrate molecules. The Kcat values of the wild-type proteins  can create about 30 folds of QNGlyα2 and QNGlyα2-2.The N-glycan chain at the N398 site had a strong influence on the protease activity of DSPAα2. The Km/Kcat ratio when fibrin was the cofactor was almost identical to the value when fibrinogen was the cofactor . Values were the mean ± SD of the three replicates. The fitted Michaelis–Menten curves are displayed in The kinetics of S-2765 hydrolysis were measured as described in the Materials and Methods Section. The kinetic parameters were derived from a nonlinear regression analysis of Michaelis–Menten plots depicting the velocity of [Over the past few years, many researchers have studied thrombolytic drugs with the intent to develop new ones. DSPAα2 has been found to be an efficient natural thrombolytic protein and is often compared with existing thrombolytic drugs due to its superior fibrin sensitivity and longer half-life. However, the focus of current studies has often on improving the expression efficiency, while the effect of N-glycosylation has not been studied. Experiments have shown that modifying the glycosylation of different expression systems is inconsistent, but this does not affect the DSPA activity . WhetherThe N398 site is highly correlated with the plasminogen activation activity of DSPAα2. After mutating site N398 to Q398, the two related mutants  showed a large reduction in activity. Site N398 is structurally wrapped in the active pocket of DSPAα2 e. The N-Pichia pastoris, a methylotropic yeast, has been developed as a host for heterologous protein expressions because of its low cost, high expression levels, efficient secretion, post-translation modification, proper protein folding, and potential for high-density cell fermentation [Pichia pastoris expression system [Cell expression systems create expensive production costs. entation . Howevern system ,51. ThisPichia pastoris expression system. For DSPAα2, N-glycosylation is critical for its plasminogen activation, especially at the N398 site. Its N-glycan chains are very close to the active pocket. The active pocket is vital for the plasminogen activation of DSPAα2. The sugar chains at this site either participate in the construction of the active center, function during plasminogen activation, or both. The N185 site is very close to the ligand-binding sites. These sites are key sites for fibrin-specific recognition. All of the mutants that had their N185 site removed exhibited low fibrin sensitivity. Moreover, the glycosylation sites introduced through homology comparison and modelling did not increase activity. We suspect that the reason for this was the proximity of these two sites (N153 and N368) to the critical active sites of DSPAα2. Through this study, we provide support for the expression strategy of DSPAα2 using Pichia pastoris.In this study, we found two N-glycosylation sites in DSPAα2 that may affect its activity using homology modelling, multiple sequence alignment, and specific mutants when using a


Multi-year El Niño events induce severe and persistent floods and droughts worldwide, with significant socioeconomic impacts, but the causes of their long-lasting behaviors are still not fully understood. Here we present a two-way feedback mechanism between the tropics and extratropics to argue that extratropical atmospheric variability associated with the North Pacific Oscillation (NPO) is a key source of multi-year El Niño events. The NPO during boreal winter can trigger a Central Pacific El Niño during the subsequent winter, which excites atmospheric teleconnections to the extratropics that re-energize the NPO variability, then re-triggers another El Niño event in the following winter, finally resulting in persistent El Niño-like states. Model experiments, with the NPO forcing assimilated to constrain atmospheric circulation, reproduce the observed connection between NPO forcing and the occurrence of multi-year El Niño events. Future projections of Coupled Model Intercomparison Project phases 5 and 6 models demonstrate that with enhanced NPO variability under future anthropogenic forcing, more frequent multi-year El Niño events should be expected. We conclude that properly accounting for the effects of the NPO on the evolution of El Niño events may improve multi-year El Niño prediction and projection. The causes of long-lasting behaviors of multi-year El Niño are still not fully understood. Here, the authors find that persistent two-way teleconnections between the North Pacific Oscillation and the tropical Pacific constitute a key source of multi-year El Niño. Typically, El Niño and La Niña episodes develop during the boreal summer, peak during early winter, and decay rapidly during the following spring, lasting about 9–12 months. However, not all ENSO events are the same6. Some La Niña events persist through the following year and often re-intensify in the subsequent winter, lasting two years or longer7. Although less frequent than multi-year La Niña events, multi-year El Niño events are also occasionally observed in the tropical Pacific (such as the 2014/15/16 El Niño event)8  is the dominant climate phenomenon in the tropical ocean affecting the global climate and extreme weather conditions12 25, may play an important role in affecting the development29, pattern32, phase transition35, and decadal variability36 of ENSO. Despite these extensive studies, it remains unclear if the NPO and multi-year ENSO events are dynamically linked, and if they are, then how.The occurrence of multi-year ENSO events results in non-cyclic evolution of ENSO and poses a challenge to the prediction of ENSO. For example, the 2014/15/16 multi-year El Niño event surprised the ENSO scientific community by its unique evolution, and most operational forecasting models compiled at the International Research Institute for Climate and Society (IRI) failed to predict the evolution of this event40 during the subsequent winter30, which in turn feeds back into the North Pacific to re-intensify the variability in the NPO, thereby maintaining El Niño conditions for another year. Our results highlight the importance of extratropical atmospheric variability in generating multi-year El Niño events.Here we use observational analyses in combination with a series of climate model experiments to show that persistent two-way teleconnections between the NPO and the tropical Pacific constitute a key source of multi-year El Niño events. The NPO during the boreal winter can induce a Central Pacific (CP) El Niño27. We denote the year when El Niño first develops as year (0), and the following two years as years (1) and (2), respectively. It is noted that the JFM(0) NPO index is greater than 1.0 in all five multi-year El Niño events  feedback45. This WES feedback excites the NPMM, which propagates positive SST anomalies from the subtropics into the central equatorial Pacific, where positive SST anomalies are conducive to the development of El Niño. In addition to the NPMM, there are at least two other mechanisms by which NPO atmospheric anomalies can lead to the onset of El Niño through the weakening of off-equatorial trade winds46 or the excitation of the off-equatorial Rossby wave47. However, studies of these ENSO extratropical precursor dynamics have focused mainly on the effects of the NPO on the onset of El Niño rather than on the potential role of the NPO in prolonging the duration of El Niño. Thus, there is a need to develop an understanding of the dynamics underlying the link between the NPO and multi-year El Niño events.Previous studies have shown that the North Pacific meridional mode (NPMM)43  El Niño onto the Aleutian Low, the CP El Niño projects onto a different SLP pattern in the North Pacific, and is associated with negative SLP anomalies over the Hawaiian region50  and the other during JFM(1)  is consistent with the warming in the central tropical Pacific associated with CP El Niño, implying that the NPO-induced central tropical Pacific SST variability can feedback into the North Pacific, generating negative SLP anomalies over the Hawaiian region (by extension of the positive phase of the NPO in the North Pacific). This process is referred to as tropical−extratropical teleconnections.We then examine whether the equatorial Pacific warming can exert an influence on the extratropics. We note that the NPO-induced Pacific SST anomaly pattern during JFM(1) resembles that of the CP El Niñofic Fig. . It has (1) Fig. . The re-41, resulting in a re-intensification of the NPMM during March–April–May (MAM)(1) (1) and propagate gradually from the subtropics into the central equatorial Pacific44  produces an anomalous southwesterly flow on its eastern flank  Fig. . This po40 or a mixed pattern40 of CP and EP El Niño in JFM(1), characterized by maximum warming near the central equatorial Pacific. Furthermore, there are negative SLP anomalies between Hawaii and western North America that are consistent with a typical pattern of the SLP response to CP El Niño SST forcing. The negative SLP anomalies weaken the off-equatorial trade winds and then strengthen the NPMM, resulting in the multi-year evolution of El Niño.Through a careful examination of each of the individual multi-year El Niño events  variability. Di Lorenzo et al.31 suggested that the NPO atmospheric variability, which acts as the extratropical stochastic forcing, may enhance the low-frequency variance of ENSO through a chain of extratropical−tropical feedback processes. The present study demonstrates that a similar dynamical chain may also validly explain the dynamics underlying the emergence of multi-year El Niño events, which are dominated primarily by interannual variations. Taken together, it would appear that NPO atmospheric forcing, through extratropical−tropical feedbacks, may contribute to enhancing the low-frequency variability in ENSO over a wide spectrum of time scales .The schematic of Fig. 29. Equatorial ocean dynamics, such as zonal advection in the tropical Pacific, can extend NPO-induced warming in the central equatorial Pacific eastwards, leading to EP El Niño events55. Several single-year El Niño events , which are also preceded by a positive NPO event during JFM(0)  into the western Pacific warm pool where the deep convection can more easily be excitedThe role of the NPO in generating multi-year El Niño events is also supported by historical (1900–1999) simulations of CMIP5/6 models . The imposed NPO forcing is represented by the observed air temperature (T) and wind  anomalies associated with the NPO, which are assimilated to constrain the atmospheric component in the CGCM model  with and without the positive NPO forcing  is followed by a distinct La Niña pattern in JFM(1) whose anomalous cold SST extends to the central equatorial Pacific region  National Oceanic and Atmospheric Administration Extended Reconstructed SST version 4 (ERSST)68; (3) Kaplan Extended SST version 2 (Kaplan SST)69; and (4) Centennial in situ Observation-Based Estimates SST version 2.9.2 (COBE SST)70. The monthly atmospheric fields including surface winds and SLP were from the National Center for Environmental Prediction (NCEP)/National Center for Atmospheric Research (NCAR) reanalysis 1 (NCEP1)71. The SLP field derived from the Hadley SLP data set (HadSLP2)72 was also employed to test the reliability of the results. All of the anomalies in this study are relative to the climatological annual cycle for the period 1981–2010.The analyzed monthly SST data are from: (1) the Hadley Center Sea Ice and SST dataset version 3 (HadISST)73 and CMIP674. According to our hypothesis, the influences of the NPO on multi-year El Niño and La Niña events involve a chain of tropical−extratropical feedback processes associated with the CP El Niño phenomenon. To successfully simulate the role of the NPO in developing multi-year El Niño and La Niña events, the model should be able to simulate both the NPO–ENSO connection and the two types of El Niño. Therefore, we first correlated the JFM(0) NPO index with the following JMF(1) Niño3.4 index over the 100-year period using the CMIP5/6 historical simulations. A total of 51 CMIP5/6 models simulate a significant connection reminiscent of the observations. We then used the correlation coefficient between the Niño3 index  and the Niño4 index  during the DJF season of El Niño events in the pre-selected 51 models to evaluate whether these models can simulate the two types of El Niño75. The correlation between the Niño3 and Niño4 indices in the CMIP5/6 models varies from −0.46 to 0.88, compared with an observed correlation of 0.48 for the period 1900–1999. Finally, we selected the 29 models  simulations from coupled general circulation models (CGCMs) participating in the CMIP5Simulations under the historical (1900–1999) and representative concentration pathway (RCP) 8.5 (2000–2099) scenarios from CMIP5/6 were also used to examine the relationship between future change in the frequency of multi-year El Niño events and change in the NPO variability. Of the selected 29 models based on their historical simulations, there are 23 CMIP5/6 models whose RCP8.5 simulations are available  for 1950–2020. The monthly Niño3.4 index is linearly detrended and smoothed with a 3-month-running-mean filter. We denote the year when El Niño and La Niña first develop as year (0), and the following two years as year (1) and year (2), respectively. A multi-year El Niño (La Niña) event is identified when the Niño3.4 index is over 0.75 (below −0.75) standard deviations in any month during October(0) to February(1), and remains above zero thereafter and exceeds 0.5 (−0.5) standard deviations again in any month during October(1) to February(2)25. The NPO index is defined as the second principal component (PC2) time series associated with the EOF2. The NPMM represents a coupled SST-surface wind pattern over the subtropical northeastern Pacific41. The NPMM index is defined as the normalized SST anomalies over the subtropical northeastern Pacific (15°‒25°N and 150°‒120°W)76. The CP ENSO pattern and index are defined by computing the first empirical orthogonal function (EOF1) and principal component (PC1) of tropical Pacific SST anomalies between 20°S and 20°N after subtracting the anomalies regressed on the Niño1 + 2 index 39, respectively. Similarly, the EP ENSO pattern and index are defined by computing the EOF1 and PC1 of tropical Pacific SST anomalies between 20°S and 20°N after subtracting the anomalies regressed on the Niño4 index (representing the influence of the CP ENSO), respectively.The NPO mode is generally defined as the second leading empirical orthogonal function (EOF2) of boreal winter (JFM) SLP anomalies in the North Pacific poleward of 15°N77 to examine whether the boreal winter NPO index before multi-year El Niño events and multi-year La Niña events is significantly different. The 135 values of the JFM(0) NPO index for multi-year El Niño events and 182 values of the JFM(0) NPO index for multi-year La Niña events in the historical simulations from the 29 selected CMIP5/6 models were re-sampled randomly to construct 10,000 realizations of the NPO index over the 29 models, respectively. Any model can be selected again in this random resampling process. The red and blue vertical lines in Fig. t-test with n − 2 degrees of freedom (n being the number of years).We used a bootstrap method78. The FGOALS-g2 is a state-of-the-art CGCM, consisting of the Grid-point Atmospheric Model of LASG/IAP version 2 (GAMIL2), the LASG/IAP Climate System Ocean Model version 2 (LICOM2), the Community Land Model version 3 (CLM3), and the improved version of Community Ice CodE version 4 by LASG (CICE4-LASG) connected with the coupler (CPL6). The atmospheric component has a resolution of about 2.8° × 2.8° in the horizontal dimension and 26 layers in the vertical dimension up to 2.194 hPa. The oceanic component has a horizontal resolution of 1° × 1°  and has 30 vertical levels with an interval of 10 m in the upper 145 m and increasing with depth.The model used in sensitivity experiments of this study is the Flexible Global Ocean–Atmosphere–Land System Model Grid-point version 2 (FGOALS-g2), one of the CMIP5 models, developed by the State Key Laboratory of Numerical Modeling for Atmospheric Sciences and Geophysical Fluid Dynamics (LASG), Institute of Atmospheric Physics (IAP), Chinese Academy of Sciences (CAS)79. The data assimilation method used in the WCDA system is the Dimension Reduced Projection Four-Dimensional Variational (DRP-4DVar) scheme80, which is an economical approach for implementing 4DVar by using the technique of dimension reduced projection (DRP). Although the ocean component of the coupled model is not assimilated, the WCDA system can transfer the assimilated atmospheric information to the ocean through air–sea coupling. Thus, we tested the role of the North Pacific NPO forcing by conducting experiments in which a positive NPO forcing is only added to the atmosphere component of the FGOALS-g2 model.Recently, a weakly coupled data assimilation (WCDA) system for constraining the atmospheric component in a coupled model was developed and applied to the FGOALS-g2 modelNPO), whereas the experiment with the climatological annual cycle of the atmosphere component is denoted as the CTRL experiment (EXPCTRL). The CTRL experiment was run for 100 years, with its atmospheric composition, solar irradiance, and land cover fixed at 1850 values. The NPO forcing experiment consisted of 60 member ensemble simulations from the CTRL, each initialized with conditions on 1 November from the last 60 years of the 100-year integration.The imposed NPO forcing is represented by monthly air temperature (T) and wind  anomalies at three low pressure levels  associated with the NPO during the boreal winter−spring (from November of year (−1) to May of year (0)), which are obtained by regressing U, V, and T anomalies at 1000, 925, and 850 hPa on the concurrent NPO index (Supplementary Fig. Supplementary InformationPeer Review File


The retention forces in the Morse taper groups were not significantly different (p > 0.05). The screw-retained hexagonal IAC showed the highest retention stability. The screw preload/retention in the conical IAC was lost over time in the group where the screws were kept in place during loading. Nevertheless, the screwless Morse taper IACs were stable for an extended service time and might represent a valid form of treatment for single-tooth replacement.The use of screwless Morse taper implant–abutment connections (IAC) might facilitate the clinician’s work by eliminating the mechanical complications associated with the retention screw. The aim of this study is to evaluate the effect of artificial chewing on the long-term stability of screwless Morse taper IACs. Thirty-two implant abutments restored with an upper central incisor zirconia crown were used and divided into four groups according to the implant–abutment assembling manner ; C2: tapped; or C3: torqued ). All specimens were subjected to a cyclic loading (98 N) for 10 million chewing cycles. The survived samples were exposed to a pull-off force until failure/disassembling of the connection. All the samples revealed a 100% survival. Regarding the pull-off test, the screw-retained internal hexagonal IAC revealed significantly higher resistance to failure/disassembling (769.6 N) than screwless conical IACs (171.6 N–246 N) ( They can be customized or prefabricated by the manufacturer and made of various materials, such as titanium, gold, aluminum oxide, or zirconium oxide ,12,13,14In general, there are two types of implant–abutment connections with two-piece implant systems  connections). Internal conical or internal butt–joint connections show significantly less deformation and more stability compared with external connections . InternaImplant-supported superstructures may be either cement retained or screw retained. Screw-retained superstructures have the advantage that they can be easily removed upon demands, so technical and the biological complications can be managed more easily. Nevertheless, correct sagittal and transverse implant positioning in the hosting bone are of importance for this type of connections. Only a proper implant position and angulation enable the screw access opening in the anterior zone to be positioned in the invisible oral area . On the However, the most common reported technical complications of implant-supported prosthetic constructions are screw loosening or screw fractures ,22,23. PIn an attempt to limit the fixation screw complications, an alternative to screw-retained implant systems  has been manufactured ,26. ThisFor this in vitro study, a total of 32 samples consisting of 32 crowns made of zirconia  mounted on two different types implant–abutment connections were evaluated. According to the implant–abutment assembly method, the samples were then categorized into four main groups. The first group of samples was screw-retained on titanium implants with an internal conical (C1) connection, and the screws were kept throughout the dynamic loading and were removed after dynamic loading before the pull-off test. The second group was a tapped-/knocked-in screwless internal conical (C2) connection (no screws were used for this group). The third group was screwless internal conical (C3) connection. The screws were used only to seat the abutments into the implants and removed before the dynamic loading. The fourth group (control group) was a screw-retained internal hexagonal (H) connection. Dynamic loading test in an artificial chewing simulator was applied to all samples. After chewing simulation, the specimens were investigated using a light microscope to observe any defects. The survived samples were subjected to pull-off test until the crowns or abutments were detached from the implants in a universal testing machine .Groups C1–C3: Morse taper internal conical connection . A cone angle of 2.8° and a cone length of 3 mm; indexed with four grooves in a cross form A.Group H: Internal hexagonal connection  characterized with parallel-walled orientation surfaces B.The implants utilized in this in vitro study were made of titanium (Grade 4) and were 14.5 mm long and had a diameter of 4.2 mm. The implants had one of the two following internal connection characters:Groups C1–C3: SICvantage Bonding Base CAD/CAM red, straight, CEREC for the internal conical connection, height: 4.65 mm (SIC Invent AG) A.Group H: SIC Bonding Base CAD/CAM, straight, CEREC for the internal hex connection group, height: 4.65 mm (SIC Invent AG) B.Prefabricated abutments were used as crown substructures. The following prefabricated abutments were used:Thirty-two anatomical monolithic zirconia crowns mimicking the first upper right incisor with a length of 12 mm and a width of 8 mm were customized from 3 mol% yttria-stabilized tetragonal zirconia polycrystal discs . In addition, two nooks were designed in the cervical  area for a precise grip of the pull-off instrument. The predesigned crown prototype was sent to a milling center , where it was scanned  and optimized in a design program  A. For th2O3 particles with 0.5 bar pressure. To cement the crowns onto the titanium abutments, a Multilink® Hybrid Abutment composite cement  was employed.The bonding surface of the crowns and the CAD/CAM abutments was air-abraded by applying 50 µm Al® Dual, DMG, Hamburg, Germany). To ensure the ISO 14,801 requirements, a distance of 3 mm of the upper part of the implant was exposed to mimic marginal bone loss. All implants were mounted perpendicularly into the PEEK tubes using a special securing device. The hosting resin had a modulus of elasticity between 8.7 and 10.3 GPa [All implants were hosted in polyether ether ketone (PEEK) tubes using a dual polymerizing acrylic resin  were made of titanium grade 5 with a length of 9.5 mm and a diameter of 1.9 mm (head) and 1.6 mm (shank and thread).Group C2: Screwless connection: To ensure a standard fixation method of the crown–abutment assemblies to the implants, a special loading force device was used to tap/knock in the samples of this group in a standardized way . This deGroup C3: All specimens of this group were assembled with a screw using the preloading torque recommended by the manufacturer (20 Ncm) to ensure the maximal anchorage of the abutments into the implants. Then the retention screws were removed before the chewing simulation.Group H: A screw-retained control group with an internal hexagonal implant–abutment connection was used. The screws were tightened (20 Ncm) according to the manufacturer’s recommendation.n = 32) were subjected cyclic mechanical loading in a mastication simulator , including water thermocycling (5 to 55 °C) [All samples (o 55 °C) ,30. A lo® V 7.1 (Zwick) software  to record the pull-off force of each assembly (measured in N).After the dynamic loading, all samples were visually assessed using a light microscope (LM)  at 7-fold magnification. To grant observing the samples for any surface deformations therearound, equidistantly at the area of the implant–abutment connection under the light microscope, a special apparatus was equipped according to a previously described procedure . As showp-values for subsequently pairwise comparisons were corrected using the Students–Newman–Keuls method in order to correct for multiple testing. Statistical significance was set at p < 0.05. All obtained data were statistically analyzed using the software STATA .For a descriptive analysis of the monotonic failure strength, the mean, median, and standard deviations of the acquired data were computed and statistically analyzed at the Institute of Medical Biometry and Medical Statistics of the Albert Ludwig University, Freiburg, Germany (KV). Boxplots were used for a graphical explanation of the data. A one-way analysis of variance was used to check for differences among the groups for monotonic failure pull-off force results. The All specimens survived the chewing simulation without any evidence of complications. This yielded an overall implant and superstructure survival rate (screwed and screwless groups together) of 100%.Hence, no differences in survival between the different retention method groups (screwed and screwless) were observed.According to the light microscope assessment, all specimens exhibited no postloading defects at the implant or abutment level. However, all specimens (100%) of group C1  showed screw loosening. The group H  did not show screw loosening after the dynamic loading. This was rated by controlling the screw retention force using a torque gauge before the pull-off test. All the specimens of group H did not show any loss of the preloading torque (20 Ncm). Decementation of zirconia crowns was detected during the dynamic loading in 4 of the 32 specimens (12.5%) (2 specimens in C2 at 9.9 million cycles and 1 specimen each in group C1 at 10 million cycles and C3 at 8.5 million cycles). The incidence rates of complications for the different groups are summarized in After the specimens were submitted to pull-off tests in the universal testing machine, they were examined to characterize the failure mode.After quasi-static pull-off, all specimens exhibited either detachment of the abutment from the implant or retention loss (decementation) of the crown from the abutment  for the statistical analysis. The mean monotonic pull-off failure forces (±SD) ranged from 171.6 N (±37.7 N) for group C2 to 769.9 N (±413.5 N) for group H. The differences were statistically significant between the four groups  stability of two implant–abutment connection (screwed vs. screwless) designs in an in vitro environment using artificial mastication, followed by quasi-static pull-off test. The current study found that the presence or absence of the retention screw during chewing simulation has no influence on the stability of the Morse taper conical implant–abutment complex while being artificially loaded. Moreover, internal hexagonal or internal conical connected implant–abutment geometries restored with monolithic zirconia crowns exhibited an equivalent performance during chewing simulation . In the present investigation, no implant fracture in both, the hexagonal and/or the conical connection, groups was reported during the chewing simulation. Although a systematic review on the clinical application of different implant–abutment connections did not find significant differences in failure rates between different connections , oral im® Plus). In that investigation, the abutment height was 5 mm, whereas it was 4.65 mm in our investigation. The results of both studies, therefore, are very comparable. In no instance did an abutment–implant dislocation occur in our investigation.In the control group, all cemented crowns came loose from the corresponding abutments during the pull-off test at a mean pull-off value of 770 N. In a recent study , the autHowever, only two samples of the C groups showed decementation of the crown from the abutment before the abutment was detached from the implant. For these two specimens, the friction between the abutment and the implant was higher than the pull-off force required to decement the crown. Possibly, there was an inaccuracy during the cementation process.On the implant level, our results contrasted an in vitro study by Ugurel et al. (2015), which reported that the mechanical strength of the screwless Morse taper implant–abutment connection is lower than that of the screw-retained ones . The autThe most common technical complications of implant-supported prosthetic reconstructions are screw loosening or screw fractures ,34,35,36On the other hand, the aforementioned vertical displacement seems to have a positive effect on the pull-off force required to detach the abutment from the implant in the conical connection groups. Our results revealed a mean pull-off force ranging from 172 to 246 N for the screwless conical implant–abutment connections without statistically significant differences between the three groups C1 to C3. Pull-off forces were previously reported with a mean value of 120 N , 77.6 N Another influencing factor besides the loading protocol might be the implant–abutment connection geometry. The implants used in the first study  were chaA standardized test protocol, as utilized in the present investigation, seems to be helpful for comparing different implant materials and implant designs . In the In summary, all but one implant and restoration survived the long-term artificial loading, irrespective of the abutment retention method or implant–abutment connection design. This indicates that the screwless implant–abutment connection, irrespective of the assembling method, is a reliable option for the presented implant system. However, this should be clinically approved. Moreover, despite the lower pull-off force required to disassemble the abutment from the implant in case of the Morse taper screwless implant abutment connection, this force remains in a clinically acceptable range.The survival rates of screw-retained and screwless abutments used in the current in vitro study are similar. The use of a screwless Morse taper implant–abutment connection represents a valid form of treatment for single-tooth replacement. Screw loosening in a Morse taper implant–abutment connection was frequent but had no effect on the (retention) stability of the connection. Nevertheless, the screwless implant–abutment connections used in our study seem to withstand average occlusal forces even after an extended interval of artificial loading.


The Antarctic continent reached its current polar location ~83 Ma and became shrouded by ice sheets ~34 Ma, coincident with dramatic global cooling at the Eocene-Oligocene boundary. However, it is not known whether the first Antarctic glaciers formed immediately prior to this or were present significantly earlier. Here we show that mountain glaciers were likely present in the Transantarctic Mountains during the Late Palaeocene (~60–56 Ma) and middle Eocene (~48–40 Ma). Temperate (warm-based) glaciers were prevalent during the Late Eocene (~40–34 Ma) and, in reduced numbers, during the Oligocene (~34–23 Ma), before larger, likely cold-based, ice masses (including ice sheets) dominated. Some temperate mountain glaciers were present during the Miocene Climatic Optimum (~15 Ma), before a widespread switch to cold-based glaciation. Our findings highlight the longevity of glaciation in Antarctica and suggest that glaciers were present even during the Early-Cenozoic greenhouse world. This study finds that glaciers have existed in the Transantarctic Mountains for the past 60 million years, and that warm-based mountain glaciers were present in Antarctica long before ice sheets came to dominate the continent. During the Cenozoic (past ~66 Ma), a gradual reduction in atmospheric CO24 pushed Earth from a greenhouse to icehouse climate6. The most dramatic period of cooling occurred at the Eocene–Oligocene (EO) boundary (~34 Ma) and led to the growth of ice sheets in Antarctica7. Given the key role of Antarctic ice sheets in shaping global climate8, and controlling global sea level (SL), their development and evolution during and after the EO-boundary has been the focus of considerable research9. However, far less is known about the evolution of early mountain glaciers in Antarctica, despite them being the first ice masses to occupy the continent, acting as inception points for ice-sheet growth12, and serving as key palaeoclimatic indicators. A better understanding of these glaciers can help to address some fundamental and unanswered questions about Antarctica’s glacial history13, including: when was the onset of glaciation? Were glaciers present during full greenhouse conditions? For how long did temperate (i.e. warm-based) mountain glaciers exist?During the Late Cretaceous (~100.5–66 Ma), the global climate was considerably warmer than present14. As a result, many previous efforts to understand the continent’s long-term glacial history are based on distal proxies, including offshore sediment records15, marine isotope data16, and indicators of eustatic SL fluctuations13. However, these records are unable to fully capture information about smaller ice masses such as those that formed at the onset of glaciation. Here, we show that glacial cirques , which are ubiquitous in Antarctica18, can help to fill this knowledge gap11, revealing information about the early glacial history—including the transition from greenhouse to icehouse conditions, a period for which there are few high-latitude terrestrial records19. In particular, cirque altitudes can provide information about glacier equilibrium line altitudes (ELAs) during former periods of small-scale (cirque-confined) mountain glaciation20, which can, in turn, be used to obtain quantitative information about past climates21.Extensive ice-sheet cover and widespread erosion have often hindered the study of former mountain glaciers in Antarctica by obscuring and eroding glacial deposits20 and using modern temperate glaciers (and the climatic conditions they experience) as analogues11, we reconstruct palaeotemperatures from these ELAs. Through comparison with published palaeotemperature data, we then establish when glaciers first formed in the TAM and estimate where and when subsequent temperate (i.e. warm-based) mountain glaciers were present .In this study, we analyse the altitudes of glacier-free cirques across the Transantarctic Mountains (TAM) and use this information to estimate the associated glacier ELAs. Assuming that these cirques developed when occupied by temperate glaciers (see the “Methods” section)We show that mountain glaciers were likely present in the TAM during the Late Palaeocene (~60–56 Ma) and middle Eocene (~48–40 Ma). Temperate (warm-based) glaciers were widespread during the Late Eocene (~40–34 Ma) and, in reduced numbers, during the Oligocene (~34–23 Ma). By the Early Miocene (~23–15 Ma) it is likely that all of the cirques in the TAM were submerged by ice sheets, and/or occupied by mostly cold-based glaciers, but that some temperate glaciers were present during the Miocene Climatic Optimum (~15 Ma), before a widespread switch to cold-based glaciation, which continues to the present day.20, the range of SL MSATs , with resulting ELAs ranging from 211 to 3888 m  is likely to have triggered a period where the majority of cirques in the TAM were occupied by temperate mountain glaciers . Some may have remained glacier-free, while those at higher elevations were likely engulfed by larger (possibly cold-based) ice masses , presumed to reflect increased erosion by temperate glaciers26.At the start of the Cenozoic, during the Palaeocene (~66–56 Ma), the Antarctic and global climate experienced warming towards the Early-Eocene Climatic Optimum   were likely occupied by temperate mountain glaciers, while all others would have been occupied by cold-based ice  it is likely that all of the cirques in the TAM were submerged by ice sheets, and/or occupied by mostly cold-based glaciers. Thus, temperate mountain glaciation in the TAM effectively ceased.At the EO-boundary (~34 Ma), global climatece Figs. . Given l4. Fossil wood from the TAM, and pollen records from the Ross Sea, imply summer temperatures of 7 ± 4 °C31, which coincided with mean annual precipitation of ~600 mm19. Results from the present study suggest that these conditions may have triggered the return of temperate mountain glaciers to some cirques in the TAM   caused a switch in glacier thermal regime from warm- to cold-based32. This was likely the termination of temperate mountain glaciation in the TAM, as the climate progressively cooled and dried through the Pliocene (~5.3–2.6 Ma) and Pleistocene (~2.6 Ma–11.7 ka)33. Some of the (now) glacier-free cirques investigated in this study may have been occupied by cold-based ice for much of this time, and only exposed subaerially due to Plio-Pleistocene bedrock uplift9 and/or comparatively recent  ice-sheet thinning34. Following subaerial exposure, the region’s extreme aridity prevented the formation of glaciers within these cirques9, and they will likely remain devoid of glacial ice until the future climate is conducive to temperate mountain glaciation.During the Mid-Miocene Climatic Optimum (~15 Ma), the Antarctic climate switched from its cryo-arid state and experienced a notable period of warming and reduced aridity1%) Fig. . This as3, and that warm-based mountain glaciers were widespread in the TAM even prior to the EO-boundary. As such, we provide insight into Antarctica’s glacial history and support previous studies which indicate early glaciation of the continent13.Overall, our findings help to address long-standing uncertainty about the timing of glacier initiation in Antarctica and demonstrate that mountain glaciers were likely present at high elevations during the Early Cenozoic when sub-tropical vegetation occupied much of the continent35. Cirques were identified as large depressions, bounded upslope by arcuate headwalls, and open in a down-valley direction36. Initially, all features classifiable as cirques in the REMA DSM were mapped, irrespective of the degree of glacier cover. For all cirques, headwalls (i.e. their arcuate upper margins) were identified and mapped following established procedures20. For cirques with minimal glacier cover, the cirque threshold (i.e. the break-of-slope which often marks a cirque’s lower limit) was also mapped, again following established procedures20. Where thresholds were not identifiable, the lower extent of each cirque was drawn as a straight line connecting the outer limits of the headwall or lateral spurs  Digital Surface Model (DSM), which has an 8 m spatial resolution and a vertical error of <1 m37, in part due to low rates of sub-aerial weathering during hyper-arid ice-free conditions38, and because they, and much of the Alpine topography of the TAM, were often occupied by minimally erosive cold-based ice40. These characteristics also apply to glacier-occupied cirques, but they are not considered here since the presence of ice of unknown thickness limits our ability to measure meaningful cirque altitudes. To assess the degree of glacier cover within each mapped cirque, we used a published rock outcrop dataset41. For most of the study region, this dataset was compiled through automated mapping from Landsat 8 satellite imagery , with an overall pixel classification accuracy of 74 ± 9%41. At higher latitudes (i.e. above ~83°S), we used a rock outcrop database compiled through manual identification and digitisation from topographic maps (available via http://www.add.scar.org41). For our purposes, all areas not classified as bedrock were classified as glacial ice. This conservative approach is likely to over-estimate glacier cover since areas covered by non-glacial ice (e.g. ice patches) and/or perennial snow will also be classified as glacial ice. Given this potential, we classified cirques as glacier free if <20% of their surface area was covered by glacial ice37.To investigate the early evolution of Antarctic mountain glaciers, we focused only on glacier-free cirques. These landforms provide a unique record of past temperate mountain glaciation  and are unlikely to have been modified extensively during subsequent glacial-interglacial periods42. From this, former mountain glacier ELAs were calculated using the toe-to-headwall altitude ratio (THAR) method. This approach assumes that a glacier’s ELA lies at a certain proportion of the altitude between its minimum and maximum points43. Given that the present study relies on cirque altitudes, which are a good indicator of former mountain glacier minimum altitudes but are likely to overestimate their maximum altitudes, we adopt a comparatively low THAR of 0.3543. Since our interest is in Antarctica’s long-term glacial history, each former ELA was modified using nine different reconstructions of palaeo topography 44. In the manuscript, we report results based on the median reconstruction of topography at 34 Ma given the focus on conditions prior to widespread ice-sheet development in Antarctica, and since the maximum and minimum reconstructions for this period represent extremes. In the Supplementary Information . Though the EO-boundary might not precisely reflect conditions during the initial period(s) of temperate mountain glaciation, this topography is more representative than the present-day, given ~34 million years of subsequent ice-sheet loading, volcanism, thermal subsidence, horizontal plate motion, erosion, sedimentation, and flexural isostatic adjustment, all of which are considered in the EO topographic reconstruction44.For each glacier-free cirque, the single lowest and single highest DSM grid cells were recorded as the minimum and maximum altitudes, respectively45, using altitude-dependent present-day lapse rates for Antarctica 46.At each modified ELA, present-day MSAT  spanning the 1981–2010 period was estimated from gridded  ERA5 reanalysis data37. Thus, we presume that the climatic conditions which facilitate present-day temperate glaciation in various regions globally also prevailed in the TAM when the ice-free cirques analysed in this study were being formed11. Following this logic, we note that, based on a global dataset of examples21, the MSAT at the ELA of present-day temperate glaciers (here defined as glaciers <60° latitude) is 3.6 ± 2.5 °C , this is partly a pragmatic simplification since ELAs are also known to vary in response to other factors including topography, aspect, and avalanching48.Because of the requirement for efficient and focused subglacial erosion , it is generally assumed that cirques primarily develop (i.e. initiate and grow) when they are occupied by comparatively small temperate (i.e. warm-based) mountain glaciersSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7


Homo sapiens. The protein networking guided by phosphorylation turned out to be prominent in humans, chaotic in yeast, and weak in flies. Furthermore, the molecular functions of GO annotation enrichment regulated by phosphorylation were found to be drastically different between flies, yeast, and humans, suggesting an evolutionary drift specific to each species.The phosphorylation of proteins affects their functions in extensively documented circumstances. However, the role of phosphorylation in many interactive networks of proteins remains very elusive due to the experimental limits of exploring the transient interaction in a large complex of assembled proteins induced by stimulation. Previous studies have suggested that phosphorylation is a recent evolutionary process that differently regulates ortholog proteins in numerous lineages of living organisms to create new functions. Despite the fact that numerous phospho-proteins have been compared between species, little is known about the organization of the full phospho-proteome, the role of phosphorylation to orchestrate large interactive networks of proteins, and the intertwined phospho-landscape in these networks. In this report, we aimed to investigate the acquired role of phosphate addition in the phenomenon of protein networking in different orders of living organisms. Our data highlighted the acquired status of phosphorylation in organizing large, connected assemblages in  Kinases elicit the inhibition and activation of a large spectrum of biological events via the addition of phosphate groups to amino acid residues. The end of the process involves the action of specific phosphatases removing the phospho group by cleavage. This appears as an essential mode of intracellular transduction pathways triggered by outside signaling inputs ,2,3,4. TOn the other hand, a large-scale analysis of the constraint of ideal linear sequence motif(s) presented by the substrates of each known kinase using a systemic approach to individually modify the order of amino acids was performed under the “Phosphorylome Project” using yeast proteome microarrays ,20. The H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe, and S. cerevisiae, and these numbers have gradually increased due to their complementation with more recent experimental datasets. The detailed annotations of proteins are accessible for each entry, and the duplication or the multiple redundancy between these separate databases coming from divergent experimental procedures constitutes a powerful method for cross examination. The CUCKOO workgroup at Huazhong University of Science and Technology has been working for some years on the development of an integrative database of protein phosphorylation in animals, plants, and fungi  where a value of +1 meant a perfect positive association between ranks, a value of 0 meant no association between ranks, and a value of −1 meant a perfect negative association between ranks. Data about the phylogenetic tree of the seven selected species were from Hedges (2002) .S. cerevisiae, we divided the data into quintiles  which made it impossible to divide it into bins of equal size. For this organism, we considered the non-phosphorylated proteins as a category and sub-divided the rest into quartiles  was used to perform the survey. Euler diagrams shown in http://degradome.uniovi.es/cgi-bin/nVenn/nvenn.cgi (accessed on 20 April 2022) [The site il 2022) .p-value generated by a differential analysis pipeline. The String database [In the qPhos database, we selected data relative to the variation of phosphorylation following EGF exposure to Hela cells measured using SILAC (stable isotope labelling by amino acids in cell culture) at six time-points: after 1 min, 5 min, 10 min, 15 min, 20 min, and 30 min. The dataset contained the variation of phosphorylation at each time-point compared to the control for multiple phosphorylation sites. For the same protein, increases of phosphorylation were observed on some sites, while some other sites showed a decrease. In order to obtain a unique value reflecting its change in phosphorylation for each protein, we counted the number of sites where the variation in phosphorylation exceed a factor of two (a Log2 FoldChange EGF vs. control ≤ −1 or ≥ 1). Our count of the number of phosphorylation variation by protein for every time point is specified in database  was used