Coscinodiscophyceae, Rhodobacteraceae (e.g. Amylibacter) and the SAR116 clade. Winter replenishment of nitrate, silicate and phosphate, linked to vertical mixing and a unique microbiome that included Magnetospiraceae and Dadabacteriales, fueled the following phytoplankton bloom. The spring-summer succession of Phaeocystis, Grammonema and Thalassiosira coincided with ephemeral peaks of Aurantivirga, Formosa, Polaribacter and NS lineages, indicating metabolic relationships. In the East Greenland Current, deeper sampling depth, ice cover and polar water masses concurred with weaker seasonality and a stronger heterotrophic signature. The ice-related winter microbiome comprised Bacillaria, Naviculales, Polarella, Chrysophyceae and Flavobacterium ASVs. Low ice cover and advection of Atlantic Water coincided with diminished abundances of chemoautotrophic bacteria while others such as Phaeocystis increased, suggesting that Atlantification alters microbiome structure and eventually the biological carbon pump. These insights promote the understanding of microbial seasonality and polar night ecology in the Arctic Ocean, a region severely affected by climate change.The Arctic Ocean features extreme seasonal differences in daylight, temperature, ice cover, and mixed layer depth. However, the diversity and ecology of microbes across these contrasting environmental conditions remain enigmatic. Here, using autonomous samplers and sensors deployed at two mooring sites, we portray an annual cycle of microbial diversity, nutrient concentrations and physical oceanography in the major hydrographic regimes of the Fram Strait. The ice-free West Spitsbergen Current displayed a marked separation into a productive summer (dominated by diatoms and carbohydrate-degrading bacteria) and regenerative winter state . The autumn post-bloom with maximal nutrient depletion featured  Microbes are fundamental for the marine biosphere and have been recognized as key components of global change biology . UnderstDue to the extreme winter conditions and remoteness, continuous observations covering the polar night have been seldom accomplished through shipboard expeditions, or performed in coastal areas , 16. NewHere, using an array of autonomous samplers and sensors, we portray microbial and oceanographic seasonality in the two major hydrographic regimes of the Fram Strait. This main deep-water gateway to the central Arctic Ocean harbors the northward, relatively warm and ice-free West Spitsbergen Current (WSC) and the southward, ice-covered and cold East Greenland Current (EGC), with some recirculation in central Fram Strait across the marginal ice zone Fig. . Our stuHere we investigated how polar day and night shape seasonality, expecting considerable differences between summer and winter microbiomes in both regions. We hypothesized that phototrophy- and heterotrophy-dominated periods in the WSC harbor markedly dissimilar microbial communities, whereas sea ice cover and polar water masses in the EGC sustain winter-type communities year-round. Our study illuminates fundamental principles of seasonality in Arctic microbial diversity, the ecological importance of the polar night, and potential effects of Atlantification. This evidence helps understanding natural variability and human impact in a region under severe threat by climate change , 37, withttps://www.awi.de/en/expedition/observatories/ocean-fram.html), Remote Access Samplers  were deployed in July 2016 on seafloor moorings F4-S-1 in the core WSC (79.0118 N 6.9648 E) and EGC-3 in the marginal ice zone (78.831 N−2.7938 E), constituting a fixed-point Eulerian approach  according to the manufacturer’s instructions, and quantified using Quantus . 16S and 18S rRNA gene fragments were amplified using primers 515F–926R  and 528i2 v4.12 [After primer removal using cutadapt , 16S and2 v4.12  respectihttps://epic.awi.de/id/eprint/43137. For chemical sensors (Sunburst SAMI-pH and Sunburst SAMI-CO2), the raw readouts are reported. Mooring data are available under https://doi.pangaea.de/10.1594/PANGAEA.904565. Sea ice and surface chlorophyll concentrations, derived from the AMSR-2 and Sentinel 3A OLCI satellites, were downloaded from the University of Bremen and the European Space Agency respectively, considering grid points within a radius of 15 km around the moorings.Temperature, depth, salinity, oxygen concentration, and oxygen saturation were derived from Seabird SBE37-ODO CTD sensors attached to the RAS, confirming consistent properties of the two water samples per date. Sensor measurements were averaged over 4 h around each sampling event, allowing to determine the relative proportions of Atlantic Water (AW) and Polar Water (PW) . Relativhttps://doi.pangaea.de/10.1594/PANGAEA.936749.Nitrate, nitrite, phosphate, and silicate were quantified using a QuAAtro Seal Analytical segmented continuous-flow autoanalyser following standard colorimetric techniques. Accuracy was evaluated using KANSO LTD Japan Certified Reference Materials, with corrections applied as required. Following quality controls, results deemed questionable or of bad quality  were excluded from further analyses (labeled NA in Supplementary Table https://rstudio.com). In short, alpha-diversity and rarefaction curves were computed on raw ASV counts using R package iNEXT [https://github.com/matthiaswietz/RAS-1617.Data analysis was done in R v4.1.1 implemented in RStudio . Hence, differences between the WSC and EGC correspond to different hydrography, ice cover as well as sampling depth. Earlier studies investigated the background of these vertical and horizontal contrasts in hydrography [Environmental conditions and microbial communities substantially differed over the year, but also between the two sampling sites Fig. , 3. At trography  and micrrography .rho = 0.6, p < 0.006).This study focuses on the seasonal shifts in microbial communities. In both the WSC and EGC, communities markedly changed in composition and diversity over the annual cycle Fig. , illustrWe contextualized major patterns in microbial and physicochemical variability Figs. −4 to delp < 0.001), whereas bacterial composition varied mostly with temperature , comparable to the global TARA microbiome study [Pirsonia and MAST-3 . This explained why some taxa correlate with seasonally changing environmental parameters, and some with polar or Atlantic conditions  compared to spring/summer (WSC) respectively . Furthermore, Dadabacteriales appeared from February (WSC) or late March (EGC)  Fig.  and mighThe increase of Syndiniales, parasitic recyclers of phytoplankton biomass , in NoveNitrosopumilaceae and Nitrospinaceae  might recycle nitrogen by fixation and contribute to a yet underestimated nitrogen source [Magnetospiraceae perform CO2 fixation and thiosulfate oxidation [Nitrosopumilaceae, Nitrospinaceae, and Magnetospiraceae during winter in both Arctic and Antarctic Oceans [Pirellulaceae and Woeseiaceae through ammonia oxidation and denitrification respectively [Winter bacteria and archaea likely contributed to nutrient replenishment. The co-occurrence of ae Figs. , 6b, thepinaceae . In addin source , 79. Furxidation . Overallc Oceans , 81. Furectively , 83. Theectively , 85. OveBacillaria and Naviculales, together with Polarella and Chrysophyceae flagellates, constituted up to 15% of sequences between February and March , suggesting that ice cover sustained these winter-type taxa into summer. As SAR406 might participate in sulfur cycling [An EGC-specific winter bacterium was the SAR406 clade, peaking at 9% sequence abundance in March and remaining detectable into summer. In addition, the frequently ice-associated genus ne Figs. . Both SA cycling , loss of(Phaeocystis and Chaetoceros) followed by araphid-pennate diatoms (Grammonema) and centric diatoms   Fig. . A compara) Fig.  suggestseae Fig. , comparaThalassiosira was specific for summer and the final bloom stage, when nitrate and phosphate declined and oxygen concentrations peaked  and concurrently decreasing chlorophyll indicates that trophic structure shifted towards heterotrophy. Increase of the roseobacter Amylibacter (formerly NAC11-7) to 15% sequence abundance emphasized the beginning transition to the autumn post-bloom where Rhodobacteraceae dominated  and Gyrodinium (Dinoflagellata) that constituted up to 35% of eukaryote sequences. Chromidina is normally considered an animal parasite, suggesting yet undescribed free-living niches in the marginal ice zone. The prevalence of mixotrophy was underlined by the earlier detection of Woloszynskia, and twofold lower flavobacterial abundances compared to the WSC ; declining nitrate and shift towards centric diatoms and mixotrophic flagellates upon increasing stratification (summer); minimum nutrients and highest temperatures when Coscinodiscophyceae diatoms and oligotrophic bacteria prevailed (autumn); and chemoautotrophic microbial recyclers and nutrient replenishment during vertical mixing (winter). Comparable observations have been made in a year-round study using Niskin-based sampling [Coscinodiscophyceae in autumn, Aurantivirga and SAR92 as first bacterial responders, and Amylibacter at the summer-autumn transition [Dynamics in the WSC illustrate key principles of microbial seasonality in the ice-free, open Arctic Ocean: sampling , illustransition . This su−1 [Colwellia, with surface phytoplankton growth mainly detected during low ice in autumn. Periods of low ice coincided with higher abundances of Phaeocystis, Thalassiosira, OCS116 and Aurantivirga (Supplementary Fig. The EGC exhibited combined effects of depth, ice cover and variable polar/Atlantic water masses, with a strong heterotrophic signature year-round. Seasonality and similarities to the WSC scaled with the extent of AW advection. At a maximum speed of 0.25 m s−1 , water fPhaeocystis [Atlantification of the Arctic may enhance early blooms of eocystis , 27 and This first assessment of microbial seasonality in the Fram Strait by autonomous sampling identified marked seasonal contrasts, distinct transition events, as well as dynamic variability linked to polar vs. Atlantic conditions. The characterization of bloom stages, ephemeral abundance peaks, and polar night characteristics promotes the understanding of the drivers and timescales of microbial seasonality in ice-covered and ice-free Arctic waters. These insights yield a number of fundamental ecological conclusions, with implications for the present and future Arctic Ocean.In conclusion, the demonstrated seasonal microbiome dynamics and drivers contribute to the understanding of Arctic ecosystem functioning over polar day and night. This evidence is particularly relevant considering the anticipated impact of climate change on polar regions.Supplementary Fig. 1Supplementary Fig. 2Supplementary Fig. 3Supplementary Fig. 4Supplementary Fig. 5Supplementary Fig. 6Supplementary Fig. 7Supplementary Fig. 8Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Methods


The increased glucose levels occurring in diabetes lead to several metabolic alterations responsible for the onset of the so-called diabetic complications, which include nephropathies, neuropathies, retinopathies, and cataract. An increased flux of glucose through the polyol pathway is considered the most relevant among these alterations. For this reason, the block of the polyol pathway, through the inhibition of the enzyme aldose reductase, is considered a valuable strategy to impair the onset of diabetic complications. However, aldose reductase also exerts a beneficial effect inside cells, since it can remove toxic aldehydes. Thus, to ameliorate the outcome of the use of aldose reductase inhibitors, the use of “differential inhibitors” has been proposed. These inhibitors should block the catalytic activity depending on the substrate the enzyme is working on, thus preserving the detoxifying action of the enzyme. In this work, derivatives of catechins are analyzed to evaluate their inhibitory action on aldose reductase. The study was conducted both in vitro on the isolated enzyme and in silico through a computational approach. Results demonstrated that gallocatechin gallate and catechin gallate act as differential inhibitors and that this action may be linked to an incomplete inhibitory effect.The inhibition of aldose reductase is considered as a strategy to counteract the onset of both diabetic complications, upon the block of glucose conversion in the polyol pathway, and inflammation, upon the block of 3-glutathionyl-4-hydroxynonenal reduction. To ameliorate the outcome of aldose reductase inhibition, minimizing the interference with the detoxifying role of the enzyme when acting on toxic aldehydes, “differential inhibitors”, i.e., molecules able to inhibit the enzyme depending on the substrate the enzyme is working on, has been proposed. Here we report the characterization of different catechin derivatives as aldose reductase differential inhibitors. The study, conducted through both a kinetic and a computational approach, highlights structural constraints of catechin derivatives relevant in order to affect aldose reductase activity. Gallocatechin gallate and catechin gallate emerged as differential inhibitors of aldose reductase able to preferentially affect aldoses and 3-glutathionyl-4-hydroxynonenal reduction with respect to 4-hydroxynonenal reduction. Moreover, the results highlight how, in the case of aldose reductase, a substrate may affect not only the model of action of an inhibitor, but also the degree of incompleteness of the inhibitory action, thus contributing to differential inhibitory phenomena. Aldose reductase  is a NADPH-dependent oxidoreductase, belonging to the aldo-keto reductase superfamily  and reprHere, we report the characterization of additional catechin derivatives as potential ARDIs. Our results increase the number of molecules exerting a substrate-dependent inhibitory action of AKR1B1 and highlight that the occurrence of an incomplete inhibition may contribute to a differential inhibitory effect.Gallocatechin gallate , catechin gallate , epicatechin gallate , catechin , gallocatechin , and epicatechin  were from Extrasynthese ; NADPH and L-idose were from Carbosynth . Bovine serum albumin (BSA), D,L-dithiothreitol (DTT), D,L-glyceraldehyde (GAL), EDTA, glycerol and glutathione (GSH) were purchased from Sigma-Aldrich ; ammonium sulfate, sodium phosphate monobasic were from J.T. Baker ; YM10 ultrafiltration membranes were from Merck-Millipore . All other chemicals were of a reagent grade.340 = 6.22 mM−1·cm−1) using a Biochrom Libra S60 spectrophotometer. The standard assay mixture (0.7 mL) contained 0.25 M sodium phosphate buffer pH 6.8, 0.18 mM NADPH, 0.4 M ammonium sulfate, 0.5 mM EDTA, and 4.7 mM GAL. One unit of enzyme activity is the amount of enzyme that catalyzes the conversion of 1 µmol of substrate/min in the above assay conditions. The same assay conditions were adopted in the inhibition studies with L-idose, HNE, or GSHNE as substrates.The AKR1B1 activity was determined at 37 °C, as previously described , monitor50 values were determined by non-linear regression analysis using GraphPad Prism version 7.04 . Values were obtained from at least six different inhibitors concentrations (each assayed at least in triplicate) and are reported with their 95% Confidence Limits (CLs). Inhibitors were dissolved either in water  or in 40% ethanol . The final concentration of ethanol in the assay was never higher than 1%. The presence of ethanol did not affect the inhibitory ability of GCG, CG, and ECG. The percentage of inhibition was calculated referring to an assay performed using the same solvent concentration in the absence of inhibitors.ICw/v) glycerol. Before use, AKR1B1 was extensively dialyzed against 10 mM sodium phosphate buffer pH 7.0.The human recombinant AKR1B1 was expressed and purified to electrophoretic homogeneity, as previously described . The speappkcat and appKM were evaluated by non-linear regression analysis of rate measurements vs. substrate concentration according to Michaelis-Menten equation. The analysis was performed through GraphPad Prism 7.04 software by a non-linear “Robust Regression” analysis in which each point is individually weighted through iterative weighing of the smallest squares , making use of previously derived equations  approaches to zero. This is the case of L-idose reduction in the presence of all the tested compounds. On the contrary, in the case of CG and GCG for HNE reduction and at least in the case of CG for GSHNE reduction, a decrease of the appKM/appkcat versus [I] is observed. Moreover, the observed trend could hardly be interpolated with a straight line.It is evident that, for all the substrates, both k+4 > 0)  .appKM and appkcat (Equations (1)–(3), see ref  data were interpolated by Equation (4) as an incomplete, uncompetitive inhibition. With such an approach [k+4 values are reported in appKM/appkcat versus [I] (Equation (3) or Equation (4)) and 1/appkcat versus [I] (Equation (1)) plots. An essentially complete inhibition occurred for all the inhibitors in the case of L-idose reduction (k+4 ranging from approximately 0.7% to 2.9% of k+2). Conversely, an incomplete inhibition by CG, GCC, and ECG, respectively, was observed for HNE reduction, with k+4 values resulting approximately 6, 12, and 9% of k+2. Additionally, for GSHNE reduction, the inhibition appears not complete, with k+4 values resulting as approximately 4, 6, and 14% of k+2 for CG, GCG, and ECG, respectively.Thus, ary data  were anaapproach , the emeiK and *iK one order of magnitude higher with respect to CG and GCG). On the other hand, ECG was the one more closely behaving as the previously studied EGCG  related to all the tested catechins acting on HNE reduction and for inhibition of GSHNE reduction by CG and GCG. These results can be fully explained by the occurrence of an incomplete inhibition of the reactions combined with an uncompetitive model of action, two conditions indicated to be necessary to observe the phenomenon [k+4 approximately 14% of k+2) and the evident mixed type of inhibition (i/KiK* approximately 2) did not generate any decrease of the appKM/appkcat versus [I]. In this case, it must be assumed that the driving force for the evolution of the enzyme–substrate complexes  towards products is counteracted by the relative stability of EI complex. Finally, no special comment is deserved for those cases where the inhibition was complete, in which the appKM/appkcat versus [I] either may increase (mixed inhibition) or remain constant (uncompetitive inhibition). This is the case of the mixed inhibition exerted by all the catechins/epicatechins on L-idose reduction (Panel A of As noticed above, peculiar evidence appearing from inhibition kinetic data  concernsenomenon . This reThe possibility that a substrate may affect not only the model of action of an inhibitor, but also the degree of incompleteness of the inhibitory action may contribute to differential inhibitory phenomena. The results from this study, while offering a characterization of catechin derivatives as powerful inhibitors of AKR1B1, confirm the potential of some molecules in displaying a differential inhibition of the enzyme depending on the substrate under transformation. Our data shed light on some inhibitory features that molecules must exploit to exert differential inhibition; however, the observed differential action remains phenomenological evidence, being structural restrictions for an ARI to become an ARDI still waiting to be clearly defined. In this regard, work is in progress to evaluate the influence of the incomplete inhibitory action exerted by the hemiacetal form of the sugars  on the d


The quality of lake ice is of uppermost importance for ice safety and under-ice ecology, but its temporal and spatial variability is largely unknown. Here we conducted a coordinated lake ice quality sampling campaign across the Northern Hemisphere during one of the warmest winters since 1880 and show that lake ice during 2020/2021 commonly consisted of unstable white ice, at times contributing up to 100% to the total ice thickness. We observed that white ice increased over the winter season, becoming thickest and constituting the largest proportion of the ice layer towards the end of the ice cover season when fatal winter drownings occur most often and light limits the growth and reproduction of primary producers. We attribute the dominance of white ice before ice-off to air temperatures varying around the freezing point, a condition which occurs more frequently during warmer winters. Thus, under continued global warming, the prevalence of white ice is likely to substantially increase during the critical period before ice-off, for which we adjusted commonly used equations for human ice safety and light transmittance through ice. Under continued global warming, lakes will increasingly be covered by white ice, in particular towards the end of the ice cover season when fatal winter drownings occur most often and light limits the growth and reproduction of primary producers. Increasing temperatures have an effect not only on the mean but also on the amplitude of the annual air temperature cycle, which in turn causes accelerated ice cover loss rates in a warmer world5, which have been documented for many lakes across the Northern Hemisphere over the past decades6. Future climate simulations indicate that the spatial distribution of lake ice cover in the Northern Hemisphere will turn more than 35,000 seasonally frozen lakes into intermittently ice-free lakes in a 2 °C warmer world, affecting nearly 400 million people7. Of those lakes, up to 5700 are projected to become permanently ice-free within this century8.The majority of lakes in the Northern Hemisphere are still periodically covered by ice in winter, but long-term records show a rapid decline in the number of days when lakes are frozen, mainly due to globally increasing air temperatures10, lake ice quality is usually not assessed and only very few studies present data on ice quality , a few weeks after ice-on in December or January. Many lakes were already ice-free in April, decreasing the number of sampling occasions during that month to ten. In our data set, the thickest ice layers were usually observed in February or March, depending on how long the ice cover lasted.During the IceBlitz campaign, the total lake ice thickness on lakes across the Northern Hemisphere varied from 1 to 103 cm, with a median of 29 cm (mean ± one standard deviation: 32 ± 18 cm). Most measurements were performed in January and February . Because winter air temperatures in the Northern Hemisphere are projected to further increase23, we expect that the trend towards thinner ice will continue into the future.The observed ice thickness during the 2020/2021 IceBlitz campaign was generally lower than we found reported in the literature Table , only 21ars Fig. . IncreasThe ice on IceBlitz lakes commonly consisted of both a black and a white ice layer where the black ice layer was the dominant ice layer when all 167 measurements were taken into consideration (mean ± one standard deviation: 70 ± 28%). There was, however, a clear seasonal trend in the ice layer composition with a substantial increase in the thickness and the proportion of white ice towards late winter and spring , and H is the total ice thickness in cm. Although Eq.  might be responsible for ice collapse despite a sufficient theoretical total ice thickness. Laboratory experiments have shown a reduction of the flexural strength of ice  by 51% for white ice compared to black ice at a temperature of −0.5 °C16. Based on these results, we suggest a modification of Gold’s equation during days when air temperatures vary around the freezing point:P is the allowable load in kg, A corresponds to 3.5 kg cm−2  is the transmittance of light through the combined black and white ice/snow layer, τ(A) is the transmittance of light through the upper white ice/snow layer, τ(B) is the transmittance of light through the lower black ice layer, ρ(A) is the reflectance of the upper white ice/snow layer and ρ(B) is the reflectance of the lower black ice layer. τ(C) approaches zero when snow covers the ice, as snow usually has a reflectance of close to one35. τ(C) also declines when the upper white ice layer becomes thicker36. Based on the data from Lake Vendyurskoe (1997–2020)36 we obtained the following model for underneath ice light conditions:Edz is the 12-hour day time mean (8.00 a.m. to 8.00 p.m.) irradiance underneath ice in W m−2, a is a solar radiation dependent constant . We attribute the lack of model improvement to the fact that the thickness of black ice was unrelated to Edz in Lake Vendyurskoe (p > 0.05). These results indicate that Edz is mainly driven by the thickness of snow and white ice, where each cm of snow and white ice reduces the under-ice irradiance by approximately 9%. Further, by analyzing the inter-annual variability in Edz in Lake Vendyurskoe in spring, we found no trend over time in Edz despite a significant decrease in total ice thickness during this period . Thus, under-ice light conditions did not improve despite thinner ice in spring. We suggest that these under-ice light conditions were driven by the thickness of white ice which did not show a significant decrease in spring from 1997 to 2020 (linear time series model: p > 0.05).White ice conditions do not only affect the stability of the ice cover but also the light regime underneath. White ice is known to have a severalfold higher reflectance than black ice, thus only small amounts of photosynthetically active radiation penetrate through white ice38. Most vulnerable to white ice conditions and snow on ice are photoautotrophs in lakes39, in particular non-motile photoautotrophs that are dependent on radiatively-driven convection to reach sufficient light 41. Motile taxa can have an advantage during low light conditions under-ice13. Mixotrophy is another strategy to adapt to low-light under-ice conditions42. Further up in the food web zooplankton has developed survival strategies to low-light under-ice conditions by feeding on mixotrophic microorganisms and allochthonous organic material, but for growth and reproduction, a diet containing nutrient-rich photoautotrophs is still essential43. Thus, despite a variety of survival strategies, snow and white ice induced changes to the photoautotroph community cascade through the food web, with substantial consequences for microbial, zooplankton, and fish populations45.Low light conditions in spring caused by a white ice layer and/or snow on ice are critical for the development of primary producers and consumers as their growth and reproduction is dependent on light and convection underneath iceOur sampling campaign focused on spatial and seasonal patterns. The most obvious and relevant result was the consistent buildup of white ice in spring until the time just before ice-off which we attribute to the increased time window when air temperatures vary around the freezing point. Under expected future global warming, many lakes in the world will become exposed to these prolonged freeze-thaw cycles. According to our study, such lakes will experience a shift in the ice layer composition towards white ice conditions. We conclude that white ice conditions in lakes need far more consideration than previously given. We suggest the use of Eq.  to re-exhttps://gleon.org) from December 2020 to April 2021. Participants of the campaign were asked to conduct at least one sampling visit at an ice-covered lake and measure  total ice thickness, the thickness of black and white ice, the thickness of snow and/or slush layers on top of the ice layer, water temperature in the drilling hole and air temperature 1.5 m above the drilling hole using a standardized protocol  and Lake Vendyurskoe . We used these data to evaluate in how far ice conditions during previous years differed from our sampling winter. Additionally, we collected literature data on ice quality, which comprised 117 ice observations from 18 lakes across the Northern Hemisphere , version 4. NASA Goddard Institute for Space Studies980 Fig. . Data frake Fig. .th and 75th quantiles and the lines that extend from the box are whiskers that represent 1.5 times the interquartile range from the top and bottom of the box. Values that fall above or below the end of the whiskers are plotted as dots. Time series analyses were performed in R 4.0.2  using linear models and checking for temporal autocorrelation, i.e., lm(formula = ice~time). Time series analyses were performed on April measurements for Lake Vendyurskoe. Data from Mozhaysk Reservoir were not used for time series analyses because ice quality was only measured during 15 out of 51 years from 1971 to 2021.All statistical analyses, except of the time series analyses, were performed in JMP, version 14.2.0., SAS Institute Inc. For the smoothing splines a lambda of 0.05 was chosen which commonly is used to estimate a functional relationship between a predictor and a response variable. Boxplots are outlier boxplots, also known as box-and-whisker plots, where the bottom and top of the box show the 25Supplementary InformationPeer Review File


Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of supercooled water only a few degrees below 0 °C. An open question is whether this ability also extends to the deposition mode, i.e., to water vapor. In this work, we used three proteins, apoferritin, InaZ (ice nucleation active protein Z), and myoglobin, of which the first two are classified as ice-nucleating proteins for the immersion freezing mode. We studied the ice nucleation ability of these proteins by differential scanning calorimetry (immersion freezing) and by environmental scanning electron microscopy (deposition freezing). Our data show that InaZ crystallizes water directly from the vapor phase, while apoferritin first condenses water in the supercooled state, and subsequently crystallizes it, just as myoglobin, which is unable to nucleate ice. Understanding SCW formation is crucial in various technological processes5, usually to avoid undesired ice formation. SCW at ambient pressure forms below the freezing point, where pure water can be prevented from freezing for long periods, e.g., in hard confinement6 or in soft confinement9. SCW becomes progressively less stable at lower temperatures. In very pure conditions, SCW can remain liquid until − 38 °C11, which corresponds to homogeneous crystallization, i.e., in this case, ice nucleation starts inside bulk water  on biphilic surfaces15, pulsed-laser–heating techniques10, and simple optical detection of freezing in ml containers16.Supercooled water (SCW) is of utmost interest for environmental processes, mainly in the atmosphere17. Merely cooling water vapor does not produce ice. Rather, ice formation requires overcoming the nucleation barrier. A possible way (depending on weather conditions) is immersion freezing, where a droplet of SCW combines with a solid particle, and subsequently freezes 21, with the droplet ice nuclei counter Zurich (DRINCZ)22, with differential scanning calorimeters (DSCs)25, and with optical microscopes equipped with various cooling systems29. The essential feature is always cooling of an aqueous solution or suspension at a controlled rate, typically at ambient pressure, to find the crystallization temperature (Tcryst). In other words, an aqueous liquid mixture is (super)cooled until it crystallizes.The significance of SCW in the atmosphere is mainly related to aerosols in connection with cloud formation. The mechanism by which water vapor condensates to form ice clouds has been investigated for decades because it is essential for understanding cloud formation, which in turn is one of the decisive factors in climate and weather30. One mechanism is the deposition of SCW on a (solid) particle, followed by ice nucleation and growth, ultimately resulting in a bulk ice crystal31. The other one is direct vapor to ice nucleation deposition on the particle. Deposition nucleation with both mechanisms is represented in Fig. 36.In contrast to immersion freezing, the deposition nucleation mode starts from water vapor, and the crystallization process can follow two different routes, as shown in Fig. 2 lattices, differs between immersion and deposition mode27. In fact, it is not even trivial to compare both modes.Although ice nucleation by immersion and deposition consists of water crystallization on a “foreign” particle, both modes are independent. They can provide different information about the nucleation properties of the particle. For example, a recent study showed that the nucleation ability of the aluminosilicate feldspar and of quartz, two essential aerosol minerals, both based on SiOPice, and the blue one is the SCW saturation pressure PSCW, respectively. We can define the relative humidity of ice as:P is the absolute pressure. Similarly, we can define the relative humidity with respect to SCW as hSCW by using PSCW instead of Pice  are widespread in nature. They induce ice formation in SCW, e.g., the bacterium Pseudomonas syringae43 employs the ice nucleation active protein Z (Ina Z) on its outer membrane to trigger freezing at temperatures up to − 2 °C44 . InaZ is the most efficient and one of the best studied INPs. It comprises ~ 1200 amino acids45 with many repeats, which are arranged in a β-helical folding, which in turn is based on stacked β-sheets. The conformation is similar to that of insect antifreeze protein structures47 (which provide freeze protection inside cells). The β-sheets result for both protein types in extended and almost flat surfaces. Further similarities are typical ice-binding sequences (TxT motifs49) and the ability of arranging the external hydration layer into an ice-like structure51, despite the opposite function for ice nucleation. However, InaZ has a unique property, namely the capacity to promote energy transfer across the water interface51. Moreover, InaZ has direct applications such as freezing technology and food preservation, and especially the induction of snow formation 52.Although immersion and deposition freezing modes are different, ice nucleation proceeds in the presence of a “foreign” solid particle. This is usually assumed to be a mineral (inorganic), but can also be composed of organic matter16 recently found that the apoferritin protein cage is a good ice nucleator in immersion freezing experiments. Apoferritin is ubiquitous in many cells from bacteria to vertebrata. It has 174 amino acids, arranged in α-helical conformation, but forms a 24-mer spherical cage (4176 amino acids), which is used by the organisms to store iron. Its ice nucleation ability is apparently not exploited by nature. It could be based on specific properties of the cage surface 53. Apoferritin has no technical use, but is of great interest for nanoscale science and nanotechnological uses, also in biomedicine.There are, however, probably many more protein structures (ternary and quaternary), which can promote ice nucleation. For example, Cascajo-Castresana et al.Myoglobin is a small protein present in high concentrations in vertebrata; its task is oxygen transport. It comprises only 153 amino acids folded into α-helices; its morphology is almost spherical with a high curvature. Myoglobin was the first protein to be characterized by X-ray diffraction. It is often used as a standard example of a globular protein in research; furthermore, it can be envisaged as the “natural color” in red meat.Here, we present experiments on ice nucleation on the above-mentioned three proteins, in both modes, immersion and deposition freezing. We investigated apoferritin, and chose InaZ as positive control because it is an excellent INP, and myoglobin, which does not nucleate ice, as negative control. Our question is whether the proteins have the same nucleation ability in both nucleation modes. To this end, we analyzed immersion freezing by differential scanning calorimetry (DSC) in solution, and studied deposition freezing by ESEM. The latter allowed us to follow the fate of liquid microdroplets in contact with the (dry) proteins, and in contact with a solid surface (copper). Our results indicate that apoferritin is a good INP in immersion mode but not in deposition mode. Therefore, the two crystallization modes are not correlated.−4 g/ml. To remove buffer salts and prevent aggregation of the individual cages, the diluted solution was dialyzed in ammonium bicarbonate (NH4HCO3) buffer for 96 h. For this purpose, the samples were suspended in 10 mM ammonium bicarbonate , pH = 7.4 to 7.6, prepared with Milli-Q water. Dialysis was carried out in 10,000 MWCO dialysis cassettes (Thermo Scientific) for 96 h, with the ammonium bicarbonate buffer replaced every 24 h54. In the vacuum of the ESEM chamber, NH4HCO3 completely decomposes into gaseous H2O, CO2, and NH3. The advantage of this procedure is that potential apoferritin disassembly in pure water  is avoided.Apoferritin  was diluted to 3.4 × 10−4 g/ml.In addition to apoferritin, we employed myoglobin and Snomax solutions as negative and positive controls for ice nucleation, respectively. Snomax is a commercial product that contains (among additives such as carbohydrates and ash) ice-active protein complexes from the bacterium Pseudomonas syringae. This snowmaking additive freezes water at a temperature close to 0 °C. Snomax is therefore considered an excellent INP. We used it without further purification. Myoglobin  is not known to nucleate ice. Myoglobin was dialyzed in 10,000 MWCO dialysis cassettes (Thermo Scientific) for 24 h in water, replacing it three times during the day, and once overnight. Both Snomax and myoglobin were diluted to 3.4 × 10DSC measurements were carried out on ∼ 10 mg specimens in a Q2000 TA Instruments, operated in standard mode. Sealed aluminum pans were used for all the samples. For the nucleation experiments, the samples were cooled at 1 K/min from 20 to − 33 °C to determine the crystallization temperature. After annealing at − 33 °C for 5 min, the samples were reheated at 10 K/min to determine the melting properties. A helium flow rate of 25 mL/min was used throughout.We employed a Quanta 250 ESEM , which provides water vapor pressures of > 2000 Pa and sample cooling by a Peltier stage to < − 20 °C. ESEM experiments were carried out in low vacuum mode (pressure limitation 200 Pa) at − 20 °C. This allows the recording of large-scale images (mm size) because a pressure-limiting small aperture at the column cone is not required.−1 K−1). For decreasing the sample surface area, too, we covered it with carbon tape, mounted above the PDMS film, leaving a window of only several mm2. Here, a droplet of protein solution was placed on the copper surface and evaporated in air. This procedure avoids ice crystallization on exposed sample edges55. Figure On the Peltier stage, a homemade copper piece  was fixed. Heat-conductive silver paste was placed between the stage and the copper piece for optimal heat transfer. The copper surface is the coldest part in contact with the water vapor, hence, condensation starts here. To achieve a highly stable temperature, it is useful to restrict this surface area. To this end, we employed a poly(dimethylsiloxane) (PDMS) film with low thermal conductivity (0.2 Wm2 particles) to remove any visible damage, and cleaned in an ultrasound bath, first with acetone, then isopropanol, and finally water. The protein solution was deposited on the upward facing surface of the copper piece by drop-casting, and dried in air (see above). Figure 56.Before every new deposition experiment, the copper stub was polished .Chamber pressure decrease to high vacuum , Fig. To characterize the copper surface  in terms of wettability and roughness, static water contact angle (WCA) and AFM measurements were conducted. Figure cp = 3.4 10−4 g/ml. Water crystallization is an exothermic process; consequently, heat is released to the surrounding. Therefore, the onset of crystallization is identified as the temperature where the heat flow starts to increase.This section investigates the crystallization of aqueous solutions of apoferritin, Snomax, and myoglobin in immersion freezing mode. Figure 11. However, the imperfections of a DSC experiment, such as the surfaces of the aluminum pans and possible water impurities, induce heterogeneous crystallization. In such a case, the onset of crystallization is shifted to between − 25 and − 17 °C, depending on the cooling rate26. A similar crystallization temperature, (− 20.6 ± 0.9) °C, was found for the myoglobin solution, a protein that does not show any ice nucleation or ice binding properties. This behavior is expected: An aqueous solution of myoglobin contains small globular molecules, which are not aggregated. Their surface is not flat but highly curved: The radius of ~ 2 nm is just above molecular radii, the shape is far from that of a perfect sphere, and each surface spot has its own specific behavior, determined by the local amino acid sequence. This is a significant difference to the proposed requirement of low curvature for INPs53.As seen in Fig. 12. Of the many interpretations, we here point out one of the most simple ones, based on a purely geometric effect58, which links ice nucleation to flat molecular surfaces. The curvatures of the large protein InaZ, with its extended stacked β-sheets, and of the large supramolecular surface of apoferritin  are relatively small. Moreover, both apoferritin and InaZ readily form aggregates16, resulting in even lower curvatures. In contrast, the curvature of a small globular protein such as myoglobin (~ 2 nm radius) is very high. Apoferritin showed several additional crystallization or re-crystallization processes at lower temperatures. The released heat was very small, so these processes might not have occurred in all parts of the sample.On the contrary, the crystallization of water with apoferritin occurred at a higher temperature (− 5.4 ± 0.1 °C), and Snomax solutions (containing InaZ) showed (− 4.4 ± 0.1) °C. These temperatures are much higher than those observed in pure water, or in water with myoglobin. This increase in the crystallization temperature is due to the ice nucleation ability of both proteins, as previously reportedIn the following, we investigate whether the ability of apoferritin and Snomax to bind ice inside a droplet of supercooled water  extends to proteins in contact with supersaturated vapor .P = 102 Pa and T = − 20 °C). Ice growth was triggered when the pressure was increased to 119 Pa . Figure  Pa Fig. b. Subseq Pa Fig. c,d. The Hence, for the InaZ protein (in Snomax), water crystallizes directly from the vapor phase, preventing the formation of SCW. We conclude that InaZ is an excellent INP since it shows an extraordinary ability to avoid SCW in both immersion and deposition modes. This result also confirms our choice of InaZ/Snomax as a positive control.hSCW = 1. Nevertheless, we detected neither ice nor SCW. We increased the pressure well above Pwater, to reach hSCW > 1. When hSCW > 1 and when crystallization was not triggered . This new front was not in contact with the protein spot, so its origin should have been ice crystallization generated on the copper substrate outside of our observation window.Figure In conclusion, myoglobin is a poor ice nucleator in both immersion and deposition. Notably, in deposition freezing, water crystallization occurs by the first mechanism, i.e., water is condensed in the form of SCW and crystallizes. This is expected, and we can employ our result as a negative control.hSCW > 1), we were able to create droplets .The characteristics of the SCW droplets observed in Fig. Figures Similar to myoglobin, SCW crystallization was triggered on the apoferritin spot during the scan, covering it completely  measurements to compare ice nucleation in the liquid phase (immersion nucleation) and from the gas phase (deposition nucleation), for three proteins. In addition, it presents electron microscopy of (liquid) supercooled water (SCW).Myoglobin was tested as a well-known standard protein, for which no ice nucleation action is documented. As expected, this is reflected in the very low nucleation temperature (− 20 °C) in the immersion test. The deposition nucleation from water vapor had not been reported before. We demonstrate the presence of some SCW on the solid protein for a short time (sub-seconds), but in general, the nucleation appears to proceed directly from vapor to solid. Hence, we postulate two crystallization routes: From vapor to solid and from vapor to SCW to solid.We chose the commercial "snowmaker" Snomax, which contains the ice nucleation protein (INP) InaZ, as test for a well-characterized INP. We reproduced the very high immersion nucleation temperature of − 4.4 °C, which is the base for snow production from SCW. We further demonstrated that InaZ is also an excellent deposition nucleator, resulting in the fast deposition of ice from water vapor onto the solid protein.Our third protein is the supramolecular cage apoferritin, which is a surprisingly good INP in the immersion mode (− 5.4 °C). We found, however, that the deposition mode involves the formation of SCW from water vapor, and only afterwards crystallization. Hence, apoferritin is a relatively poor deposition mode nucleator.In comparison, apoferritin and InaZ differ primarily in the deposition mode: Apoferritin is a relatively poor INP for deposition. It develops SCW as an intermediate between gas and solid phase, i.e., first, SCW condensates from vapor, and, with some delay, it freezes. This would be a slow analog to the fast SCW freezing on myoglobin. Another interpretation is that the formation of SCW droplets cannot be avoided on apoferritin; therefore, this protein, although being a good INP, cannot follow the direct vapor route to nucleation of solid ice. Hence, the two crystallization modes are not correlated.The ESEM observation of SCW also entailed imaging of SCW droplets, whenever they were preserved for sufficient time (minutes). We present a reproducible method to produce SCW, based on a (hydrophobic) polished and solvent-cleaned copper surface, and a water vapor pressure jump at a low temperature. As expected, SCW droplets on our hydrophilic proteins show a very different behavior: They have an irregular shape, cover more area, and feature smaller gaps between droplets, i.e., they nicely wet the surface of a protein spot. Once the crystallization of SCW on proteins is triggered, it is very fast and covers the complete mm-sized protein spot in seconds. The ice propagates from the rim of the spot, where it bridges the protein spot with the copper surface.61. Once could circumvent this problem by employing complex biological surfaces such as the cell wall of Pseudomonas syringae43, from which InaZ is isolated.Our results demonstrate that ESEM is helpful for experiments in a broad range of water/ice-related topics, specifically for investigating freezing and sublimation on the microscale. This scale should be expanded to the nanoscale, thus providing new insights into the processes leading to ice cloud formation on small nuclei. In addition, the ice growth on airplane wings, a critically dangerous consequence of SCW condensation on flat surfaces, might require investigating submicron surface features. Unfortunately, extending the studies to living cells and biological antifreeze processes, i.e., working with living matter, is not straightforwardSupplementary Information 1.Supplementary Video 1.


Cip1/Waf1, p27Kip1, and p57Kip2 bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signaling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular-dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis, and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs) p21 Cell proliferation is driven by the activation of Cyclin/Cyclin-dependent kinases (Cdks). Cyclin/Cdk activity is tightly regulated to prevent unscheduled activation, which can promote tumorigenesis. Three conserved inhibitors bind to Cyclin/Cdks: p21, p27, and p57. Inhibitor activity is modified by their phosphorylation downstream of pro-proliferative signaling. We systematically investigate the effect of inhibitor phosphorylation on the structure of Cyclin/Cdk/inhibitor complexes and the implications for Cdk activity. Despite structural similarity, the three inhibitors respond in non-intuitive ways to phosphorylation, leading to diverse effects on Cdk activity, providing insight into how proliferation can be regulated in a context-dependent manner. Scrutinizing Cyclin/Cdk/inhibitor interactions also contributes to our understanding of the mechanism of action of clinically relevant Cdk4/6 inhibitors, which may act by displacing Cdk inhibitors.During development and in tissue homeostasis, the controlled release of cells from a quiescent  state into proliferative cycles is vital to achieve and maintain a healthy state. Dysregulation of these pathways underpins abnormal development and tumorigenesis. Cell proliferation can be induced by multiple signals, including mitogenic growth factors, the extracellular matrix, and cell-cell contacts. Despite differences in upstream signaling, common to signals that promote proliferation is the downstream activation of Cyclin-dependent kinases (Cdks) that drive entry into and through the cell cycle   are intrinsically disordered proteins that fold upon binding to Cyclin/Cdk dimers to inhibit Cdk activity  and accelerated molecular dynamics (aMD) simulations have been applied to investigate the folding and binding of p27 in solution  and revetivation , 30, 31.The starting structure of p27/Cdk2/CyclinA was taken from the crystal structure (1JSU) , of resoEnergy minimization was performed for 2000 steps using combined steepest-descent and conjugate-gradient methods. Following minimization, 20 ps of cMD was performed in the constant temperature, constant volume ensemble using a Langevin thermostat  to regulWe simulated a total of 20 molecular systems using cMD and aMD, totalling over 20 μs of simulation, of which the details are given in The per-residue decomposition of the binding free energy was calculated using the molecular mechanics Poisson-Boltzmann surface area method , where thttps://www.orthodb.org) for p21, p27, and p57 based on their corresponding UniProt identifiers  and downloaded the ortholog group that contained all three proteins (1595421) at the Eukaryota level (1595421at2759). Orthologs were then aligned using MAFFT (mafft-linsi). The resulting protein-level alignment was used to compute a maximum likelihood phylogeny using RAxML (v.8.1.16 -f a -m PROTCATAUTO).We searched OrthoDB v.10 (a). The notable exceptions are p21 orthologs in non-mammalian tetrapods, which all lack Y88 (a and b). However, Y88 remains present in p20 (CDKN1D), perhaps suggesting that mammalian p21 functionality might be partitioned between p21 and p20 in these species (a). Y74 is generally present in p27 orthologs, and the single Cip/Kip copy in non-vertebrates, and is particularly well conserved in mammals but is largely absent from p21 and p57. Finally, Y89 is restricted to eutherian mammals, i.e., absent from marsupials and monotremes as well as non-mammalian vertebrates. Altogether, our analyses suggest that Y88 phosphorylation may have similar roles in regulating the functions of all Cip/Kips across eukaryotes, whereas other tyrosine phosphorylation sites may play more Cip/Kip-specific roles.Tyrosine phosphorylation plays key roles in cellular signaling, including in cell cycle control. Proteins regulating the cell cycle are highly conserved across eukaryotes. Therefore, to investigate the conservation of tyrosine phosphorylation and its effects on the structure of Cip/Kip inhibitors and bound Cyclin/CDK complexes, we first took an evolutionary approach to determine the gain and loss of these key signaling hubs. p21, p27, and p57 bear distinct signatures of confirmed and putative phosphorylation sites. Focusing on tyrosine residues known to be important for p27 function , we note that only Y88 is almost universally conserved across vertebrate Cip/Kip family members a. The nlack Y88 a and b. species a. Y74 ib). We restricted our analysis to S/T-P sites, which can be phosphorylated by Cdks, MAP kinases, and GSK3B, all kinases implicated in mitogenic signaling  between Cdk2 and CyclinA, mediated by hydrogen bonding between Cdk2 R150 and CyclinA E268/E269, 2) between Cdk2 and CKI, predominantly through interactions between Cdk2 E81, K20, and V18 and the CKI, and 3) between CyclinA and CKI, via CyclinA Q254 and W217 and the CKI RXL motif . While tE268 and Cdk2R150, compared with bound p27 (0.56) and p57 (0.57). Furthermore, when p21 binds CyclinA/Cdk2, we see the formation of a long-lived hydrogen bond between Cdk2N23 and p21D62, which we do not observe in the corresponding positions in p27 or p57. This increased affinity at close proximity to the ATP-binding site moves Cdk2 in such a way that we observe the loss of long-lived interactions between the CyclinA/Cdk2 interface. This could represent an additional mechanism by which p21 might influence Cdk2 activity that differs to p27 and p57.Despite the three significant areas of interaction stated previously persisting in all CKI-bound complexes, we observe notable differences in the intermolecular interactions between molecules. We measure the fraction of the lifetime of a hydrogen bond as a probability, e.g., P(bound/unbound). If the contact is present for 1 ns or longer, it is deemed to be bound, and if not, unbound. When p21 is bound, we observe a reduction in the fraction of the lifetime of the hydrogen bond between CyclinA and Cdk2 (0.69–0.39), namely CycAY63 binds in an equivalent position to p27F62 in the trimer, we consider p57Y63 as analogous to p27Y74. Moreover, while not a tyrosine residue and so not explicitly considered in our analyses, ERK, JNK, and GSK3 phosphorylation of p21T57 decreases p21 stability by promoting its degradation  in each state of tyrosine phosphorylation we have modeled, relative to the starting structure . We showY74 is phosphorylated, p27 shows a significant deviation from the equilibrium average just before 400 ns . As this is not seen in the RMSD of the inhibitor alone (b), it appears that this deviation occurs in the CyclinA/Cdk2 dimer as a consequence of p27Y74 being phosphorylated. An alignment of trajectory frames taken at 500 ns for p27 and p27Y74, shown in Y2P74 complex, we see the establishment of longer-lasting hydrogen bonds upon Y74 phosphorylation between CyclinA E268 and F267 and Cdk2 R157 and R150. We see little deviation in Y88, Y89, or doubly phosphorylated Y88/Y89 p27 from the steady-state equilibrium in these classical MD simulations, indicating that these structures exhibit few conformational changes on these timescales (a). Phosphorylation of Y88 is known to drive the ejection of the When p27or alone b, it apmescales a. Phospive site ,31. Howeb and d). Unlike what we observed for p27, for p21, we see CyclinA/Cdk2/p21 jumping from one state to another, happening at around 60 and 340 ns (c).These shifts seem to occur in the CyclinA/Cdk2 dimer, as they are not observed in the p21 inhibitor (d). The jump between two RMSD states appears to be due to the two conformations of the Cdk2 N-lobe, as shown in a. Phosphorylation of p21 Y77, which sits in the Cdk2 active site, abolishes these shifts between states (c). Closer inspection of CyclinA/Cdk2 bound to p21 phosphorylated at Y77 shows long-lasting intermolecular hydrogen bonding between all three molecules (see b). For p57 Y91 phosphorylation, which also sits in the Cdk2 active site, we see the opposite effect to that of p21 Y77 phosphorylation, where tyrosine phosphorylation promotes transitions between states, compared with the unphosphorylated protein (c). This deviation appears to occur in p57 (d) and in part represents the ejection of the Y91 is phosphorylated.As the p21 and p57 sequences were templated on to the crystal structure for p27, it is not unexpected that they deviate more than that of p27 compare b and d.d 340 ns c.These nhibitor d. The jn states c. Closeules see b. For p protein c. This r in p57 d and inY74 phosphorylation, which appears to promote a change in the CyclinA/Cdk2 structure, phosphorylation of p57 at Y63 leads to a deviation in the structure of p57, not the CyclinA/Cdk2 complex (c and d). This may be because, as mentioned, p57Y63 does not bind in the same position as p27Y74 in the trimer  for phosphorylation when p27Y88 or p57Y91 are phosphorylated. There is also a trend in that the Y2P91 and p21Y2P77.Unlike the hydrogen-bonding-network analysis that is based on donor-acceptor distances and angles, the Gibbs free energy of binding is calculated from the force-field energies. The energies of the solvated ligand and receptor are subtracted from the complex to leave the free energy of binding. The energy decomposition in ated p57 . In addiY88, p21Y77, and p57Y91 residues, we generated animations of the unphosphorylated and phosphorylated forms  in tyrosine-phosphorylated p27 and p57 complexes, but not in phosphorylated p21 trimers.In summary, although all Cip/Kip proteins share conserved tyrosine residues, our cMD simulations suggest that phosphorylation of these residues has different effects on CyclinA/Cdk2/CKI complexes. While the individual effects are described above, this is also significant as it means that we should remain cautious when extrapolating changes that have been observed to occur in p27 and bound CyclinA/Cdk2 complexes after p27 tyrosine phosphorylation to the other conserved Cip/Kips.A question that arises when considering the phosphorylation of protein residues is whether the amino acid is exposed and accessible to kinases. While solvent exposure alone is not the only criterion for a residue to be phosphorylated, it is reasonable to assume that the more exposed a residue is, the greater is its likelihood of being phosphorylated by specific kinases. Many residues are not solvent exposed in the unphosphorylated state, which begs the question of how such residues are modified . AlthougTo determine how solvent exposed each tyrosine phosphorylation site is in all three Cip/Kips, in the presence and absence of other phosphorylated tyrosines, we calculated the solvent-accessible surface area (SASA) using the linear combinations of pairwise overlaps algorithm . From oua shows the potential for Y88 in p27, and the corresponding positions in p21 and p57, to be phosphorylated in the presence and absence of phosphorylation of other tyrosine residues (listed on the right-hand side). The SASA of tyrosine at this position  remains close to zero in all models, telling us that throughout these cMD simulations, these residues remain solvent inaccessible and buried and in this state are unlikely to be phosphorylated. b shows the SASA of Y89, which is only present in p27. Unlike its neighbor Y88, Y89 fluctuates between accessible and inaccessible states in otherwise non-tyrosine phosphorylated p27 . When Y74 or Y88 are phosphorylated, Y89 remains in a solvent-accessible state (see around 280–400 ns in b), suggesting cooperativity between these phosphorylation events.We first considered the tyrosine residues that sit in the Cdk2 active site. ated p27 b, red. Y74 remains solvent accessible in its canonical, unmodified, form, whereas p57Y63 appears to be buried and inaccessible. p27Y88, p57Y91, and p21Y77—that these residues are buried and inaccessible to solvent, which would imply that they are not available for modification by phosphorylation under these conditions and on these timescales (a). p27Y89 resides in an accessible state in our simulations (b).To quantify the potential for tyrosine phosphorylation of these residues, we calculated the density of SASA taken from three independent cMD simulations . This comescales a. p27Y8ulations b.Y88, p21Y77, and p57Y91) are inaccessible to phosphorylation. The exception to this is p27Y89, which remains accessible. p27 is the only Cip/Kip to have a double YY motif in this position. Since experimental data have shown that these sites can be phosphorylated, our simulations indicate that phosphorylation must occur under modifying conditions. For p27 Y88, that could be brief exposure “dynamic anticipation” of the residue , and so ejection of the Our models suggest that the only circumstances in which the  effects . This pr solvent c, and sY91 phosphorylation . These models, together with published experimental data  as a two-dimensional LigPlot . Together, these data suggest that inhibition of Cdk2 by Cip/Kips and inhibitory kinases are mutually exclusive events, which has implications for Cdk2 activation during S-phase entry.In Cdk2 that is unphosphorylated at T14 and Y15, the N-terminal domain of Cdk2 is not free and exposed. The red time series in is shown a as a t LigPlot  of resid diffuse c. TogetY88, p21Y77, and p57Y91 binding is the dominant interaction in the Cdk2 models, representing interactions between the Q77, p57T80, and p21E66 bound to Cdk4R24. This may explain why no ordered structure for this helical region was observed in the electron density map for the CyclinD/Cdk4/CKI structure , which is not observed in p27. It appears that this additional interaction leads to a change in the hydrogen bonding between CyclinD/Cdk4—effectively pulling apart the Cdk4L48-CyclinDE141 interaction  residue, the less active Cdk4 is.Unlike CyclinA/Cdk2, which is active in the dimeric complex, CyclinD binding alone does not induce catalytic activity in Cdk4 . Compareb shows the distance between p27Y74, the corresponding amino acid in p21 (which is not a tyrosine), p57Y63, and the center of mass of Cdk4 . This distance tells us whether the tyrosine residue is tightly bound to the Cdk4 molecule, suggesting that the Cdk4 molecule is inactive. Of the models studied, unphosphorylated p21 and p27 are tightly bound at this position, suggesting that Cdk4 is inactive (b). If Y74 is phosphorylated in p27 (green line), Y2P74 transitions from bound to unbound at around 150 ns, and we predict that Cdk4 would become more active—consistent with published results . This is a measure of the “openness” of the active site and a proxy for Cdk4’s ability to accept ATP. Without one of the Cip/Kip family of inhibitors, CyclinD/Cdk4  exhibits the smallest distance between the C helix and the catalytic lysine over the course of a 500 ns simulation, suggesting that Cdk4 is inactive. Considering both of these distances together, that is, the distance between the catalytic lysine and C helix and between p27Y74 and Cdk4 (shown in a), allows us to predict Cdk4 activity in these inhibitor-bound states. Taking both measurements into account, we suggest the following order of activity for CyclinD/Cdk4-bound complexes: p21 (inactive) = p27 (inactive) and p27 Y2P74  A second measure of the potential for CyclinD/Cdk4 to be active is the distance between the Cdk4 C helix and the catalytic lysine residing in the active site b. This inD/Cdk4 c, red eshown in a, allowOur MD simulations are consistent with published experimental data  showing How mitogenic signaling can regulate context-dependent cell proliferation is not fully understood. Here, we have tackled this problem by investigating how mitogens, via the activation of tyrosine kinases, regulate structural changes in Cyclin/Cdk/CKI trimers that ultimately drive Cdk activation and cell proliferation. Our findings reveal that despite shared structural features, tyrosine phosphorylation of CKIs promotes non-conserved and non-intuitive changes in Cyclin/Cdk/CKI complexes and, therefore, Cdk activity. Our models have generated a number of predictions, and below, we discuss the significance of these predictions and how they could be experimentally tested in the future.Y77 in the Cdk2 active site does not promote ejection of the Y74 residue and so remains a Cdk4 inhibitor on mitogen stimulation, our analyses could explain how p21 is able to maintain cell-cycle arrest in the presence of DNA damage and ongoing mitogenic signaling  induces a conformational change in p21 that promotes helix ejection. These model predictions can be experimentally tested, for example, by solving the crystal structures of p21 and p57 bound to CyclinA/Cdk2 and of CyclinA/Cdk2/p21 in the absence or presence of Abl kinase.Cip/Kip inhibitors are intrinsically disordered proteins that fold upon binding to Cyclin/Cdk dimers. Our simulations show that notable differences exist in the strength of hydrogen bonds between Cyclin/Cdk/CKI proteins. For example, p21 binding to Cyclin/Cdk appears to destabilize interactions between CyclinA/Cdk2 and CyclinD/Cdk4, which may be an additional mechanism of Cdk inhibition by p21. p21 also has an additional strong hydrogen bond to Cdk2, with N23 in the Cdk2 active site, that is not present in p27 or p57. This, together with our observations that phosphorylation of p21ignaling ,59. Expenhibitor . Our anaY89, all Cip/Kip tyrosine residues that sit in the Cdk2 active site are inaccessible to phosphorylation. Yet, these sites can be phosphorylated. For p27, previous work demonstrated that p27 exhibits intrinsic flexibility, such that p27 can briefly exist in states where Y88 is exposed for sufficient time to be phosphorylated (Y74 or p57Y63 phosphorylation increasing solvent accessibility of p27Y88 or p57Y91. Conversely, it was recently reported that p27Y88 phosphorylation increased the exposure of p27Y74 29, which we did not observe in our simulations. In the models in (Y89 was mutated to p27Y89F to mimic previous experimental conditions, and therefore this may have affected how p27 behaved in the simulations. Therefore, we favor a combination of intrinsic flexibility and kinase-mediated conformational changes promoting tyrosine exposure.Our SASA measurements demonstrate that, with the exception of p27orylated . SimilarY74 and p27Y88 to investigate p27Y89 accessibility and flexibility, would determine if our model prediction is correct.p27 is the only Cip/Kip to have two adjacent tyrosine residues, Y88 and Y89, in the y of Y89 . HoweverY91, a prediction that is experimentally testable using biophysical methods such as NMR. p57 is expressed at high levels during embryogenesis, and its expression decreases, becoming much more restricted at embryonic day 13.5 and only being expressed in a limited number of adult tissues (Our simulations of Cip/Kip inhibitors bound to CyclinD/Cdk4 were able to recapitulate recently reported features of Cdk4 activation and inhibition by p27 and p21 . We were tissues . This isRecent experimental data have suggested that Cdk4/6 inhibitors being used in the clinic in the treatment of Her2-negative/estrogen-receptor-positive breast cancer, for example, Palbociclib, may promote cell-cycle arrest by forcing the redistribution of Cip/Kip inhibitors from Cdk4 onto Cdk2 . This unConceptualization, J.B.S. and A.R.B.; methodology, J.B.S., K.L.M., and T.W.; investigation, J.B.S.; visualization, J.B.S. and K.L.M.; supervision, A.R.B. and T.W.; writing, J.B.S. and A.R.B.


Glacial deposits offer discrete snapshots of glacier extent but cannot provide the continuous records required for detailed interhemispheric comparisons. By contrast, lakes located within glaciated catchments can provide continuous archives of upstream glacial activity, but few such records extend beyond the last glacial cycle. Here a piston core from Lake Junín in the uppermost Amazon basin provides the first, to our knowledge, continuous, independently dated archive of tropical glaciation spanning 700,000 years. We find that tropical glaciers tracked changes in global ice volume and followed a clear approximately 100,000-year periodicity. An enhancement in the extent of tropical Andean glaciers relative to global ice volume occurred between 200,000 and 400,000 years ago, during sustained intervals of regionally elevated hydrologic balance that modified the regular approximately 23,000-year pacing of monsoon-driven precipitation. Millennial-scale variations in the extent of tropical Andean glaciers during the last glacial cycle were driven by variations in regional monsoon strength that were linked to temperature perturbations in Greenland ice cores Analysis of a continuous and independently dated record of glaciation in the tropical Andes spanning 700,000 years shows that Andean glaciation follows patterns of global ice volume change, with a periodicity of approximately 100,000 years. However, because about 80% of global ice volume change over glacial–interglacial cycles occurred in the middle to high latitudes of the Northern Hemisphere (NH)5, the marine δ18O record tells us little about the timing and extent of glaciation in the Southern Hemisphere (SH) and in all tropical mountain ranges. Radiometric dating of moraines in the SH7 confirms that the local last glacial maximum occurred roughly at the same time as the last peak of global ice sheet volume . However, moraine records are inherently discontinuous, with younger glacial advances commonly obliterating evidence of older ice positions, making it challenging to make interregional comparisons of the timing of ice build-up and decay over several ice-age cycles using moraine chronologies alone. By contrast, lake sediment records can provide continuous archivesof up-valley glaciation8, but long lacustrine records of tropical glaciation have thus far been limited by a lack of sufficient independent age control and/or a lack of clarity about the glacial signal preserved10.The δ4 (ref. 11). Millennial-scale perturbations in Andean glacier extent during the last glacial cycle were similar in timing to both regional cave records of precipitation and to temperature oscillations recorded in Greenland ice cores1; this pattern of change may have also occurred during previous glacial cycles. Thus, persistent multiscaled interhemispheric climatic teleconnections affected tropical Andean glaciers during much of the past 700 ka.Here we report an approximately 700 thousand year ago (ka) continuous and independently dated lacustrine record of alpine glaciation from the central Peruvian Andes that is directly comparable with records of extratropical temperature change, global ice volume and atmospheric greenhouse gas (GHG) concentrations. We show that tropical Andean glaciers waxed and waned with the NH on orbital timescales but that the relative amplitude of glacial–interglacial cycles has not been constant. During much of MIS 7–11, tropical Andean glacier extents were enhanced relative to global ice volume, and this enhancement may have been related to increased precipitation coupled with reductions in the concentration of atmospheric CH10Be exposure ages from these moraines indicate that they span several glacial cycles6, but at no time during the past 700 ka has the lake been overridden by glacial ice. Thus, Lake Junín is ideally situated to record cycles of tropical glaciation12. During the local last glacial maximum, alpine glaciers descended from cirque headwalls at about 4,700 masl to moraine positions about 4,160 masl, within several kilometres of the modern lake shoreline6. A seismic reflection survey ) is a seasonally closed-basin lake located in the uppermost Amazon basin between the eastern and western cordilleras of the Peruvian Andes  were extracted from the lake’s depocentre , 12 U-Th-dated intervals of authigenic calcite from five carbonate intervals between about 21 and 71 m (ref. 14) and 17 geomagnetic relative palaeointensity (RPI) tie points between 24 and 88 m (ref. 15) . As the age model is not orbitally tuned, the Lake Junín record enables an independent comparison of the pacing of tropical glaciation with that of LR04 (ref. 3), which is a proxy for global ice volume and benthic ocean temperature  Fig.  Fig. 2)13) were ure Fig. .Fig. 2Ag−3), magnetic susceptibility (MS) and concentrations of elements  derived from erosion of the non-carbonate fraction of the regional bedrock . Seven glacial–interglacial cycles are evident in the GI and, within the uncertainty of the age model15, they mimic changes in LR04 and in the δD of Antarctic ice18, a proxy for high-latitude SH air temperature  and the hydrologic balance of the upper Amazon basin and Altiplano21. Given the orders-of-magnitude difference in response times between alpine glaciers and NH ice sheets, the close match between the GI and LR04 23 linking orbital forcing, NH ice sheets, GHGs and tropical glaciation must have been relatively rapid, as evidenced by the lack of measurable lag time in tropical ice core records of the last deglacial temperature rise24.The Lake Junín GI record over the past 677 ka shows a strong similarity to LR04  records using nine tie points  for the Junín GI and LR04 for the full record is 0.62; for the past 140 ka, it is 0.74. The similarity between the records for the MIS 1–6 window does not support the conclusion25 that MIS 4 was an anomalously large glacial cycle based on cosmogenic radionuclide ages on moraines from the middle and low latitudes of both hemispheres. The index of tropical glacier enhancement  variations in TGE may be driven by differences in tropical versus global ice volume and/or by long-term changes in basin sensitivity to record clastic sediment, such as through the progressive progradation of glacial outwash fans   Fig. .1  Pacupahuain Cave26 and also with the warming episodes recorded in Greenland ice cores, known as Dansgaard–Oeschger (DO) events27  amount in response to instabilities in North Atlantic temperatures. This is consistent with established linkages between North Atlantic climate and the hydrologic balance of Altiplano lakes21. Whereas glaciers in the inner tropics of the Andes are especially sensitive to temperature because of sustained precipitation year-round, glaciers in the outer tropics, such as those at the latitude of the Junín basin, experience greater seasonality of precipitation and are twice as sensitive to changes in precipitation as those in the inner tropics30.The last glacial cycle in the Junín region was punctuated by intervals of abrupt glacial retreat, as evidenced by lowered values of Ti, MS and sediment density, and lowered lake level, as recorded by the accumulation of organic-rich sediment in the centre of the Junín basin18O record from nearby (about 25 km south-east of Lake Junín) Huagapo Cave31 and a synthetic Greenland δ18O record for MIS 6 (ref. 32) further suggests long-term forcing of the hydrologic balance and, thus, glacier extents in the SH tropics by instabilities in North Atlantic climate31  in the Junín core beyond 50 ka Fig.  preclude34, drove atmospheric temperatures in the tropics over ice-age cycles, whereas instabilities in North Atlantic climate and monsoon precipitation acted as secondary effects on tropical ice extent. The mass balance of the small alpine glaciers of the tropical Andes rapidly integrated the effects of both air temperature and moisture flux with short response times35. Because mid-tropospheric tropical temperature is nearly uniform with longitude owing to weak Coriolis force at low latitudes36, this record may be broadly representative of tropical glaciers around the world over the past 700 ka.This continuous, independently dated record of tropical glaciation confirms the role of fast-acting teleconnections, many apparently originating in the high latitudes of the NH over the past 700 ka. Global GHGs, modulated by orbitally forced NH ice sheet growth45.The topography of the Junín basin Fig.  is based46). The air gun seismic survey imaged over 100 m of nearly horizontal sediment with no evidence of deformation. Details of the stratigraphy were obscured by ringing in the shallow water column, but several prominent, continuous reflectors were observed that could be traced across the lake basin. One of these, at a depth of approximately 100 m below the lake floor, was especially prominent and could be mapped throughout the survey area and was noted to be flatter than the modern lake floor. This suggests broad-scale basin subsidence and a lack of local tectonic deformation. Drill cores showed that this reflector marks a transition from fluvial sand and gravel to finer-grained lacustrine sediment. At the time this deep reflector was formed, the lake was apparently larger than at present. No evidence was observed in the central part of the lake for large-scale lake-level fluctuations, such as wave-cut shorelines, fluvial channels or low-stand deltas.High-resolution (4–24-kHz), shallow-penetration data were collected in 2008 and an air gun survey was conducted in 2011 . Similarly, TIC was determined by acidifying samples using an Automate acidification module and measuring the resultant CO2 by coulometry. We calculated the weight percentage TOC from TOC = TC − TIC. We measured the biogenic silica (bSiO2) content of a total of 65 samples obtained randomly from all facies present in the sediment core following the procedure outlined in ref. 47. Siliciclastic flux (Fluxclastic) was calculated as:−1) using the age model of ref. 15, BD is the bulk density (g cm−3), TOM is the weight fraction total organic matter of the bulk sediment and TCC is the weight fraction CaCO3 of the bulk sediment. We calculated TOM (%) from TOC (%)/44 to reflect the molar ratio between plant cellulose (C6H10O5)n and TOC (%), and we calculated TCC (%) from TIC (%)/12 to reflect the molar ratio between TIC (%) and CaCO3. Because of the presence of both authigenic and detrital CaCO3 in the sediment core, the removal of all CaCO3 in the estimation of Fluxclastic results in an underestimation of the total detrital flux during intervals of high clastic input, and this, in turn, reduces the amplitude of change in clastic flux between intervals of high and low glacigenic sediment input. Although we do not explicitly remove the bSiO2 in the calculation of Fluxclastic, the average weight percentage bSiO2 for all 65 facies samples is 0.92 ± 1.12% (±1 sigma), and—thus—is negligible. Because Fluxclastic is affected by sedimentation rates and because short-term changes in sedimentation rate require closely spaced age control, the highly variable Fluxclastic of the past 50 ka  at the University of Minnesota . Density was measured on the whole-round cores by gamma ray attenuation using a multisensor core logger and MS was measured by a Bartington MS2E point sensor on the split core surface. Furthermore, density was measured from the air-dried mass of 1-cc samples taken every 2.5 cm above 6.665 m and every 8 cm below 6.665 m. X-ray fluorescence (XRF) scanning was performed at the LacCore XRF Lab, University of Minnesota Duluth  Large Lakes Observatory using a Cox Analytical ITRAX with a Cr tube, 5-mm resolution and 15-s dwell time. Total organic and inorganic carbon  was measured on samples taken every 2.5 cm above 6.665 m and every 4 cm below 6.665 m. Total carbon was determined by combusting samples at 1,000 °C using a UIC CM5200 automated furnace and analysing the resultant CO13. Intervals were sampled with 8-cc discrete palaeomagnetic cubes centred at 93.80, 93.98, 94.65 and 94.78 m and measured at the Oregon State University  Paleo- and Environmental Magnetism Laboratory using a 2G Enterprises model 755-1.65UC superconducting rock magnetometer. Samples were demagnetized in an alternating field (AF) of 10, 15 and 20 mT to remove the relatively soft drilling overprint15 and show the primary direction recorded by a (post-)depositional remanent magnetization. Palaeomagnetic directions stabilized during demagnetization using the three peak AF fields following 20-mT AF demagnetization; inclination values of the four samples were −24.0°, −11.0°, +4.7° and −9.4°, respectively. These largely negative inclination values are consistent with, and/or are slightly shallower than, predicted inclination values for normal polarity of −21°, assuming a geocentric axial dipole field. Consistent relative declination values of 157°, 183°, 157° and 154°, respectively, suggest that the shallower inclination values deeper in the core reflect normal secular variation rather than variability driven by proximity to the Brunhes–Matuyama polarity reversal. As a result, it is probable that all four intervals were deposited during the Brunhes chron and are younger than 773 ka (ref. 16).To provide a basal limiting date on the Junín record, four samples were obtained from core JUN15-1D-35H, the deepest core in the continuous stratigraphic splice8) showed a close correspondence among the distribution of radiometric ages of moraines and variations in clastic sediment flux and concluded that the primary control on clastic sediment delivery to alpine lakes in the tropical Andes is the extent of glacial ice cover; basin-specific factors that might complicate this relationship, such as changes in sediment storage or paraglacial lag times, are not important on 104-year timescales8. The absence of any evidence of notable clastic sediment delivery to Lake Junín during the current interglacial period  suggests that precipitation events alone are unable to transport clastic sediment to the lake depocentre, and that it is the production of fine-grained glacial flour that is essential for the delivery of such sediment to the lake depocentre. Furthermore, fringing wetlands may prevent the small amounts of clastic sediment that may be transported from the Junín catchment from entering the lake today12 and, by extension, during previous interglacial intervals. The lithostratigraphy  of 0.70.Downcore variations in MS and the ratio of the elements Ti to Ca are sensitive to the concentration of lithogenic material. Higher values of these parameters occur in glacial periods, related to the increased generation and delivery of clastic material to the lake during times when glaciers occupied the Lake Junín catchment. A compilation of sediment records from tropical Andean lakes from 2° S to 16° S (ref. 37 of the 250-year-time-step GI dataset used a Morlet basis function in the PAST software package48, with the p = 0.05 significance level contoured in black (Fig. The GI dataset was selected for time-series analysis to examine the evolution of periodicities throughout the length of the record. Wavelet analysisack Fig. .Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04873-0.


Compared to the DHS+P and ASNIS models, the X-PIN+P model presented, respectively, increases of 51.6% and 64.7% for Max P, 85% and 247% for Min P, and 18.9% and 166.7% for von Mises stress. Max P1 did not differ between the models, but Max P2 was 55% and 50% lower for X-PIN+P than ASNIS and L, respectively. All displacement values were lower for X-PIN+P than the other models. In this FEM testing, the X-PIN+P was superior to the other models, which was due to improvement in all parameters of stress distribution, displacement, and von Mises stress compared to models using a lateral plate (DHS and DHS+P) or not (ASNIS and L).Femoral neck fractures in young adults are uncommon, resulting from high-energy trauma. Despite their infrequency in this population, there is higher rate of complications, especially in the more vertical fracture line, classified by Pauwels as a type-3 femoral neck fracture. The implant type is of paramount importance for maintaining anatomical reduction, since it must resist the deforming forces that act on the fracture. We comparatively evaluated two constructions of the novel locking autocompression implant (X-PIN and X-PIN+P) using the finite element method and previously established methods for treating Pauwels type-3 femoral neck fractures. Six fixation models were developed for the study: a dynamic hip screw (DHS), a DHS with an anti-rotation screw (DHS+P), the inverted triangle multiple cannulated screws construction (ASNIS), the multiple cannulated screws in an L-configuration (L), and the two models of the novel locking autocompression screw (X-PIN and X-PIN+P). Under the same conditions with a load of 2100 N, the following parameters were evaluated using SIMLAB Femoral neck fractures (FNF) in young adults are uncommon, accounting for 3% of all hip fractures, and usually result from high-energy trauma ,2. TreatThe implant type is of paramount importance for maintaining anatomical reduction, since it must resist the deforming forces that act on the fracture focus . The morCurrently, the sliding hip screw, combined or not with an anti-rotation  screw, is considered the standard implant in P3 FNF ,10. SeveDespite the sliding hip screw’s superior mechanical strength to other extramedullary implants, problems related mainly to its inability to control rotation, especially when an additional derotational screw is not used, with varus subsidence and femoral neck shortening, which alter hip offset, have been reported in the literature . This isThe main objective of this study is to evaluate the biomechanical behavior of a locking autocompression screw, called X-PIN, and a variant (X-PIN+P) in P3 FNF using a finite element model (FEM). The secondary objective was to compare the results of this model with clinically established fixation methods for FNF.A fourth generation 3908 virtual model of the femur  was used, which corresponds to a physical model with 17 pounds per cubic foot and characterizes the model as a young adult femur . A full The X-PIN model features a main screw with 12.7 mm of distal diameter and 13 mm of proximal diameter, both extremities with threads of different pitches and areas, allowing compression between the fractured fragments, and a 4.7 mm locking screw that crosses the main screw through a smooth hole and anchors bicortically. The only change in the X-PIN+P model was a fully threaded 4.7 mm screw, positioned from posterior to anterior along the femoral neck, transfixing the fracture without a sliding tunnel, thus acting as a position screw .For biomechanical comparison, we used a 135° dynamic hip screw (DHS) and a 7.0 mm cannulated screw, both manufactured by Hexagon Ltd. , in conformity with models approved for clinical use by the Brazilian National Regulatory Agency .The models were abbreviated according to implant type: DHS, DHS with anti-rotation screw (DHS+P), inverted triangle multiple cannulated screws (ASNIS), multiple cannulated screws in an L-configuration (L), and X-PIN and X-PIN+P (the new autocompression system) .For the simulations, the material properties, modulus of elasticity, and Poisson’s coefficient of each of the parts of the digital models  were previously defined. All metallic models in this study shared a common alloy .X, Y, and Z axes were selected. For the study, a 2100 N load was applied in the Z axis (which corresponds to the stress applied to the femur of a young adult weighing 70 kg in single-leg stance); no loads were applied to both the X and Y axes. Subsequently, the movement restriction regions (fixations) were delimited, marked in all directions of the X, Y, and Z axes to guarantee the stability of the system. A tetrahedral mesh formation was adopted for the meshes, and the models were tested with a 10° inclination in the Z axis  and a 9° inclination in the Y axis (posterior), with the load applied perpendicular to the ground in the superior region of the femoral head , localized maximum P1 , localized maximum P2 , total principal minimum (Min P), presented as a negative value to represent the application axis, total displacement, localized displacement (displacement of the fracture focus), rotation deviation, and distribution of von Mises stress.The Max P1 and P2 values were calculated using the mean tension obtained in the nodes of the most affected region for each group. The rotation displacement was calculated by observing two coincident elements on opposite sides of the fracture before and after loading to evaluate their displacement .The von Mises stress was captured in the synthesis material of the models and all the results are presented in absolute values and percentiles between the models.All results are presented for the DHS, DHS+P, ASNIS, L, X-PIN, and XPIN+P models in The Max P values were 607, 711, 654.3, 501, 582, and 1078 MPa . The X-PThe Min P values were “−765”, “−517”, “−276”, “−597”, “−856”, and “−959” MPa , with thThe Max P1 values were 29, 24, 23, 19, 28, and 25 MPa A. The moThe total displacement values were 8.4, 7.1, 9.2, 8.7, 9.0, and 2.4 mm. A, with tFinally, the von Mises values were 859, 1005, 448, 393, 955, and 1195 MPa, with the X-PIN+P results 18% and 166% higher than DHS+P and ASNIS, respectively .FNF in young adults have been the subject of several studies, especially P3 femoral neck fractures, due to the inherent instability of the vertical fracture line. Although the sliding hip screw is considered standard for this fracture pattern, the mechanical and biological failure rates are still not very acceptable. In addition, a large amount of bone is removed making later reconstructions difficult if required and there is a higher possibility of damaging the femoral head blood supply if the implant is imperfectly placed or there is a mechanical failure with varus collapse of the head . Thus, iIn this scenario, the X-PIN model was developed and tested in vitro using FEM, a fundamental tool for biomechanical investigations in orthopedics ,24. FEM X, Y, and Z axes, cranial and axial migration of screws within the femoral head, or failure under vertical load. They concluded that a femoral neck system provides comparable biomechanical stability to the DHS for treating displaced femoral neck fractures in young adults. Unfortunately, their conclusion cannot be extrapolated to the more vertical P3 FNF. Davidson et al. [Recently, other systems in which the sliding screw is locked by another screw have been biomechanically and clinically investigated. Moon et al.  comparedn et al.  conducteRegarding distribution of the main maximum stresses in our study, the X-PIN+P was similar to the DHS and the DHS+P, but with better distribution. One hypothesis for this is the different cephalic screw locking system, with a screw inserted obliquely, more medially, and bicortically. Freitas et al.  found thIt is interesting to note that the lack of a lateral plate on the femur (ASNIS and L) increased the load on this cortex, producing local weakness that is normally observed in clinical studies using only screws to fix P3 FNFs ,32,33. TThe main limitation of our study is the comparison between our findings with those of other biomechanical benchtop and clinical studies that did not use previous FEM analysis, which could be subject to several types of bias. The results of FEM can be influenced by several factors, such as the software, the types of meshes and elements, the minimum differences in conditions, and the contours. On the other hand, the results of benchtop tests have not always matched the clinical experience and a computer simulation might be indicated instead of idealized, simple models often used in experimental tests . Augat eThe main strength of our study is that stress distribution was better in X-PIN+P than DHS or DHS+P and, thus, was apparently superior to implants developed to treat the difficult condition of P3 FNF in young adults. Although we have not compared the X-PIN and X-PIN+P with the more contemporary implants such as the Synthes Femoral Neck System, the Rotationally Stable Screw-Anchor (RoSA) and the InterTan nail, biomechanical studies have shown that both mean axial stiffness and mean torsional stiffness of these implants were comparable to that of the DHS, especially when fractures were stabilized using the sliding hip screw system with a blade ,36,37.In FEM testing, the X-PIN+P was superior to the other models, which was due to improvement in all parameters of stress distribution, displacement, and von Mises stress compared to models using a lateral plate (DHS and DHS+P) or not (ASNIS and L).