Samples for single-particle cryo-electron microscopy can be routinely vitrified using only liquid nitrogen at its boiling temperature, allowing sample handling workflows to be simplified and sample stresses that contribute to beam-induced motion to be reduced. −1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitrogen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitrogen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitrogen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitrogen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitrogen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s Major investments in new cryo-EM facilities and development of easy-to-use software  . The sample is then transferred from ethane to liquid nitrogen (LN2), loaded into grid boxes, transferred to additional containers and finally to a storage Dewar. Samples are then removed from the storage Dewar and grid boxes, and loaded into a cold microscope stage. Alternatively, samples may be ‘clipped’ before or after cooling for eventual loading into a cold sample cassette compatible with automated grid handlers. The stage or cassette is then loaded into the TEM.As in cryo-crystallography, the key challenges in single-particle cryo-EM are associated with sample preparation and handling. The basic principles and methods in current use were developed in the 1980s  for initially cooling and vitrifying samples; propane, ethane/propane and related hydrocarbons with large differences between their melting and boiling temperatures have also been used. In his 1988 review  – the minimum cooling rates required for sample vitrification – depend on the maximum tolerable or detectable ice fraction in otherwise vitrified samples . The ethane level is typically at a millimetre or more below the top of its cup. The top of the ethane cup may be just above or well below the top of the LN2 container. A layer of cold gas at least a few millimetres thick – often revealed via water droplet and ice crystal ‘fog’ – forms along the sample plunge path to the liquid ethane. With a predicted cooling rate for the sample + foil in dry N2 gas at ∼100 K of ∼200,000 K s−1, the sample need only travel ∼1.4 mm – half the grid diameter – through this gas before it has vitrified, a distance comparable to or smaller than any plausible cold gas layer thickness. The presence of  10–20 µm-thick grid bars reduces cooling rates nearby, but cooling rates of sample + foil near the center of grid openings may be of this order  were used for single-particle imaging experiments. The first (grid Type A) was a commercial UltrAuFoil R1.2/1.3 300 mesh grid with a Au grid and Au foil, from Quantifoil . The second (grid Type B) was an in-house developed 300 mesh Cu grid/Au foil prototype. The third grid type (Type C) combined a 300 mesh EMS Au grid  and an in-house-made Au foil. Details of grid and foil fabrication are given in the supporting information.Three different types of grids  for 2 min.3.2.et al.  vertical sample translation stage, a gas management manifold containing a plunge bore, an insulated tank filled with boiling LN2 beneath the gas manifold, an automated sample carousel in the tank and an LN2 level control system. The gas management manifold  uses a combination of heaters, dry room temperature N2 gas and suction to completely remove cold gas above the LN2 within the bore and to maintain the temperature within the bore above 273 K to within <50 µm of the LN2 surface. This ensures that nearly all sample cooling occurs once the sample enters the LN2. The gas management manifold also isolates all cold surfaces from ambient air to minimize or eliminate frost accumulation. The sample carousel accepts four standard 16-sample storage and shipping ‘pucks’ used in automated handling at synchrotron source beamlines and automatically positions an empty sample slot beneath the plunge path after each plunge. Using thermocouples with ∼30 µm beads, measured cooling rates in boiling nitrogen using NANUQ are in excess of 50 000 K s−1.Samples were cryocooled using the MiTeGen NANUQ automated liquid nitrogen-based cryocooler for cryocrystallography , which is based on insights into the physics of cryocooling described by Kriminski  al. 2003 and Wark al. 2006. Briefly2 within the plunge bore is removed and replaced with dry ambient-temperature gas, the sample is plunged into the LN2, and then the sample is translated and released into the storage puck.To use NANUQ in cryocrystallography, a nylon or microfabricated ‘loop’ attached to a magnetic steel goniometer base is held using a magnetic wand. Crystals are looped or scooped out of solution onto the loop, and then the wand, base, loop and crystals are loaded on the NANUQ vertical translation stage. When the access door to the vertical stage is closed, the cold gas above the LNTo use this instrument for cryocooling cryo-EM grids, two generations of prototype grid holders consisting of custom forceps attached to standard crystallography goniometer bases were fabricated. A grid was grasped by a grid holder, which in turn was held by the magnetic wand, and then the grid + holder + wand were loaded on the NANUQ vertical translation stage. After cooling, samples were automatically transferred into one of two standard sample holding ‘pucks’ used in cryocrystallography: a UniPuck for the first-generation grid holder and a cryovial-containing CombiPuck for the second-generation grid holder .3.3.w/w NaCl solution. With a 400 mesh Quantifoil R 2/2 holey carbon grid attached to a first-prototype grid holder and wand in the NANUQ ready-to-plunge position, and with the grid in ambient air having ∼50% relative humidity (r.h.), 2 µl of solution was pipetted onto the foil side of the grid. The grid was manually blotted for 2–3 s using Whatman no. 1 paper, and then immediately plunged at 2 m s−1 into LN2 and automatically transferred into a UniPuck immersed in the LN2. When sample plunging was complete, the UniPuck was removed from NANUQ and transferred to an LN2-filled insulated container, and samples were removed from the puck one by one and released into standard cryo-EM grid boxes. These grid boxes were then transferred to an LN2-filled Dewar for storage and transport to the TEM.Initial experiments in summer 2020 used a protein-free 0.5% −1 equine spleen apoferritin  in 50 mM HEPES buffer with pH 7.4. The initial grid position and its plunge path were humidified to 90% r.h. reducing the film evaporation velocity to ∼5 nm s−1, making the timing of blotting and evaporation less critical. Second-generation prototype grid holders improved ease of gripping and grid perpendicularlity to the LN2 surface during the plunge. A 3 µl volume of solution was pipetted onto the foil side of either a Quantifoil (Type A) or MiTeGen prototype  grid, and the excess was blotted by hand with Whatman no. 1 paper for between 2 and 8 s. Each grid was then immediately plunged at 2 m s−1 into LN2 at 77 K, and then automatically translated into cryovials held within a CombiPuck. After all samples in a run had been cooled, the CombiPuck was removed from the NANUQ carousel, the cryovials removed from the CombiPuck, the grid and holder removed from each cryovial using a standard wand, and the grid released into a SWISSCI cryo-EM grid box. The grid boxes were then stored in a MiTeGen cryo-EM puck, which was loaded into a Worthington CX100 shipper with a MiTeGen cryo-EM transport cane.Subsequent experiments used a solution comprised of 5 mg ml3.4.Cryo-EM grid screening and data collection were performed using a Thermo Fisher Scientific Talos Arctica cryo-TEM operating at 200 kV and equipped with a K3 direct electron detector operating in electron counting mode and a Bioquantum energy filter (Gatan). Grid screening (but not data collection) was also performed using an FEI Tecnai 12 BioTwin TEM operating at 120 kV and equipped with a Gatan Orius 1000 dual-scan CCD detector. Both TEMs were housed at the Cornell Center for Materials Research.3.5.et al., 2018Approximate ice thickness on the grids was determined by comparing the intensity measured with and without the 20 eV energy filter. The apparent mean free path for this microscope was measured to be 290 nm by comparing the log of the intensity ratio of unfiltered over filtered images to the absolute thickness determined by tomography  using tilt-series tomography . Tilt series in 3° increments with a tilt range from −60 to +60° were collected using 3.6.− Å−2 and exposure time of 3 s. The exposure rate at the detector over vacuum was 28 e− pixel−1 s−1. Data acquisition was automated using the SerialEM software  was used to make figures. Statistics for the two models are given in Tables 1 and S1 of the supporting information.Model building and refinement were performed using each map . A high-resolution crystal structure of apoferritin  and local beam-induced motion. The local per-particle motion was then analyzed by computing the RMS particle displacement (in-plane component) as a function of fluence, where the mean is taken over all picked particles.In-plane particle trajectories were determined using a local motion job in 4.4.1.2 using NANUQ. Both thick and thin ice across large areas of the grid were fully vitrified. The minimum cooling rate to vitrify a 0.5% NaCl solution is ∼200 000 K s−1, based on a visual ice assay that has an ice fraction detection limit of roughly 1%  and frequent foil damage. These likely reduced cooling efficiency. Grid bending during plunging should grow with distance traveled through liquid cryogen. In NANUQ, the plunge distance is >3 cm, much larger than in typical ethane-based cryo-EM plunge-coolers. The resulting transient bending sometimes exceeded the elastic limit of the grid, causing permanent deformations that provided direct evidence for plunge-induced bending that otherwise would be difficult to detect.In these initial experiments using first-prototype grid holders, the main factor (aside from excessive sample thickness) preventing vitrification was deviation of the grid plane from absolute perpedicularity to the LN4.2.−1 apoferritin solution. In the first batch, 16 grids were plunge-cooled, 12 were screened and of these 3 had good ice suitable for high-resolution single-particle imaging. In the second batch, 16 grids were plunge-cooled, 12 were screened and 8 had good ice. Good ice was achieved reproducibly using both the Quantifoil grids and the MiTeGen grid prototypes. ‘Good ice’ here means that there were many grid squares with little or no visible crystalline ice and little or no evidence of ice in image FFTs or diffraction patterns . For sample 2, FFTs of only 11% of hole images show evidence of ice, and only 5% showed strong intensity consistent with either a few or multiple ice particles. Fig. 24.4.Model building and refinement based on the 2.64 Å cryo-EM apoferritin reconstruction from sample 1 gave a final model with good statistics and no artifacts Table 1. The mod4.5.− Å−2), determined as described in Section 3.9cryoSPARC local motion correction and samples at 0° tilt; a map of the particle displacements; and both a diffraction mode image and an image FFT, all for the same hole. Sample 3 was incompletely vitrified, showing a similar extent of ice in image FFTs as sample 1. Sample 4 was largely vitrified, similar to sample 2. Sample 3 shows smaller net particle motions than sample 4, despite showing a larger fraction of crystalline ice. This could be a factor contributing to the somewhat higher resolution particle reconstruction obtained using sample 3.Fig. 4i.e. FFTs of most hole images show no ice or only weak diffuse ice rings), the displacement has a steep initial increase with fluence followed by a more gradual increase at fluences beyond ∼5 e− Å−2, as is typically observed. The initial slopes of displacement versus fluence for these samples range from ∼2 to ∼6 Å3/e−. Sample 7 in Fig. 53/e−, similar to that of the vitrified samples cooled in boiling LN2. These values for boiling LN2 and ethane-cooled samples compare with a value of ∼1.9 Å3/e− reported for an ethane-cooled sample on a foil with 1.2 µm diameter holes  and the ‘free’ volume fraction of water (i.e. water not involved in hydration of solutes) small enough that cooling rates required for vitrification may be substantially reduced  it does so on/in a foil that is under tensile stress. The tensile foil stress is released as the grid bars cool towards the liquid cryogen temperature, placing the sample within the foil holes under compressive stress. Radiation-induced creep  foil strain released between when the sample initially vitrifies (on the tensile-stressed foil) and when the grid and foil have both cooled to the temperature of the liquid cryogen. The overall magnitude of the observed motion is consistent with rough estimates of grid-foil temperature differences during cooling  to 163 K, both reduce initial beam-induced motion by a factor of 4  proportional to the diameter of the hole  is slightly less than that of vitreous ice (∼0.944 g cm−3)  is proportional to the product of the plunge speed and cooling rate. Consequently, increasing the cooling rate reduces the width of this band, which increases the maximum thermal gradient within the band. Non-uniform grid bar contraction due to this thermal gradient creates a trapezoidal deformation of the grid openings and of the foil that spans it, as shown schematically in Fig. S9. Since the foil + sample cools faster than the grid bars, the sample will vitrify on the distorted foil. As cooling continues and the thermal gradient across the grid diminishes, the foil distortion will diminish. This will tend to create a primarily uniaxial compressive stress in the vitrified sample oriented perpendicular to the plunge direction that will again drive radiation-induced creep and beam-induced motion, but not in the radially symmetric way expected if the grid cooled uniformly.Smaller cooling rates may reduce another, related component of sample stress that may drive beam-induced motion. During plunge-cooling, as the grid + sample enters the liquid cryogen, cooling from 6.2 to just above its freezing temperature (∼63 K) via thermal contact with evaporatively cooled LN2. Somewhat larger cooling rates might be achievable using ethane with improved cooling instrument and grid designs. However, single-particle cryo-EM reconstructions are insensitive to small crystalline ice fractions that may result from smaller cooling rates, and slower cooling is likely to reduce sample stresses that drive resolution-limiting beam-induced motion. By eliminating the use of flammable ethane, all-LN2 cooling may simplify cryo-EM sample workflows and cold chains and simplify the design of automated sample-cooling instruments, with no compromise in data quality.The present results establish the feasibility of routine vitrification of single-particle cryo-EM samples using only boiling liquid nitrogen and show that these samples can yield high-resolution particle reconstructions and refined structures, comparable to those achieved when samples are cooled in liquid ethane. Cooling rates perhaps four times larger (Hua & Xu, 20007.supporting information: Marr et al. (2014The following reference is cited in the  al. 2014.10.1107/S2052252521008095/eh5013sup1.pdfSupporting figures and tables. DOI:


Across two developmental paradigms we find a distinct switch from high i3’UTR expression for stem cell related genes in proliferating cells to high CDS for these genes in newly differentiated cells. Unbiased transcriptome analysis across multiple gene sets shows that regardless of tissue, genes with high 3’UTR to CDS ratios belong predominantly to gene ontology categories related to cell-type specific functions. In contrast, the gene ontology categories of genes with low 3’UTR to CDS ratios are similar across tissues and relate to common cellular functions. We further show that, at least for some genes, traditional transcriptional start site genomic elements correspond to identified RNAseq 3’UTR peak regions, suggesting that some i3’UTRs may be generated by de novo transcription. Our results provide critical information from which detailed hypotheses for individual i3’UTRs can be tested, with a common theme that i3’UTRs appear poised to regulate cell-specific gene expression and state.Mature mRNA molecules are expected to be comprised of a 5’UTR, a 3’UTR and a coding region (CDS). Unexpectedly, however, there have been multiple recent reports of widespread differential expression of mRNA 3’UTRs and their cognate coding regions (CDS), reflecting the expression of isolated 3’UTRs (i3’UTRs); these i3’UTRs can be highly expressed, often in reciprocal patterns to their cognate CDS. As with other long non-coding (lncRNAs), isolated 3’UTRs are likely to play an important role in gene regulation, but little is known about the contexts in which they are deployed. To illuminate the functions of i3’UTRs, here we carry out  Sox11 CDS compared to 3’UTR, but switch and then maintain high Sox11 3’UTR levels [In contrast to the canonical view that an mRNA molecule is comprised of a 5’UTR, a 3’UTR and a coding region (CDS), we and others have found widespread stable expression of mRNA 3’UTRs in the absence of their cognate coding regions (CDS) –4 and CDR levels . Thus, cin vitro, and in vivo data on the non-random and dynamic i3’UTR expression of select genes in progenitor cells, followed by in silico, unbiased transcriptome-wide analyses of open source RNAseq genes sets. The data suggest a strong proclivity for high 3’UTR to CDS expression in cells and for genes involved in transition states, during both proliferative and developmental maturation events. We also report that for some genes, traditional transcriptional start site genomic elements correspond to identified RNAseq 3’UTR peak regions, suggesting a potential mechanism for generation of i3’UTRs for those genes.Historically there are a few reports of biological roles for 3’UTRs independent of their cognate CDS that were documented, when i3’UTR’s were intended as a control, but where their overexpression or deletion produced a biological effect –10. MoreSox gene family members we observed overlapping, but distinct patterns for each probe pair (3’UTR and cognate CDS) in serial adjacent sections, such as for Sox9 and Sox11 in the developing head  [Sox11, 12 and 4, each with unique, and finely tuned distinct expression . These data show that our probes and hybridization procedures result in specific signals. In addition, the use of cognate 3’UTR and CDS probes allow us to uncover previously unseen biological subtleties for closely related family members such as the Sox genes. These differences may be critical during important developmental windows.ing head ; arrows.0071526) , 20. How neurons ; carrot r probes, finding indistinguishable hybridization patterns between the two (data not shown) and verified that co-hybridization with identical probes labeled with the two different fluorophores-digoxygenin or fluorescein- resulted in the essentially all positive cells appearing yellow, as expected from signal overlap (not shown). We did find slight non-overlap at tissues edges; thus, all data herein is from non-edge tissue regions. We verified that ISH signals replicated bilaterally, across sections and in multiple experiments and that dissimilar genes in serial sections show unique hybridization patterns, as in the developing kidney  neuron development but these data suggest a wider role in the nervous system, as well as a role in other tissues (see Discussion).We further compared in-house and custom RNAscopeg kidney . We furtula with . Analysi cortex, . In the These data demonstrate that our ISH procedure is specific and sensitive and can distinguish differential signal in closely juxtaposed cells . Furtherin vivo previously [Map2 and Actb are restricted to the nucleus, but in migrating neurons the Map2 (but not Actb) CDS localizes to the cytoplasm  expression in cells that line specific axons detected with the marker TAG1 (Otx2 (with Wnt1 (not shown) show high i3’UTR expression within the axons (with no or low CDS)   show unique 3’UTR to CDS patterns across the OlfE with Sox2 displaying graded differential 3’UTR to CDS expression, and Ascl1 with more coincident 3UTR/CDS expression-both show identical patterns bilaterally  into the basal regions  , as wellm (OlfE)  and 3, wm (OlfE) . At low lly with ;*. Withi regions . For Mycr region .Myc shows equal 3’UTR to CDS in the proliferative epithelial region , Shh 3’UTR and CDS are both expressed: with Myc shows much greater 3’UTR compared to CDS expression  at this line, with high 3’UTR compared to CDS expression in the Ki67+ cells, shifting to high CDS compared to 3’UTR in Ki67- pre-cortex cells (with Myc showed more coincident expression with somewhat higher 3’UTR than CDS in Ki67+ cells compared to pre-cortex (with Sox9 and three other Sox genes showed similar 3’UTR to CDS throughout (Shh and Ntn1 (used as a control) both showed asymmetric localization, in subsets of the Ki67+ cells  (Actb) CDS compared to 3’UTR, whereas the cells at the perimeter all show high 3’UTR and low CDS; this pattern replicates exactly across more than 30 circles/chip  cells were plated on prepatterned glass chips  was highly colocalized with P-body markers  become sequestered within cells. For this we examined the co-localization of various probe sets to specific antibody markers for the nuclear membrane (Lamin), P-body phase regions , endosomes (Lamp1), and apoptotic cells marked by activated Caspase3. Unlike Ki67 which is highly correlated with the i3’UTR for many genes  tend to show a high, or low 3’UTR to CDS ratio. We first examined two tissues: hindbrain and heart across multiple ages. Each sample had two biological replicates  and we asked, for each tissue and age, whether the GO categories corresponding to Hi  to 1 (high 3’UTR) or Lo 3’UTR genes (defined as < 0.4) (see methods for details) are different. We found, first, that, regardless of tissue or age, the top GO category for Hi CDS (Lo 3’UTR) genes is consistently “translation”, followed by “ribonucleoprotein complex biogenesis” ; left. OWhile intriguing, we wanted to see if this result held more generally and in the adult, and therefore examined this across 18 adult tissues. We observed similar results across all 18 tissues, as the top GO categories for Hi CDS genes were “respiratory electron transport” and “oxidative phosphorylation” ; top, whSox9; gray arrows) and as previously shown in [Sox11) we examined gene sequence elements upstream, or aligned with, these peaks and valleys. Using data collected from the UCSC browser  we aligned the following elements: conservation across species, transcription factor (TF) binding sites, CpG islands, H3K27ac peaks, DNase 1 hypersensitivity sites, and miRNA binding sites- to 3’UTR RNAseq peaks and valleys. Remarkably, for some genes, each of these elements shows tight alignment with discrete 3’UTR peaks and valleys. For example, for Sox11 , CpG islands, H3K27ac peaks, and transcription factor (TF) binding clusters are located and confined to the valleys immediately upstream of both peak 1 and 2 within the Sox11 3’UTR  corresponding to the CDS alone , we do not find strict gene element and peak alignment. It may be for some genes, differential 3’UTR to CDS expression is not regulated from within the genome by gene elements, but is post-transcriptional.In summary for some genes, we find very high 3’UTR conservation across species with gene sequence elements that are perfectly aligned perfectly with observed RNAseq peaks and valleys. For other genes, even though we observe highly differential 3’UTR to CDS expression by ISH  and parts or all of their 3’UTR sequences. This occurs in a non-random fashion, and within a tissue, gene ontology analysis shows that genes displaying high 3’UTR to CDS expression are likely to be involved in the particular functions of that tissue, whereas genes with more equal CDS and 3’UTR expression, or higher CDS than 3’UTR expression, are likely involved in more general cell maintenance functions. In addition, 3’UTR to CDS ratios are not fixed but are dynamic and change as a cell changes state or developmental age. Within a 3’UTR we (and others) , 2, findNurr1 3’UTR expression in both developing and adult tissues, often in the absence of high or even detectable levels of CDS. Genes that are thought to have very restricted expression based on their protein or CDS location may have widespread and high levels of 3’UTR expression in other cells. Nurr1 is one of three orphan steroid receptors sub-family members that are all suggested to be tumor suppressors [Nurr1 3’UTR levels and Nurr1 protein add to this contextualization. Here we showed high levels of Nurr1 isolated 3’UTR sequences in proliferating cells (in the Ki67+ cells of the deep hair follicle) and high levels of Nurr1 CDS in just adjacent Ki67- cells. Nurr1 is known to affect cell cycle [The use of multiple controls shows our ISH procedure is robust and sensitive, and the i3’UTR expression patterns allow interesting conjectures. By analogy to the known functions of lncRNAs, , isolated 3’UTRs may bind to specific miRNAs and RNA binding proteins (RBPs) , 32, andpressors –35, howepressors . It is ill cycle , and it Importantly we found many instances of isolated 3’UTR expression in the nucleus. Further, while proteins are often localized to cells with equal levels of cognate 3’UTR and CDS, or higher levels of their cognate CDS, we find multiple notable exceptions: certain transcription factors are found to preferentially colocalize with their cognate i3’UTR sequences in the nucleus in some cells and some instances. While we cannot verify that the CDS is entirely absent from these regions, what is clear is that there is less CDS than in nearby cells, so we can conclude in such instances that there is preferential localization of protein with i3’UTR sequences compared to CDS sequences. It is interesting to speculate that 3’UTR sequences in the nucleus may interact with their cognate protein in some way, e.g., to stabilize/and or sequester it to delay degradation or temporarily prevent function. Further studies are required to examine this. The specific localization of high levels of 3’UTR sequences to either the nucleus, or to axons, and the dynamic nature of these localizations have significant implications not only for nervous system axonal guidance synaptogenesis and plasticity, but for other cellular functions as well.Sox genes has been elusive due to imprecise knowledge about their gene and protein expression, as well as partial biological redundancy as evidenced by only small phenotypes after gene deletion in some cases. Knowledge and manipulation of the distinct 3’UTR to CDS expression patterns of members of these gene families provides an additional handle with which to examine their function in neural and other development processes. Dual ISH of Sox mRNA expression reveals intricate patterns for each Sox gene in the nervous system with precise and abrupt changes in the use of the 3’UTR and/or the CDS during neuronal development. Extremely intriguing Sox gene 3’UTR to CDS patterns were observed in other tissues as well, and require more detailed exploration.Pinning down the precise or even general role of large gene families such as the Unc5d 3’UTR in blood vessels suggests that perhaps this alternate family member with highly expressed 3’UTR sequences may in some way enhance or modulate the specific known interactions of Netrin-1 and Unc5B in this tissue [In addition, the Netrin ligand-receptor family contains multiple distinct receptors, including four members of the Unc5 family, which have roles in neuronal and vascular development as well as tumor suppression , 38. Thes tissue , 40.Shh and Ascl1) show more restricted patterns, and with more coincident 3’UTR to CDS expression. This is notable for two reasons, first PpG gene expression has not been highly documented outside of pluripotent stem cells. In agreement with other studies, we find that PpG protein is low in these systems (not shown), but their mRNA levels are robust, and protein is reliably detected in a reasonable cohort of cells expressing the cognate mRNA. We expect that the precise regulation of the 3’UTR to CDS ratio, and thereby regulation of PpG protein expression might be critical for stem/progenitor cell function, possibly affecting the onset, duration and magnitude of proliferation and quiescence. It may be that PpG protein is used sparingly, but that cells maintain specific 3’UTR to CDS expression levels, possibly to provide precise and titrated levels of protein. Secondly, we find abrupt changes in 3’UTR to CDS ratios in closely apposed but morphologically distinct tissues, such as Sox2 and Myc in the whisker dermal papillae and a number of genes in Ki67+ vs Ki67– cells in the hair follicle. Sox2 is critical in DP development [Myc, known for its role in proliferation, shows high 3’UTR. Interestingly, DP cells serve as a reservoir of stem cells  network in progenitor cells. Importantly, in adjacent sections, non-PpG genes; . Conversely, the identification of i3’UTR genes may inform us about a tissue at hand, either in chronic (cancer) or acute  disease states.In sum, we have shown the widespread and non-random use of differential 3’UTR to cognate CDS expression across tissues, development and cell state. This data shows there is a strong proclivity for high 3’UTR to CDS in cells and for genes involved in transition states, during both proliferative and developmental maturation events.Animal procedures were approved by the Stanford University Institutional Care and Use Committee and followed the National Institutes of Health guidelines for care and use of laboratory animals. All work was given written approval by IACUC protocol APLAC-32091, with expiration date 11/18/22. For this 1–2 animals were sacrificed/month in order to harvest tissue for in situ hybridization and immunohistochemical analyses. Decapitation was only used for embryos harvested from recently euthanized pregnant female. Pregnant females were sacrificed by CO2 inhalation followed by cervical dislocation, with embryos dissected from the carcass on ice and decapitated before dissection and harvesting embryonic tissue for histological analysis. For tissue harvested from animals older than E18, animals were anesthetized at various postnatal ages by ketamine/xylazine IP injection, and sacrificed via transcardial perfusion of paraformaldehyde.Two color fluorescence ISH was performed as previously described , using t’C in DMEM, 10% FBS and 0.5 mg/ml Penicillin Streptomycin Glutamine. Sodium azide (Sigma/Aldrich) was added to cell media 30 min before fixation.NIH3T3 cells were grown at 376) was evenly plated onto chips lain in 6-well plates for two days in complete DMEM medium before fixation and ISH.Micropatten CYTOO  chip cell culture was performed as previously described . Patterns of 800, 500, 225, 120 and 80 um circles are dispersed over the full surface, with 25, 144, 576, 900 and 1296 colonies of each size respectively. Briefly, 700 ul drops of 20 mg/ml Laminin (L20202)(Sigma) in PBS without calcium and magnesium (PBS-/-) in were spotted onto parafilm lain in a 15 cm tissue culture plate. After one wash in PBS-/-, chips were inverted on top of the drops for 2h at 37°C. They were then washed 5x with PBS-/- and a single cell suspension of 3T3 cells (2 x 10http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All raw reads were aligned to mm10 reference genome using STAR (v2.7.3a) with default settings and aligned files were filtered with minimum mapping quality 20 using samtools (v1.11). Alignment files (BAM) from ENCODE were downloaded directly. To obtain expression matrices for 3’UTR and CDS sequences, we quantified gene coverage for 3’UTR and CDS respectively using the bedtools (v2.17.0) function coveragebed, with the alignment file (BAM). From this output, we were able to obtain gene effective length per gene as the number of bases with non-zero coverage, and read count /per gene. CDS or 3’UTR expression was normalized by their library size as reads per million total mapped reads and quantified as transcripts per base pair using the following formula: Genes with > = 5 mapped reads are considered expressed.Raw data was downloaded from the ENCODE or GEO databases and quality control (QC) was carried out for all FASTQ files using the FastQC tool (CDS or 3’UTR expression = mapped reads assigned to each gene’s CDS or 3’UTR/(gene effective length (kb) x total mapped reads (millions)).http://metascape.org/gp/index.html) [For each gene, the relative abundance of 3’UTR to CDS is quantified and expressed as the fractional ratio of (3’UTR mean coverage)/ (3’UTR mean coverage + CDS mean coverage) which ranges from 0, (highest ratio of CDS to 3’UTR) to 1, (highest ratio of 3’UTR to CDS). Gene ontology (GO) enrichment analysis was performed using the Metascape online tool (ex.html)  and the S1 File(ZIP)Click here for additional data file.


The equatorial tropics house some of the earliest rock art yet known, and it is weathering at an alarming rate. Here we present evidence for haloclasty  from Pleistocene-aged rock art panels at 11 sites in the Maros-Pangkep limestone karsts of southern Sulawesi. We show how quickly rock art panels have degraded in recent decades, contending that climate-catalysed salt efflorescence is responsible for increasing exfoliation of the limestone cave surfaces that house the ~ 45 to 20-thousand-year-old paintings. These artworks are located in the world’s most atmospherically dynamic region, the Australasian monsoon domain. The rising frequency and severity of El Niño-induced droughts from anthropogenic climate change , combined with seasonal moisture injected via monsoonal rains retained as standing water in the rice fields and aquaculture ponds of the region, increasingly provide ideal conditions for evaporation and haloclasty, accelerating rock art deterioration. While human health and security4 have rightly been research focuses, climate change equally impacts the long-term survival of physical remnants from the human past, our cultural heritage7. Here we argue that climate fluctuations over recent millennia and especially in recent decades, have been, and are increasingly, a major catalyst for the deterioration of Pleistocene cave art in Maros-Pangkep, a limestone karst area on the Indonesian island of Sulawesi. Uranium-series dating of rock art found in the caves and rock shelters of these near-coastal lowland karsts has demonstrated it is some of the earliest yet known anywhere on the planet, dating to a minimum 45.5 ka and including hand stencils and figurative depictions of animals, human/animal composites (therianthropes), as well as possibly the earliest known narrative scene in prehistoric art11. With more than 300 cave/shelter sites in the region known to preserve this Pleistocene style parietal art11, it is clear that the Maros-Pangkep assemblage rivals the celebrated ‘ice age’ cave art of western Europe, where scholars had until recently thought the oldest cave art traditions first emerged. We contend that climate-catalysed salt efflorescence  is responsible for the exfoliation of the older, case-hardened limestone surfaces of Maros-Pangkep’s cave/shelter sites, a process that is widespread throughout older karst cave surfaces in the broader Indonesian region15. Moreover, the exfoliation process, which destroys the rock surfaces or “canvases” on which the Late Pleistocene art was created, appears to have worsened in Maros-Pangkep in recent decades—a trend we believe is set to accelerate with warming ambient temperatures and increasingly frequent/severe El Niño events16. The extent of salt efflorescence in the 11 Maros-Pangkep sites we investigated, coupled with conservative forecasts for a 1.5 to 2 °C raise in global temperatures and accompanying extreme weather events18, have grave implications for the conservation of this globally significant cultural heritage. Aside from continuing limestone quarrying for the burgeoning domestic cement and marmer (marble) industries, global warming should be regarded as the greatest threat to the preservation of the ancient rock art that survives in Sulawesi and other parts of tropical Indonesia11.The effects of human-induced climate change are increasingly expressed through environmental disasters across the globe19. The lowland ‘tower’ karst of Maros-Pangkep sits at the southern end of the Tonasa Formation, an isolated platform sequence of Eocene to late Miocene marine-deposited limestones dominated by photic zone benthic foraminifera20. The karst landscape covers an area of ~ 450 km2 between 4°7′S and 5°1′S and is situated on an alluvial plain close to the western shoreline of Sulawesi’s southwestern peninsula, bounded to the east by the volcanic mountains of the Western Dividing Range. The isolated karst towers and plateau-like hill masses of Maros-Pangkep range from 150 to 300 m in height and 1 to 10 km in diameter, with networks of footcaves, overhangs, and high-level cave passages21 housing an extensive rock art assemblage first reported by archaeologists in the 1950s22. More than 300 cave sites or rock shelters harbouring parietal imagery have been documented in these karsts, with new rock art sites found each year during ongoing field surveys  shown in outline profile with irregular infill. The second, more recent rock art phase is found on fresher cave surfaces  and is assumed to be only a few thousand years in age. This phase is attributed to the Neolithic farming communities colloquially known as ‘Austronesians’ who colonised Sulawesi ~ 4000–2000 years ago. This younger art phase is usually executed in black pigment  and is characterised by small images of anthropomorphic figures and domesticated fauna such as dogs, as well as an array of geometric motifs and abstract signs/symbols. We conducted direct Accelerator Mass Spectrometry (AMS) radiocarbon (14C) dating of a charcoal drawing of a human figure, typical of the Austronesian-style, located on the fresher surfaces of the ceiling panel at Leang Bulu Bettue. This yielded an age of 1583‒1428 calBP ; to our knowledge this is only the second absolute age determination reported for such a motif23 , a region characterized by major ocean–atmosphere interactions24. The case-hardened surfaces of the Maros-Pangkep caves, co-created by biofilms including plentiful microbial mats28 . While case-hardening makes the surface layer more resistant to weathering, the sub-surface zone immediately underneath is weakened by loss of cement matrices, the void spaces becoming susceptible to the accumulation of evaporites such as geological salts, especially where the outer crust has been breached29. From the earliest published descriptions of the Maros-Pangkep rock art over half a century ago, exfoliation of the older case-hardened surface of panels has been noted—termed “blistering” in early reports22 (p. 30). Also evident is the execution of the second phase of art production on more recent surfaces and/or overlying small areas of remnant case-hardening. In many instances, such as at Leang Bulu Bettue, Austronesian style rock art was created on long-exfoliated cave surfaces that preserve residual hand stencils of presumed Late Pleistocene age, suggesting that in some cases the early rock art was already in a state of advanced deterioration several thousand years ago. Similarly, at the cave art site of Leang Lambatorang Dutch graffiti signed with dates as early as AD 1769 is visible on “fresh” limestone surfaces exposed by exfoliation of early phase rock art (SI). A mounting body of quantitative and anecdotal evidence suggests that the rate of exfoliation is increasing (SI). Indonesian cultural heritage professionals, local academic archaeologists, and site custodians from nearby communities  report that the destruction of rock art through spalling has accelerated, with more panel loss from exfoliation over recent decades than at any other time in living memory11.In almost all sites containing early art, the hand stencils and figurative motifs are heavily affected by exfoliation of the limestone cave wall/ceiling surfaces that comprise the artists’ “canvas” . Human-induced environmental changes have accelerated in the Holocene with the compound, cumulative effects of agriculture (including rice cultivation in Asia from 6.5 ka and on Sulawesi from 3 ka), colonial migration and expanded human settlement (the late 1400 s onwards), industrialisation (1760s onwards), nuclear and thermonuclear testing (1940s on), and today’s persistent greenhouse gas emissions35. Although the Pleistocene rock art of Maros-Pangkep has existed throughout tens of thousands of years of climate variation in response to the earth’s orbital geometry, there is no doubt that global carbon cycling and greenhouse gasses are now significantly amplifying climatic responses36 (p. 24), and, we infer, hastening the deterioration of the unique, irreplaceable record of early human artistic culture in a little-understood region, one that continues to provide important insights into the culture of the first peoples of IMC21.Studies of human–environment interactions suggest changing climates were key factors in the evolutionary emergence of our species in Africa36. The Australasian monsoon domain within which Sulawesi is located (from the equator to 10° S latitude and from 110° E–130° E longitude) is the most atmospherically dynamic region on Earth37. Monsoon intensity has been the dominant feature of the Australasian paleoclimate37 (p. 110) with recent regional reconstructions beginning to disentangle the varied, localised responses to climate fluctuations across the IMC36 (p. 22). Generally speaking, a dry background climate state dominated the Australasian monsoon region during the Late Pleistocene (40–12 ka). At the time the earliest rock art in Maros-Pangkep was being created (at least 45–35 ka), climatic conditions were cooler and moister/more humid, resulting in lower evaporation36 (pp. 26–27). This cool trend increased from ~ 32 ka throughout the glacial period when the tropics experienced generally stable dry conditions, peaking at the Last Glacial Maximum (LGM) 22–18 ka as a result of sea levels dropping to at least ~ 125 m lower than today, reducing Sea Surface Temperatures (SST) by 1–3 °C and constricting the Indo-Pacific Warm Pool (IPWP). Current evidence suggests the monsoon disappeared entirely over northern Australia at this time, but there were at least periodically wet conditions over the IMC36 (pp. 27–28). The monsoon appears to have undergone a process of reinvigoration  from 15 ka and was well-developed by 13–12 ka; oxygen isotope data throughout the tropics records relatively stable temperatures, even during the Arctic Cool Reversal (14.5 to 12.5 ka) and Younger Dryas (12.85–11.65 ka)37 (p. 102), before a return to generally wetter, warmer conditions across the IMC with the inundation of the Sahul Shelf to the south 12–11 ka, and the Sunda Shelf to the west ~ 9.5–8.5 ka, warming SSTs and increasing evaporation37.The tropics have always been integral to the global climate system, essentially acting as a “heat engine” driving millennial-to centennial-scale oscillations36 (p. 30)37 (p. 103). The IPWP reached thermal maximum 6.8–5.5 ka when coccolith records from the Banda Sea east of Sulawesi suggest a significant shift in the position of the monsoon (~ 6 ka) owing to low-latitude insolation forcing37. Localised aridity from El Niño characterises much of Australasia from 5 ka36 (p. 29)37 , though more extreme and longer dry events are recorded 2.5–1.7 ka37 (p. 103). Modelling of ambient temperatures over the last millennium suggests peak pre-industrial warmth in Australasia 0.8–0.65 ka38 (p. 5581). Terrestrial and marine records show dry El Niño dominated conditions on Sulawesi over the past 400–300 years39 coincident with the advent of rice cultivation in the south of the island, dating to at least the fourteenth century41, though rice has been grown in West Sulawesi for more than 3 ka35. Minimum temperature anomalies are recorded at the time of the Northern Hemisphere’s so-named ‘Little Ice Age’ in the 16th and early nineteenth centuries38 (p. 5381). These data complement stable isotope records from the Great Barrier Reef of Australia that document ENSO intensifying from the early 1600 s, with extended droughts in the mid-1760s to 1780s37 (concurrent with dated Dutch graffiti at Leang Lambatorang—SI). During these drier conditions, extensive deforestation of the fertile plains in Maros-Pangkep is likely to have occurred in step with the emergence of the earliest agrarian kingdoms (complex chiefdoms) in South Sulawesi in the late thirteenth century, and certainly from the fifteenth century onwards with the rise of the Makasar Goa-Talloq kingdom41. More extreme wet and dry years occur in the current era, which many scientists refer to as the ‘Anthropocene’ (< 1965)37 (p. 103)34, precisely because of the prevalence of human induced environmental change. There is a clear trend toward more frequent ENSO activity in recent decades compared with past centuries or millennia16 (SI). These more intense and prolonged dry climate cycles, interspersed with moister recharge from monsoonal rainfall—combined with abundant evaporative water sources in the inundated rice fields at the base of the tower karsts and brackish aquaculture ponds toward the coastline—are providing highly favourable conditions for the deposit of the evaporite salts causing exfoliation of ancient cave/shelter surfaces.Though less frequent or extreme than it is today, the El Niño Southern Oscillation’s (ENSO’s) El Niño phase became a dominant climate feature 7–5 ka42, with Indonesia identified as a high-risk area in terms of drought-induced food security and extreme weather events such as tsunamis, flooding, and bush fires impacting densely populated coastal areas18. The specific physiogeography of the Maros-Pangkep region makes it highly susceptible to ENSO. Located in the IPWP, Sulawesi’s hydroclimate is influenced by the changing position of the Intertropical Convergence Zone (ITCZ) and varying Indo-Pacific Walker circulation, with rainfall highest on the western coast compared with the rest of the island43. The highly seasonal precipitation in Maros-Pangkep is intensified by the central mountain range dividing the southwestern peninsula or ‘arm’ of Sulawesi, which preferences rainfall from southward monsoon migration—delivering ~ 80% annual precipitation December-March, while preventing astral winter rainfall from reaching the region as the ITCZ migrates north44 (p. 11). Weather station data from Sulawesi shows 13 El Niño events between 1972 and 2012 with dry days  increasing by more than two months in El Niño years compared with typical La Nina conditions43. Rainfall observations between 1967 and 2015 indicate that southwest Sulawesi is most highly affected by El Niño, experiencing longer dry spells, and, in the case of the Maros-Pangkep region, the most consecutive dry days45. It is this combination of seasonal rainfall with alternating prolonged dry conditions under El Niño that provides ideal conditions for salt efflorescence and other forms of evaporite deposition.The extensive shorelines of the IMC make the region sensitive to climate change9  and humidity induce salt migration, precipitation, crystal growth, hydration, and expansion, triggering mechanical stress on stone surfaces at both the “macro” level  and on a “micro”’ scale in the form of granular disintegration. Beginning at the solute/air interface, high relative humidity and/or high ambient temperatures trigger precipitation and evaporation, causing salt crystals to grow, filling pore and void spaces in stone until successive crystallisation pressure generates cracks; crystallisation continues in the form of columns which lift spall flakes, detaching them from the surface29 (p. 227) as we observed, for instance, at Leang Timpuseng  (× 2) and halite (sodium chloride) at Leang Lompoa, Leang Balang Pakalu 5 and Leang Timpuseng (SI). These salts epitomize different aspects of the weathering processes: flaking and granular disintegration. With hydration, gypsum can increase in volume by 63%, creating accompanying swelling pressure, while the dehydration (phase transition to anhydrite) induces a 39% volume decrease29 (p. 228); however, halite, a deliquescent salt found on the surfaces at Leang Timpuseng, has a smaller hygrometric morphology that tends to form a granular crust on damp substrates29 (p. 227). Notably, Leang Timpuseng is one of two studied sites in closest proximity to the extensive wet rice fields (sawah) characteristic of Maros-Pangkep. The identification of halite at Leang Timpuseng is also important because the higher the concentration of sodium chloride in solutes, the lower the temperature required for hydration/dehydration transitions (shrink and swell) of calcium sulphide crystals that catalyse the flaking/spalling of rock surfaces29 (p. 230). This indicates a far greater risk for accelerated degradation of Pleistocene rock art in proximity to sawah and also empang—the extensive networks of saline aquaculture ponds in the lower reaches of the Rammang-Rammang River, the watershed boundary at the heart of the Maros-Pangkep region.Elevated sulphur abundances were recorded throughout the 11 Maros-Pangkep rock art panels examined Figs.  and SI, 17. Here we have outlined the increasing deterioration of the Maros-Pangkep rock art assemblage as a consequence of climate change over at least the past four centuries, with apparent rapid acceleration over the past 40 years associated with anthropogenic climate forcing. The challenges facing the preservation of Pleistocene rock art in the tropics are being accelerated by climate change, with urgent action now required. If sustained net zero human CO2 emissions were reached (and non-CO2 reduced elevating radiative forcing) it may still be possible to halt anthropogenic global warming on a multi-decadal timescale17. The dramatic CO2 emission reductions achieved as a result of restricted mobility during global Covid-19 pandemic shutdowns show what is possible1.Global warming has already impacted natural and human systems to varying degrees of severity as greenhouse gas emissions raise global temperatures an average of ~ 0.2 °C per decade. Unabated, conservative predictions are for global warming to 1.5 °C above pre-industrial (> 1750) levels within decades , with areas of climate sensitivity such as the tropics set to experience up to 3 × higher temperature increases. This trend in higher global temperature accompanies increased frequency and intensity of extreme weather events in the mid-latitudes (i.e. the tropics) where hot days have already increased by ~ 3  ͦC49. Our study of the materiality of the Maros-Pangkep rock art panels shows that geological salts are ubiquitous, something borne out across the IMC by emerging physicochemical work on rock art in adjacent Indonesian islands50. Loss of the painted limestone “canvas” from salt efflorescence enhanced by El Niño conditions is the most pressing threat to rock art preservation in this region – aside from the industrial-scale quarrying of limestone (with associated pollution yet to be quantified)53. Long term physical and chemical monitoring has been ongoing in the Franco-Cantabrian rock art sites of Lascaux and Altamira since 1963 and 1997, respectively, with detailed climatic studies currently underway at Chauvet, Orgnac (Ardèche), Esparros (Hautes-Pyrénées), and Villars caves54. While a small-scale monitoring program has recently been initiated in Maros-Pangkep by the Makassar-based cultural heritage agency Balai Pelestarian Cagar Budaya, more support for this initiative is required, especially infrastructure investment. As some of the earliest known examples of artistic creativity on the planet, the Pleistocene rock art of Maros-Pangkep warrants monitoring and conservation efforts on par with those carried out over decades in Europe. The exceptionally old cave art of Indonesia is located within a dynamic tropical environment that renders it particularly vulnerable to the destructive impacts of climate change, adding unique urgency to this call for further research.Understanding the environmental context of early rock art is crucial for designing effective management strategies to help preserve these irreplaceable images from the human pastPortable X-Ray Fluorescence (pXRF) assay locations were selected for features of interest presenting the pXRF aperture to minimise any air gaps and associated X-Ray attenuation. Assay were collected at the height of an El Niño induced drought in late 2015, with strong winds noted in the latter part of the field program. The exceptionally dry conditions were perfect for evaporite deposition and it is likely that the preceding hot, dry climatic period contributed to the ubiquity of geological salts on the cave/shelter surfaces.Small (< 2.5 cm diameter), thin flakes that were near spalling were collected from the case-hardened cave surfaces for use in laboratory-based analyses 2017. At Leang Timpuseng the art panel is on the ceiling. Spall flakes from the edge of the ceiling art panel near its junction with the back wall of the rockshelter were collected. At Leang Lompoa and Leang Balang Pakalu 5 spall flakes from the rock art panels, including black and red pigments from motifs (SI), were collected.21.Pigment characterizations were undertaken using a Brucker TitanS1 800 pXRF instrument, equipped with a silicon drift detector, Rh target X-ray tube  and five position motorized filter changer. Two beam phases collecting for 90 analytic seconds were taken for each assay (180 s per spectrum). Phase 1 parameters: 45 kV, 10.45 µA with a Ti 25 µm, Al 300 µm filter in the beam path. Phase 2 parameters: 15 kV, 31.55 µA without a filter. Relative element abundances are derived from the manufacturer’s empirical parameters calculations corrected for limits of detection . Spectra were additionally manually interrogated using Bruker Artax software (V8). Certified reference materials were used as internal standards for the dataset to monitor instrument stabilityField emission SEM was undertaken on a Joel 7100 JSM. Energy Dispersive Spectrometry (EDS) of relative element abundances measured in spot, area and element mapping modes using an Oxford X-Max 80 probe and Aztec software. Sections of the collected spall flakes were mounted on stubs  and gold coated to facilitate the analysis of both the case-hardened outer surfaces and the underlying salt covered surfaces being mechanically pushed free of the limestone massive.10.Fragments of the spall flakes were crushed into homogenized powders manually using an agate mortar and pestle. Once powdered, they were placed into 0.3-mm-diameter borosilicate capillaries and mounted on the Australian Synchrotron beamline. Diffraction data were collected at the at a wavelength of 0.77412(3) Å, calibrated using a NIST SRM 660b, from 5° to 85° 2Theta, with a Mythen microstrip detector with an inherent step size of 0.002°, using two detector positions and a collection time of 5 min per position. Samples were rotated at around 1 Hz during data collection to ensure good powder averaging. Phase identifications were undertaken using Panalytical Highscore with the ICDD PDF4 database55 applying atmospheric data from Hogg et al. 202056.Black pigment samples were removed from the rock face using a sterile blade, with material collected directly into clean Teflon vials. Pigment from two locations within the same motif, demarcated subsamples LBB 3 (shoulder) and LBB 4 (head) Fig. , were coSupplementary Information 1.Supplementary Information 2.


After ultrafast laser irradiation, a target enters a poorly explored regime where physics of a solid state overlaps with plasma physics and chemistry, creating an unusual synergy—a warm dense matter state (WDM). We study theoretically the WDM kinetics and chemistry in a number of group III-metal oxides with highly excited electronic system. We employ density functional theory to investigate a possibility of nonthermal transition of the materials into a superionic state under these conditions. Atomic and electronic properties of the materials are analyzed during the transitions to acquire insights into physical mechanisms guiding such transformations. A thermal one is initiated via electron–ion (electron–phonon) coupling equilibrating a highly excited electronic system with the ionic one2. This coupling results in a noticeable lattice heating when an amount of energy transferred from electrons to atoms becomes significant, typically at timespans > 1 ps3.A high-intensity femtosecond laser irradiation of solids excites an electronic system of a material, whereas an ionic system is initially unaffected. Two main mechanisms govern structure transformations in materials after the laser pulses7. Nonthermal mechanism is induced by a significant energy density deposited into the electronic system, usually corresponding to the electronic temperature of a few eV8. Such excitation of the electronic system temporarily changes a potential energy surface of the atomic system in a solid. Appearing uncompensated forces initiate movements of atoms trying to find their new equilibrium positions and can even cause nonthermal structure transitions. In some materials, depending on the excitation level, these new equilibrium positions may not exist, and nonthermal melting ensues even when the atomic temperature does not exceed the melting point of the material10. Nonthermal atomic movement dominates at timescales under 0.5–1 ps, until the thermal channel starts to play a significant role1.The second one is a nonthermal channel, which may occur at much shorter times11.Materials under such conditions are one of the types of manifestations of the warm dense matter state (WDM)—an intermediate state between a cold solid and a hot plasma12. One of such unusual phases is the superionic one13. It consists of one subsystem of a compound in a liquid state, whereas the other one is in a solid phase, simultaneously. In conventional superionic materials such as metal-lithium compounds, the high ionic diffusion conductivity is a result of sparse atomic structure14. In contrast, under extreme conditions, an entire sublattice may be destabilized exhibiting a liquid behavior. Such superionic state was recently produced by laser-induced dynamical shock compression of water ice15. Presumably, a similar superionic ice can be found naturally in giant planets interiors such as Uranus, Neptune, or other exoplanets16.It may lead to exotic structures and phases , that are not achievable at equilibrium conditions12. This stable during hundreds of femtoseconds state with liquid oxygen sublattice and solid aluminum one may be produced in irradiation spots of free-electron lasers19.Superionic alumina was predicted to form after fs-laser irradiation12 because only α-Al2O3 (corundum) was investigated. Understanding the formation mechanism of such states should inspire experiments and theoretical research aimed at production of new superionic materials including those under less extreme conditions. It may open up opportunities for potential applications in areas requiring materials with controlled atomic ordering.However, general mechanisms leading to superionic state formation in materials were not discussed in Ref.2O3, Ga2O3, In2O3, and In2S3 for comparison, to gather some statistics of materials demonstrating superionic behavior under extreme electronic excitation. These materials also have the same R-3c group symmetry as that of α-Al2O3 and their electronic structure is formed by overlap of ns2np1 and ms2mp4 atomic orbitals. Additionally, we check polymorphs of these materials for the existence of a superionic state. By analyzing such material properties as group symmetry, chemical composition and electronic energy levels, we investigate which of the parameters may be driving forces of the transition into the superionic state.In this paper, we study a possibility of nonthermal transitions in a few oxides formed by group III metal: AlWe identify thresholds of electronic excitation required to produce the superionic states in some of these compounds. These thresholds should be reasonable reference points for future experiments. We also note that superionic behavior in some materials may realize as a transitional state to a stable phase, which will form at much longer timescales than those investigated in this paper.20. We use norm-conserving pseudopotentials from the QE library and Perdew–Burke–Ernzerhof (PBE) exchange–correlation functional21. Although PBE functional does not contain explicit dependence on the electronic temperature  we use it since only a few developed Te-dependent functionals23 were applied mostly for metals and their capability to reproduce correct band gaps is unclear. Non-hybrid functionals are known to underestimate the band gap value at ambient conditions, however, in a case of high electronic temperatures they perform much better24. To validate our application of the PBE functional, we performed a cross-checking simulation for one material with—one of the most advanced and modern R2SCAN meta-GGA functional25.For all presented simulations, we use the density functional molecular dynamics (DFT-MD) simulation within the Quantum Espresso (QE) simulation packageTi = 300 K via DFT-MD with unperturbed electronic system (Te = 300 K set via Fermi–Dirac smearing) during 500 fs with 0.5 fs time step. After that, we instantly elevate electronic temperature to a certain value assuming that electrons adhere to the Fermi–Dirac distribution. At this step, next DFT self-consistent cycle automatically adjusts atomic forces and electronic band structure in accordance with new electronic temperature. Then we proceed DFT-MD simulation within 1000 fs with 0.5 fs time step.To study atomic and electronic structure dynamics during nonthermal transitions, we implement the following algorithm. After the standard procedure of a geometry optimization, the lattice is allowed to thermalize at the room temperature 26. This allows us to apply a thermalized distribution for the electronic ensemble at all times avoiding complex consideration of a short-living nonequilibrium stage that cannot be treated in DFT.Within this procedure, we assume that electron cascades after laser irradiation can be neglected since they take a few femtoseconds in a typical FEL spot except for hard X-rays and does not significantly affect lattice dynamics2O3 that demonstrated electron-lattice equilibration due to electron–ion coupling requires at least a few picoseconds1. We note, however, that all simulations beyond 500 fs in the presented work are performed only in order to confirm nonthermal transition behavior when it is unclear at shorter timescales for some compounds investigated here.We further assume that the electronic temperature can stay constant throughout the simulation since electron energy loss via kinetic energy transfer to the lattice or via spatial dissipation is minor within ~ 500 fs in the central area of an irradiated volume. These assumptions are supported by the nonadiabatic tight-binding MD simulations in AlWe do not consider excitonic effects as well, since we assume that a significant level of electronic excitation and induced atomic perturbations studied here do not allow excitons to form.27.An NVT-ensemble  is used for the electronic system and an NVE-ensemble  for the atomic system. This choice corresponds to the conditions in a bulk achieved after irradiation with an FEL pulse, where the unperturbed media maintains a constant volume of the target’s excited part for times sufficiently longer than those modeled here28 with a target pressure Ptarget = − 600 kbar and atoms kept in the ideal lattice positions. For this analysis, the initial state of a material was set to its ambient structure and zero atomic and electronic temperatures (the latter was set via Fermi–Dirac distribution at Te ≈ 30 K). At each molecular dynamics step, energy levels were extracted and shifted to zero chemical potential (Fermi level). At the last step, the gamma-point projected electronic density of states (PDOS) was calculated.Apart from the DFT-MD simulations, further analyses were performed. In order to identify affiliation of obtained energy levels to atomic orbitals, a dependence of gamma-point energy levels on the interatomic distance was constructed via Parrinello-Raman variable-cell molecular dynamicsa0 − 1) Å to (a0 + 1) Å, where a0 is a coordinate  of the equilibrium position, with 0.1 Å step. For each point of the grid, a self-consistent calculation was carried out. Then, the total energy of the electronic system representing atomic potential energy was calculated (excluding pseudo-nuclei repulsion term that becomes noticeable only at much shorter interatomic distances than those considered here).Atomic potential energy surfaces were constructed in a series of calculations. For example, for one Ga or one O atom  a uniform 3-dimentional grid was set on Cartesian coordinates from (Ecut ≈ 952 eV (70 Ry) for nonthermal transitions simulations, potential energy surfaces construction and PDOS calculations for materials at ambient conditions. For energy levels of the expanding cell and corresponding PDOS calculations, the parameter was set to Ecut ≈ 1360 eV (100 Ry) since increasing interatomic distance requires more plane waves in the DFT basis set to describe electronic states becoming more localized around nuclei. For all calculations, supercells consisting of 80 atoms were used.Energy cutoff parameter controlling the size of the plane wave basis in DFT simulations was set to Calculated thresholds for nonthermal phase transitions in studied materials are presented in Table 2O3 discussed below) we observed the same profile of mean atomic displacements. During the first ~ 250–300 fs, both metal (Me) and O atoms are rapidly moving away from their ambient equilibrium positions. After that, displacements of Me atoms saturate at values ~ 0.6 Å, while O atoms keep moving demonstrating diffusive (liquid-like) behavior.For all materials becoming superionic during nonthermal transition  and atomic trajectories in a supercell are presented in Fig. 2O3. It is also clearly seen that at the threshold temperature Ga atoms (green) oscillate around their ambient equilibrium positions, while O atoms (purple) move more chaotically unbound from their former equilibrium positions. For clarity, these Figures represent 640 atoms supercells—translated original simulated supercells of 80 atoms. This and the choice of R-3c phase of Ga2O3 was done for the best visualization of a superionic state.In Fig. 30 and obtained values of some tens of mS/cm which is in a range of typical conductivities for superionic materials31.Additionally, we estimated ionic conductivities of materials becoming superionic via Nernst–Einstein relationIn the Supplementary Information we present detailed information about each transition as well as a comparison of the mean displacements calculated with R2SCAN and PBE functional. R2SCAN predicts slightly higher electronic threshold temperature due to a larger band gap. However, the mean displacements calculated with PBE and R2SCAN are in a good agreement at the threshold temperatures. This confirms applicability of the used PBE functional for the purposes of this work.2O3, nonthermal transition to the superionic state at Te = 2 eV can be clearly identified only at times > 1 ps which is far longer than times at which the nonthermal transition channel can exist without noticeable interference from the thermal one; we thus expect that it may not be observable in experiments at the threshold temperature. However, increase of the electronic temperature to Te = 2.25 eV results in much faster transition within < 500 fs, which should be observable.We note that for Ia-3 phase of In2S3 nonthermally melts, while other compounds with the same space group become superionic. Y2O3 (Ia-3 space group) in our previous study also did not demonstrate existence of a superionic state4.Table 2O3 exhibits melting, while all other studied materials with oxygen turn into superionic state.A mere presence of oxygen atoms in a structure does not guarantee appearance of the superionic state, since C2/m phase of GaNevertheless, Table 2O3 and In2S3 from other materials and that could be interpreted as an unambiguous indicator of an ability or inability of a material to exhibit transition to the superionic state. This suggests that electronic energy levels structure itself does not determine a type of a transition. Instead, a complex interplay of various material properties affects formation of the superionic state.We compared calculated projected electronic density of states (PDOS) of each ambient material from Table 2O3 on the interatomic distance. It is clear that the upper energy levels of the valence band and the lower levels of the conduction band are formed from oxygen p-orbitals. This means that at elevated electronic temperatures triggering the transition into the superionic state, electrons are mainly excited from oxygen bonding energy levels that are shifted below those of isolated atom to oxygen antibonding (shifted above) levels while occupation numbers of metal bonding and antibonding levels remain almost untouched. Moreover, electronic temperature and atomic movement cause these levels to shift  to become bonding. By the end of the simulation (500 fs) only ~ 1.8% of simulated electrons are occupying “Ga-p” antibonding levels. All this indicates that the electronic excitation is affecting potentials in a way that may lead to oxygen melting while preserving metallic sublattice.Figure 12.Further electronic temperature increase should involve more Ga energy levels both in the valence and conduction bands resulting in nonthermal melting of the material or at least significant changes in the Ga sublattice. This is exactly what happened with alumina in our previous study2O3 as well as In2S3 should also have been transformed into a superionic state if the above reasoning is sufficient. Considering that these phases demonstrate melting instead of the superionic behavior, it means more than one material property affects the superionic state formation.Energy levels behavior from Fig. 2S3 is a very unstable material that is starting to turn into β-In2S3 already at 40 °C32. This means that atoms in ε-In2S3 are in a very shallow potential well. In such a case, almost any significant external perturbation would lead to destabilization of the material.Analyzing properties of materials from Table 2O3, while Ia-3 phase is more “exotic” and less stable at ambient conditions (it turns into ε-Ga2O3 at > 500 °C)33. Nevertheless, C2/m phase nonthermally melts in contrast to Ia-3 phase exhibiting solid-superionic phase transition. This difference may arise because of asymmetry in Ga potential surface that may result in preferential direction for atomic movement. In combination with thermal oscillations and interatomic potential changes after laser irradiation, this may be a source of easier destabilization of Ga lattice and consequently may lead to melting instead of a transition into superionic state after the electronic temperature elevation.In contrast, C2/m is the most stable phase of Ga2O3 phases, we constructed potential energy surfaces of Ga atoms for Ia-3 and C2/m polymorphs  in all pictures are wider for O atoms. This means that the oxygen potential well is slightly shallower than the gallium one allowing for easier displacements of oxygen atoms.Increase of the electronic temperature to the values around the superionic threshold itself does not seem to change the potential energy surface qualitatively—it simply makes the potential well shallower see Fig. . It is i35. Moving oxygen atoms change the potential energy surface profile of metal atoms “dragging” them away from the equilibrium positions of an ambient crystal. This “dragging” continues until metal atoms find positions not hindering further oxygen atoms flow. After that, although the mean displacement of metal atoms remains almost constant, they may strongly oscillate around new positions as it is seen from mean displacements along the lattice vectors (XYZ axes).Combining obtained information, we can deduce the following path of nonthermal transitions into a superionic state in the investigated compounds. The transition starts from a fast displacement of O atoms because of shallow and highly asymmetric potential wellFor the observed materials exhibiting transition into a superionic state, the mean displacement saturation level at the threshold electronic temperature for metal atoms is usually around ~ 0.6 Å while oxygen atoms are displaced to larger distances > 0.8 Å at 500 fs after the electronic temperature increase. This saturation of the metallic atoms displacements may result from energy levels changes during the transition: when a sufficient amount of metal levels turns its behavior from antibonding to bonding, metal atoms slow down settling in a new sublattice.36 or even contradictory information exists about their crystal structure37. This makes them challenging for first-principles calculations38 and requires a separate dedicated study.We presume that non-oxide III–VI group materials should be capable of transforming into a superionic state. However, the majority of these compounds have defective structuresWe established threshold electronic temperatures triggering nonthermal phase transitions and their types (solid–liquid or solid-superionic) in a number of group III-metal oxides in the warm dense matter state. We demonstrated that the majority of considered materials exhibit nonthermal transitions into a superionic phase where oxygen exhibits a liquid-like behavior in contrast to the metallic sublattice remaining in a solid state.We analyzed electronic structures of these materials and concluded that a unique combination of bonding and antibonding states may be responsible for such a behavior. A transition into a superionic state occurs when an asymmetry of oxygen potential energy surface induces liquid-like flow of oxygen atoms.Supplementary Information.


There are approximately 1 million total knee arthroplasty (TKA) and total hip arthroplasty (THA) procedures performed annually in the United States. With this number projected to increase, it is vital for orthopedic surgeons and health-care systems to properly anticipate healthcare utilization related to TKA and THA. Google Trends (GT) is a free, open source tool that provides customizable analysis of search terms entered into the Google search engine. We aim to explore the relationship between public interest in TKA and THA as determined by GT data and volume of TKA and THA procedures.GT data were compiled for ten search terms related to TKA and ten search terms related to THA from January 2009 to December 2017. Annual case volumes for TKA/THA procedures were obtained from the Healthcare Cost and Utilization Project National Inpatient Sample from 2009 to 2017. Trend analysis was performed using univariate linear regression of GT data and TKA/THA case volumes.There was a statistically significant positive correlation between GT data and procedure volume for 14 of the 20 search terms studied. Seven TKA-related search terms with a positive correlation to procedure volumes include “total knee replacement,” “knee replacement,” “knee osteoarthritis,” “knee ache,” “knee swelling,” “knee stiffness,” and “chronic knee pain.” Seven THA-related search terms with a positive correlation to procedure volumes include “hip arthroplasty,” “total hip replacement,” “hip replacement,” “hip osteoarthritis,” “hip ache,” “hip swelling,” and “chronic hip pain.”GT may provide a high utility as a convenient and informative data set for orthopedic surgeons to analyze public interest in TKA and THA procedures. The data provided by GT have the potential to provide real-time, actionable information that may help surgeons and health systems to characterize public interest in TKA and THA and to best identify and address patient needs.The GT tool can be used to measure public interest in TKA/THA, which can inform physician expectations for the patient encounter and lead to the creation of decision aids that better inform the public about the risks and benefits of TKA/THA. Total knee arthroplasty (TKA) and total hip arthroplasty (THA) are two of the most common orthopedic procedures, with more than 1 million TKA and THA surgeries performed each year in the United States ,3. In faThere have been many recent attempts to monitor and characterize public interest in surgical procedures to better forecast surgical case volumes ,6. HowevThe purpose of our study is to explore the relationship between public interest in TKA and THA as determined by GT data and the volume of TKA and THA procedures performed in the United States. We also describe important temporal and seasonal patterns in public interest related to TKA and THA in the United States, providing insight that may help inform important staffing and resource allocation decisions that maximize productivity and improve patient outcomes.GT analysis for a given search term can be filtered by time period and geographic location. After a search term is entered into GT and the appropriate temporal and geographic constraints specified, GT generates visuals that reflect the volume of a given search term relative to its peak popularity within the defined time period, which is assigned a value of 100. The data are presented as relative search volume (RSV), which is computed as the percentage of searches of a term in a location during a specific time period. An RSV value of 100 indicates the largest ratio between searches for a specific topic and the total amount of Google queries. A value of 0 indicates that at the specified time point, the proportion of queries for the search term was <1% of its peak RSV (RSV 100) .Search terms related to TKA and THA were chosen using the “related queries” feature of GT. Databases of search volumes over time were collected for 20 search terms that were divided into four subgroups. Two subgroups were devoted to each procedure, with one subgroup focusing on different variations of the name of the procedure itself and one subgroup focusing on common symptoms that may indicate the given procedure is necessary. Search terms in the TKA procedure category were as follows: “total knee arthroplasty,” “knee arthroplasty,” “total knee replacement,” “knee replacement,” and “total knee replacement surgery.” Search terms in the TKA symptom category were as follows: “knee osteoarthritis,” “knee ache,” “knee swelling,” “knee stiffness,” and “chronic knee pain.” Search terms in the THA procedure category were as follows: “total hip arthroplasty,” “hip arthroplasty,” “total hip replacement,” “hip replacement,” and “total hip replacement surgery.” Search terms in the THA symptom category were as follows: “hip osteoarthritis,” “hip ache,” “hip swelling,” “hip stiffness,” and “chronic hip pain.” Colloquial terms as well as technical terms related to both TKA and THA were included in our analyses. We used GT’s customizable filters to include results for searches within the United States from January 2009 to December 2017 for all selected terms based on availability of case volumes for the years included in the study.Annual case volumes for both TKA and THA were obtained from the Healthcare Cost and Utilization Project (HCUP) National Inpatient Sample (NIS) annual reports from 2009 to 2017 using proper International Classification of Diseases, Ninth or Tenth (ICD9/10) Revision codes .P<0.0050 because of the increased risk of a type 1 error when performing multiple linear regressions. In addition, seasonal trends for all search terms with a statistically significant correlation to TKA and THA case volumes were observed using a month-by-month analysis of variation from yearly GT means. All statistical and trend analyses were performed using Microsoft Excel Version 15.21.1 and SPSS Version 26.0.0.1.Univariate linear regression was used to evaluate the correlation between average annual GT data and annual volume of TKA and THA procedures, mimicking the protocol of other GT studies that examined the association between Google search volumes and case volumes -13,18. WP=0.0010, R2=0.876), “knee replacement” , “knee osteoarthritis” , “knee ache” , “knee swelling” , “knee stiffness” , and “chronic knee pain” . No significant correlation between GT search data and TKA case volumes was detected for the search terms “total knee arthroplasty,” “knee arthroplasty,” and “total knee replacement surgery” , “total hip replacement” , “hip replacement” , “hip osteoarthritis” , “hip ache” , “hip swelling” , and “chronic hip pain” . No significant correlation between GT search data and THA case volumes was detected for the search terms “total hip arthroplasty,” “total hip replacement surgery,” and “hip stiffness”  consisted of the search terms “total knee replacement,” and “knee replacement.” Group 2 (TKA Symptom Terms) consisted of the search terms “knee osteoarthritis,” “knee ache,” “knee swelling,” “knee stiffness,” and “chronic knee pain.” Group 3 (THA Procedure Terms) consisted of the search terms “hip arthroplasty,” “total hip replacement,” and “hip replacement.” Group 4 (THA Symptom Terms) consisted of the search terms “hip osteoarthritis,” “hip ache,” “hip swelling,” and “chronic hip pain.” Within each group, average monthly RSV was calculated for each month from January 2009 to December 2017.Peak interest in TKA Procedure search terms (Group 1) was observed in the months of October (+3.5%), February (+3.3%), and June (+2.1%). Minimal interest in TKA procedure search terms (Group 1) was observed in the months of January (−3.5%), December (−3.2%), and August (−3.0%), with no clear seasonal patterns .Peak interest in TKA Symptom search terms (Group 2) was observed in the months of June (+8.5%), July (3.4%), and August (+2.6%). Minimal interest in TKA symptom search terms (Group 2) was observed in the months of February (−7.8%), December (−6.1%), and January (−3.3%), with a clear pattern of decreased search traffic in the winter months of December-February and increased search traffic in the summer months of June-August .Peak interest in THA Procedure terms (Group 3) was observed in the months of October (+7.2%), April (+5.8%), and November (+2.7%). Minimal interest in THA procedure search terms (Group 3) was observed in the months of December (−8.0%), January (−5.6%), and August (−3.1%), with a less pronounced pattern of decreased interest in the winter months of December-February when compared with the rest of the year .Peak interest in THA Symptom terms (Group 4) was observed in the months of October (+6.6%), November (3.1%), and July (+1.9%). Minimal interest in THA symptom search terms (Group 4) was observed in the months of January (−3.3%), June (−2.2%), and September (−2.0%), with no clear seasonal patterns .Our study demonstrates the value of GT in monitoring and characterizing patient interest in TKA and THA procedures over time. Univariate linear regression demonstrated statistically significant positive correlations between GT data and TKA/THA procedure volumes for 14 of the 20 search terms studied. Our results add to the growing body of evidence suggesting that GT is a tool that can be used to characterize trends in case volumes for surgical procedures -13,18,19The previous studies examining the association between GT data and surgical case volumes have discussed the importance of careful section of search terms ,19. Our et al. [The ability to use GT to monitor and characterize public interest in both TKA and THA has important consequences for orthopedic surgeons and healthcare systems alike. A review of medical claims data from 2010 to 2017 by the Blue Cross Blue Shield Association (BCBSA) discovered that knee replacements and hip replacements increased by 17% and 33%, respectively, from 2010 to 2017 [et al.  recentlyet al. . GT dataet al. .In addition to staffing decisions, trends in GT data can also be used to optimize OR scheduling. Operating rooms are typically a hospital’s greatest revenue source . As a reThe ability to characterize public interest in TKA and THA will be critical to orthopedic surgeons and health-care system that wish to anticipate healthcare resource utilization by patients who endure TKA and THA. The previous literature discusses the increased utilization of health-care resources by patients with osteoarthritis compared to individuals without osteoarthritis -30. IncrThe power of the GT tool to characterize public interest in medical conditions and procedures in real-time has many clinical implications. For one, GT may help to track public interest in more controversial procedures/therapies that have not yet been proven effective, which can lead to policy changes or recommendations. For example, the previous reports indicate an increase in public interest for both stem cells and platelet-rich plasma to treat hip/knee osteoarthritis, despite the fact that limited evidence exists for the use of either therapy ,17. WhenIn addition to informing resource allocation decisions in the present, seasonal GT data may also provide answers to weather-related joint pain questions in the future. Our results indicate that although peak GT interest for TKA and THA-related search terms varied, seasonal trends indicate decreased search volumes for all TKA and THA-related search terms in the winter months of December-February compared with the March-November months in the spring, summer, and fall seasons . These rThere are several limitations to our study. First, GT provides RSV rather than raw values of Google searches for search terms that are entered into the Google search engine. In addition, Google provides very limited demographic data about the users whose search traffic data are represented in this study. As a result, it is difficult to discern how many unique users are being reflected in the GT data. More specific demographic information about the age of users searching for information about TKA and THA would be beneficial to future GT study. Furthermore, our analysis is limited by the fact that GT only provides data about search trends for search terms with sufficient search traffic to report. There are other search terms related to TKA and THA that did not populate in the “related queries” feature of GT due to insufficient search traffic and that may help explain some of the trends observed in our study. Finally, we must be careful not to confuse correlation with causation when examining the relationship between GT search volumes and actual procedure volumes for TKA and THA. Previously, many factors including the overall health of the economy and insurance status have been shown to influence whether or not a patient proceeds with elective surgery ,43. The GT is a free, open-source tool that has previously been shown to be useful in characterizing case volumes for many elective surgical procedures; however, there has been little study about the association between GT data and case volumes for orthopedic conditions such as TKA and THA. Our study demonstrates that search volumes associated with many GT search terms related to TKA and THA exhibit statistically significant positive correlations with annual case volumes. This information may provide valuable insight to orthopedic surgeons and surgical centers that hope to characterize public interest in TKA and THA procedures for both staffing and resource allocation purposes.The authors declare no conflicts of interest.