The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels. Due -β folds –8. At thβ/alpha units connected by variable loops [The TIM barrel (TIMB) fold is a critical target of these efforts due to its ubiquity in nature across highly diverse functional families, suggesting its broad utility as a design scaffold –11. In ple loops , 12–14. le loops . Here, wle loops –18.β-α loops play an important role in ligand recognition and catalytic residue positioning and exhibit a broad spectrum of lengths and conformations [β barrel plays a key role in the positioning of active site residues in a way that is not simply rectified by altering loop conformations, and the ability to control and vary β barrel curvature allows for insertion of new, more complex residue combinations and networks that may not be possible in a circular TIMB. Natural TIMBs exhibit great diversity in this element, ranging from “circular” TIMBs to elongated or “ovoid” TIMBs, as well as other nonsymmetrical arrangements in between. Indeed, the eponymous TIMB enzyme, triosephosphate isomerase, exhibits an ovoid β barrel curvature. In sTIM11 and all subsequent variants, the β barrel is circular and has limited structural designability due to its central relationship to the fold architecture, determining in part both the lengths and orientations of α helices and connecting loops. This problem is exacerbated by the fact that TIMB stability is heavily dependent on hydrophobic packing and charge interactions in the β barrel [β barrel have made it challenging to create functional variants of the de novo TIMB.However, exploring beyond the principles learned in the design of the original de novo TIMB is important for generating structural variants better equipped for installing function. Several features inherent to the design of the de novo TIMB limit its capacity for supporting functional elements. In natural TIMBs, the rmations , 20; in β barrel , 22. Botβ barrel to adapt new geometry through mutations and sequence redesign have remained futile, suggesting that the principles deduced from the circular TIMB design may not be generalizable to alternative geometries. This is despite significant progress in β barrel design, including an idealized syntax for the TIMB fold and successful “single-wall” de novo β barrel proteins [β barrel shape in TIMB design. Because the β strands play a smaller role in determining TIMB architecture , strategies that induce control over β barrel shape, such as β-bulges and glycine corners, are difficult to effectively transfer from single-wall β barrels to TIMBs. Similarly, the fourfold symmetry of sTIM11 makes it difficult to induce symmetry breaking and ovoid curvature using sequence mutations alone. Taken together, this indicates that defining a new twofold symmetric syntax via de novo design is necessary for control over TIMB β barrel curvature.Thus far, while additions and modifications have been made to the original de novo TIMB , 18, attproteins , 15, 23.β barrel could be altered through de novo design while also eliminating structural loops and improving stability. To this end, we report the computational design and experimental validation of a de novo ovoid TIMB protein, with a completely novel syntax and sequence. The designed protein exhibits an elongated β barrel architecture with loops which are not structurally involved and a more developed hydrophobic core. In accordance with our design goals, the protein folds solubly and is highly stable. The crystal structure is in close agreement with the design model, demonstrating the effectiveness of our design hypotheses. The extended shape of the ovoid TIMB allows the installation of more diverse residue identities and combinations, facilitating several tests of stability. The interior of the barrel is highly robust to mutation, supporting buried polar and charged residues and even ablation to polyalanine. Our de novo designed ovoid TIMB represents a highly improved version of the original TIMB with many more potential avenues for downstream functionalization.Here, we wanted to investigate whether the curvature and eccentricity of the central β1-α1-β2-α2 unit, each successive secondary structure element was sampled individually: the β3 unit was sampled with β1-α1-β2-α2 held constant, and a promising design was selected, then the α3 unit was sampled with β1-α1-β2-α2-β3 held constant, and a promising design was selected, and so on with β4. Promising designs were selected by manual inspection of conformity to structural ideality . To sample α4, we sampled the α4 helix jointly with the entire second half of the structure using the -repeat_structure option of the RosettaRemodel, conditioned on all previous elements.  In this step, promising designs were selected by first ranking all designs by α/β barrel probability under a fold-classification model  [RosettaRemodel  was usedesidues)  and thenβ barrel curvature. Once the backbone architecture was stabilized, we refined the sequence using three rounds of an “iterative enrichment” protocol [β barrel were enforced to maintain structural fidelity. Example Rosetta flags/options and Remodel blueprints for sequence design are provided in Fig. ab initio structure prediction, for each initial design, 3-mer and 9-mer fragments were picked with Rosetta and 25,000 ab initio prediction trajectories were initiated and run with the AbInitioRelax protocol [With ideal backbones identified, de novo sequences were designed by interleaving rounds of side chain and rotamer sampling with gradient-based Rosetta energy minimization. First, automated design was conducted with the relax/minimization step constrained to starting coordinates, to prevent changes to the protocol . Each reprotocol .Genes encoding the designed sequences and a N-terminal 6xHis tag were ordered from Integrated DNA Technologies and Twist Biosciences and cloned into the pET24a expression vector using Gibson assembly. Resulting plasmids were sequenced to confirm fidelity to the design and transformed into the E. coli BL21 (DE3) strain. Single colonies were picked and used to inoculate 5 mL starter cultures, which were grown to saturation and used to inoculate 0.5 L or 1 L main cultures. Both starter and main cultures were grown using 2xYT media  with 30 mg/L final concentration kanamycin. Main cultures were induced in late log phase with 0.5 mM final concentration IPTG, and the protein was expressed overnight at 16°C.g for 30 min. The supernatant was separated from the pellet and loaded on to Ni-NTA beads (Thermo Scientific) in gravity columns at room temperatures, washed with phosphate-buffered saline  with 50 mM imidazole, and eluted with stepwise 1 mL fractions of PBS with 0.5 M imidazole. Protein expression was assessed by SDS-PAGE and Coomassie SafeBlue staining (Thermo Scientific) or absorbance on a gel reader (Bio-Rad). Fractions containing protein of interest were then pooled and concentrated on Amicon Ultra-4 10000 Da MWCO centrifugal filters. This was followed by fast protein liquid chromatography on an AKTA Purifier system (GE Life Sciences) using a 1 mL HiTrap Q HP anion exchange column (GE Life Sciences) and/or a Superdex 75 10/300 GL size exclusion gel filtration column (GE Life Sciences). All proteins underwent immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC); only protein preparations which were used for crystallography underwent anion exchange, which was after IMAC and before SEC. For anion exchange, the sample was injected in 0.5× PBS with 250 mM imidazole and eluted in a linear gradient from 50 mM to 800 mM NaCl over 15 column volumes.The cells were pelleted and resuspended in 30 mL of 50 mM Tris, pH 7.5, and frozen at -80°C and then thawed and lysed at room temperature with 0.6 mL 600 mg/mL lysozyme , 0.6 mL 50 mM PMSF  (Roche), and 4.2 mL 4 M NaCl , followed by sonication or microfluidization on ice. Cell debris was then removed by centrifugation at 20,000 Circular dichroism (CD) spectra were collected on a Jasco J-815 CD spectrometer. Protein in PBS solution (Santa Cruz Biotechnology) was diluted to approximately 0.2 mg/mL and placed in a cuvette with a pathlength of 1 mm. The CD signal was measured at 20°C at each unit wavelength from 200 to 260 nm at 1 nm/s and a D.I.T. of 1 s. A spectrum with a blank sample of pure buffer was taken before taking the protein spectra to measure the background signal; if significant background was measured, the cuvette was cleaned by heating and washing with Contrad-10 detergent. For thermal denaturation, the sample was then heated from 20°C to 95°C at a rate of 1°C/min, measuring the CD signal at each degree change. A spectrum was measured after reaching 95°C and then another time after allowing the sample to cool to 20°C again. For chemical denaturation, a cuvette with a pathlength of 1 cm was used. A constant volume of purified protein in PBS was mixed with varying amounts of PBS and denaturing buffer  to give a range of denaturant concentrations and held at room temperature. GdmCl concentration was checked by measuring refractive index with a refractometer. After two days, we observed no change in CD signal compared to two hours after adding denaturant, and the signal at 222 nm after two days was used to plot the unfolding transition curve.μL was mixed with an equal volume of crystallization buffer using the Morpheus screen. Crystals were grown using the sitting drop vapor in an incubator at 12°C. Crystallization experiments were set up using a Douglas Oryx8 Nanodrop dispensing robot . Within one week, many crystals appeared from several different crystallization conditions. We set up a focused screen centered on Morpheus well A10 where the best crystals seemed to appear . Crystals were harvested and cryocooled under liquid N2. In general, crystals harvested even from a single crystallization condition showed a huge variation in X-ray diffracting power, and therefore, a large number were screened for initial data quality assessment. Then, a full dataset collection was performed. Data collection to a minimum Bragg spacing of 1.92 Å was performed at 100 K using Stanford Synchrotron Radiation Lightsource (SSRL) beamline 9-2  [2(H2O)6, 0.03 M CaCl2(H2O)2, 0.1 M Tris/Bicine, 10% PEG-8000, and 20% ethylene glycol at pH 8.5. The structure was solved by the molecular replacement method with Phaser  using the OT3 design model as the search model, and the search was carried over the orthorhombic P222 crystal class to fix screw translations. The crystal belonged to the space group P 21 21 21 and contained one polypeptide chain per asymmetric unit. Residues 1-228 were unambiguously traced in the electron density maps. Throughout refinement with PHENIX [Purified OT3 protein was concentrated to 7 mg/ml in 20 mM Tris, 150 mM NaCl, pH 8.0, and 0.15 CA, USA) . Diffrach PHENIX  manual ah PHENIX , trimminh PHENIX , 32. Hydh PHENIX , and pach PHENIX , 35.β barrel curvature. Such a TIMB would ideally exhibit the maximum twofold symmetry while adhering to universal TIMB structural parameters, for example, the number of strands  residues at the minor faces of NanC. Additionally, the flattened major face appears to correlate with the presence of the longest β strands in the structure. We recognize that this unique feature has not been described previously in the literature as a topologically derived mechanism to control β barrel geometry [β strands. A post facto inspection of native TIMB structures revealed a potential agreement with the long-strand scheme. This second design hypothesis, in which we enrich for longer β strands along the major or elongated axis of the barrel, guided our modification of the syntax from that of the earlier circular de novo TIMB ; we retained the original loop in design OT6 and the original “flipped strand” sequence (but included the new loop) in design OT5. We made other mutations to fine-tune solvent-exposed positions and hydrophobic packing, yielding designs OT2-4 , indicating that the designed twofold symmetry is self-consistent and viable  and the design model and disagreement between the seventh helix (α7) and the design model  and α7 are in agreement with the design model. However, residues from D178 to E182 adopt a different conformation from the design, likely driven by the packing of I179 into a void space near the eighth β-α turn  compared to sTIM11 . These trends mirror those found in the DeNovoTIMs, which also exhibit consolidated clusters, increased buried contacts and area, and increased stability relative to sTIM11  as well as packing quality , limiting the space of possible packing solutions. This is further supported by the observation that these gaps are reduced but not eliminated in the DeNovoTIMs  in Since many functional sites in proteins contain polar and charged residues, and often in solvent-occluded pockets, the ability to introduce similar residue identities and pockets into designed proteins without causing excessive destabilization or unfolding is important for designing functional proteins. The high stability of the OT proteins led us to investigate the robustness of these scaffolds to destabilizing mutations, which might provide information on their viability as scaffolds for functional design. We designed two sets of mutants: one with buried polar or charged residues in the core and one ablating different regions of hydrophobic packing by mutating them to alanine. Since functional mutations are often destabilizing , 48, ourFor the polar/charged mutations, we mutated either one or two core residues to one of lysine, glutamate, or serine. PV1 (Polar Variant 1) has an I60K mutation with a sterically compensating I174A mutation in the same position on the opposite repeat subunit; PV2 has an I174E mutation; and PV3 represents the combination of these two (I60K and I174E) and is modeled as a buried salt bridge , we mutated different “layers” of the hydrophobic core to alanine to simulate the effect of carving a binding pocket out of the core. Each layer is formed by alternating strands of the β barrel . Since tβ barrel can also be sufficient for high thermal stability. Furthermore, we expect these mutations to be far more drastic than typical mutations made to install functional motifs such as active sites, which may involve ablating only a few hydrophobic packing residues and installing only partially occluded ionizable residues rather than the mutations we have tested here.We expressed and purified these proteins and found that all but HV1 purified as soluble monomers. HV1 eluted at an earlier volume during size exclusion chromatography and appeared to purify in a dimeric form Fig. . We measβ barrel in TIMBs, we have increased the accessible structural space of the original de novo TIMB and developed it into a powerful scaffold for various protein engineering and design efforts. The computational “flexibility” of the barrel curvature observed in design suggests that our new syntax could be used to explore differing degrees of eccentricity and enable fine control over TIMB curvature. In comparison to earlier de novo protein design studies, where large numbers of designs were tested with a success rate ranging from 25 to 40 percent, our high rate of soluble, stable expression (100 percent) among only a small set of experimentally tested designs suggests that our understanding of protein design has matured. However, as evidenced by the reduced yield of some designs, further work is needed to understand the dynamic process of protein folding and how sequence determines the traversal of the folding energy landscape.This work illustrates the potential of deriving simple structural hypotheses from studying natural proteins that can be implemented through de novo protein design. By uncovering design principles behind the curvature of the core Our work suggests ways in which de novo design can expand beyond the natural framework of evolution for generating new proteins. In natural TIMB proteins, stability appears to be driven by hydrophobic packing in the core region . Howevera modulation, and other effects. The regularly placed and oriented β barrel positions in the de novo TIMB, which our results suggest may be able to assume any combination of amino acid identities, present a unique and powerful system in which to design these hydrogen bond networks–almost like a molecular “breadboard.” We anticipate that this system will be a useful scaffold in which many different functional proteins can be designed.The ability to support one or more polar amino acid at different positions in the core of the protein is important for many different functional applications. Hydrogen bond networks (buried and unburied) are important in molecular recognition and enzyme catalysis, conferring increased affinity, interaction specificity, charge stabilization, pK


To compare construct stability of cement augmented sacroiliac screws using two different cementation sites in a biomechanical fragility fracture model of the pelvis.A fracture model with an incomplete fracture of the sacral ala and complete fracture of the anterior pelvic ring mimicking a FFP IIB fragility fracture of the pelvis was established in five fresh frozen human cadaveric pelvises. Sacral fracture stabilization was achieved with bilateral 7.3 mm fully threaded sacroiliac screws. Cement augmentation was performed at the tip of the screw  on one side, and at the midshaft of the screw  on the contralateral side. Biomechanical testing was conducted separately on both sides comprising cyclic loading of axial forces transferred through the tested hemipelvis from L5 to the ipsilateral acetabulum. Combined angular displacement in flexion and internal rotation (“gap angle”), angular displacement of the ilium in relation to the screw (“screw tilt ilium”), and screw tip cutout were evaluated.p < 0.001), and for “screw tilt ilium” , respectively. No significant difference was indicated for screw tip cutout between the two groups .Relative interfragmentary movements were associated with significantly higher values in group A versus group B for “gap angle” (2.4° vs. 1.4°; The present study demonstrated less fragment and screw displacements in a FFP IIB fracture model under physiologic cyclic loading by cement augmentation of sacroiliac screws at the level of the lateral mass compared to the center of vertebral body of S1. In older adults fractures of the pelvis are mainly caused by physiological loads during activities of daily living, induced by the patient’s own body weight or by low-energy trauma, in case of an extreme reduction in bone mass (e.g. severe osteoporosis) –4. MoreoOnce osteoporotic pelvic fractures have occurred, the destabilized pelvis causes a sudden onset of immobilizing pain. The loss of mobility determines the length of hospital stay and the discharge type; patients with pelvic fractures are representative for long hospitalization periods . The amoDecision making for treatment is guided by the inability of the pelvic ring to withstand physiological loads without displacement as determined by the degree of instability of various fracture types , 12. TheAll pelvises were from donors who bequeathed their corpses to Science Care  for use in medical science during their lifetime. Written consent is available. No local or national ethical approval was necessary. All experiments were carried out under the relevant guidelines and regulations.n = 5, three female; age 82 ± 9 years (mean  ± standard deviation), range 75–98 years) of donors with no history of skeletal disease were considered for biomechanical testing. Computed tomography (CT) scans  were used to rule out any differences between specimens in (i) volumetric bone mineral density (BMD) of the sacrum at the level of S1 by a phantom , (ii) presence of pre-existing deformations, fractures, neoplasms or in (iii) degenerative changes in the sacroiliac joint (as previously published [Fresh-frozen ( – 20 °C) human cadaveric pelvic specimens (ublished ).In all specimens, both sides of the sacrum were used to directly compare two operative techniques in the same bone quality. The left and right anatomical sites of each pelvis were randomly assigned in alternating manner for sacroiliac screw instrumentation with additional bone cement augmentation of either the screw tip , or the midshaft region . The left hemipelvis of each specimen was instrumented and tested first, keeping the contralateral right side intact. A total of three specimens were assigned for screw tip augmentation (group A), whereas two specimens were assigned for midshaft augmentation of their left hemipelvis (group B). After biomechanical testing of the left hemipelvis, the procedures were repeated for contralateral right sides after instrumentation with a sacroiliac screw including the complementary augmentation technique. Consequently, each group consisted of five specimens.Prior to preparation and biomechanical testing, the specimens were thawed at room temperature for 24 h. The soft tissue was completely dissected leaving only the bony pelvic structures with anterior and posterior sacroiliac, sacrospinosus, sacrotuberosus, and iliolumbar ligaments intact. Whereas the lumbar spine was dissected between L4 and L5 leaving L5 in continuity with the pelvis, the femora were completely removed. To generate a FFP IIB fracture with an “anterior ring disruption and posterior undisplaced sacral crush injury” a stable vertical paraforaminal sacral fracture was created in the lateral mass of the sacrum (zone 1 according to Denis classification ) anterioFully threaded self-tapping stainless steel 7.3 mm cannulated sacro-iliac (SI) screws  were used in this study, prepared for augmentation by means of two perforations measuring 1.6 mm in diameter and having an angular offset of 180° to each other. The perforations were custom drilled either at 7 mm and 12 mm distance from the screw tip for the screw tip augmentation (group A), or at 35 mm and 40 mm distance from the screw head for the midshaft augmentation (group B). The site for the perforations were defined based on preoperative CT scan measurements to identify the distance of the external cortex of the ilium at the screw entry point and the sacral lateral mass, which was found consistent and independent from individual specimens. The screw length was selected individually from these scans for each site separately. Due to the bi-lateral test design, care was taken that the final position of the screw tip did not cross the midline to avoid screw interdigitation or cement transgression to the contralateral side during the second test of the right, contralateral side.Sacroiliac screws were placed using the standard technique previously reported in detail , 28. In The L5 vertebral body was embedded in polymethylmethacrylate  cylindrical form, which served as machine anchorage point. The bone-PMMA fixation was enhanced via three 5.0 mm screws. Finally, optical marker sets were attached to the ilium, the sacrum, and the screw for optical motion tracking. Biomechanical testing was performed on a servo-hydraulic material testing system  equipped with a 5 kN load cell. For that purpose, the specimens were oriented and mounted to the machine in simulated upright standing position Fig. . The L5 Machine data in terms of axial displacement (mm) and axial load (N) were recorded from the controllers at 128 Hz. Initial stiffness was calculated from the ascending slope of the load–displacement curve of the quasistatic test ramp within a load range 40–80 N. Two optical cameras  continuously recorded the marker positions at 20 Hz for motion tracking, operating at resolution of 12 megapixel and maximum acceptance error of 0.004 mm . Based oStatistical analysis among the parameters of interest was performed using SPSS software . Mean and standard error of the mean (SEM) were calculated for each parameter of interest. The outcome measures were pooled over the five investigated time points for each parameter of interest and treatment group separately and reported in terms of median values and 95% confidence intervals (CI). Based on these pooled data sets, significant differences between the two treatments were assessed with Wilcoxon Signed-Rank tests, accounting for non-parametric data distribution. In addition, the reproducibility of each technique was assessed by pooling the SEM's of all investigated outcome measures—gap angle, screw tilt ilium, and screw tip cutout—for each group separately, and by statistically comparing them with the Wilcoxon Signed-Rank test. All p values < 0.05 were considered significant.3) or the midshaft (81.1 ± 15.6 mgHA/cm3) augmented, p = 0.267. Screw augmentation was associated with cement leakage into the first neuroforamen following screw tip augmentation (group A) in one case and in all cases in the fracture gap but not into the true pelvis in group B. The initial axial construct stiffness was comparable for screw tip (37.1 ± 7.7 N/mm) or midshaft (39.5 ± 7.1 N/mm) augmentation, p = 0.729.Morphometrically, BMD in the massa lateralis was homogeneously distributed between the sites having the screw tip (90.7 ± 12.5 mgHA/cmp < 0.001). Similarly, the pooled outcome measure “screw tilt” was associated with significantly higher values following instrumentation of group A  versus group B . However, for “screw tip cutout” no significant difference was obtained between group A  and group B . Finally, comparison of the reproducibility revealed significantly higher pooled SD's for group A  versus group B .All outcome measures analyzed over the five time points after 10,000, 20,000, 30,000, 40,000, and 50,000 cycles are summarized in Table Pelvic (fragility) fractures resulting from low-energy trauma are increasingly frequently observed in patients older than 60 years of age . MoreoveFragility fractures of the pelvis with moderate instability (FFP IIB) account for the majority of these injuries and surgical treatment with sacroiliac screws is the clinical practice. However, failure rates of sacroiliac screws occurred in 14% to 17% of patients , 38. AccTo our knowledge, the site for cement augmentation of sacroiliac screws in FFP IIB fractures providing the highest construct stability has not been defined clearly yet. We hypothesized that cement augmentation of sacroiliac screws at the weakest part  enhances construct stability compared to current practice (body of S1). The rationale for that hypothesis and the presented study is given by previous consistent findings as follows: The lowest bone quality was noted in the lateral portions of S1 which contain yellow marrow and an alar void, associated with minimal regional bone density , 39–41. The presented biomechanical analysis with cyclic loading demonstrates that augmentation of the lateral mass compared to the one in the center of the vertebral body of S1 results in less angular displacement between the sacral fragments medial and lateral to the fracture, as well as between the screw and the pelvic bone itself. In summary, the screws with a cement-augmented lateral mass provided a construct being more stable under cyclic loading.The results confirm the hypothesis of the study, as the cementation at the weakest part of the sacrum enhanced stability. This finding might parallel the noted so-called “windshield-wiper effect” in reconstruction surgery of the anterior cruciate ligament (ACL) as follows: Transverse motion of the ACL graft in the proximal tibial bone tunnel results in widening of the osseous tunnel at risk for failure of ACL reconstruction surgery , 45. AccAnother explanation might be that the osteosynthesis in these pathologic fractures acts comparably with a compound osteosynthesis . Using tA limitation of the study might be concerns about the clinical application of cement injection at the sacral lateral mass using fully threaded screws with the potential risk for cement leakage in the fracture gap and/or no compression at the fracture site preventing fracture healing. However, the following arguments might abolish these concerns: (1) Cement leakages are observed (e.g. as “retrograde” leakage) regardless of the site of cement application , (2) surThe strengths of the study are: (1) we used fresh-frozen human cadavers to reflect the clinical situation rather than synthetic bone models without any ligaments attached to the bone, (2) to ensure homogeneity and comparability of these specimens clear inclusion criteria were defined and analyzed using CT scans; including a phantom to detect differences in bone mineral density, (3) to allow for direct comparison between the two groups, both techniques were tested within the same bone quality within one cadaver, (4) a FFP IIB fracture was tested by generating “a posterior undisplaced sacral crush injury with an anterior ring disruption” to reflect the most frequent fracture pattern, (5) for fixation, we used fully threaded screws as these provided more stability in osteoporotic bone by improved anchorage and by fixation within the cortical bone of the sacroiliac joint; accordingly, we obtained a high fixation strenght at baseline before application of cement, (6) all osteosynthetic constructs were tested under cyclic loading to reflect the physiological loading as much as possible with shear stresses concentrated at the sacral lateral mass.The presented study demonstrates less fragment and screw displacement in a FFP IIB fracture model under physiologic, cyclic loading by cement augmentation of sacroiliac screws at the level of the lateral mass compared to the center of vertebral body of S1 for the first time. The study results provide the rationale to assess the potential benefit of this technique in a clinical setting.


Extreme waves are intense and unexpected wavepackets ubiquitous in complex systems. In optics, these rogue waves are promising as robust and noise-resistant beams for probing and manipulating the underlying material. Localizing large optical power is crucial especially in biomedical systems, where, however, extremely intense beams have not yet been observed. We here discover that tumor-cell spheroids manifest optical rogue waves when illuminated by randomly modulated laser beams. The intensity of light transmitted through bio-printed three-dimensional tumor models follows a signature Weibull statistical distribution, where extreme events correspond to spatially-localized optical modes propagating within the cell network. Experiments varying the input beam power and size indicate that the rogue waves have a nonlinear origin. We show that these nonlinear optical filaments form high-transmission channels with enhanced transmission. They deliver large optical power through the tumor spheroid, and can be exploited to achieve a local temperature increase controlled by the input wave shape. Our findings shed light on optical propagation in biological aggregates and demonstrate how nonlinear extreme event formation allows light concentration in deep tissues, paving the way to using rogue waves in biomedical applications, such as light-activated therapies. Rouge waves have been observed in a number of complex systems, but not in biological structures. Here, the authors report the observation of optical rouge waves manifesting in tumor-cell spheroids when illuminated by randomly modulated laser beams. Generally associated with unpredictable occurrences and catastrophes, RWs can also represent a route to transport and to localize energy in disordered systems. Their formation is supported by a variety of linear and nonlinear physical processes13, which encompass chaos14, turbulence16, wave scattering18 and topology19, and whose interplay and unification is actively debated21. Their appearance is strongly dependent on the microscopic properties of the system, a fact that stimulates a wide-ranging research effort aimed at taming and exploiting RWs in a variety of applications, including neuromorphic computing22.Many complex systems, including oceans23, features that make the concentration of visible light in deep tissues a critical problem24. In this context, RWs could be exploited as noise-resistant localized wavepackets that act as a source of intense light spots to probe, activate, and manipulate biochemical content. However, their formation in biological structures has not been reported yet.Biological systems are complexity-driven, forming a potentially rich and hereto unexplored field for optical extreme waves. In particular, living biomatter is an optically thick, agitated, absorbing, and highly scattering environmentHere, we observe optical extreme waves that form in a dense spheroid of living tumor cells. The RWs propagate through the bioprinted three-dimensional tumor model (3DTM) for several millimeters, delivering extreme optical intensity and causing the transmitted light to have heavy-tailed statistics. The probability density of the intensity is found to be well modeled by a Weibull distribution with signature scale and shape parameters. We identify extreme events with spatially localized modes of enlarged transverse size. Importantly, the RWs exhibit complex dynamics when the input power is varied, which indicates the anomalous statistics originates from nonlinear effects. Measurements of the transmission matrix show these intense filaments constitute addressable channels with super transmittance. We analyze the RWs to achieve controllable large local temperature increases within the tumor spheroid by shaping the input excitation, thus enabling their possible application in photo-thermal therapy. These findings unveil a phenomenon able to transport massive optical power through strongly scattering biological matter.25 (see Methods—Tumor spheroid growth). Tumor spheroids, namely 3DTMs, are widely used as cancer surrogates to study cell proliferation and drug response, and an increasing interest is currently emerging on their physics29. 3DTMs are formed by cells that are densely packed into an elastic network permeated by intercellular fluid. Unlike biological suspensions where cells are free to move under the action of light32, tumor spheroids optically behave as a complex multiparticle assembly that strongly scatters visible radiation33.We investigate laser light propagation in millimeter-sized tumor spheroids fabricated via bioprinting from human pancreatic cellsλ = 532 nm. The input beam is richer in wavevectors than a collimated laser. We use a phase-only spatial light modulator (SLM) to shape randomly the wavefront of the optical field incoming on the tumor spheroid. Figure 34. At the operating wavelength, the measured absorption coefficient of the bioprinted spheroids without culture is ≈0.25 cm−1, comparable with similar pancreatic tissues that are strongly scattering35. We hence expect the wave propagation to be described by a random Gaussian process for the field amplitude , which results in the Rayleigh statistics36. Depending on either the input beam power, size, and shape, two distinct scenarios are observed. At low input power (P < 15 mW), the speckle intensity distribution contains spots following RWT . Exceeding this optimal power region, the statistics returns to approach RWT. At higher power (P > 100 mW), the output becomes unstable (non-stationary). Interestingly, when strong RWs occur, the scale and shape parameters have specific power-independent values, λRW = 0.16 ± 0.07 and γRW = 0.45 ± 0.04  that corresponds to the optimal power region observed in Fig. 37. Weakening and suppression of RWs at very high power is also a sign of their nonlinearity. In fact, in nonlinear disordered media, instability induces fast temporal dynamics and hence wave randomization38. The RW phenomenon is thus profoundly different from the long-tail statistics reported for biological tissues in optical coherence tomography39 and diffuse reflectance40, where the anomalous PDF comes from the complex shape of the biological scatterers and the multi-scale nature of the tissue structure. The observed phenomenological picture suggests the presence of optically-induced thermal effects and structural deformation of the cell network. It is known that cell nuclei regulate their shape and volume in a highly temperature-sensitive manner, and can exhibit volume transitions for laser-induced temperature variations of just a few degrees41. Observations in Fig. 29.To understand the RWs origin, we capture a RW generated at high power and observe its evolution as the laser energy is gradually decreased. The behavior, reported in Fig. 42. We first identify each anomalous wave by using the oceanographic criterion extended to optical data43, which defines the RW threshold as IRW > 2IS, where the significant intensity IS is the mean intensity of the highest third of events. Figure IRW = 5.7. We evaluate the Euler Characteristics (EC) for the amplitude field Uηh, with Uηh the excursion set of η of level h. In fact, for high threshold h, EC(Uηh) gives the probability that a wave peak exceeds h44. The observed EC is compared with the EC expected for a linear (Gaussian) and nonlinear (Tayfun) wave surface . The agreement with the nonlinear wave model indicates that nonlinear interaction intervenes in RW generation45.The nonlinear nature of the extreme waves is confirmed by analyzing the spatial extremes of the wave field in analogy with studies on oceanic sea statesd between the 3DTM and the input focal plane. Given an input field having a discrete wavevector spectrum kj determined by phase modulation, via the optical geometry we alter the typical size of the modes impinging on the 3DTM. When the sample is placed after the focal region, modes of a wider transverse size excite the 3DTM. RWs appear in this condition , while they are suppressed in the weak coupling case (d = 0), with the PDF that closely follows RWT . In Fig. 48. Generally, this approach is performed to study the transmission properties of well-controlled scattering systems, such as thin diffusers49 and two-dimensional (2D) disordered waveguides50, but still holds for nonlinear random media51. Here, importantly, we are applying the eigenchannels method to a macroscopic biophysical system.To understand to what extent we can control RWs in 3DTMs, we measure the transmission matrix (TM) and analyze the transmission eigenchannelsP = 60 mW is reported in Fig. t, with complex coefficients tnm, for N = 64 (M = 900) input (output) modes, for weak and strong coupling. We consider the transmission eigenvalues τn of the matrix tt†, which coincides with the squared values of the singular values of the TM, τn that correspond to high-transmission modes. The measured τn are reported in Fig. kRW exciting RWs. Similar peaks are found for the 2nd and 3rd eingenchannel  emerge (“Methods—RW-induced temperature increase”). Figure T produced by each randomly modulated input. The most intense RWs gives temperature spikes exceeding 10 °C. The input phase mask that generates an extreme wave , which inhibits cell replication. We observe no systematic differences in the intensity statistics and RW properties. This suggest that RWs in tumor cells are robust to the presence of drugs.To conclude, we have observed that macroscopic tumor spheroids, bioprinted from human pancreatic cells, support optical extreme waves. This demonstrates nonlinear optical rogue events in biophysical structures, opening a new scenario for the application of extreme waves. Our observations reveal that a Weibull distribution characterizes laser transmission in tumor models, and demonstrate intense, localized, naturally-occurring light transport over millimeters through dense cell aggregates. These findings may introduce new optical approaches for biomedical applications. Among these, the exciting possibility of treating cancer using tsunamis of light.λ = 532 nm and tunable output power up to 2W (LaserQuantum Ventus 532), linearly polarized along the experimental plane (x-axis), is expanded and impinges on a reflective phase-only SLM . The SLM active area is divided into N = 64 squared input modes by grouping 120 × 120 pixels, with each mode having a phase ϕj. The available phase levels are 210, linearly distributed within . The phase-modulated beam is spatially filtered and focused by a plano-convex lens  into a 3DTM sample positioned along the beam path (z-axis) utilizing a sample holder equipped with a three-axis translational stage. The input power P is measured close before the sample. The 3DTM is immersed in its fresh culture solution in a 10 mm long optical-quality quartz cuvette. The average diameter of the 3DTM samples is 3 ± 0.2 mm. The spheroid position d with respect to the input focal plane is varied in the 0.5–5 mm range to change the optical coupling. The transmitted intensity on the cuvette output facet (transmission plane) is imaged by an objective lens  on a CMOS camera (Basler a2A1920-160umPR) with 12-bit sensitivity (4096 gray-levels). The entire setup is enclosed in a custom-built incubator kept at the constant temperature of 35 °C and under constant 5° CO2 influx.A picture of the experimental setup is reported in Supplementary Fig. L = 640 transmitted intensity distributions, each for a different randomly-modulated input wave. We refer to this dataset as the outcome of a single experiment. Data are obtained sequentially at 10 Hz sampling rate by loading on the SLM the L phase masks. Every mask is made by blocks with random phases ϕj drawn from a uniform distribution. Within the measured image, we select a 200 × 200 μm2 region of interest (ROI), a size that ensures we analyze a speckle pattern that is homogeneous on a large scale. To position the ROI, we adopt the average position of the intensity center of mass (CM) as the origin of the xy-plane. Variations of the CM for a set of input phase masks are shown in Supplementary Fig. For fixed experimental conditions , we collect We carry out three replicated experiments  for a given setting, showing similar results. Then we change the input power, and the coupling geometry, but keeping the same 3DTM sample. The whole set of experiments is repeated over five different 3DTM samples . This measurement campaign is performed within a day. It has been repeated for five days within a week, each day using a new set of 3DTM samples. No qualitative differences are found between the results of different days. Microscopy of different 3DTM samples shows similar features. Parallel experiments have been performed on a line of 3DTMs treated with chemotherapy (Gemcitabine 100 μm).IRW > 2IS, with IS the mean intensity of the highest third of events. Measured intensity data are normalized to the mean intensity 〈I〉. Data (matrices of size 200 × 200) are processed to find local maxima, which form the series of events used to evaluate IS for that set of experimental parameters. The PDF in Fig. I〉 to remove the incoherent component (background). In this case we count approximately 2700 RWs  = CC − H, being CC the number of connected components and H the holes. It is computed on the binary matrices Uηh in MATLAB. The expected EC for a random wave field (Gaussian) is ECξG = h/σ, with σ the standard deviation of η, and NS the number of waves on the area. The nonlinear EC (Tayfun) is ECμ. For further details see ref. 45. The maps in Fig. I * = 10.In oceanography, RWs are defined as waves whose trough-to-crest height exceeds twice the significant wave height, the mean height of the highest third of waves. In optics, the definition becomes RWs Fig. a. For thtnm connect the amplitude and phase of the optical field between the m-th output and the n-th input mode, L = 10 × N = 640 random phase masks as independent realizations of the input field on N input modes, and we select M = 900 output modes within the camera ROI. Output modes are obtained by binning over a few camera pixels, and they have a size comparable with the spatial extent of a speckle grain (7 ± 1 μm). The TM is reconstructed from the entire set of intensity data using a phase retrieval algorithm55. When we vary only the coupling conditions . For this purpose, 3 × 106 cells/mL were mixed with alginate (Sigma-Aldrich) at 5% w/v on a syringe with a 1:1 ratio. Cells mixed with the hydrogel were loaded on a bioprinting cartridge. Spheroid droplets were released on a 96-well round bottom (Corning) previously filled with 100 μL of 2% CaCl2 to induce crosslink of alginate. Droplets were incubated for 5 min at room temperature (25 °C), then CaCl2 was replaced with fresh complete culture medium. Spheroid droplets were then incubated at 37 °C, 5% CO2 for further treatments.Three-dimensional tumor models (3DTMs) have been prepared from human pancreatic cells. The cancer cell line PANC-1 was purchased from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum , 2% penicillin-streptomycin and 2% L-glutamine (Sigma-Aldrich). Cells were cultivated in T75 flasks and kept at 37 °C, 5% CO56. Optical and fluorescence microscopy were carried out by using Cytation 3 Cell Imaging Multi-Mode Reader (BioTek). We collected bright-field data in a time span of 30 days and processed them via the Fiji software. The growth of spheroids was monitored over time in terms of radius and cell density. Representative confocal microscopy images were obtained with an inverted microscope . For these measurements, bioprinted 3DTMs were stained with Calcein-AM at a final concentration of 10 μM, and incubated at 37∘ C, 5% CO2 for 15 min. Microscopy results are reported in Supplementary Fig. We monitored the growth of the bioprinted spheroids by a specifically-developed microscopy analysis tool54. This corresponds to a typical biomedical situation where hot NPs are injected into a small cavity inside a massive tissue and optically pumped. When the pumping beam is modeled as a uniform optical filament with spot area Ab, radius Rb, optical lenght lb, and total optical intensity I0 (W/cm2), the local temperature change produced by the laser beam isnNP is the average density of NPs of radius RNP. cw a constant that depends on NP plasmonic response and water thermal conductivity54. To evaluate Eq.  and laser illuminated. NPs absorb light and generate heat dissipated into the environment, giving a macroscopic temperature increase. The effect is analyzed via a thermal transfer model that neglects convection and assumes uniform background heat conductivityFurther information on research design is available in the Supplementary InformationPeer review fileReporting Summary


Cellular protrusions are fundamental structures for a wide variety of cellular behaviors, such as cell migration, cell–cell interaction, and signal reception. Visualization of cellular protrusions in living cells can be achieved by labeling of cytoskeletal actin with genetically encoded fluorescent probes. Here, we describe a detailed experimental procedure to visualize cellular protrusions in medaka embryos, which consists of the following steps: preparation of Actin-Chromobody-GFP and α-bungarotoxin mRNAs for actin labeling and immobilization of the embryo, respectively; microinjection of the mRNAs into embryos in a mosaic fashion to sparsely label individual cells; removal of the hard chorion, which hampers observation; and visualization of cellular protrusions in the embryo with a confocal microscope. Overall, our protocol provides a simple method to reveal cellular protrusions in vivo by confocal microscopy. Cellular protrusions are fundamental structures that play pivotal roles in various aspects of developmental biology such as cell migration, cell-cell communication, and signal reception . These pOryzias latipes) is a freshwater fish with beneficial features for developmental biology, similar to zebrafish; daily spawning and external development of transparent embryos as well as a small genome size enable investigation of cellular events taking place during embryogenesis  d-rR strain. Can be ordered from NBRP, JapanMedaka (https://shigen.nig.ac.jp/medaka/strainDetailAction.do?quickSearch=true&strainId=5666). Adult medaka should be kept at 26–28 °C in a room with a 14:10 h light/dark cycle to promote spawning(AC-TagGFP2 (pMTB--TagGFP2 . Availabα-bungarotoxin (Addgene Plasmid #69542) (pMTB- #69542) AvaI . 10× CutSmart buffer is included in the productEcoRV . 10× CutSmart buffer is included in the product0.5% phenol red in DPBS Penicillin-streptomycin solution https://shigen.nig.ac.jp/medaka/strain/hatchingEnzyme.jsp). You can also make hatching enzyme by yourself [see pp. 254–255 in Kinoshita et al. (2009)]Hatching enzyme (store at -80 °C). Can be ordered from NBRP, Japan Agarose, low melting point (LMP) Wizard SV gel and PCR clean-up system mMESSAGE mMACHINE SP6 transcription kit . 2× NTP/CAP, reaction buffer, and enzyme mix are included in the kitRNeasy mini kit Fish-scooping net with fine mesh9 cm plastic dish, Asnol Multidish, 4 wells, Nunc Glass bottom dish https://www.shapeways.com/); choose “Smoothest Fine Detail Plastic” for the materialInjection mold. You need to make the mold by yourself; to design it, see pp. 279 in Kinoshita et al. (2009). You can order a plastic mold using the design through the Shapeways website Microcapillary tip Forceps Pasteur pipette Yamamoto’s Ringer’s solution (store at 4 °C) (see Recipes). Dilute 50 mL of 10× Yamamoto’s Ringer’s solution in 450 mL of water, autoclave it, and add 5 mL of penicillin-streptomycin solutionHatching buffer (store at room temperature) (see Recipes). Dilute 100× hatching buffer in waterHeating dry bath incubator Heating chamber Microvolume spectrophotometer Capillary puller Microinjector Manipulator Magnet stand for the manipulator Iron plate for the magnet stand Incubator Stereomicroscope External light source for fluorescence excitation Inverted confocal microscope 25× water immersion objective with a long working distance 40× water immersion objective with a long working distance https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html)Zen  (Fiji (e/fiji/) mRNA synthesismRNA is synthesized in vitro from a plasmid containing both a promoter sequence for SP6/T7/T3 RNA polymerase and an SV40-derived polyA signal. Here, we use the pMTB vector, which was originally designed for zebrafish transgenesis as well as in vitro mRNA synthesis . pCS2+ iAC-TagGFP2 and pMTB-α-bungarotoxin plasmids with restriction enzymes. To do so, mix the following solutions and incubate at 37 °C for 2 h:Linearize the pMTB-AC-TagGFP2 or pMTB-α-bungarotoxin), 2 μgPlasmid , 1 μLRestriction enzyme (EcoRV for pMTB-Water up to 20 μLRun electrophoresis with 1 μL of the reaction solution to verify that the plasmids have been fully linearized.Purify the linearized templates with Wizard SV gel and PCR clean-up system kit as per the manufacturer’s instructions. Elute the DNA in 25 μL of water.Assess the concentration of the eluents with a NanoDrop spectrometer. The concentration should be approximately 100 ng/μL.Synthesize mRNA using the mMESSAGE mMACHINE SP6 transcription kit. After thawing the kit solutions at room temperature (except the RNA polymerase enzyme mix), mix the solutions listed below (from top to bottom) at room temperature and incubate at 37 °C for 2 h:Nuclease-free water up to 10 μL 2× NTP/CAP, 5 μL10× reaction buffer, 2 μLTemplate, 0.5–1 μgEnzyme mix, 1 μLWhile handling RNA, be sure to avoid RNase contamination by wearing gloves.Digest the DNA templates. Add 1 μL of TURBO DNase (included in the kit) and incubate at 37 °C for 15 min.Purify the mRNA with RNeasy mini kit following the manufacturer’s instructions and elute the mRNA in 30 μL of nuclease-free water.280/A260 ratio of purified mRNA is typically approximately 2.0. The quality of mRNA can be further validated by agarose gel electrophoresis, whereby a single band with little smear will appear unless mRNA is degraded or unsuccessfully synthesized. The solution can be stored at -80 °C for months.Assess the concentration of the mRNA eluents with a NanoDrop spectrometer. The concentration should be 100–500 ng/μL. The APreparation of injection toolsInjection gelPour 20 mL of melted 1% agarose dissolved in the hatching buffer into a 9 cm plastic dish and apply the injection mold onto the agarose before it solidifies. Trapping the air bubbles under the mold should be prevented by slowly sliding the mold onto the agarose surface with an angle of 45° .Once the agarose is solidified, remove the mold and pour the hatching buffer on the gel to prevent drying out . The agaInjection needlesstep 1 mode. This configuration provides an ideal needle shape for microinjection into medaka embryos.Pull the glass capillaries using a Narishige capillary puller. Adjust the No. 2 heater adjustment parameter to 57.5 and set the mode selector knob to the Store the needles in a container so that the fragile tips do not touch anything .Microinjection into medaka eggs In the evening of the day before injection, pair adult medaka for mating in fish tanks. Separate females from males using transparent partitioners.In the morning of injection day , remove the partitioner to enable the pairs to start mating. Mating will happen typically within 5–10 min.While waiting for the eggs (approximately 10–30 min), prepare the injection solution by mixing the following ingredients:Actin-Chromobody-TagGFP2 mRNA, 1.5 μgα-bungarotoxin mRNA, 0.25 μg0.5% phenol red, 0.75 μLNuclease-free water up to 10 μLCollect eggs from the female and transfer them into the hatching buffer.Medaka eggs tend to stick together because of long attaching filaments on the chorion. To separate eggs, transfer them into a fish-scooping net and gently roll the eggs with the net between your fingers until the egg clumps disintegrate and all eggs are separated –2C. DuriTransfer the eggs onto the injection gel, which is covered with the hatching buffer, and gently align the eggs within the wells using forceps .Load the prepared glass needle with ~3 μL of the injection solution using a Microloader tip and attach the needle to the injector.Initiate the injector and adjust the injection pressure (Pi) and compensation pressure (Pc) to appropriate levels. During the injection process, the needle should neither take up the embryo medium nor eject the injection solution too fast. Pressure values vary between individual needles, but we typically set Pi and Pc to ~500 and ~150 hPa, respectively.Break the tip of the needle by gently scratching the surface of a chorion, until injection solution slowly leaks out of the tip.When the embryos reach the 4-cell stage , inject Incubate the injected embryos in the hatching buffer at 23–32 °C.DechorionationDechorionation should be performed on the day of injection to maximize the chances of obtaining healthy embryos. Although dechorionation can be done later, digestion of the chorion is more likely to be incomplete, potentially leading to damaging the embryo with partially digested chorion pieces.Dilute the hatching enzyme 1:5–1:20 with 1/2× Yamamoto’s Ringer’s solution. The dilution ratio should be determined based on the enzymatic activity of the enzyme lot; when you use the lot for the first time, we recommend that you begin with a 1:20 dilution and examine how long it takes to digest most of the chorion (see step D3).Transfer embryos to a container with a small bottom area, e.g., a 4-well dish, and remove the culture medium .Apply the diluted hatching enzyme onto the embryos and incubate them at 28 °C until the chorion is mostly digested –3D. The Carefully transfer the embryos to a glass dish filled with Yamamoto’s Ringer’s solution to rinse off the enzyme. Most of the embryos are still surrounded by the thin layer of the chorion, but some may be fully out of it. Transfer all embryos to a new glass dish filled with Yamamoto’s Ringer’s solution. Since physical contact with the bottom surface of a brand-new plastic dish could damage the embryo, keeping the embryos in a glass dish is preferred.Incubate the injected embryos at 23–32 °C until they reach the desired developmental stage. For stage 28, it takes approximately three days at 26 °C .Mounting embryos in agarose for imagingIn this protocol, we focus on embryos from stage 28 onward.When the embryos reach the desired stage, screen for embryos exhibiting GFP fluorescence at the region of interest (ROI), such as somites or epidermis. For the screening, use a fluorescent stereomicroscope and transfer suitable embryos into a new dish filled with Yamamoto’s Ringer’s solution. Using forceps, gently remove the remaining chorion of the selected embryos.Dissolve 1% LMP agarose in hot Yamamoto’s Ringer’s solution and keep it at ~40 °C using a heating incubator.Pour a few drops of the LMP agarose solution to cover the entire glass part of a glass bottom dish and immediately transfer 2–3 embryos into the agarose. Mount the embryos before the agarose solidifies. Orient the embryos with forceps in a way that the ROI is in contact with the glass bottom . This alImaging cellular protrusions using an inverted confocal microscopeUsing an objective with a long working distance is critical for imaging cells located inside the embryo.Start the confocal microscope and the microscope software Zen. Use the 25× water immersion objective and select the Locate tab on the software to allow observation through the eyepieces.Place the dish with the mounted embryos on the microscope stage and find the ROI by visual inspection through the eyepieces. Screen the embryos by their GFP fluorescence at the ROI.Once a good candidate is found, select the Acquisition tab on the software and start imaging the focal plane. Overall, tissue structures can be captured with this magnification.To obtain subcellular resolution, switch to the 40× water immersion objective and acquire images . TimelapAddition of a scale bar to the confocal imageOpen the image in Fiji.Go to Analyze > Tools > Scale bar… and choose from displayed options. A scale bar with the defined length will appear on the image.10× Yamamoto’s Ringer’s solution (store at room temperature)100× Hatching buffer (store at room temperature)


Direct observation on initial electron transfer to CO2 to form surface-bound CO2•− radicals is lacking due to the technical challenges. Here, we use picosecond pulse radiolysis to generate CO2•− via aqueous electron attachment and observe the stabilization processes toward well-defined nanoscale metallic sites. The time-resolved method combined with molecular simulations identifies surface-bound intermediates with characteristic transient absorption bands and distinct kinetics from nanosecond to the second timescale for three typical metallic nanocatalysts: Cu, Au, and Ni. The interfacial interactions are further investigated by varying the important factors, such as catalyst size and the presence of cation in the electrolyte. This work highlights fundamental ultrafast spectroscopy to clarify the critical initial step in the CO2 catalytic reduction mechanism.Time-resolved identification of surface-bound intermediates on metallic nanocatalysts is imperative to develop an accurate understanding of the elementary steps of CO 2•− radicals bound to the surface. Here, the authors use pulse radiolysis to directly observe the stabilization process of CO2•− radicals at nanoscale metallic sites from nanoseconds to seconds.Understanding the activity and selectivity of metal catalysts requires elucidating the dynamics of CO However, the high activation energy associated with the change in orbital hybridization and geometry of CO2 upon the first electron attachment, the generation of CO2 radical anion (CO2•−), makes its reduction a significant challenge. As a result, the successful operation depends heavily on the viable catalysts that can accelerate the reaction. In most electrolytic, photolytic, and radiolytic processes, metal-based catalysts primarily contribute to CO2 transformations6. Active metals can be categorized into several groups based on their product distribution. For example, Ag and Au predominately yield CO, while Ni and Fe exclusively produce H2 with low activity, and Cu has an unusual capacity to synthesize hydrocarbon/multi-carbon products9. These findings raise questions about the importance of reaction intermediates binding at the interface on the efficiency. Although great progress has been made in understanding the underlying surface chemistry, the reaction sequence remains incomplete, partly due to the lack of direct experimental evidence for unraveling the fast dynamics of CO2•− during the initial stages of complex multi-electron processes. On the other hand, advances in synthetic methodology have made well-controlled metallic nanoparticles and nanoclusters available, and these nanostructured materials exhibit better performance in CO2 conversion and lower cost. Strategies such as surface engineering, morphology control, and composition manipulation have been developed to enhance the efficiency of CO2 activation12. In this regard, the characterization of CO2•− coordination complex with metal centers could provide a basic descriptor to establish the structure-property correlation, where any transient state changes with variables of interest at nanoscale spatial resolution could be detected. However, detecting these active sites directly from experiments has been impossible thus far16.The conversion of planet-warming carbon dioxide  attachment has recently been recognized by plasma or radiation-driven catalytic and nanodiamond-assisted photolytic CO2 reduction24. Different catalytic systems have several rate-determining steps (RDS), such as initial electron transfer to CO2 to form surface-bound CO2•− radicals, the first proton-coupled electron transfer, or the reduction of other bound intermediates21. Due to the large structural reorganization of the bound radical anion, the radical anion CO2•− formed by the first electron reduction occurs at very negative potentials. Even though CO2•− formation lacked experimental evidence, many studies suggested that the process is likely to be one of the critical RDS for CO2 reduction15. If the CO2•− radicals stabilization on catalytic surfaces is kinetically non-favorable, the overall activity for CO2 transformation will be low. This hypothesis is further supported by theoretical studies that developed a contemporary model for various *CO2•− coordination structures, including *COOH or *OCOH, uncovering the selective electrocatalytic pathway toward either CO or formate30. In situ surface-enhanced Raman scattering has reported the presence of this surface species on an operating Cu surface, but the dynamic resolution remains unresolved32. For nanocatalysts, operando X-ray absorption and photoelectron spectroscopy are sensitive to surface oxidation evolution and to the accurate active site of metals such as Cu, Pd, Zn, and Sn during CO2 reduction34. Few studies have been dedicated to the initial step of CO2 reduction at a nanoscale catalytic surface, owing to the challenges of forming well-defined nanocatalysts and CO2•− intermediate. Additionally, unlike carbon monoxide, which is frequently used as a mechanistic probe, CO2•− radicals decay rapidly in aqueous solutions, with a short lifetime of microseconds. The existing operando techniques are limited to the resolution of seconds or sub-seconds range, which cannot access the first elementary reactions occurring at nanosecond timescale.The existence of CO2 activation, pulse radiolysis provides a time-resolved prerequisite to insight into the general transient absorption kinetics of CO2•−. Radiolysis is often referred to as “an electrolysis process without electrodes”. The high-energy radiation (X-rays/accelerated e−) with sufficient energy ejects electrons directly from the water. Unlike electrochemical or photochemical reduction, where electrons are provided by the external electric field or photo-induced carriers  separation in semiconductor materials, the radiation-driven approach produces electrons in bulk solutions via water ionization, known as the eaq− with characteristic transient absorption at 715 nm. With the standard potential of −2.87 VSHE, eaq− can easily overcome the first CO2 reduction step barrier35. The picosecond pulse radiolysis coupled with transient absorption spectroscopy  could form eaq− in CO2-saturated aqueous conditions within 7 ps, and eaq− reacts directly and quantitatively with CO2 forming CO2•− radicals with an almost diffusion-controlled rate (8 × 109 M−1 s−1) within 10 ns. Until now, the current in situ/operando spectroscopic measurements on CO2 reduction were performed with limited time resolution in a few studies with subsecond time resolution based on infrared, Raman, MS, and XAS38. Compared to these spectroscopic techniques based on molecular vibration or photoelectron excitation, transient absorption profiles can disclose the electronic transition of surface-bound intermediates on metallic nanocatalysts with the nanosecond time resolution, which allows for the direct observation of the reaction between CO2•− and nanoparticles (NPs) as well as their stability kinetics on the surfaces. The method avoids external potential supported catalysts perturbation and the complexity associated with processes involving photosensitizers or electrodes. Therefore, pulse radiolysis measurements allow us to gain critical insights into the first elementary step of CO2 reduction, such as the intermediate binding and transformation, as well as charge delocalization on nanoscale metallic surfaces. This methodology would set up a model to investigate the factors in operating conditions, such as cations in electrolytes and the nano-size effect of catalysts, which are important but under intense debate in CO2 reduction because the existing in situ spectroscopic measurements often deal with combined signals caused by various possible consequences including solution effects, competing reactions, and surface structure evolution or catalysts oxidation.For initial CO40, we present direct transient spectroscopic evidence of CO2•− radical stabilization process on the surface of nanoscale catalysts, leading to the formation of surface-bound CO2•−. The characteristic absorption spectrum of surface species and density functional theory (DFT) simulations revealed the coordinated structure and interactions between metal clusters and CO2•− radicals. The Tafel analysis and electrochemical catalytic performance with the same NPs used in the pulse radiolysis observations are also performed. As a result, our distinct transient kinetics on various well-defined metal  nanoparticles (NP), which are commonly included in electrocatalysis, provided fundamental insights to revisit the activity and selectivity of metal catalysts in CO2 reduction.Herein, by using pulse radiolysis2 radical formation in CO2-saturated formate aqueous solution in the absence of any catalyst. Immediately following the electron pulse, the typical broad band in the 400–720 nm range corresponds to eaq−. The appearance of a new absorption band below 420 nm on the nanosecond timescale, which is associated with eaq− decay, indicated the rapid formation of CO2•− radicals via eaq− reduction . Meanwhile, 0.1 M formate was used to convert •OH and •H into CO2•− radicals , thereby converting all water radiolysis radicals into CO2•− radicals. Subsequently, CO2•− radicals underwent a recombination reaction  and lived for approximately 10 µs under the experimental conditions  metal NP by an established radiolytic reduction method with surfactants to regulate the size44 . Our results ruled out the possibility that the new absorption band is caused by the reaction of CO2•− radicals in solutions  activity and strong binding capabilities with CO intermediates. Nevertheless, our results on CO2•− radical kinetics suggest that the activity of metal sites can be predetermined, well before the binding of intermediates from CO2 dissociation.In the Ni case, COncy Fig. . At the ncy Fig. , underly2•−)ad radicals, we extended the observation of transient kinetics to the sub-millisecond range. Notably, (CO2•−)ad radicals on both Au and Cu surfaces maintain their specific spectral features even after 800 µs, with intensity dependent on the concentration ad radicals.To probe the structure evolution of surface-bound , once the next reaction happens, a second intermediate species is observed without noticeable bleaching of the absorption band in the case of Cu NPs. Nevertheless, the bleaching of the surface plasmon absorption band in the Au system persisted up to 900 ms, accompanied by the decay of 2•− radicals  in our work . In contrast, 2•− and Cu atom. The DORI analysis reveals two covalent interaction sites. Moreover, this larger electron sharing between the two entities is reflected by a Mayer bond order and an ELF valence basin population of 0.85 and 2.5 electrons for Cu-C and 0.54 and 2.2 electrons for Cu-O, respectively. The total spin density of the systems at the ground state was computed and represented in Supplementary Fig. 2•−, so it remains a radical species due to a weakly covalent interaction. For Cu, the result highlights a large delocalization of the spin density as a sign of the stronger covalent interaction in To better understand the nature of distinct absorption bands from COmentclasspt{minima50. The absorption spectra of 2•− in solutions, with a band maximum at 358 nm for Au and 293 nm for Cu, in agreement with experimental observation  due to the greater density of surface-bound CO2•− radicals. DFT simulations were performed on the larger size of Au NP with more CO2•− radicals  and similar to the above results for 2•− reflect that More importantly, the increase in Au NP size reduces the intensity ratio between two bands  electric field, buffering of the interfacial pH, and the stabilization of intermediates  through local field effects. To verify the above assumptions, we carried out experiments in solutions without sodium cations, the samples were centrifugated, and formic acid was used to adjust the concentration of proton. Compared with the above results in the presence of sodium cations, the kinetics of CO2•− radicals stabilizing on Au surfaces was retarded, and the absorption intensity of surface-bound CO2•− radicals on Au NPs remarkably decreased ) that obstruct the stabilization of CO2•− radicals on Au surfaces. Also, the trend in transient absorption spectra intensity follows the order Li+ < Na+ < K+ for the same concentration  is the same as whatever is produced by radiolysis or direct reduction of CO2 on the surface of the electrode. The surface stabilization process of CO2•− radicals on Au and Cu surfaces includes the initial adsorption and the structure optimization process, which extend substantially the lifetime of (CO2•−)ad radicals by at least 100 times compared to CO2•− radicals in solutions, thereby laying the groundwork for the subsequent multi-electron transfer reaction. Most notably, the respective (CO2•−)ad radical transient kinetics and characteristic spectra across Au, Cu, and Ni systems suggest diverse stabilization behavior and structures of (CO2•−)ad radicals on various metal surfaces. Extensive research in electrochemistry has resulted in categorizing metal electrodes into four distinct groups based on their primary products. The different selectivity of CO2 reduction between metals has been explained by their binding energy to key intermediates, including *OCHO, *COOH, and *CO. Therefore, we assume that the stabilized structure and kinetics of the first key intermediate, surface-bound CO2•− on metallic sites, influence the catalytic properties and the product selectivity. Au, Ni, and Cu NPs distinctly represent the individual catalytic selectivity and activity of each group. We observed very clearly the difference between the three nanocatalysts. In the same conditions, we found that Ni cannot stabilize the CO2•− radicals, which corroborates its slight activity of CO2 reduction reported in the literature; however, Au and Cu can stabilize the CO2•− radicals weakly and strongly, respectively, in agreement with the results obtained by steady-state electrocatalysis. The importance of our work lies not only in explaining the reaction mechanism in electrocatalysis but also in deciphering the function of the metal in CO2 reduction. This selective stabilization mechanism provides deeper insight into the intrinsic mechanism of CO2 reduction, extending from steady-state intermediates to transient elementary reactions. Our results are a significant advance in the development of time-resolved techniques for optimizing catalyst design and system performance.In summary, pulse radiolysis allows us to acquire a complete image of the stabilization process of CO2 (99.999%) and Ar (99.999%) were purchased from Air Liquide Industrial Gases Company.Cetyltrimethylammonium chloride , sodium formate (99%), copper(II) sulfate (98), nickel(II) chloride (98%), sodium tetrachloroaurate (99%), and cetyltrimethylammonium bromide (98%) were purchased from Sigma-Aldrich. All chemicals were used without further purification. Ultrapure water with a resistivity of 18.25 MΩ cm was obtained from a water purification system. CO2O. Then, 10 mL copper(II) sulfate (1 mmol L−1) solution was added to form homogeneous solutions. The solutions were saturated with Ar and sealed in a homemade cuvette. The cuvette was irradiated at a 60Co gamma source  at a dose rate of 2.2 kGy h−1 at ambient conditions. The dose rate was calibrated using the Fricke dosimeter, and the total absorbed dose reached 6.6 kGy. The resulting Cu nanoparticle solution was directly used for pulse radiolysis experiments and absorption spectroscopy characterization.All metal nanoparticles were synthesized by radiolytic methods: In a typical synthesis for Cu nanoparticles, 20 mmol sodium formate and 0.5 mmol CTAC were added to 10 mL Hg for 24 h using an ultracentrifuge  to remove the NPs.For Ni or Au nanoparticles, the precursor was replaced with nickel(II) chloride or sodium tetrachloroaurate. For size modification, it was controlled by varying the concentration of CTAB, with higher concentrations resulting in larger particles. The synthesized 1.7, 5.3, and 6.2 nm Au nanoparticles correspond to 1, 2.5, and 10 mM CTAB concentration systems. The supernatant solution was centrifuged at 164,700×Particle morphology and size distribution were examined by high-resolution transmission electron microscopy . UV-vis spectra were recorded on a UV-spectrophotometer  in the range from 190 to 800 nm.39. Laser (260 nm) driven Cs2Te photocathode allowed the production of short electron pulses with a typical half width of 7 ps, a charge of ≈6 nC, and energy of ≈7.8 MeV at a repetition rate of 10 Hz. During irradiation, metal NP solutions were contained in a homemade cell with a path length of 1 cm. The diameter of the electron beam was 3 mm, and the irradiated volume was less than 0.1 mL.Pulse radiolysis experiments were carried out employing the picosecond laser-triggered electron accelerator, ELYSE, coupled with a time-resolved absorption spectrophotometric detection system40.Absorption spectral measurements were performed using the white light from a homemade Xenon flash lamp. The light was focused on the sample parallel to the electron beam with a smaller diameter and then directed onto a flat field spectrograph , which disperses the light on the entrance optics of a high dynamic range streak-camera  to obtain an image resolved in wavelength and time. The kinetic data and absorption spectra were extracted from three series of 250 resulting images. In this work, the transient spectra were measured from 290 to 720 nm at 1 µs, 10 µs, 20 µs, 100 µs, and 1 ms64. The geometry optimization calculations were performed using the PBE functional, the SDD pseudopotential  for the metal atoms, and the def2-TZVP basis set for carbon and oxygen atoms. Dispersion corrections were added to the functional used in the D3 framework proposed by Grimme with the addition of the Becke–Johnson damping (D3BJ) in all cases65. 2•− radical with solvent effects represented by a dielectric continuum model. The orientation and nature of the interaction between the radical and NP have been probed by several quantum chemical analyses. Numerical frequencies were calculated to ensure the structures corresponded to energy minima. For Au and Cu, the 38-atom nanoparticle is considered in the singlet spin state, and the complex with the radical results in a doublet spin state. The CPCM implicit solvent model was used to represent the water environment. The electronic spectra were computed in the TD-DFT framework using the CAM-B3LYP functional and the implicit solvent. Between 600 and 800 excited states were included in the calculations to obtain the spectra of the different metal systems. The TD-DFT spectrum of the isolated CO2•− radical was obtained at the CAM-B3LYP/aug-cc-pVTZ level by calculating 200 excited states. The structure of the 92-atom Au NP with 5 CO2•− radicals was optimized at PBE-D3BJ level (with the SVP basis set for C and O atoms) using an implicit solvent. A simplified calculation of the electronic spectrum was computed in vacuum using the simplified sTDA66 approach and the wB97X functional. The quantum chemical topological and electronic transition analyses were performed with the Multiwfn code by analyzing the electron density generated from ORCA67. The analyses of both covalent and non-covalent interactions were calculated based on the Density Overlap Regions Indicator (DORI)68. The electron localization function (ELF) was calculated to determine the valence basins69.DFT calculations were done using the ORCA 5.0.3 softwareFurther information on research design is available in the Supplementary informationPeer Review FileReporting SummarySource Data


Polymeric materials possessrich phase transitions that can be directly tuned by their chemicalstructures, which has enabled researchers to engineer versatile adaptivematerials that can respond to targeted external stimuli. Reportedherein are suspensions of (readily prepared) micrometer-sized polymericparticles with accessible glass transition temperatures (Tg) designed to thermally control their non-Newtonian rheology.The underlying mechanical stiffness and interparticle friction betweenparticles change dramatically near Tg.Capitalizing on these properties, it is shown that, in contrast toconventional systems, a dramatic and nonmonotonic change in shearthickening occurs as the suspensions transition through the particles’ Tg. This straightforward strategy enables the in situ turning on (or off) of the system’s abilityto shear jam by varying the temperature relative to Tg and lays the groundwork for other types of stimuli-responsivejamming systems through polymer chemistry.Suspensions of polymericnano- and microparticles arefascinatingstress-responsive material systems that, depending on their composition,can display a diverse range of flow properties under shear, such asdrastic thinning, thickening, and even jamming (reversible solidificationdriven by shear). However, investigations to date have almost exclusivelyfocused on nonresponsive particles, which do not allow  in situ turning on (or off) of the system’s ability to shear jamby varying the temperature.Suspensions consisting of polymer particles that can undergoglass transition are designed to enable the  This highly tunable approach makes it possible to regulatethe collective behaviors in colloids comprising such particles, suchas their macroscopic packing and various transport properties.The structure–property relationshipsestablished for polymershave enabled the design of adaptive materials that can undergo rapidchanges upon exposure to an external stimulus such as temperature,pH, or light.13 On account of the prevalence of complex interactions that involveinterparticle contact forces in addition to hydrodynamic couplingforces, dense suspensions can display drastic non-Newtonian rheologythat in extreme cases resembles a reversible liquid to solid transition.20 As the contact interactions are highly sensitive to the compositionand surface structures of the particles, the stress response of thesuspension can be tailored by varying the fluid and the particle mechanicalproperties.26 For example, suspensions consisting of soft particles (E′ = 102–104 Pa) can exhibit dramaticshear thinning and provide platforms for designing novel lubricants.13 On the other hand, dense suspensions of rigid particles  can exhibit a dramaticincrease in viscosity or even solidify under shear (shear jamming),thereby finding applications in impact mitigation.29 While suspensions consistingof diverse classes of materials have been investigated to map outthe structure–property relationships that connect particleproperties to suspension rheology, material systems that allow for in situ control of suspension flow behavior have receivedlimited attention.31 One prototypical example is provided bymicrogel suspensions of soft thermoresponsive particles.30 In such systems, the lightlycross-linked polymer network can be driven to deswell through triggeringa solubility transition at certain temperatures, leading to a drasticdecrease in the volume fraction of the particles and a decrease inthe suspension yield stress under shear.23 While this strategy is effective for tuning shear thinning, systemsrelying on solubility transitions are limited to a few selected solvents.Dense suspensions consist of a large volume fraction (ϕ)of small solid particles dispersed in a carrier fluid. Such materialsdisplay rich rheological behaviors and provide intriguing opportunitiesto design material systems with applications complementary to solidpolymeric materials.Tg). When a polymeric material is heated through Tg, within a span of just a few degrees the molecularmobility increases by over 10 orders of magnitude,34 elastic modulidrop by 2–3 orders of magnitude,37 and friction exhibitsa distinct temperature anomaly.44 Remarkably, while these changes around Tg potentially have a large influence on the interparticle forces indense suspensions, their role in controlling, and tailoring, the suspensionflow behavior remains largely unexplored due to a lack of experimentalstudies in the vicinity of Tg.In contrast to solubility transitions, the polymer glass transitioncan result in a dramatic change in particle stiffness without theaid of a solvent. As one of the most fundamental properties of polymers,a considerable amount of research has been aimed at characterizingpolymeric interfaces around the glass transition temperature  of the material’sability to shear jam by varying the temperature relative to Tg and lays the groundwork for switchable jammingsystems that leverage a diverse range of polymer chemistry.Thiswork considers the interplay between polymer glass formation/devitrificationand shear-jamming phenomena and how that can be leveraged to arriveat suspensions with adaptable shear-jamming characteristics. Morespecifically, introduced herein is a generally applicable strategythat relies on the design of suspensions of particles with targeted Tg) that is well above ambient conditions (Tg ∼ 100 °C). Such temperatures are rarelyaccessed in rheological measurements.46 Furthermore,non-cross-linked PS or PMMA particles can suffer from irreversibleplastic deformations and dissolution above Tg,48 and randomly cross-linked polymers can exhibitbroadened Tg’s that can be difficultto characterize.51 These and other features serve to underscore thatstudying the effect of Tg in suspensionrheology requires the careful choice of the particles’ chemistry.A vast majority of polymericparticles reported in the dense suspensionliterature are based on poly(styrene) (PS) or poly(methyl methacrylate)(PMMA), which exhibit a glass transition temperature  the glass transition temperature range is narrow, (3) the Tg of the polymer can be precisely tuned by varyingthe monomer structures, and (4) the reaction is facile and relativelyinsensitive to moisture or air.54 Three types of particles , namely, P-9, P-29, and P-50 were synthesizedby thiol-Michael dispersion polymerization at a stoichiometric thiol/vinylratio following general procedures reported by Bowman and co-workers.53 In each case, a tetrathiol, namely, pentaerythritol tetrakis(3-mercaptopropionate), was mixed with monomer(s) containing multipleMichael acceptor functionalities, namely, trimethylolpropane triacrylate, divinyl sulfone , or1,3,5-triacryloylhexahydro-1,3,5-triazine ,at a vinyl to thiol ratio of 1:1. By varying the type and ratio ofthe different Michael acceptor-containing monomers, it was possibleto tailor the Tg of the particles . Images fromscanning electron microscopy (SEM) demonstrate that the particleswere uniform in size .To access a series of cross-linked particles with a range of distinctand accessible monomers 1A and B.articles 1C–E. Thearticles S1–S3. ImMn) 200 g/mol (PEG200)was used as the carrier fluid for all the particle systems . The Tg’s of both the dry particles and the suspensionswere determined by differential scanning calorimetry (DSC), as shownin Tg of the particles immersedin the carrier fluid drops by 15–25 °C below that of thedry particles, suggesting that the carrier fluid acts as a plasticizerthat can accelerate the polymer dynamics in the bulk and on the surface.26 Nevertheless, the particle dimensions stay roughlythe same in the dry or wet state on account of their high cross-linkingdensity .To create suspensions, poly(ethylene glycol) ofmolecular weight in both thedry  and carrier-fluid-swollenstates (53 The ratio of loss to the storage modulus (tan δ) providesa measure of the mechanical losses due to dissipation at the molecularscale.55 In general, the peak in tan δ is seenaround Tg because polymer chains juststart to become mobile at that temperature but still experience highmolecular friction, leading to higher dissipation than in the glassyor rubbery states. The thermomechanical Tg of the materials, designated by the peak in the dissipation factortan δ, is comparable to that from the DSC measurements for thedry and immersed film . Importantly, the tensile storage modulus (E′)changes by three orders of magnitude from around 109 Pain the glassy state to 106 Pa in the rubbery state forall three materials.As the thermomechanicalproperties of these networks representa critical aspect of this work, they were evaluated by preparing monolithic nstates 2 fora bT) on therheology, steady-state flow curves for particle suspensions with fixedparticle volume fractions were measured at different temperaturesas a function of shear stress (τ). The suspension viscosityη can be normalized by the viscosity η0(T) of the Newtonian carrier fluid to define a relative viscosityηr = η/η0(T) . Critically, the strengthof shear thickening can be parametrized by the power law exponentβ (ηr ∝ τβ),which represents the slope of the curve during the shear thickeningregime on a log–log plot, with β = 0 corresponding toa Newtonian fluid and β = 1.0 signaling the occurrence of discontinuousshear thickening (DST), which is a precursor of shear jamming in steady-staterheological measurements.56To examine the effect of temperature .61 In contrast to the behavior of these traditional suspensions, the P-9 particle suspension shows a strong sensitivity to temperature,as demonstrated in r for a volume fraction of ϕ= 53% shows a significant change in shear thickening strength as T is varied from −15 to 45 °C. Here, a strongand nearly discontinuous thickening with β ≈ 0.9 wasobserved below 15 °C. These findings should be contrasted withmeasurements at higher temperatures, for example 35 and 45 °C,where the suspension shows only mild thickening (β < 0.7). Interestingly, note that the secondarythinning that follows the thickening, as predicted by Jamali and Wagner,was not observed, which could be a consequence of limitations on theshear rates accessible by parallel plate rheometers.63 The change in β over a temperature range of ∼20 °Csuggests that control of shear thickening should be possible simplyby modulating temperature, thereby providing new avenues for engineeringresponsive fluids or for facilitating suspension processing, whichis typically challenging without the use of additives in the carrierfluid.In typical suspensionsof hard spheres, such as silica particles,the ηT, acommon way to probe molecular dynamics in a polymer and molecularglass transition is to investigate how viscosity varies with temperaturefor a given stress.37 A temperature rampexperiment under constant shear stress  was conducted for P-9, P-29, and P-50 suspensions . In all cases, the particle volumefraction was held at ϕ ∼ 50%. To normalize the temperaturedependency of the carrier fluid viscosity (η0), ηr = η/η0(T) is shownin r can be regarded as a measureof the additional resistance due to the presence of the particles.For conventional hard sphere suspensions, the curve is expected tobe approximately flat with a slight decreasing trend with temperature.61 For all systems measured in this study, it was found that the relativeviscosity of the suspension clearly shows a local peak near each particle’s Tg, which further supports that the maximal shearthickening occurs around the glass transition. Interestingly, theobserved behavior in viscosity bears similarities to studies of polymerdispersions that undergo microphase separation, such as that in blockcopolymer solutions64 and polymers aroundtheir lower critical solubility temperature.65 In those studies, a peak in η is observed at a temperaturewhere the conformation of polymer chain is changing. Here, the peakin ηr suggests maximal constraints on the suspensionflow. It is worth noting that this peak can be observed in both heatingand cooling experiments , implyingreversibility in the interactions induced by Tg.As an alternative to measuringβ vs pensions S12. In aTg, further experimentswere carried out on suspensionsof P-29 and P-50. While the three typesof particles have different sizes (ranging from ca. 1 to 3.5 μm),the effect of particle size is well understood and has been shownto mainly affect the onset shear stress of thickening but not thetrend in β.67 For these higher Tg suspensions, thenonmonotonic behavior in β is found to be similar for all particlecompositions. The peak, however, is shifted to higher temperatures,consistent with the changes in Tg . Critically, inall three cases the peak in β can be well aligned with tan δin T = 15°C, suggesting that the temperature dependence is directly relatedto the thermal glass transition of the particles . An offset ΔT may arise from two effects: (1) Polymer glass transitionis a kinetic event where the dynamics can start to slow down at temperaturesas much as 50 °C higher than Tg(Tg.42 (2) Polymerinterfaces under confinement and, in particular, with different deformationrates can exhibit dynamics different from the bulk.35 This result motivates further research to quantify how polymer stressrelaxation at the interface can influence the constraints on particlerelative motions.67 For the three materialsconsidered here, the fact that the maximum observed β stronglytracks with Tg offers a general yet simpleapproach to controlling the shear thickening characteristics of densesuspensions by simply tuning the Tg ofthe cross-linked particles.To confirm whether this phenomenon is indeed relatedto the particles’ r than Tg and the T varies for differentvolume fractions (ϕ), detailed studies were carried out using P-9 suspensions at four additional ϕ values that spannedweak to strong shear thickening regimes . In all cases, β is a nonmonotonic function of T, with a peak near 15 °C for all volume fractions studied . Although this number appears small in magnitude, it is about100 times larger compared to the deformation in the glassy state andmay affect the constraints on particle motions. With this in mind,it is hypothesized that deformability may lead to the observed attenuationin shear thickening at temperatures much above Tg: as the temperature increases, the particle surfaces becomemore compliant, discouraging the formation of sample-spanning rigidforce chains under shear.68To explore how β vs imes see S10. In aTg then βshould also increase.56 However, it was found that αof the particles is comparable to that of the carrier fluid . For all suspensions, the calculateddrift in ϕ is minimal (Δϕ < 0.05% for changing T by 10 °C) and therefore unlikely to be the main causeof the peak in β .The observednonmonotonicity leads us to consider other effectsof temperature on the suspension. First, the thermal expansion coefficients(α) of the particle materials were evaluated, since if ϕincreases significantly near Tg.44 Different from hard surfaces, friction between polymer surfacesoriginates from dissipative interactions between polymer chains.44 Around Tg, adhesive interactions increaseon account of an increase in the true contact area, which may leadto stronger cohesive “sticky” forces and enhanced rollingfriction.41 On accountof the viscoelastic nature of the interface between two particlessheared into contact, the friction coefficient (μ) can eitherincrease monotonically with T at moderate to highsurface deformation rates or show a peak if the deformation rate isslow enough to cross over into the time scale of surface relaxation44 (see the Supporting Information for anestimate of the Deborah number). AFM measurements of surface adhesionand friction on dry P-9 films were carried out at varying temperatureswith a silicon probe. Consistent with the literature, it was observedthat the apparent stiffness decreases while adhesion and frictioncoefficients increase when heating through Tg . Increased adhesive interactions above Tg are consistent with the observation that the P-9 suspension shows shear thinning at 35 and 45 °C for the low-stressregime . In contrast,the P-50 system shows brittle fracture and large Fmax all the way up to 45 °C, with a liquid-likeresponse occurring only above 45 °C  for targeted material applications.80From a theoretical perspective, this work raises many excitingquestions that warrant deeper investigation. For example, the contactmechanics at a polymer–polymer interface are fundamentallydifferent from those at conventional rigid particle surfaces. It remainsto be seen how the interfacial polymer dynamics  affect the formation and destructionof force chains. Future work that utilizes advanced scattering techniquessuch as X-ray photon correlation spectroscopy (XPCS) can contributeto a more detailed understanding on the suspension microstructurethat bridges single-particle properties with macroscopic rheologyand how these microstructures respond to a change in the particleproperties.Conversely, given thatDST and SJ are exquisitely sensitive tothe strength of particle–particle contact interactions, densesuspension rheology provides a powerful lens with which to observemacroscale consequences that are a direct result of interfacial polymerdynamics at molecular length and time scales.