Moreover, oxtr−/− fish showed impairments in the maintenance of social preference. Social isolation prior to testing led to impaired maintenance of social preference in both wild-type and oxtr and oxtrl knock-out fish. Knocking-out either of the Oxytocin receptors also led to increased group spacing and reduced polarization in a 20-fish shoal at 8 weeks post fertilization, but not at 4. These results show that the development and maintenance of social behavior is influenced by the Oxytocin receptors and that the effects are not just pro- or antisocial, but dependent on both the age and social context of the fish.Zebrafish are highly social teleost fish and an excellent model to study social behavior. The neuropeptide Oxytocin is associated different social behaviors as well as disorders resulting in social impairment like autism spectrum disorder. However, how Oxytocin receptor signaling affects the development and expression kinetics of social behavior is not known. In this study we investigated the role of the two oxytocin receptors, Oxtr and Oxtrl, in the development and maintenance of social preference and shoaling behavior in 2- to 8-week-old zebrafish. Using CRISPR/Cas9 mediated  In order to optimize cohabitation within a group, different forms of social behavior evolved.Many species, including humans, live in groups to enhance their fitness—their lifetime reproductive success. Living in a social context offers many benefits like improved predator and food detectionDanio rerio), a small teleost fish, is a powerful animal model used in biomedical research, including drug discovery4, developmental biology6 and neurobiology8. Furthermore, zebrafish exhibit a variety of behaviors including avoidance9, foraging and hunting10, responses to stress11 and different forms of sociality16. Examples of sociality include mating behavior, aggressive behavior and other simpler behaviors that occur in groups. Zebrafish prefer to swim in cohesive shoals, a tendency that develops within the first weeks of age17. Swimming in close proximity to conspecifics, also called social preference, starts to develop as early as 1–2 weeks-post-fertilization (wpf)19. Although the development of shoaling behavior has been correlated with age-dependent changes in the dopaminergic and serotonergic system20, the mechanisms underlying the development and maintenance of social preference and shoaling behavior are not well understood.The zebrafish (21. The zebrafish orthologue Isotocin (abbreviated Oxt) differs by only two amino acids from human Oxytocin21. In addition to its role in parturition22 and lactation23, Oxytocin has been described in the context of memory consolidation24 and nocifensive behavior28. Intracerebral Oxytocin levels also influence anxiety in mice31, rats33 or humans35. Moreover, the association between Oxytocin and social behavior has been demonstrated in multiple studies38 and the human Oxytocin receptor may influence social traits affected in neurodevelopmental disorders such as autism spectrum disorder40.The nonapeptide Oxytocin is a highly conserved neuropeptide, present in humans and with only minor alterations in most other animals41, aggression42 and depressive-like behavior43. Gilles and Polston44 reported that a two-week isolation of rats starting after weaning  led to increased pro- and antisocial behavior. They further described an inverse correlation between the number of oxytocinergic neurons in the anterior parvicellular division of the hypothalamic paraventricular nucleus (PVN) and the level of antisocial behavior44. After long-term social isolation, the application of Oxytocin was shown to reduce depression-like behavior of prairie voles43 or aggressive behavior in male mice42. Furthermore, different studies using rats described the number of oxytocinergic neurons in PVN to be reduced45 , increased46  or unchanged44  following social isolation. Isolation has also been reported to reduce the activity of Oxytocin receptors (OTR) in the nucleus accumbens of rats46 and to affect the expression of OTR in specific brain regions to different degrees: a four-week long social isolation of prairie voles led to reduced hypothalamic expression of OTR47, whereas six- and eight-week social isolation did not change the expression of OTR in murine hippocampus48.Social isolation for multiple weeks has an impact on different rodent behaviors including anxiety49. As teleost fish like zebrafish experienced a round of whole genome duplication approximately 320–350 million years ago50, they possess two orthologous Oxytocin receptors, the Oxytocin receptor (Oxtr) and the Oxytocin receptor like (Oxtrl). Several studies53 investigated how Oxytocin receptors influence social recognition or social preference in adult zebrafish by blocking both Oxtr and Oxtrl with antagonists52 or knocking out the oxtr53. Often however, these studies came to contradictory conclusions, underscoring the complexity of Oxytocin’s effects. The discrete impact of Oxtr or Oxtrl on the development and maintenance of social behavior in socially or isolation reared zebrafish remains to be elucidated.In mice, the Oxytocin receptor is expressed in different brain regions including the hippocampus, amygdala, suprachiasmatic nucleus and prelimbic cortexoxtr and oxtrl knock-out lines, the primary objective of this study was to investigate the role of the Oxytocin receptors in the development and maintenance of social behavior, specifically social preference and shoaling behavior in zebrafish (Danio rerio).Making use of CRISPR/Cas9 generated oxtr−/− and oxtrl−/− fish and their wild-type controls in two behaviors: social preference  and shoaling.To investigate the role of the Oxytocin receptors in development and maintenance of social behavior we compared the 54, although not significant (p = 8.03 × 10−1). The oxtr−/− fish, by contrast, exhibited an early peak of maximal social behavior at 3 wpf. In contrast to the wild-type fish, this high sociability was not maintained but rather significantly (p = 4.95 × 10−2) decremented over 4–8 wpf  altered in the oxtrl−/− fish, which also exhibited an early onset of social preference, reaching a peak precociously at 3 wpf  wild-type, oxtr−/− and oxtrl−/− fish from 2 days post-fertilization (dpf) to the day of experiment  affect the onset kinetics of social preference, but it dramatically diminished the maintenance of social preference typically observed at 8 wpf  social preference. In contrast to socially reared wild-type fish, the increase in social preference from 2 to 4 wpf was significant after isolation rearing (p = 1.22 × 10−2). However, a direct comparison of same age socially and isolation-reared wild-type fish revealed no significantly different levels of social preference at 2 to 4 wpf. Isolation rearing had a similar effect on the oxtr−/− fish: isolation did not alter the onset kinetics of social preference but led to an accelerated decline in social preference measured at 8 wpf  or decreased (2 wpf) social preference. Another example is the significantly reduced social preference of 4-week old isolation reared oxtrl−/− or oxtr−/− fish with an average swim speed comparable to wildtype zebrafish. Taken together, these data indicated that changes in swim speed induced by the loss of either Oxytocin receptor do not provide a straightforward explanation for the changes in social behavior observed.An analysis of swim speed revealed significant differences between wildtype and mutant fish at 2 and 3 wpf . To quantify shoaling, we analyzed the nearest-neighbor distance, inter-individual distance and farthest-neighbor distance of each fish as well as the cumulative shoal distance and the polarization parameter obtained from principal component analyses of individual videos (see “oxtr+/+ vs. oxtr−/−) = 4.74 × 10−1 (a), 4.09 × 10−1 (b), 4.74 × 10−1 (c) and 8.88 × 10−1 (d), p(oxtrl+/+ vs. oxtrl−/−) = 3.17 × 10−1 (a), 2.59 × 10−1 (b), 1.29 × 10−1 (c) and 4.80 × 10−1 (d))  = 1.64 × 10−3 (a), 1.38 × 10−2 (b), 1.91 × 10−2 (c) and 4.95 × 10−2 (d), p(oxtrl+/+ vs. oxtrl−/−) = 4.31 × 10−2 (a), 2.01 × 10−2 (b), 1.91 × 10−2 (c) and 4.95 × 10−2 (d))  was deleted, the development of social preference was accelerated, reaching its maximal level precociously at 3 wpf. Although Autism spectrum disorder is caused by multiple factors like mutations in the Fragile × mental retardation 1 (FMR1) gene62, intranasal application of Oxytocin can ameliorate some of the social impairments of autistic patients40 and additionally, specific single-nucleotide polymorphisms (SNPs) of the human OXTR39 gene are frequent across the ASD population and have been described as potential genetic risk factor for ASD63. We have shown here that the development of social preference is affected by Oxytocin receptors in a distantly-related vertebrate, the zebrafish. It is interesting to note, that a recent study reported that the development of social preference is also sped up in fmr1−/− zebrafish64.Many studies65 described decreased social preference in about 40% of fish after isolation rearing. They used wild-type fish with the genetic background AB and a behavioral chamber described previously19, which, in contrast to the chamber used here, does not allow simultaneous visual access to both stimulus areas. Unfortunately, the age of fish was not specifically defined, but they classified the experimental fish as “juvenile”, suggesting an age between 30 and 89 dpf66. In our experiments, isolation-reared wild-type fish exhibited social preference that was comparable to socially-reared fish at age 14 to 28 dpf, but they were less social after 8 weeks of isolation. These data suggest that reduced social preference is a response to isolation periods longer than 28 dpf but differences in experimental design  can influence the results of social preference tests.Isolation rearing of zebrafish did not affect the time course of the development of social preference, but significantly affected its maintenance. A recent study by Tunbak and colleaguesoxt expression in socially- and isolation-reared fish revealed that isolation led to significantly reduced oxt levels at 8 wpf, but not at 4 wpf  knock-out zebrafish line. Difference in the results obtained between Ribeiro et al. and our study may be due to the difference in experimental condition or age as they used a different behavioral chamber for the social preference test and three- to six-month old fish. oxtrl knock-out fish were not tested in their study. In another study, Ribeiro and co-workers described a non-significant tendency for less social preference in adult oxtr wild-type and knock-out fish after rearing in presence of oxtr knock-out conspecifics69. In our experiments, we reared wild-type fish with wild-type and knock-out fish with knock-out conspecifics. Different studies73 with Oxtr knock-out mice have shown decreased social behavior and impaired social recognition with enhanced aggression. Remarkably, the deficits in social behavior were also present in heterozygous Oxtr+/− mice73. In these studies, adult mice were tested. In a very recent study74, Nunes, Gliksberg and colleagues observed impaired social affiliation in adult zebrafish after chemical ablation of oxytocinergic neurons in the dorsorostral part of the neurosecretory preoptic area at early developmental stages (4–6 dpf and 12–14 dpf), but later ablation (20–22 dpf and 90 dpf) did not affect social preference of adult fish. As the ablated oxytocinergic neurons fully recovered within 42 days74, the described impaired social preference is likely due to the requirement of these oxytocinergic neurons at early developmental stages. The receptor knock-out fish we used in this study lacked Oxtr and Oxtrl, respectively, during all developmental stages. For oxtr−/−, we observed a significant reduction in social preference between the peak of maximal social preference at 3 wpf and 8 wpf. It is possible that this significant decrease persists until adulthood, resulting in impaired social preference of adult oxtr−/−, but this remains to be tested with knock-out fish. In comparison to oxtrl−/− fish, the maintenance of social preference was impaired at higher degrees in oxtr−/− fish, suggesting that this receptor might be more important for the Oxytocin influenced development of adult affiliative behavior in larval stages.Our results differ from the work of Landin and co-workers, who tested the social preference of adult and 3-week-old zebrafish after application of the Oxytocin receptor antagonist L-368,899, a specific inhibitor of both Oxtr and Oxtrloxtr−/− and oxtrl−/− fish at the ages tested, as two indicators of anxiety—boldness , shoal size  and the behavioral chamber . Furthermore, in Landin et al., both Oxytocin receptors were blocked by antagonist injection 1 h prior to experiment whereas in our experiments only one of the two Oxytocin receptors was knocked-out since the time of fertilization. Although not significant, the first signs of reduced oxtrl−/− shoaling capability were visible as early as 4-weeks of age. As shoaling behavior develops continuously until adulthood17, the most parsimonious explanation for both sets of data is that Oxytocin receptors are likely to be important for shoal organization at later stages of development, whereas other signaling pathways may be able to compensate at 4 wpf. Similar to the social preference, both Oxytocin receptors modulate shoaling behavior, but to different degrees. Since shoaling parameters are also influenced by predator avoidance77 and experience78, shoaling should not be considered as an exclusive expression of social behavior. Moreover, shoaling and schooling are regulated by both visual and mechanosensory79  inputs.As 80 the shoaling behavior of 90 CRISPR/Cas9 generated knock-out lines (for different genes) was compared . This study revealed the influence of multiple genes on collective behavior of zebrafish—in particular on swim speed, group spacing and polarization. Tang et al. further showed that a single gene does not necessarily affect swimming speed, group spacing and polarization, but can influence only one or two of these shoaling parameters. Our data are consistent with this finding as we did not detect significant changes in the cumulative shoal distance at 8 wpf . This threshold was calculated for each age group by determining the average swimming speed of each genotype group  and multiplying the smallest with 0.6. The fish of three genotype groups  were reared either in isolation or with conspecifics at 10–15 fish/L density and were tested for their social preference at age of 2, 3, 4 or 8 wpf. To keep the testing conditions as similar as possible, the different genotypes were tested without unnecessary delay  and the isolation and socially reared fish of one genotype were tested on the same day, whenever possible.For the investigation of social preference, we planned to test 18 fish per group. The sample size was determined prior to experiments using the E-equation methodoxtr+/+;oxtrl+/+  = “oxtr+/+” or “oxtrl+/+”, oxtr−/−;oxtrl+/+  = “oxtr−/−“and oxtr+/+;oxtrl−/−  = “oxtrl−/−”). In contrast to social preference, we detected a significant difference in some shoaling parameters between the homozygous wild-type cousins of oxtr−/− and the homozygous wild-type cousins of oxtrl−/−. Therefore, we analyzed the wild-type zebrafish as two genotype groups, “oxtr+/+” and “oxtrl+/+”, respectively. We tested 23–28 socially reared (10–15 fish/L) shoals per group.In the 30-min long shoaling experiment, shoaling parameters were analyzed in a shoal of 20 fish at age of 4 or 8 wpf in three different genotypes  it was excluded from further analysis.oxtr (ZDB-GENE-110805-2/NCBI mRNA Reference Sequence: NM_001199370.1) and oxtrl (ZDB-GENE-110805-1/NCBI mRNA Reference Sequence: NM_001199369.1) genes were mutated by Ajay Mathuru and Caroline Kibat using the sgRNA:Cas9 system described in 83. Two CRIPSR targets  for each gene were identified using ZiFit (http://zifit.partners.org/zifit/). Customized sgRNAs with 20 nucleotide sequence complementary to a target site were synthesized by first cloning the target sequences into the expression construct pDR274  and then in vitro transcribed using T7 promoter according to the manufacturers protocol (Thermo Fisher # AMB13345).The Cas9 mRNA was transcribed from linearized plasmid MLM3616  following the manufacturers protocol (Thermo Fisher # AMB1344). The sgRNA:Cas9 RNAs cocktail (containing 12.5 ng/μL sgRNA and 300 ng/μL Cas9) were injected into single-cell embryos of AB wild-type background. The efficiency of the CRISPR targets and quality of the Cas9 endonuclease were determine 24 h post injection of sgRNA:Cas9 by PCR on 10% of the injected embryos. The remaining embryos were raised to adulthood and genotyped at three months post injection. A total of 64 individuals were genotyped. The sequence of the genotyping primer set is in detailed in the genotyping methods section. The PCR products were cloned into pGEMT (Promega #1360) and sequenced to verify the mutations. The F0 mutants were then then outcrossed to Danio Reds (https://doi.org/10.1016/S0006-291X(03)01282-8). F1 fish were in-crossed to establish germline transmitting homozygous mutant lines.The oxtrync02 and oxtrlync03, in this paper abbreviated as oxtr−/− and oxtrl−/−, respectively. The homozygous wild-type cousins of these fish were used as wild-type control. Moreover, these wild-type cousins were used for regular out-crossing of the homozygous knockouts (KO) to keep the genetic background of the experimental fish as similar as possible. We could not test oxtr−/−oxtrl−/− double KO fish as their generation by breeding failed, possibly because the presence of at least one oxytocin receptor is vital at specific developmental stages. When evaluating the expression of the knocked-out receptor using specific primers by real time PCR, amplification signals were detected in the wild-type but not in the mutant fish, indicating the successful knock-out. Furthermore, a compensation of the knocked-out receptor by its orthologue Oxytocin receptor was not detectable , 8 mm (3 wpf), 10 mm (4 wpf) and 14 mm (8 wpf). No differences in development or growth were detected comparing mutant and wild-type fish. In the social preference test the stimulus fish (two per test fish) had the same age and similar size than the test fish. As it is difficult (juveniles) to impossible (larvae) to distinguish males and females at the developmental stages used in this study, fish became experimental fish independent of their sex.Experimental fish were bred by incrossing homozygous mutant or wild-type fish in a 1:1 sex ratio per batch. Each group consisted experimental fish of multiple clutches (up to 17) and the clutch size was 300–400 eggs (no difference between wild-type and mutant fish). On the day of experiment the test fish were 2, 3, 4 or 8 wpf old, depending on the age group. We used experimental fish with a total body length comparable to the total body lengths described in the Zebrafish Book2, 0.33 mM MgSO4] per 22 mm diameter well) or kept in pertri dishes  in groups of 50.Up to 5 dpf, the larvae were kept in a 28.5 °C incubator with a 14/10 light–dark cycle. At 2 dpf, eggs were either individually isolated in a 12-well plate , CAVIAR 50–100 (SAFE) and crushed Gemma Micro 75 (Skretting), from 10 to 20 dpf with vinegar eelworms, brine shrimp  and CAVIAR 50–100, from 21 to 56 dpf with brine shrimp and CAVIAR 100–200 (SAFE).From 5 dpf on, zebrafish were housed in a ZebTEC Active Blue Stand Alone System, with the water temperature of 28.5 °C ± 1 °C, the pH = 7.4 ± 0.3 and the conductance = 650 µS ± 100. The light–dark cycle in the fish facility is 14 h-light/10 h-dark with a 30 min twilight phase. Fish were fed three times per day: Up to 10 dpf with vinegar eelworms  for testing social preference consisted of the test area (25 × 75 mm (for 2 to 4 wpf) and 50 × 75 mm (for 8 wpf)) and the stimulus areas (8 × 75 mm (for 2 to 4 wpf) and 16 × 75 mm (for 8 wpf)). The behavioral chamber was placed on a screen, providing white backlight illumination and a camera positioned above recorded the fish’s movements with 20 (3–8 wpf) or 30 (2 wpf) frames per second using the program “Pylon recorder”. To reduce visual and acoustic disturbances, the setup was surrounded by a black, sound-absorbing box (LBH: 450 × 670 × 850 mm). Prior to the experiment the test and stimulus fish were moved from their home tank to a 1L breeding tank  with a nursery insert  and kept in a 28.5 °C incubator (test and stimulus fish in different tanks). After each test, the behavioral chamber was cleansed with hot water (~60 °C) and refilled with fresh ZebTEC Stand Alone system water (28.5 °C). The fish were moved using a disposable 3 ml pipette (the tip was cut off to generate a sufficient big diameter) by elevating the nursery insert and then transferred to the center of test area (test fish) or edge of stimulus area (stimulus fish) of the behavioral chamber. At 8 wpf the chamber was covered with the lid of a 145 mm dish to prevent fish from jumping out. The test fish habituated to the chamber without any other fish for ten minutes followed by a 12 min test phase in which two stimulus fish of same age and size were placed to one of the stimulus areas. A transparent wall between test and stimulus area allowed visual access to the stimulus fish whereas a white opaque wall determined the rear side of the stimulus area.The shoaling setup . Additionally, each experimental fish was genotyped after the experiment. The following primer sequences were used.In order to identify homozygous knockouts (KO) and homozygous wild types (wt) for generation of experimental fish, fin clip PCR was performed according to the ZIRC genotyping protocols (oxtr) or 2% (oxtrl) agarose gel and run at 100 V for 30 min (oxtr) or > 2 h (oxtrl). But as the difference between oxtrl+/+ and oxtrl−/− is only 10 base pairs, PCR products were also sequenced using the forward primer.The PCR products were loaded on a 1% (67 and using the QuantiTect Reverse Transcription Kit (QIAGEN Cat. No. 205311) 200 ng of RNA was transcribed into cDNA. 5μL of 1:10 diluted cDNA template were mixed with 1.3μL primers (10 μM) and 6.25 μL SYBR Green PCR master mix (Applied Biosystems/Thermo Fisher Art. No. 4309155). The cycling parameters were 10 min at 95 °C, followed by 40 cycles of denaturation (15 s at 95 °C) and amplification (60 s at 60 °C). A Real Time PCR System (Applied Biosystems/Thermo Fisher Art. No. 4376600) was used for qRT-PCR. The fluorescence threshold was set to 0.9 for all experiments and genes and we used TATA box binding protein (tbp) as reference gene. We used the ΔCT method and the following primer sequences.RNA was extracted from isolated brains as described beforeIn the social preference test, the location of the test fish was measured in each frame of habituation and during the last 10 min of test, as the stimulus fish needed up to 2 min after being transferred to the stimulus area to exhibit normal social behavior. During analysis the test area was divided into two regions of interest (ROI). The “social ROI” was an area next to the stimulus fish whereas the “antisocial ROI” was on the opposite site of the test area. The percentage of total time was determined, in which the fish was located in the “social ROI”. The average swimming speed in absence (habituation) or presence (test) of stimulus fish was calculated and used as exclusion criteria for fish with reduced motion (e.g. because of freezing).84 (tracking threshold 15 (4 wpf) and 50 (8 wpf)) and the nearest-neighbor distance, the inter-individual distance and the farthest-neighbor distance of each fish were calculated and cumulative shoal distance and variance explained as parameter for coordinated swimming were determined. For determination of the variance explained, x- and y-components of all individual trajectories  were first assembled in a 40 × 54,000 (30 min with 30 frames per second) matrix, whose covariance matrix was then eigen decomposed to obtain principal components (PCs). We found the first two PCs corresponded to the x- and y-component of the shoal centroid trajectory over time. Principal component analysis (PCA) is a dimensionality reduction approach that can be used to quantify the degree of linear correlation in a dataset. If individual features are highly correlated, they can be compressed in one PC while virtually no data are lost. This can be measured with the variance of the original dataset that is explained by the derived PCs. In our case, the higher the variance explained by the first two PCs, the higher the correlation between the individual fish’s movement and the movement of the shoal overall . The statistical analyses were carried out using python  or MATLAB . In the social preference test n = 332 fish were included in the analysis (79 excluded), in the shoaling experiment n = 184 shoals were included in the analysis (18 excluded).84 plus a custom written python script for shoaling) to provide an unbiased data analysis. The test fish were chosen randomly from their home tank group and in the social preference test the location of stimulus fish was changed between replicates in a random order. The experiments were performed from 8:00 AM to 5:00 PM and the order of genotype groups per day was changed between experimental days to minimize a potential circadian confounder. Furthermore, the position of tank during rearing was chosen in a way that all replicates of one experimental group were reared in all possible heights/light conditions.The investigators were not blinded to the genotype of the experimental fish, but both experiments were recorded with an overhead camera and analyzed using a tracking software .Supplementary Information.


Modern humans expanded into Eurasia more than 40,000 years ago following their dispersal out of Africa. These Eurasians carried ~2–3% Neanderthal ancestry in their genomes, originating from admixture with Neanderthals that took place sometime between 50,000 and 60,000 years ago, probably in the Middle East. In Europe, the modern human expansion preceded the disappearance of Neanderthals from the fossil record by 3,000–5,000 years. The genetic makeup of the first Europeans who colonized the continent more than 40,000 years ago remains poorly understood since few specimens have been studied. Here, we analyse a genome generated from the skull of a female individual from Zlatý kůň, Czechia. We found that she belonged to a population that appears to have contributed genetically neither to later Europeans nor to Asians. Her genome carries ~3% Neanderthal ancestry, similar to those of other Upper Palaeolithic hunter-gatherers. However, the lengths of the Neanderthal segments are longer than those observed in the currently oldest modern human genome of the ~45,000-year-old Ust’-Ishim individual from Siberia, suggesting that this individual from Zlatý kůň is one of the earliest Eurasian inhabitants following the expansion out of Africa. Homo sapiens individual from the site of Zlatý kůň, Czechia. Although radiometric dating of the human remains was inconclusive, the authors were able to use molecular methods to demonstrate that she was probably among the earliest Eurasian inhabitants following expansion out of Africa.The authors present the genome sequence of a >45,000-year-old female  A complete genome has been produced from the ~45,000-year-old remains of Ust’-Ishim, a Siberian individual who showed no genetic continuity to later Eurasians3. This contrasts with the ~40,000-year-old East Asian individual from Tianyuan whose genome is more closely related to many present-day Asians and Native Americans than to Europeans4. From Europe, only the partial genome of an individual called Oase 1 and dated to ~40 ka has been recovered, and this showed no evidence of shared ancestry with later Europeans5. However, Oase 1 carried more Neanderthal ancestry (6–9%) than other modern human genomes sequenced to date, owing to admixture with Neanderthals that occurred within the six generations before the individual lived.Only three genomes have been recovered from individuals that fall close in time to the settlement of Europe and Asia more than 40 thousand years ago (ka)7 12, a recent craniometric analysis that included a virtual reconstruction of the Zlatý kůň skull supports that the individual lived before the last glacial maximum13.Here, we study genome sequences generated from a largely complete ancient skull that was discovered alongside other skeletal elements in 1950 inside the Koněprusy cave system in present-day Czechiabp; MAMS-36077) compared with the first direct date. A third date, comprising a solvent pre-wash treatment followed by ultrafiltration on the same bone fragment, produced a younger date of ~19 ka 14. The large discrepancies between the three direct dates suggest that the Zlatý kůň specimen is highly contaminated and that radiocarbon dating on bulk collagen may be unreliable . However, we suspect this is also artificially young due to the presence of trace exogenous contaminants derived from animal glue, as supported by genetic analysis .We extracted DNA from ~15 mg bone powder from the Zlatý kůň petrous portion of the temporal bone and first enriched for and sequenced the mitochondrial genome (mtDNA) to ~150-fold coverage (Methods). Around 4% of the mtDNA sequences were estimated to stem from human contamination  were covered at least once after genome-wide enrichment (capture dataset). In addition, we sequenced ~4 billion random DNA fragments from the same DNA library of Zlatý kůň, resulting in ~3.8-fold genomic coverage (shotgun dataset). In line with the sex assignment based on morphology13, the X chromosome and autosomes showed similar coverage, indicating that Zlatý kůň is female  19 in a phylogenetic analysis  with our capture and shotgun datasets. We first compared Zlatý kůň with present-day European and Asian individuals using an African population (Mbuti) as an outgroup and found that Zlatý kůň shares more alleles with Asians than with Europeans 31 and compared the genetic length of the 100 longest segments in Zlatý kůň with those identified in other early Eurasian hunter-gatherers using the same method  yielded comparable results 33. Applying this method to the Zlatý kůň shotgun dataset yielded an estimate of 63 generations since admixture (s.e. = ±0.6), whereas Ust’-Ishim was estimated to have lived 84 generations after the admixture (s.e. = ±1.3).To estimate the time of admixture independent of calling Neanderthal ancestry segments, we applied a previously published method that is based on the correlation of the state of Neanderthal informative sites over increasing distances28. To test whether the Neanderthal ancestry in Zlatý kůň shows the same relationship, we used D statistics to compare the sharing of alleles with the high-coverage genomes of a European and an Asian Neanderthal in addition to five published low-coverage genomes from late Neanderthals from Europe34. Zlatý kůň showed no significant difference compared with Ust’-Ishim in its sharing of alleles with late Neanderthals, in line with the similar proportion of Neanderthal ancestry in these two hunter-gatherers . After indexing, the library was amplified for 30 cycles followed by a reconditioning PCR to remove heteroduplexes. The resulting library was diluted and shotgun sequenced on an entire flow cell (eight lanes) of an Illumina HiSeq 4000 platform for 1 × 75 cycles.All laboratory procedures were conducted in the dedicated ancient DNA facilities of the Max Planck Institute for the Science of Human History in Jena, Germany. The Zlatý kůň cranium was sampled from the base of the cranium with a dentist drill after removal of a thin layer of bone powder. Two aliquots of ~15 mg bone powder were sampled and one was used to extract DNA using an established protocol43. Adapter sequences were trimmed, filtered for a minimum length of 30 base pairs (bp) and mapped to the hg19 human reference genome using BWA44 with the following parameters: -n 0.01 -l 16500. Approximately 10% of sequences mapped with an average fragment length of 49 bp. These sequences were filtered for a minimum mapping quality of 30 and duplicates were removed using Dedup43. Sequences showed C to T exchanges to the human reference, indicative of ancient DNA damage, with a frequency of ~13% at both terminal positions46. The first and last two bases of shotgun sequences were masked (set to N) to reduce the effect of damage-associated substitutions on subsequent analyses.A total of ~580 million paired-end reads from the double-stranded library matched the correct indices, allowing for up to one mismatch per index, and were further processed with EAGERAn additional ~3.355 billion single-end reads were produced from the single-stranded library on a dedicated run. All sequences were processed using EAGER with the same parameters as above, except the mapping quality filter was set to 25. Around 15% of reads mapped to the human reference, with an average fragment length of 46 bp and 30% of C to T substitutions at the 5′ molecule termini.5 and ~1.24 million nuclear SNPs (1240K capture)16 followed by HiSeq paired-end sequencing and index filtering, resulting in 600,000 and 20 million reads, respectively. MtDNA capture and 1240K capture data were mapped against the mtDNA reference sequence (the Revised Cambridge Reference Sequence (rCRS)) using CircularMapper43 and the human reference genome (GRCh37/hg19), respectively, with the same parameters as above and the mapping quality filter set to 30.The indexed library was further amplified to perform targeted enrichment of both the complete mtDNA (mtDNA capture)https://github.com/stschiff/sequenceTools). The calling of transversions among the 1.24 million target SNPs considered full sequences, whereas 2 bp at both termini of sequences were trimmed before calling transition SNPs. Finally, the resulting calls were merged with a large genotype dataset of ancient and present-day individuals for the same set of ~1.24 million SNPs .A random allele was drawn from the 1240K capture data using PileupCaller in pseudohaploid mode (Further information on research design is available in the Supplementary InformationSupplementary Sections 1–10, Figs. 1–15 and Tables 1–32.Reporting Summary


These sensing properties make the MXene aerogel feasible for monitoring ultra-weak force signals arising from a human’s deep-lying internal jugular venous pulses in a non-invasive manner, detecting the dynamic impacts associated with the landing and take-off of a mosquito, and performing static pressure mapping of a hair.There is an urgent need for developing electromechanical sensor with both ultralow detection limits and ultrahigh sensitivity to promote the progress of intelligent technology. Here we propose a strategy for fabricating a soft polysiloxane crosslinked MXene aerogel with multilevel nanochannels inside its cellular walls for ultrasensitive pressure detection. The easily shrinkable nanochannels and optimized material synergism endow the piezoresistive aerogel with an ultralow Young’s modulus (140 Pa), numerous variable conductive pathways, and mechanical robustness. This aerogel can detect extremely subtle pressure signals of 0.0063 Pa, deliver a high pressure sensitivity over 1900 kPa The fabrication of pressure sensors with both ultralow detection limits and ultrahigh sensitivity is still challenging. Here, the authors propose a design strategy for fabricating a soft polysiloxane crosslinked MXene aerogel with multilevel nanochannels inside its cellular walls for ultrasensitive pressure detection. There is an urgent demand for pressure sensors that can monitor extremely weak blood pressure waveforms  from deeply embedded veins in the human body, detect faint acoustic wave transmission in the air , and recognize tiny signals induced by small movements or ultralight weights  in intelligent healthcare, soft robot, and human–machine interface applications5. In addition, to enable the accurate sensing of subtle force signals, highly sensitive pressure sensors are needed because a low sensitivity often severely deteriorates the sensing resolution and leads to poor noise resistance, which limit the applications of such sensors in readout electronics7. Thus, the development of pressure sensors that combine an ultralow minimum detectable pressure limit with an ultrahigh sensitivity will offer exciting new opportunities for sensing applications, but significant materials-related challenges must be overcome.The real-time sensing of subtle mechanical force signals is a key requirement for many next-generation cutting-edge intelligent applications8. In principle, electrical resistance changes are governed by the structure and electrical features of piezoresistive materials, while material deformation critically depends on their geometry and mechanical properties9. Over the past few years, extensive investigations have focused on improving the detectability and/or sensitivity of piezoresistive sensors by optimizing the structural and electrical properties of sensing materials14. While conventional piezoresistive sensors made of bulk composites of conductive fillers and insulating polymers exhibit a low sensitivity15, recent studies have demonstrated that hierarchical nano/microstructured materials  provide decent sensing performance (sensitivity >100 kPa−1). This is because when a piezoresistive material is compressed, more conductive pathways can be created in hierarchical nano/microstructures than in bulk structures, resulting in greater changes to the contact and/or internal resistance in hierarchically structured sensing materials22; however, the minimum pressure detection limit for most of these devices is still low (~1 Pa) due to the relatively large modulus of the rubbery substrates or soft matrixes23. Moreover, most of these hierarchically structured sensing materials are made of stiff conductive nanomaterials such as graphene, carbon nanotubes, MXenes, or nanostructured conducting polymers27. Such conductive materials usually possess intrinsically high Young’s moduli and tend to show strong resistance to elastic deformation, further lowering their ability to detect subtle pressures28.Piezoresistive pressure sensors that rely on electrical resistance changes induced by material deformation in response to an applied pressure are currently the most promising force-sensing technology due to the broad selection of active materials and the simple fabrication and integration processes9, conductive aerogels have recently been investigated as an ideal alternative piezoresistive material for sensing subtle pressure changes due to their low modulus (or low mass density)26. Mathematically, the critical stress value (σc ∝ (t/l)3) that triggers the buckling or bending of cellular walls in aerogels is determined by the thickness (t) and length (l) of the cellular walls27. Soft conductive aerogels can be obtained by decreasing the crosslinking (or junction) density and mass density and increasing the aspect ratio (l/t) of cellular walls in the microcells. Previous work has reported that a minimum detectable pressure limit of 0.082 Pa was achieved using an elastic graphene aerogel with an ultralow density of 0.54 mg/cm3 and a large aspect ratio in the cellular walls25. Nevertheless, this ultralight graphene aerogel delivered a low sensitivity of ~10 kPa−1 due to a significant reduction in its conductive pathway density. To retain their mechanical robustness and elasticity, insulating components are often compounded into conductive aerogels, which further diminishes their subtle pressure detectability and sensitivity29. Overall, although intense recent research efforts have advanced the development of piezoresistive materials for sensing applications30, the simultaneous achievement of both an ultralow detection limit and ultrahigh sensitivity in piezoresistive materials is still challenging11.Because the sensitivity of piezoresistive pressure sensors is generally inversely proportional to their Young’s modulus3), which significantly reduced the critical stress value that triggers material deformation. The shrinking of the multilevel nanochannels under compression increased the contact area between neighboring MXene nanosheets, which led to the formation of numerous new conductive paths and a considerable resistance change31. The formation of covalent crosslinks between the MXene and polysiloxane afforded the hierarchical aerogel with excellent mechanical robustness and compressive elasticity (up to 80%). Due to this sophisticated design, the resulting piezoresistive bottlebrush polysiloxane and MXene based aerogel could rapidly respond to an extremely low pressure of 0.0063 Pa with an ultrahigh sensitivity (>1900 kPa−1). Moreover, this subtle pressure sensing performance, including the sensitivity and detectability, was retained even after 10,000 compress–release cycles. This piezoresistive aerogel can be assembled into flexible pressure sensing arrays for diverse cutting-edge applications that require the ability to detect extremely weak mechanical signals.Herein, we propose a strategy to design a piezoresistive sensor for ultrasensitive subtle pressure sensing using a conductive sensing material based on an elastic and ultrasoft MXene-based aerogel with a hierarchical multilevel cellular wall structure. The core innovation of this design—the easily shrinkable multilevel nanochannels inside the cellular walls of the aerogel—was achieved by intercalating ultrasoft bottlebrush polysiloxane into MXene interlayers via covalent crosslinking. This hierarchical structure, together with the low-density structure of the MXene aerogel, endowed the piezoresistive material with an ultralow Young’s modulus 32. The piezoresistive sensing mechanism of MX-AG was dominated by external resistance changes (RE) in the cellular walls induced by the bending or bucking of the cellular walls in the aerogel under pressure  of the cellular walls in the aerogel  as the precursors to form composites with a variety of functional properties. GPDMS was selected to compound with MXene nanosheets based on three considerations. First, GPDMS can be hydrolyzed and polymerized into bottlebrush-like poly(3-glycidoxypropyldimethoxymethylsilane) (PGPDMS) with a soft polysiloxane main chain and flexible short side-chains  can be obtained through a simple fabrication process consisting of a hydrothermal reaction, directional freeze-drying, and mild thermal annealing . PGPDMS can act as a spacer for intercalation into the MXene interlayer, which prevents the restacking of MXene nanosheets41. This can facilitate the formation of easily shrinkable nanochannels inside the cellular walls  the spaces between nanochannels, which, in turn, leads to considerable resistance changes inside the cellular walls (denoted as an RI change)31. The total resistance changes  of BBP–MX–AG in response to external forces have two primary contributions: RE and RI  through the assembly and restacking of Tiins Fig. . Second,lls Fig. 21. Such  RI Fig. . In addi3 and with an initial MXene-to-silane weight ratio of 5:1 in the precursor mixtures. For comparison, we also fabricated MXene aerogels intercalated with an interchain-crosslinked poly(3-glycidoxypropyltrimethoxysilane) (PGPTMS) network, which is abbreviated as ICP-MX-AG (ICP represents interchain-crosslinked PGPTMS)  in the BBP–MX–AG was made of multilayer MXene with an average nanochannel spacing (dA) of 1.8 nm, as confirmed by HRTEM  in MX–AG22. This confirms the successful intercalation of PGPDMS and PGPTMS into the MXene interlayer at the molecular level to form parallel nanochannel structures inside the cellular walls of BBP–MX–AG and ICP–MX–AG, respectively41. The Fourier-transform infrared (FT-IR) spectra , high-resolution transmission electron microscopy (HRTEM), and X-ray diffraction (XRD). Figure TEM Fig.  and XRD TEM Fig. . The nanTEM Fig. . Energy-TEM Fig. . By cont3, respectively. These values indicate their ultrasoft characteristics. These Young’s modulus values are more than two times lower than that of MX–AG  was observed after the first compression–release cycle with a maximum strain of 80%  of a cellular wall containing 5 MXene layers (indicated by the red line) decreased from 7.1 nm (1 s) to 5.01 nm (19 s) when an external force was applied, and then recovered to 6.87 nm (28 s) when the stimulating force was released. This cellular wall thickness change was induced by a simultaneous spacing change in the nanochannels of the cellular walls, as confirmed by digital micrograph images  between two MXene layers decreased from an initial value of 1.78–1.6 nm and 1.25 nm at 15 and 19 s sequentially during the loading process, which then increased back to 1.72 nm at 28 s when unloaded . Instead, these changes are dominated by the shrinking of the nanochannels inside the cellular walls (RI). Significantly, the pressure sensitivity in the pressure range of 0.025–0.1 Pa was calculated to be 1476.7 kPa-1 with good linearity  in the aerogel. Moreover, the detection limit of the pressure sensors assembled from ICP–MX–AG was only 0.1 Pa  exhibited nearly the same pressure detection limit (0.0063 Pa) as the one with a density of ~10 mg/cm2  and internal jugular vein (IJV) in a non-invasive manner Fig. . Central5. Significantly, the unique shape of the IJV pressure waveform from a healthy subject can be obtained by our sensing device , C peak , and V peak (venous filling and venous pressure increase), and two valleys: X  and Y 5. The stable, uniform, and identifiable waveform signals indicate that the BBP–MX–AG sensor can continuously and accurately monitor cardiovascular events in a non-invasive manner.Figure ice Fig. . The typice Fig.  features2 and a total device area of 3 × 3 cm2  was purchased from 11 Technology Co., Ltd. (China). 3-Glycidyloxypropyldimethoxymethylsilane (GPDMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS) were purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. , with purity of 99.9%.Titanium aluminum carbide (Ti3C2Tx) MXene was synthesized by the selective etching of aluminum in titanium aluminum carbide  and delamination using minimally-intensive layer delamination (MILD)63. First, lithium fluoride (2 g) and hydrochloric acid  were mixed via stirring for 30 min. Then, Ti3AlC2 (1 g) was added to the solution. After heating at 35 °C with stirring for 24 h, the solution was poured into a centrifuge tube and then centrifuged at 3500 rpm for 10 min. The supernatant was removed and distilled water was added, and these two steps were repeated until the pH of the supernatant was >5. Then, the Ti3C2Tx layers precipitated at the bottom were dispersed in ethanol by ultrasonication for 30 min. Then, ethanol was removed by centrifugation at 16,770 × g for another 30 min. Next, with the addition of distilled water, the remaining precipitate was centrifuged at 3500 rpm to form a uniform Ti3C2Tx suspension. Finally, the resulting homogeneous MXene solution was freeze-dried to obtain MXene powder.Titanium carbide , and v is the volume of the aerogel .First, an MXene solution (10 mg/mL) was prepared by dispersing TiFirst, an MXene solution (10 mg/mL) was prepared by dispersing MXene powder in distilled water via oscillation. Next, the obtained solution was poured into a designed container and then placed in a cold source with a uniform temperature until it was completely frozen. The MXene aerogel was obtained after freeze-drying.W is the specific work, ɛ1 and ɛ2 are the initial and final compressive strains, respectively, and σ is the compressive stress64.Compression experiments were performed using a compression testing machine  at a strain rate of 5 mm/min. Before the compression test, the sample was pre-compressed with 80% compression. The Young’s modulus was calculated during the first 40% of the compression process. The energy loss coefficient was calculated by dividing the work loss by the compression work. The value of work was determined by the following formula: P) was defined as P0 and LdB denote the reference sound pressure in air (20 µPa) and the measured sound pressure level, respectively65.To evaluate the pressure sensing performance, a pressure sensor was constructed by integrating a monolithic aerogel on top of a flexible interdigital electrode-coated PET substrate. Subtle pressure stimuli were applied in two ways: (1) using a mechanical testing stage  equipped with a force sensor with an ultrasensitive force gauge (with a limit of 0.025 Pa); (2) using a loudspeaker box whose precise volume was determined by a decibel meter. A computer-controlled homemade loudspeaker box was used to produce sound or acoustic waves with various frequencies. The sound source was positioned 5 cm over the sensing device and was faced directly at the sensor surface. The output sound wave is edited by “GoldWave” software, and the sound wave template is in “freq” mode under “wave”. The volume of the output acoustic signal was controlled by computer, and the precise volume or sound level reaching the sensor surface was calibrated and determined by a decibel meter. The resistance and current changes were measured using a Keithley 2000 digital multimeter. To prevent environmental noise interference, both the sound source and pressure sensor were installed inside a noise isolation chamber. The sound pressure .The morphology of the aerogel was characterized by SEM . HRTEM images were obtained using a transmission electron microscope . XRD measurements were performed using a Rigaku Smart Lab 3 kW diffractometer . Fourier-transform infrared spectroscopy was performed using a Bruker TENSOR27 FT-IR spectrometer in the 4000–400 cmThe in-situ TEM samples were prepared by focused ion beam using an ultrahigh-resolution dual-beam scanning electron microscopy system (TESCAN GAIA3 model 2016). During in situ TEM  observations, an external force was applied using a tungsten blunt head operated by a NanoFactory system (EP1000). The distance change between the layers under pressure was analyzed by Digital Micrograph 1.6.2 software.To avoid unexpected movements during IJV measurements, a volunteer was instructed to lie still on a chair or bed with controllable lying angles during the test. Then, to remove the interference from breathing signals and precisely extract the carotid pulse signals and IJV waveforms from the retrieved data, the as-measured waveforms were filtered by fast Fourier-transform with a bandpass filter from 1.0 to 4.0 HzSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


In this work, we systematically studied morphological and signaling responses of population fibroblasts in an interconnected cell monolayer and uncovered that, besides the natural NF-κB oscillation frequency of 1/90 min−1, collective signaling response emerges in the cell monolayer at 1/20 min−1 TNF-α input periodicity as well. Using a customized microfluidic device, we independently induced dynamic chemical stimulation and cytoskeleton reorganization on the stand-alone cells to exclude the effect of cell-cell communication. Our results reveal that, at this particular frequency, chemical stimulation is translated into dynamic intracellular mechanical cues through RAC1-medicated induction of dynamic cell-cell connections and cytoskeleton reorganizations, which synergize with chemical input to facilitate collective signaling responses.Collective behavior emerges in diverse life machineries, e.g., the immune responses to dynamic stimulations. The essential questions that arise here are that whether and how cells   •Dynamic intracellular mechanical cues facilitate collective cellular responses•The dynamic chemical stimulations are translated into intracellular mechanical cues•The synergy between dynamic mechanical and chemical signal plays crucial roles Mechanobiology; Cellular Physiology; Cell Biology Collective behavior is the outcome of interactions among individual cells. The short-range cell-cell and cell-extracellular matrix (ECM) interactions allow the living tissues to undergo drastic behavioral transitions for biological functions such as migration, embryogenesis, and tumorigenesis . The intIn the crowded cellular environment of biological tissues, the local tissue architecture  and the −1 TNF-α inputs and only at the natural frequency of NF-κB signaling cascade for SA cells . Meanwhile, at the particular frequency of 1/20 min−1, the dynamic loss and re-establishment of cell-cell connections mediated by elevated RAC1 expression generates intracellular mechanical cues, which causes collective nuclear shape changes in ICM. Using a customized microfluidic device, we reconstructed the cytoskeleton reorganization and simultaneously introduced dynamic TNF-α stimulations to the SA cells, which sophisticatedly excludes the effect of chemical information exchange. It is surprising that we found the collective behaviors within the biological tissues is a consequence of the synergy between cell-cell interactions, mechano-signaling, and NF-κB dynamics. Phase mismatching among those factors leads to disrupted collective behavior. We, therefore, conclude that the on-off process of cell-cell connections provides a feedback loop, where dynamic intracellular mechanical cues caused by TNF-α-induced cytoskeleton reorganization facilitate collective cellular responses to dynamic chemical stimulations. This investigation reveals a novel cascade process linking the morphological and signaling collective behaviors and provides opportunities to develop novel therapeutic strategies, utilizing controllable mechanical cues to manipulate tissue function in vivo.In this work, we studied the effects of dynamic intracellular mechanical cues, which result from the collective morphological responses, on the signaling activities of population cells in an interconnected cell monolayer (ICM). By tuning the TNF-α input periodicities, we observed that fibroblasts in ICM show substantially different morphological and signaling responses as compared with the SA cells. Collective signaling activities emerge in ICM at both 1/20 and 1/90 min2, the landscape of mechanical cues from one cell are transmitted over distances spanning multiple cell size , the size of the ICM remains unchanged during periodic TNF-α stimulation. Instead, collective oscillatory movement of nuclear and actin filaments emerges, which suggests dynamic variations in the local stress of actin networks , and E-cadherin antibody to the ICM. The addition of RAC1 inhibitor disrupts the collective movement of actin filaments G, but th−1 TNF-α input frequency, but not the collective movement  inhibitor Y-27632, which plays central roles in mechano-transduction  and cytomovement A and 3B.−1 TNF-α stimulation, one or multiple Rho-associated signaling pathways get activated and entrained, leading to elevated RAC1 expression level, which destabilizes E-cadherin-mediated cell-cell adhesion   , NF-κB dSA cells , whose oSA cells . Accordiα stimulation increases in the ICM as well , and JASP, which disrupt the collective NSF at 20 min as well I. In conTo investigate the regulatory effects of chemically induced intracellular mechanical cues, we designed a microfluidic device in which forces mimicking those in ICM can be directly applied on SA cells B. In theα inputs of 1/20, 1/60, and 1/90 min−1 periodicities and dynamic collagen remodeling of 1/20 to 1/90 min−1 frequencies were then simultaneously introduced to SA cells. We observed that NF-κB oscillation in responses to 1/20 min−1 TNF-α stimulation is only slightly enhanced under certain conditions, i.e., when SA cells are repeatedly compressed at 1/20 min−1 frequency or mode hopping between 1/20 and 1/30 min−1 , the adhesion area will rapidly decrease when the pressure is released at t = 10 min  leads to increased NF-κB oscillation amplitude comparable with the signaling responses in the ICM  causes decreased nuclear NF-κB level. In the meantime, the otherwise random NF-κB dynamics of SA cells, as shown in α input, showing a shared frequency of ~1/20 min−1  leads to coordinated NF-κB oscillation more frequently than the natural frequency of 1/90 min−1  may be caused by the participation of other dynamic signals, e.g., the TNF-α-caused contractile action of ICM and proceeding of mechano-signaling pathways, all of which cause changing mechanical forces on the nucleus (stage ④). As the consequence of dynamic mechanical cues feeding back to the ICM, the collective NSF synergizes with the dynamic chemical input, leading to the collective cellular responses at a frequency far beyond the natural value of the signaling cascade (stage ⑤).Taken together, our results suggest a feedback loop intrinsic to the crowded cellular environment in the ICM, where dynamic mechanical cues caused by cytoskeleton remodeling facilitate collective cellular responses . In stagα stimulations at input frequencies of 1/20, 1/60, and 1/90 min−1 were introduced to either SA cells or the ICM. The correlation between the intracellular mechanical loads and collective signaling responses was investigated by simultaneously monitoring the cytoskeleton reorganization and NF-κB oscillation dynamics in response to periodic TNF-α stimulations.In this study, we quantified the collective morphological and signaling responses of population cells in an ICM, which models the crowded cellular environments in the biological tissue. Using a microfluidic chip and live cell imaging system, dynamic TNF-κB oscillation gets coordinated with its amplitude reaching maximum under only 1/90 min−1 TNF-α stimulation, which is consistent with the reported natural frequency of NF-κB signaling cascade, whereas in the crowded environment of the ICM, NF-κB oscillation gets maximum coordination at both 1/90 and 1/20 min−1 of TNF-α input frequencies. In the meantime, at the particular TNF-α input frequency of 1/20 min−1, the RAC1 expression level is unexpectedly elevated. This leads to enhanced transition from mature to dynamic cell-cell connections and more frequent on-off of cell-cell contacts, which cause a decrease in the overall extension of actin filaments in the cell. Subsequently, the properties  of intracellular mechanical cues are altered, leading to an amplified morphological and signaling response of the ICM to 1/20 min−1 TNF-α stimulation. These results indicate an intracellular mechanism facilitating the collective signaling responses of population cells in the ICM, in which chemical stimulations are translated into dynamic mechanical cues through RAC1-mediated active cytoskeleton reorganization.We found that, for the SA cells, NF-α stimulation periodicities. This supports our finding that NF-κB dynamics in ICM is only enhanced at 1/20 min−1 stimulation periodicity, where NSF reflecting the intracellular mechanical cues and the TNF-α input frequency are most coordinated. The emergence of collective morphological and signaling responses at a defined TNF-α input frequency of 1/20 min−1 implies that there exists a feedback loop in the ICM, in which the periodic TNF-α stimulation leads to entrainment in the RAC1-associated signaling pathways and cytoskeleton reorganization. Subsequently, the changing mechanical loads on the nucleus cause the collective NSF, which synergizes with the dynamic TNF-α stimulation and facilitates collective cellular responses.The intracellular mechanism determining the collective morphological and signaling responses was further studied by isolating SA cells in a customized microfluidic device, which can induce remodeling of cytoskeleton networks and independently generate dynamic chemical signals. Excluding the effect of cell-cell communication, we demonstrate that, regardless of the input frequencies of mechanical and chemical stimulations, responses of SA cells on par with the ICM can be accomplished as long as the induced dynamic cytoskeleton remodeling phase matches with the TNF-in vivo and get a coherent downstream response. Furthermore, our studies demonstrated the capacities of the presented microfluidic system in generating phase mismatching between the dynamic chemical and physical signals at milliseconds accuracy, which allows us to systematically investigate the importance of synergy between various environmental cues in regulating collective cellular responses.Our findings reveal a novel strategy that allows the biological tissue to generate phase-locked responses to acute inflammatory signaling far beyond its natural frequency. Beside its biological significance, the regulatory effects of dynamic mechanical cues on collective cellular responses suggest new therapeutic strategies, in which dynamic mechanical signals can be induced by acoustic or magnetic field to minimize the cell-to-cell variability Regarding the dynamic mechanical cues, which regulate intracellular signaling activities, the limitation of this study is that we have not yet quantified these forces. One of the remaining questions is how nucleus as the mechanosensor distinguishes diverse mechanical cues. For example, nuclear deformation was often observed during cell-cell collision. Would the transduced mechanical forces affect cellular responses?zhangce.univ@gmail.com). Materials availability This study did not generate new unique reagents.Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ce Zhang (All relevant data are available from the authors upon request.All methods can be found in the accompanying


Ventilatory efficiency was assessed by the VE•VCO2−1 slope, the oxygen uptake efficiency slope and the partial pressure of end-tidal carbon dioxide (PetCO2). The two experimental conditions were controlled using a randomized, double-blind crossover design. The resistance exercise test involved repeating the same routine twice, which consisted of wall ball shots plus a full squat (FS) with a 3 min rest or without a rest between the two exercises. A higher weight lifted was detected in the FS exercise after BJ intake compared with the PL during the first routine (p = 0.004). BJ improved the VE•VCO2−1 slope and the PetCO2 during the FS exercise in the first routine and at rest (p < 0.05). BJ intake improved the VE•VCO2−1 slope and the PetCO2 coinciding with the resistance exercise performance. The ergogenic effect of BJ could be induced under aerobic conditions at rest.The assessment of ventilatory efficiency is critical to understanding the matching of ventilation (VE) and perfusion in the lungs during exercise. This study aimed to establish a causal physiological relationship between ventilatory efficiency and resistance exercise performance after beetroot juice (BJ) intake. Eleven well-trained males performed a resistance exercise test after drinking 140 mL of BJ (~12.8 mmol NO The accumulation of lactate and [H+] is mainly due to intracellular glycolysis [2 [NO is a powerful vasodilator ,42 that acidosis . The VCOycolysis ,46 and bycolysis ,48. Sevelysis [2 ,50.2−1 slope in response to adequate VE and poor perfusion (VE/perfusion mismatching). Compared with other studies, the mean values of the VE•VCO2−1 slope  and the OUES  were worsened in our athletes than in highly trained athletes  [2−1 slope ~19 to 30; OUES, ~2.6) [2−1 slope exceeding 34 have been used as a reference to determine ventilatory impairment in diseases [2−1 slope was worsened in the FS exercise during the first routine  and the second routine  as the anaerobic environment was raised , coinciding with a decrease in weightlifting performance.In this anaerobic environment, it was expected that there would be an elevated VE•VCOS, ~2.6) ,52. The diseases ,54 or todiseases . The VE•s raised . The obv2−1 slope and exercise performance during the first routine (with a rest) after BJ intake could be linked to the biochemical and physiological properties of NO. An elevated VE•VCO2−1 slope was verified in both experimental groups (BJ and the PL); therefore, the mismatching of perfusion/VE was increased during the high intensity resistance exercise in both experimental groups [3−–NO2− conversion to NO after BJ intake. We suggest that NO could have contributed to an increased blood flow and pulmonary vasodilation reversing a possible vasoconstriction [2 output and arterial PCO2 [A rational explanation for the coincident improvements in the VE•VCOl groups . This phl groups . A 3 mintriction ,58 especial PCO2  after in3− intake on the VE•VCO2−1 slope and the exercise performance remains controversial. Several studies have proposed that ventilatory efficiency was unchanged in cardiorespiratory diseases after acute dietary NO3− intake; however, exercise performance was improved in patients with heart failure but not in patients with chronic obstructive pulmonary disease [2−1 slope in patients with systolic heart failure [2−1 slope was not evaluated after BJ ingestion. Given the limited scientific evidence, the possible effect of the increased circulating plasma NOx levels on the VE•VCO2−1 slope during endurance and resistance exercises in well-trained athletes warrants further investigation.The effect of dietary NO disease ,60. Exog failure . Unfortu2−1 slope throughout the test. BJ intake produced an ergogenic effect at specific times depending on the anaerobic conditions of the resistance exercise.It is possible that the perceived differences between studies were due to the proposed protocol, the type of exercise and the checkpoints established during the data analysis. It was evident that the aerobic-anaerobic conditions and the established rest elicited a varied response of the VE•VCO2−1 slope was reduced to normal values (BJ and PL < 25) during a rest between exercises and routines in the two experimental conditions; instead, the OUES remained elevated in the two experimental conditions (BJ and PL > 5.2). Surprisingly, the VE•VCO2−1 slope decreased during the rest between both routines after BJ intake. The reduced VE•VCO2−1 slope was maintained after the improvements in the FS exercise. It appeared that increased plasma NOx levels caused an ergogenic effect on the VE•VCO2−1 slope during a rest. This strengthened the theory of the possible vasodilator effect of BJ to regulate VE to remove CO2, improving the matching of VE/perfusion during a rest.A novel finding observed in this study was that the VE•VCO2 was evaluated to corroborate the ergogenic effect of BJ on the VE/perfusion relationship. It has been demonstrated that the PetCO2 decreased during a high intensity exercise coinciding with a reduced blood flow and blood velocity [2 in cardiac diseases [Finally, the PetCOvelocity ,62. Furtdiseases ,63.2 and time trial performance during normoxia and hypoxia conditions in trained male cyclists [2 and the proportion of physiological dead space to tidal volume in patients with systolic heart failure [2 in patients with pulmonary hypertension [2 during high intensity resistance exercises after dietary NO3− intake. We speculated that BJ improved pulmonary blood flow and cardiac output resulting in better perfusion of the alveoli and increased PetCO2 [Oral nitrate supplementation has shown no effect on the PetCOcyclists . Other s failure ; howeverrtension . There ad PetCO2 .This study presents a few limitations that should be considered. Minimal changes in performance and physiological responses are usually observed in well-trained athletes ; therefo2−1 slope and the PetCO2 coinciding with an enhanced resistance exercise performance in well-trained athletes. The positive effect of increased plasma NOx levels on the VE•VCO2−1 slope and PetCO2 could be induced, at least in part, when the aerobic conditions were gradually recovered during a rest. It is likely, therefore, that a rest after an acute resistance performance plays a key role in improving NO3−–NO2− conversion to NO during severe anaerobic exercise with low oxygen availability, enhancing its vasodilator effect at a pulmonary level.Acute BJ intake improved the VE•VCOThe findings reported here open new lines of research for understanding the physiological mechanisms that link the effects of NO with lung function and exercise performance during high intensity resistance exercises, especially during a rest.


In this work, the analogous reactivity with isostructuralAu–X complexes  is computationally explored.We demonstrate that a kinetically and thermodynamically favorablereactivity with CO2 may only be expected for the gold–aluminylcomplex. The Au–Al bond nature, which features the most (nonpolar)electron-sharing character among the Group 13 anions analyzed here,is responsible for its highest efficiency. The radical-like reactivityappears to be a key ingredient to stabilize the CO2 insertionproduct. This investigation elucidates the special role of Al in thesehetero-binuclear compounds, providing new insights into the peculiarelectronic structure of aluminyls, which may help for the rationalcontrol of their unprecedented reactivity toward carbon dioxide.Anionic aluminum(I)anions  are themost recent discovery along Group 13 anions, and the understandingof the unconventional reactivity they are able to induce at a coordinatedmetal site is at an early stage. A striking example is the efficientinsertion of carbon dioxide into the Au–Al bond of a gold–aluminylcomplex. The reaction occurs via a cooperative mechanism, with thegold–aluminum bond being the actual nucleophile and the Alsite also behaving as an electrophile. In the complex, the Au–Albond has been shown to be mainly of an electron-sharing nature, withthe two metal fragments displaying a diradical-like reactivity withCO The reactivity of gold−aluminyl,−gallyl,and −indyl complexes with carbon dioxide is predicted to befavorable only for gold−aluminyl complexes. The increased polarityof the Au−Ga and Au−In bonds makes these compounds weakernucleophiles in a radical-like reaction mechanism. Among these, CO2 capture withtransition metal (TM) complexes is surely one of the most interesting,due to the well-known ability of TMs of activating kinetically andthermodynamically inert CO2.2 The relative structural simplicity of TM complexes bearing CO2 also offers an ideal playground for characterizing in detailthe CO2 activation mechanisms.Theproblems connected with the increasing concentration of carbondioxide in the atmospheretBu3PAuAl(NON)] -2,7-ditert-butyl-9,9-dimethylxanthene, complex I)was recently reported, in which I was easily capableof capturing carbon dioxide at room temperature by inserting it intothe Au–Al bond, yielding insertion product II  activation of CO2 with theAu–Al bond being the actual nucleophilic site for the reactionand Al also assisting the activation with the electrophilicity inducedby its vacant valence 3pz orbital. Weshowed that the Au–Al bond is of the electron-sharing type,with Au and Al cooperatively inserting CO2 with a radical-likereactivity.9Since a mechanistic description for the formationof 2 forming an insertion product analogousto II.10 Following our work,a combined experimental and theoretical paper on the insertion ofCO2 into Cu–Al, Ag–Al, and Au–Al bondsappeared in the literature, showing a very similar reaction mechanismalso for Cu and Ag,11 in agreement withthe TM–Al bond behaving as a nucleophilic site.In accordance with abovereactivity, a zinc–aluminyl complexfeaturing a highly covalent electron-rich Al–Zn bond was recentlyfound to react with CO12 surely representpromising species for this aim, analogue Group 13 anions  have been known for a longer time:12 The first five-membered heterocyclic gallyl anion was reported morethan 20 years ago.13 Notably, examplesof molecular gold–gallyl complexes have been also reportedin the past15 and very recently the silver–gallylanalogue of I ]complex) has been characterized.11 Formallyanionic boryls have also been known for years,17 and recently, the nucleophilic reactivity of a nonheterocyclic gold–borylcomplex toward multiple polar bonds has been reported.18 Notably, a copper–boryl complex has beenreported in the past to catalyze the reduction of CO2 toCO.20 Concerning the heavier analogue, indium,a six-membered heterocyclic indyl anion ]−, ArNON = [O(SiMe2NAr)2]2–, Ar = 2,6-iPr2C6H3) has also been recently reported,21 bearing the same heterocyclic backbone of the diamido aluminyl[Al(SiNON)]− (SiNON = [O(SiMe2NDipp)2]2–, Dipp = 2,6-iPr2C6H3).22Thisnovel and unexpected reactivity of aluminyls raises questions,mainly about the peculiar chemical nature of the aluminyl and itsimplications for a rational design of similar bimetallic compoundscapable of reacting with carbon dioxide in analogous conditions. Whilethe very recent aluminyls24 For instance, heterocyclic boryls are found to have a very smallsinglet–triplet energy gap,24 which impliesa remarkably low stability, making their experimental isolation verydifficult. Indeed, six-membered heterocyclic boryls have not beensynthesized yet.26 However, aluminyl, gallyl, andindyl have a much larger singlet–triplet energy gap,24 which makes the synthesis of the corresponding heterocyclic anionscomplexes) much more promising even to explore additional patternsfor the bimetallic cooperative TM–X reactivity toward CO2.The electronic properties of Group 13 heterocyclic anionshavebeen widely investigated theoretically, highlighting that a “gap”separates boryls from their heavier homologues.2 in themodel complexes IX (SiNON)]−, X= Al, Ga, and In) combinedwith a common [tBu3PAu]+ gold moiety.It is worth reminding that the aluminyl and indyl ligands in complexes IAl and IIn have been actually synthesized and characterized experimentally22 and that similar heterocyclic structures appear to be reasonablyaccessible for Ga. Analogous boryl complex IB has not been included in this work since the constrainedsix-member heterocyclic ring structure ([B(SiNON)]−) represents a poorly realistic model complex, notdirectly comparable to the aluminyl, gallyl, and indyl analogues.We mention that the experimentally accessible coinage-metal–borylcomplexes possess mainly acyclic (such as the very recent gold borylcomplex bearing a boryl with two o-tolyl substituents)18 or five-membered structures.27 A comparative study of the electronic structureof boryl/aluminyl anions, including a systematic analysis of the structuraland substituent effects, is currently under way in our laboratory.The aim of this work is to analyze and characterizesystematicallyany analogy/difference between aluminyls and their heavier Group 13analogues in the framework of the carbon dioxide insertion reaction.We computationally study here the insertion of COlexes IX 1. These 2 insertionand carrying out an extensive electronic structure analysis, we willhighlight that despite all the complexes  proceed toward the CO2 insertion with the same mechanism observed for complex I]complex I,3 we found a two-stepmechanism characterized (i) by a nucleophilic attack to the CO2 carbon atom performed by the Au–Al bond also assistedby the electrophilic Al “empty” p orbital, followed(ii) by a rearrangement driven by an electrophilic attack to the oxygenatom of CO2 by the aluminum center, leading to the formationof the insertion product where the CO2 carbon atom is coordinatedto gold and both the CO2 oxygen atoms are coordinated toAl . Analogous to [tBu3PAuAl(NON)], complexes IX have been slightly simplified at the SiNON site by replacing the two Dipp substituents on the nitrogen atomswith phenyl groups (denoted as SiNON′). This modelinghas been shown to give good agreement with available experimentalgeometrical data for complex I in ref ] complex reported in ref (tBu3PAuAl(NON′)]and [tBu3PAuX(SiNON′)]  complexes are sketched with selected geometricalparameters in Figures S1–S4. Calculations of the singlet–triplet energy gap in [X(SiNON′)]−  anionsshow that these systems are stable. In particular, it is large for[Al(SiNON′)]− (1.47 eV) and evenincreases from [Ga(SiNON′)]− to[In(SiNON′)]− .28The free energyprofiles for all systems are shown in d in ref . Optimiz2 carbon atom has arelatively low activation free energy barrier for all the complexes,with the lowest value for complex IAl , thehighest for complex IIn , and intermediate for complexes I and IGa  . With the exception of TSIAl, the transition state structuresof the different systems are very similar: The carbon atom of CO2 is both close to Au (distances 2.4–2.5 Å) andat a relatively short distance from Al, Ga, and In (2.6–2.4Å). A substantial bending of CO2 and asymmetry betweenthe two C–O bonds are also found. In TSIAl, CO2 presents a larger asymmetric coordination,where the Au–C distance increases up to 2.899 Å and theO–Al distance reduces to 2.320 Å. Notably, in this casethe CO2 distortion is significantly decreased, passingfrom a bending angle of about 144–146° found for the othersystems to 160°. Thus, the structure of TSIAl is quite unique and apparently difficult torationalize, both in terms of structure and energy profile along thewhole reaction path, particularly in light of its similarity to complex I. We will return on this interesting point at the end ofthis section.The nucleophilic attack to the COINTX shows substantial differences between systemscontaining Aland those involving the heavier Group 13 elements. INTX is stabilized with respect to TSIX by 20.5 and 17.1 kcal/mol for I and IAl, respectively, andby only 6.2 and 0.2 kcal/mol for IGa and IIn, respectively,thus resulting in an exergonic step for I and IAl, an endergonic step for IGa and a highly endergonic step for IIn. Nonetheless, the INTX structures share some common features:(i) a slightly increased Au–X bond distance and (ii) an almostlinear coordination of the tBu3PAu moiety tothe carbon atom of CO2. Similar structures are also observedfor transition states TSIIX , in which the Au–X bondis significantly elongated and the second oxygen of CO2 is approaching X. However, while for TSII and TSIIAl similar lower activation barriersare found , TSIIGa and TSIIIn lie at a much higher energy, with corresponding higher ΔG# values ,suggesting that the INTX to PCX conversion would be less favorablefor IGa and IIn. Remarkably, the reverse step from INTX to RCX is expected to be kinetically favorable for Ga and, particularly,for In, predicting that the reaction of IGa and IIn with CO2 to give insertion products IIGa and IIIn is not feasible neitherkinetically nor thermodynamically. Product complex PCX  and highlyunstable for IGa and IIn .In our previous study, we investigated the possibility that the productcomplex PC (complex II in 3 and we found that the resulting oxide complex[tBu3PAuOAl(NON′)][CO] is highly unstable. Here, the corresponding[tBu3PAuOX(SiNON′)][CO]  complexes have been also calculated to be unstablewith ΔG = 14.9, 37.2, and 46.3 kcal/mol, respectively,consistent with the recent results reported in ref . This point is particularly interesting sincethe degree of activation of CO2 is often monitored by followingits bending distortion along the reaction path. For instance, in heterogeneouscatalysis studies, the bending of the OCO angle in the surface-adsorbedCO2 molecule relative to the gas-phase value of 180°(linear) has been proposed29 and widelyaccepted as a good indicator of activation. The interpretative frameworklies on the fact that upon reduction the gas-phase CO2 acceptselectron charge in its LUMO, which is of antibonding (π*) characterand becomes energetically more favored in a bent structure.2 However, this is not the case here, since a one-to-onemapping between the OCO angle in TSIX  andthe activation barrier (and stability) is not found .To clarify this issue, we explored the topology of the potential energysurface (PES) around each TS, by varying the Au–C and Al–Odistances in the 2.90–2.40 and 2.60–2.30 Å ranges,respectively. The PES around TSI is shown in It can be clearlysurmised that upon substitution of the Group13 element the reactivity with COTSI arereported in Figures S5–S7, and all the numerical values are reportedin Tables S2–S5.We also explored the PES of carbondioxide constrained at the geometryof the corresponding structure. The results for 2 activation occurs in a PES which is very flat around TSI in a wide range of interfragment distances, indicatingthat similar energies correspond to very different structures. Indeed,the energy varies overall in the tight 3 kcal/mol ca. range. Thisbecomes emblematic when considering the structure of CO2 in each structure sampled along this PES cut . In other words, in differentstructures the orbital interactions between I and CO2 always efficiently counterbalance the variable and increasingdistortion penalty of CO2.The analysis of the PES in  PES cut 4. The COI (TSI′) with a very different structurewith respect to TSI  which lies extremely close inenergy  and is almost isostructural with TSIAl. Visualization of the vibrational modes associatedwith the imaginary frequency of TSI′ (−212.5cm–1) indicates a concerted transition state associated with a vibrational mode involvingAu–C, Al–O, and Al–C interactions. We shouldalso mention that in ref is also flat: Along the same scannedrange of Al–O and Au–C distances around TSIAl, an overall variation of 6 kcal/mol isobserved, and as can be envisaged by the numerical data reported in Table S3, this range is even tighter right around TSIAl. In the 2.550–2.900 Åand 2.300–2.600 Å ranges for the Au–C and Al–Odistances, respectively, two structures with substantial geometricaldifferences (particularly concerning the CO2 structure)have been found with a variation of less than 3 kcal/mol in the electronicenergy. Concerning the PESs for TSIGa and TSIIn, less flatPESs are found, with a variation of 7 and 8 kcal/mol in the overallscanned range, respectively, showing a steeper topology in the closestregion to TSIX , andsuggesting that the orbital interactions between IGa and IIn and CO2 may not be strong enough to efficiently counterbalancethe geometrical distortion of CO2.The PES around X structure .9 Clearly, this indicatesthat, as for the intermediate structure, the bending angle of CO2 does not represent a good parameter for quantitatively evaluatingthe capability of the different complexes of activating carbon dioxide,probably due to the cooperative role of Au and X in reacting withCO2 (vide infra). Very interestingly,these results are in nice agreement with the recent finding that thedecrease of the OCO angle is not an appropriate indicator of CO2 activation on semiconductor oxides.30 As a final remark, we should note that despite the change in thecoordination at Al from a tridentate N,O,N′– to a bidentateN,N′– scaffold in I and IAl very similar free energy reaction profilesand PES topology at the TSI have been found. Previous computationalresults on the electronic structures of the parent naked aluminyls12 suggested a possible role of the oxygen atomin the [Al(NON)]− anion and a reduced N-to-Al πdonation (“folded” nonplanar [Al(NON)]− vs planar [Al(SiNON)]−), which seemhere to have only a negligible effect on the reactivity of their goldcomplexes. A detailed analysis of the N-to-Al π donation issuein the aluminyl scaffolds is still lacking, and it certainly deservesto be further investigated.Notably, as shownin To furtherinvestigate the effect of the substitution of the Group 13 elementon the reactivity discussed above, a comparative analysis of the electronicstructure of the transition states and stationary points has beencarried out. Several theoretical methods have been applied. In particular,we employed the Activation Strain Model (ASM), Energy DecompositionAnalysis (EDA) in combination with ETS-NOCV, Charge Displacement (CD)analysis, and dual descriptors for chemical reactivity.RCX with the corresponding TSIX and INTX using the ASM,33 which decomposes the path into two contributions:the distortion penalty ΔΔEdist of the increasingly deformed reactants and the interaction ΔΔEint between these deformed reactants (see the Supporting Information for details). The ASMallows to get insights into the factors involved in the stabilization/destabilizationof TSIX and INTX structures. The main results are reported in E energy profile, ΔΔEdist, and ΔΔEint in panels a–c,respectively) and discussed below. All the numerical ASM results arereported in Tables S6 and S7.We startby decomposing the electronic energy reaction path connecting each IAl is the lowest  due to anoverallsmall distortion penalty , which is efficiently counterbalancedby a small interaction stabilization . For IGa and IIn, the interaction stabilization  is not able to efficiently counterbalancethe larger distortion penalty ,yielding higher activation barriers , consistentlywith the ΔG# values discussed inthe previous section. As a comparison, the electronic energy activationbarrier for I  was found to result froma 21.8 kcal/mol distortion penalty efficiently counteracted by a −12.9interaction stabilization.9 A complementarypicture arises from the analysis of the differences between the threecomplexes at INTX, where CO2 presents a similar degree of distortion. Indeed, as it canbe surmised from INTX isonly favored for X = Al , whereas formation of INTGa and INTIn is energeticallydisfavored .This is due to the increased distortion penalty which is differentlycounterbalanced by the interaction stabilization for the three complexes.As it can be seen from RCX to INTX a similarly increased distortionpenalty is observed for all the three complexes, with IIn having a slightly less increased penaltyat INTIn with respect to INTAl and INTGa , consistent with the slightlyless bent CO2 structure in INTIn (see INTAl (ΔΔEint = −100.9) to INTGa  and INTIn .Upon inspection of NTIn see 2. The in2 approaches IX,we resort to the use of the EDA,35 and we appliedthe ETS-NOCV36 approach also in combinationwith the CD39 function on both TSIX and INTX structures.All the results are reported in Tables S8–S9 and Figures S8–S14. The isodensity pictures of the mainNOCV deformation densities associated with INTAl are reported in To shedlight on the nature of the interactions taking place whenCOTSIX and INTX, the interaction scheme betweenCO2 andthe [tBu3PAuAl(SiNON′)] isqualitatively unaltered, following the same scheme reported in ref . As an example, for INTAl in 2 and IX consists mainly of twoopposite charge fluxes: a charge transfer from the Au–X bondtoward the LUMO of CO2 (Δρ1′)and a charge transfer toward the vacant valence npz orbital of the Group 13 element from the HOMO of CO2 (Δρ2′). We mention that the presenceof these two active sites, namely, the Au–X bond and the npz orbital of X as nucleophilic and electrophiliccenters, respectively, can be also visualized in the plot of the dualdescriptors (nucleophilicity and electrophilicity) for chemical reactivity,introduced by Morell et al.40 .By analyzing the nature of the interactionsoccurring at the three d in ref  for TSI 1′ , in all cases the main acceptor MO is theLUMO of CO2, while the main donor MOs are high-lying σbonding molecular orbitals of the Au–X complex . However, a quantitative inspection of the atomiccomposition of these donor MOs reveals that the donor features ofcomplexes IGa and IIn differ from those of IAl. Indeed, as shown by the data reported in Table S10, while the energy of the MOs whichmainly contribute to the donor NOCV is comparable for all three complexes,their atomic composition varies significantly. The plot and compositionof the main donor MO of IAl  clearly suggest an Au–Al-centeredσ MO . However, the HOMO–1of IGa and HOMO–2 of IIn  are less centeredon Au and Ga and on Au and In ,thus indicating a delocalization on the [(SiNON′)]2– backbone and suggesting possibly less electron-rich(and less nucleophilic) Au–Ga and Au–In bonds. We quantitativelyinspected the extent of the interaction and charge transfer occurringbetween the CO2 and different complexes at INTX by relying on the EDA, ETS-NOCV, and CD-NOCVapproaches. The results are reported in S9 and Figure S14.The decomposition into thedonor and acceptor NOCV orbitals providesa clear picture of the nature of the molecular orbitals (MOs) involvedin the interactions depicted above and points out significant differencesbetween the systems under study. Concerning the NOCV deformation densityΔρIAl over IGa and IIn. A much stronger interaction between IAl and CO2 is observed at INTAl  with respect to IGa and IIn . The same trend canbe noticed for the orbital interaction energy ΔEoi1 and the Au–X bond-to-CO2 charge transfer |CT1| values , in Eoi2 and |CT2| values.These results show that the interaction between Au and the Group 13element does change on descending along the group. The decompositionof NOCVs into MOs presented above suggests that Ga and In may featurea more polarized Auδ+–X δ− bond, which would be consistent with the weaker activation/insertionproduct stabilization ability in a diradical-like reactivity withCO2.9 An appropriate bondinganalysis is needed to quantitatively assess this hypothesis. The resultsare presented in the next section.From a quantitative perspective,data in IX with the CD-NOCV and ETS-NOCV approaches in order to identifythe basic nature of the Au–X in these complexes and the degreeof polarization on descending along the Group 13. This method hasbeen successfully applied to complex I where revealedthe existence of an electron-sharing Au–Al bond. (ii) We assessthe variability in the electronic structure of the [X(SiNON′)] fragments, by relying on the tools of conceptual DFT42 . (iii) To assess the radical-like behavior of complexes IX (if any) and its impact on theformation of the products IIX, we model the reactivity of the radical fragments [X(SiNON′)]· and [tBu3PAu]· withcarbon dioxide.The analysis of thefeatures of the bond between gold and the Group 13 fragments may shedlight into the differences in the reactivity observed above. To thisaim we use a three-pronged approach: (i) We analyze the nature ofthe Au–X bond in complexes tBu3PAu] and [X(SiNON′)] fragments) forthe most accurate description of the bond. Such an assessment, carriedout with the protocol reported in refs ]· since thisfragmentation provides both the smaller orbital interaction and totalinteraction energies. Application of the NOCV-CD approach allows toquantify the differences between the three Au–X bonds, as shownin Table S14 and Figures S16–S21.In order to apply the CD-NOCV and ETS-NOCV approachesin a consistent way it is important to determine the most suitablefragmentation tBu3PAu] tBu3PAu]· and [X(SiNON′)]·fragments does not change upon substitution of the Group 13 elementX. It consists mainly of two components that act in opposite directions:a gold-to-X (Δρ1α′) and an X-to-gold(Δρ1β′) charge transfer of spherical(σ) symmetry. In addition to these, two dative gold-to-X πback-donation components are also envisioned in each case , although their contributionis considerably smaller.From a qualitative perspective, the bond picture between the · fragment,we see indeed that the two fluxes are practically equivalent ,resulting in an overall very small net charge transfer (−0.01e) that can be ascribed to the presence of a slightly polarized electron-sharingAu–Al bond. This picture is also very similar to that of the[tBu3PAu]–[Al(NON′)] bond reportedin ref .SiNON′)]·fragment, while the extent of the Ga-to-Au donation remains practicallyunaltered (0.29 e), the Δρ1α′component related to the Au-to-Ga charge increases (−0.37 e),thus representing a more polarized Auδ+–Gaδ− bond (net charge transfer −0.08 e).For the [tBu3PAu]·–[In(SiNON′)]· bond, the difference is even more pronounced,with a CT1α of −0.42 e for the donation towardindium and a more negative net charge transfer (−0.16 e). Theassociated ΔEoi1α values follow the same trend, with IAl having the less negative value  whichincreases for both IGa and IIn .Forthe bond involving the · and [In(SiNON′)]·are more likely to retain the negative charge with respect to the[Al(SiNON′)]· fragment. For instance, the electrophilicity(ω–) index peaks at the indium fragment (2.58)and descends toward gallium (2.15) and more rapidly toward the [Al(SiNON′)]· fragment (1.68), consistent with theincreased tendency of gallium and indium fragments to accept electronsfrom gold. The nucleophilicity (N) index follows,coherently, the opposite trend, decreasing from Al toward In .The different tendency of Al, Ga, andIn to form electron-sharingtype bonds with Au can be further inferred by relying on the toolsof conceptual DFTSiNON′)]− anions also givesan idea of the different nature of these species.45 On the basis of the gas phase proton affinity and the correspondingHOMO energy and composition properties, the [Al(SiNON′)]− fragment emerges as the most reactive and basic anion]− and[In(SiNON′)]−, respectively, see Table S16). The peculiarity of the [Al(SiNON′)]− lone pair with respect to thoseof [Ga(SiNON′)]− and [In(SiNON′)]− is immediately evident byinspection of the isodensity pictures of the corresponding HOMOs,reported in The evaluation of the strength and basicity of the lone pair ofthe [X(SiNON′)]− HOMO is verydiffuse and mainly centered at the Al site, whereas on descendingtoward Ga and In, the HOMO becomes much less diffuse and more delocalizedon the (SiNON′)2– ligand. TheHOMO energy and nature for these anions are also consistent with thistrend and with the proton affinities trend: the HOMO energy for thealuminyl is the highest (−0.356 eV), whereas gallyl and indylanions have more stabilized HOMOs , thus reflecting the higher basic character of thealuminyl. Consistently, while the aluminyl HOMO has atomic contributionsmostly from s and p orbitals of Al (more than 80%), the gallyl andindyl HOMO contains analogous contributions, but to a much lesserextent (in both cases below 50%), thus indicating more ligand-centeredMOs, coherently with their lower basic power and reactivity (see Table S16). On the basis of these results, twocanonical resonance structures can be drawn for explaining the changingbonding scheme, as depicted in The  complex,9 the electron-sharing character of the Au–Albond was strictly related to the two fragments behaving cooperativelylike radicals for the carbon dioxide insertion, leading to the [tBu3PAuCO2Al(NON′)] product. Sincea similar insertion product has been also calculated for complexes IAl, IGa, and IIn, an analogousgeometric and energetic assessment of the radical-like behavior canbe carried out.At this stage, it isworth investigating if such differences inthe Au–X bond reflect into differences on the radical-likebehavior of the fragments ]· leads to structuresthat are closely reminiscent of the in-adduct structures of the insertionproducts [tBu3PAuCO2X(SiNON′)], with the main structural parameters of the CO2 coordination being quantitatively similar . On the basisof dissociation/association reactions (i–iii) involving theseopen shell species interacting with each other and with carbon dioxide,we were able to quantitatively shed light on the differences betweenthe three Group 13 species, as displayed in From a structural perspective, optimizationof the open shell radical[COSiNON′)]· and [tBu3PAu]· fragments, with the formation of the [CO2X(SiNON′)]· species, points out that the [CO2Ga(SiNON′)]· and [CO2In(SiNON′)]· fragments are less stable with respectto [CO2Al(SiNON′)]·. Indeed, onlyfor X = Al do we observe a negative ΔE forthe exchange , while positive values are calculatedfor X = Ga and In . This isconsistent with the much lower affinity of the Ga and In radical fragmentstoward CO2 , respectively) with respect to Al . Notably, the results for reaction(iii), which depict the favorable formation of the [tBu3PAuCO2]· species  are clearly consistent with the cooperativeradical-like behavior of Al and Au.Calculation of the energies for the exchange reaction(i) betweenthe [X(SiNON′)]· fragmenthas a higher affinity toward CO2, consistent with the exergonicformation of the insertion product and with the spin density of theradical , for which the unpairedelectron is practically entirely localized on Al (0.97). However,the [Ga(SiNON′)]· and [In(SiNON′)]·radicals have a decreased affinity toward CO2, consistentwith a more delocalized spin density  and with the endergonic formation of the correspondinginsertion products. These findings are fully coherent with those recentlyreported for lithium– and zinc–aluminyl complexes.10 Indeed, the latter, featuring a highly covalentand electron-rich Zn–Al bond, has been shown to react withCO2 leading to an insertion product very similar to II and IIX, thus furthercorroborating the importance of electron-rich M–X bonds witha highly electron-sharing character for the reactivity with carbondioxide.These results clearly pointout that while in all the cases theobserved product is in accordance with a diradical-like cooperativereactivity of Au and X the  complexes reaction have been carried out usingthe Amsterdam Density Functional (ADF) code47 in combination with the related Quantum-regions Interconnected byLocal Description (QUILD) program.48 ThePBE49 GGA exchange-correlation (XC) functional,the TZ2P basis set with a small frozen core approximation for allatoms, the ZORA Hamiltonian52 for treating scalar relativisticeffects, and the Grimme’s D3-BJ dispersion correction wereused.54 Solvent effects were modeled employing theConductor-like Screening Model (COSMO) with the default parametersfor toluene as implemented in the ADF code.55 The same computational setup has also been used for the EDA, CD-NOCV,and ASM calculations and for computing the radical reactions between[X(SiNON′)], [CO2], and [tBu3PAu] fragments. Gas-phase calculation of conceptualDFT descriptors and proton affinities have been carried out by excludingsolvent effects from the same computational protocol. This protocolhas been used successfully in refs (tBu3PAuAl(NON)] and[tBu3PAuCO2Al(NON)] complexes. Forfurther details and description of the methods used in this work,see the “Methodology” section in the Supporting Information.All geometry optimizations and frequency calculationson optimizedstructures (minima with zero imaginary frequencies and transitionstates with one imaginary frequency) for the CO