Developing physical models of complex dynamic systems showing emergent behaviour is key to informing on persistence and replication in biology, how living matter emerges from chemistry, and how to design systems with new properties. Herein we report a fully synthetic small molecule system in which a surfactant replicator is formed from two phase-separated reactants using an alkene metathesis catalyst. The replicator self-assembles into aggregates, which catalyse their own formation, and is thermodynamically unstable. Rather than replicating until the reactants are fully consumed, the metastable replicator is depleted in a second metathesis reaction, and closed system equilibrium is eventually reached. Mechanistic experiments suggest phase separation is responsible for both replicator formation and destruction. Understanding self-replication and persistence in an out-of-equilibrium state is key to designing systems with new properties mimicking “living systems”. Here, the authors developed a synthetic small molecule system in which a transient surfactant replicator is responsible for both an autocatalytic aggregation pathway and a destructive pathway. While thermodynamic stability and the Second Law of Thermodynamics drive chemical processes, additional considerations are required for the richer, dynamic behaviour of life-like systems. However, understanding how energy is consumed, used, and dissipated to drive biological assembly and function remains challenging7. A synthetic model of a living system would likely be held out-of-equilibrium by the consumption of energy, and crucially involve self-replication to produce a persistent replicator population which could evolve8. Strategies to develop far-from-equilibrium processes resembling functions in living systems, such as dissipative self-assembly to mimic guanosine triphosphate-driven microtubule assembly, have also recently emerged10. Other dissipative self-assembling structures have also been reported15, including the generation of transient fibres whose formation/destruction occurs concurrently17.Understanding the basic principles and properties that govern living systems is a first step towards the creation of synthetic life. Many have concluded that a combination of standard physical laws and new concepts are required to understand what separates biological and simpler chemical matter18. Artificial self-replication induced by templates has been observed in small molecules capable of recognition20 and the formation of large supramolecular aggregates22 in which the products act as seeds for their own formation. Systems based on physical replication, where aggregates of an amphiphilic product catalyse an interfacial reaction to form their own components24 have been explored from an origins-of-life perspective as this replication mechanism forms compartments which allow for localisation and enclosure of chemical components and reactions. In the cases described so far, the most efficient replicators come to dominate the system to yield either the thermodynamic or kinetically trapped products.Achieving non-biological self-replication alone in small molecule systems is non-trivial and has given rise to several approaches26, which appear to work on similar principles. Simple physical autocatalytic systems are based on bond-formation or bond-cleavage between two phase-separated reactants A and B, one hydrophilic and one hydrophobic, that react across the interface to generate an amphiphilic product C  are resupplied to the reaction mixture in a second or third batch  and transmission electron microscopy (TEM), and the critical micelle concentration (CMC) was determined using an established fluorimetric method . TLC analyses were performed on Merck Kiesegel 60 F2O with a gradient of 5:95 → 95:5 over 5 min was used as mobile phase. Calibration showing linear fitting was obtained  H-Class system with photodiode array (PDA) detector. Instrument control and data processing were performed using Empower software. Acquity UPLC BEH C18 column, 2.1×50 mm with a 1.7 µm size particle was used. A mixture of MeOH:H3a and 3b34. Analyses were performed using an Edinburgh Instruments Spectrofluorometer FS5 model. Instrument control and data processing were performed using Fluoracle software. Measurements were done using an equilibrated heating probe at 60 °C in quartz cuvettes with 3.0 mL of sample solution. Excitation wavelength was 358 nm and emission wavelength was 430 nm as reported in the literature using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent molecule. The CMC for alkenes 3a and 3b can be extracted from the representation of the Emission vs. Concentration (Supplementary Figures An established fluorimetric method was used for the CMC determination of alkenes Analyses were performed using a Malvern Zetasizer Nano ZEN5600 model system recording particle and molecule size. Instrument control and data processing were performed using Zetasizer software. Disposable plastic cuvettes were used with 1.0 mL of sample solution. Three repetitions of ten measurements were done for every concentration, starting from 20 mM with subsequent dilutions. Measurements were done using an equilibrated heating probe at 60 °C, setting the appropriate parameters for water (Supplementary Figures Images were produced using negative staining. An aliquot of 10 µl of sample was applied to freshly glow discharged carbon Formvar 200 mesh copper grids for 2 min, blotted with filter paper and stained with 2% uranyl acetate for 10 s, then blotted and air dried. Grids were imaged in a FEI Tecnai 12 TEM at 120 kV using a Gatan OneView CMOS camera (Supplementary Figures 1H NMR and 13C NMR spectra were recorded on a 400 MHz or 500 MHz spectrometer in CDCl3 or CD3OD and referenced to residual solvent peaks (Supplementary Figures All reported data are available from the authors on request.Supplementary Information


Myxococcus xanthus possesses a form of surface motility powered by the retraction of the type IV pilus (T4P). Additionally, exopolysaccharide (EPS), the major constituent of bacterial biofilms, is required for this T4P-mediated motility in M. xanthus as the putative trigger of T4P retraction. The results here demonstrate that the T4P assembly ATPase PilB functions as an intermediary in the EPS regulatory pathway composed of the T4P upstream of the Dif signaling proteins in M. xanthus. A suppressor screen isolated a pilB mutation that restored EPS production to a T4P− mutant. An additional PilB mutant variant, which is deficient in ATP hydrolysis and T4P assembly, supports EPS production without the T4P, indicating PilB can regulate EPS production independently of its function in T4P assembly. Further analysis confirms that PilB functions downstream of the T4P filament but upstream of the Dif proteins. In vitro studies suggest that the nucleotide-free form of PilB assumes the active signaling conformation in EPS regulation. Since M. xanthus PilB possesses conserved motifs with high affinity for c-di-GMP binding, the findings here suggest that c-di-GMP can regulate both motility and biofilm formation through a single effector in this surface-motile bacterium. Myxococcus xanthus is a motile bacterium well adapted to life on a solid surface. While a unicellular organism, it possesses a social or multicellular life style during both its vegetative and developmental phases2. During vegetative growth, groups of M. xanthus cells swarm over solid surfaces to digest other bacteria and organic macromolecules as nutrients3. Its developmental program, which is triggered by nutrient limitation, involves the aggregation of ~105 cells on surfaces by directed cell movement5; these multicellular aggregates eventually mature into fruiting bodies when vegetative cells within cease to move and differentiate into dormant spores embedded in an exopolysaccharide (EPS) matrix7. When environmental conditions become conducive for growth, spores can germinate to resume proliferation and swarming. Motility is critical to this bacterium because both vegetative predation and developmental aggregation require cells to move on solid surfaces.M. xanthus entails social (S) and adventurous (A) gliding systems8. Mutations inactivating one system rarely affect the other, indicating the existence of two sets of motility machineries11. Phenotypically, A motility is functional even when cells are well isolated, whereas S motility requires cells to be in close proximity or in groups9. There is evidence that traveling motor complexes within cells drive A motility15. The motor for S motility is the type IV pilus (T4P)19. The T4P protein filament, which mostly localizes to one cell pole in M. xanthus at a given time21, is also known to power the related twitching motility in other bacteria23. The distal end of a T4P first attaches to an anchor and its ensuing retraction at the proximal end pulls the cell forward25. In M. xanthus, the preferred anchor for T4P retraction is EPS either associated with the cell surface or deposited on a solid substratum by this bacterium27. This preference explains the “social” aspect of T4P-mediated motility in M. xanthus because a cell requires its EPS-bearing or EPS-depositing neighbors to anchor and activate the retraction of its T4P motor. EPS, which is a key constituent of the matrix for biofilms and for the organization of M. xanthus fruiting bodies7, has been shown recently to affect M. xanthus motility behavior independently of its role as anchors for T4P retraction in S motility28.The surface motility of M. xanthus is regulated by a signal transduction pathway consisting of the Dif chemosensory proteins as well as the T4P machinery (T4PM)31. DifA, DifC and DifE resemble the methyl-accepting chemoreceptor proteins (MCPs), the scaffold CheW, and the histidine kinase CheA, respectively27. These three proteins form a transmembrane (TM) signaling complex that positively regulates EPS production through the kinase DifE33. DifD, a CheY-like substrate of DifE phosphorylation, functions as a phosphate sink to negatively regulate EPS production32. DifG, a homologue of the CheC phosphatase, is a negative regulator that can dephosphorylate DifD-phosphate32. The occurrence of T4P correlates closely with that of EPS31. T4P− mutants are EPS- whereas the hyperpiliated pilT mutant is EPS+. Since mutations in dif are epistatic to those in T4P or pil genes, T4P has been proposed to function as a sensory apparatus that perceives and transmits signals to the Dif proteins downstream. The communication between T4P and Dif is mediated in part by the negative regulator StkA, a DnaK-like protein35 that acts downstream of T4P but upstream of Dif36. Many questions remain concerning the mechanism of EPS regulation by this pathway although the transcription of eps genes does not appear to be the target of this regulation37.The level of EPS in M. xanthus T4P assembly ATPase PilB21 functions in a regulatory capacity in signaling EPS production independently of T4P assembly. Genetic studies uncovered that mutations in pilB can suppress the EPS defects resulting from the deletion of the pilin gene pilA. A mutation known to eliminate the ATPase activity of PilB and its ability to support T4P assembly21 was found to strongly suppress the EPS defect of the pilA deletion strain. This observation indicates that the role of PilB in EPS regulation can be independent of its role as the T4P assembly ATPase. Analysis in vitro suggests that it is the nucleotide-free or the apo form of PilB that actively signals EPS production. Our results support the conclusion that PilB functions in a signaling capacity with dual roles in the regulation of motility and biofilm formation in M. xanthus.We demonstrate here that the M. xanthus, the pil genes encoding the T4P structural proteins were targeted in a genetic screen for suppressors of a pilA deletion (ΔpilA)38. For this suppressor screen, 11 genes in three clusters at the M. xanthus pil locus  genes in one cluster, pilG, pilH and pilI (pilGHI) as well as pilM, pilN, pilO, pilP and pilQ (pilMNOPQ) in two other clusters. We first deleted pilBTC, pilGHI and pilMNOPQ as individual clusters in the WT and the ΔpilA backgrounds. These mutants were confirmed to be defective in both S motility and EPS production  marker carried by the integrative plasmid. The other three, which must have occurred elsewhere42, were not pursued further in this study.The three plasmids were mutagenized in an pilBTC gene cluster from the two EPS+ strains were identified by cloning and DNA sequencing as described in Methods. The same G to A transition mutation in pilB was found in both suppressor strains. This G to A mutation, referred to as pilB* hereafter, occurred at the third position in codon 388 of pilB, resulting in a methionine (M) to isoleucine (I) substitution (M388I)  Fig. . PilB isbox Fig. . Since npilB* mutation was solely responsible for suppression of ΔpilA in the suppressor strains, the pilB M388I mutation was reconstructed by targeted mutagenesis in the plasmid containing the WT pilBTC gene cluster. When this plasmid with the M388I mutation was transformed into the pilABTC quadruple deletion (ΔpilABTC) mutant, the resulting strain had its EPS production restored  in a ΔpilA background is EPS−  ΔpilB mutation can support EPS production in a ΔpilA background. The pilB* mutation is therefore likely gain-of-function (GOF) in EPS regulation instead of WT or LOF. If so, it should be dominant over pilBWT with regard to the EPS phenotype. To test this, a pilBWT/pilB* merodiploid was constructed in ΔpilA background by transforming the pilB*-containing plasmid into a ΔpilA mutant. As shown in Fig. pilB alleles was EPS+, indicating pilB* is dominant over pilBWT and confirming that pilB* is indeed a GOF mutation with regard to EPS regulation.A strain with either the ΔPS− Fig. , indicatpilB* mutation on M. xanthus S motility in an otherwise WT background was examined as well. The pilB* and pilBWT alleles were introduced into a ΔpilB single mutant. The resulting strains were examined for S motility  43, the WA box forms the C-terminal part of an α helix and a loop that connects the helix to a β strand. This strand is part of an antiparallel β sheet that lines one side of this α helix. M388 in the WB box is positioned in a strand in the center of this β sheet. The side chain of this methionine packs tightly against the α helix encompassing WA. Substituting this methionine with a bulkier isoleucine in MxPilB or TtPilB is expected to create clashes that would need to be resolved by altering the relative position between WA and WB. Despite its location in the WB box, the M388I substitution therefore may disturb the structure, orientation or conformation of both WA and WB.The WA and WB boxes of PilB, while separated by 56 amino acids in the primary sequence Fig. , are pac48. Although the M388I mutation apparently does not eliminate the ATPase activity of MxPilB, it may still affect its ATP binding and/or ATPase activity. To test whether the ATPase activity of PilB has an effect on EPS regulation, we constructed mutations known to have more drastic effect on the ATPase activity of PilB. The strictly conserved residues K327 in WA and E391 in WB  and E391A (pilBWB) substitution mutations, respectively, and confirmed that neither mutation supported M. xanthus S motility. As shown in Fig. pilBWA but not pilBWB restored EPS production to a ΔpilA mutant. pilBWA is in fact a more robust suppressor than pilB* because its presence in the ΔpilAB background results in a higher EPS level when compared to pilB* . We henceforth expressed and purified the T. thermophilus PilBWT, PilBWA and PilBWB equivalents  around 64–65 °C and 79–81 °C, respectively. In contrast, PilBWA unfolded over a broader temperature range with an apparent Tm around 76 °C. The obvious differences in thermal stability indicate that the structure of PilBWA, but not that of PilBWB, is significantly altered in comparison with PilBWT. We also examined the effect of AMP-PNP, a non-hydrolyzable ATP analogue54, on PilB thermal stability , which measures the thermal denauration of a protein over a temperature gradientity Fig. . This nu55, the results indicate that the secondary structure content is unaffected by the mutations in PilBWA and PilBWB or by ligand binding. In the near UV range (260–320 nm), changes in the CD spectra are related to protein conformation55. The signals in the 260–285 nm range are contributed primarily by phenylalanine and tyrosine residues55, which the TtPilB contains 14 each  spectra of these protein variants were collect to further explore their structural differences. In the far ultraviolet (UV) range (200–240 nm), all three proteins showed similar spectra with or without ATP or ADP Fig. . As the ach Fig. . There iach Fig.  and S3A.56. The fluorescence of this nucleotide is enhanced by a more hydrophobic environment frequently associated with its binding to a protein. Figure d) for PilBWT and PilBWB were similar with values of 0.17 μM and 0.19 μM, respectively. The Kd for PilBWA is 1.23 μM and this increase in Kd represents a significant reduction in its nucleotide binding affinity. These results suggest that the diminished response of PilBWA to nucleotides is attributed to its reduced affinity for its ligand. The in vitro studies 59 are the two other cases where ATPases have been proposed as signaling proteins. XB24 is a small cytoplasmic ATPase in rice with a role in immunity and defense against bacterial pathogens. It physically associates with and modulates the activity of the kinase XA21, which is a receptor for recognition of pathogen-associated molecular patterns (PAMPs). The ATPase activity of XB24 is required for its function in vivo and it is proposed that the signaling activity of XA21 is regulated by XB24. NKA is ubiquitous in the plasma membrane of all animal cells. It establishes and maintains the ion homeostasis essential for cell viability. All NKAs contain a binding site specific for steroid inhibitors such as ouabain and digoxin. There is evidence that NKAs can associate with and affect the activity of signaling proteins such as the Src kinase61. It has been proposed that NKAs are receptors and signal transducers to regulate downstream targets in a signal transduction pathway.As far as we are aware, XB2463 and signaling ATPases58, we hypothesized initially that it was the PilB in the ATP bound form that actively signaled EPS production. This was tested by mutating E391 in WB, a key catalytic residue for ATP hydrolysis48  genes, binds c-di-GMP with high affinity. FlrA/FliQ is the master regulator of flagellar genes and the binding of c-di-GMP impairs its ability to activate the transcription of the flagellar operon.The regulation of EPS is the key aspect of bacterial biofilm formationV. cholerae MshE, the PilB equivalent in the Msh pilus system70. Sequence alignment with MshE shows convincingly that PilB from M. xanthus and many other T4P systems contain this c-di-GMP binding motif at their N-termini  and Rosetta (Novagen), the Escherichia coli strains used for plasmid construction and protein expression, were grown and maintained at 37 °C on Luria-Bertani (LB) agar plates or in LB liquid medium75. Unless noted otherwise, plates contained 1.5% agar. Kanamycin and ampicillin at 100 μg/ml and oxytetracycline at 15 μg/ml were added to media for selection when appropriate.M. xanthus strains. The first set were constructs to delete single or multiple pil genes; these include pWB525 (ΔpilA), pWB581 (ΔpilB), pWB555 (ΔpilBTC), pWB556 (ΔpilAGHI), pWB605 (ΔpilGHI) and pWB557 (ΔpilMNOPQ). The second were for the expression of pil gene clusters and pilB alleles in M. xanthus; these constructs, which integrate at Mx8 att site76, include pWB559 (pilGHI), pWB565 (pilMNOPQ) and pWB566 (pilBTC) as well as pWB571 (pilB), pWB572 (pilB*), pGD5 (pilBWA) and pGD6 (pilBWB). The third includes the plasmid pWB606, which was used for the replacement of wild-type pilB with pilB*. The first and third sets were derivatives of pBJ11377. The second set used pWB425 as the cloning and M. xanthus expression vector39. pWB425 contains a BspHI/ApoI fragment from pZero-2 (Invitrogen) with the kanamycin resistance (KanR) gene and its promoter. The expression of all pil genes cloned into pWB425 is driven by this promoter because the multicloning site is immediately downstream of the KanR gene in this plasmid.Three sets of plasmids were generated to construct M. xanthus strain construction are described here. Fragments with in-frame deletion alleles of pilA, pilBTC, pilGHI, pilAGHI and pilMNOPQ were generated by a two-step overlap PCR as described previously29. These fragments were cloned into pBJ11377 to construct pWB525 (ΔpilA), pWB555 (ΔpilBTC), pWB556 (ΔpilAGHI), pWB605 (ΔpilGHI) and pWB557 (ΔpilMNOPQ). In these plasmids, the ΔpilA allele deleted the codons from 7 to 218 of pilA, ΔpilBTC from 8 of pilB to 409 of pilC, ΔpilAGHI from 7 of pilA to 250 of pilI, ΔpilGHI from 5 of pilG to 250 of pilI and ΔpilMNOPQ from 7 of pilM to 896 of pilQ, respectively. The pilB deletion allele from DK1041618 was PCR amplified and cloned into pBJ113 to produce pWB581. For the allelic exchange of wild-type pilB with pilB*, the fragment from pWB572 (see below) was cloned into pBJ113 to create pWB606.The details for the construction of plasmids used for M. xanthus chromosome at Mx8 phage attachment site (att)78 for ectopic expression were constructed using pWB42539 as the vector. pWB559, pWB565 and pWB566, which contain the respective pilGHI, pilMNOPQ and pilBTC gene clusters from pDW7979 or pSWU25717 in pWB425, were also used for mutagenesis in E. coli. With reference to the coding regions  contains DNA from 73 bp upstream of pilG to 328 bp downstream of pilI, pWB565 (pilMNOPQ) from 120 bp upstream of pilM to 19 bp downstream of pilQ, and pWB566 (pilBTC) from 22 bp upstream of pilB to 5 bp downstream of pilC. In addition, pWB571 (pilB), which contains from 22 bp upstream to 142 bp downstream of pilB, was derived from pWB566. This was achieved by digestion with SacI (site in pilT) and BamHI (site on vector), treatment with T4 DNA polymerase and religation; this removed pilC and the bulk of pilT. pWB572 (pilB*), pGD5 (pilBWA) and pGD6 (pilBWB) were constructed using pWB571 as a template by a two-step overlap PCR using primers containing the M388I (pilB*), K327A (pilBWA) and E391A (pilBWB) mutations.Plasmids that can integrate into ons Fig. , pWB559 M. xanthus strains constructed in this study. For the construction of the pil deletions and the pilB* chromosomal replacement mutant, the plasmids with these mutant alleles were electroporated into DK1622 and used for allelic exchange as previously described81. These strains are YZ690 (ΔpilA), YZ1636 (ΔpilBTC), YZ1637 (ΔpilMNOPQ), YZ1639 (ΔpilAGHI), YZ1865 (ΔpilGHI) and YZ1875 (pilB*). The deletion strains YZ1638 (ΔpilBTC ΔpilA), YZ1640 (ΔpilMNOPQ ΔpilA) and YZ1682 (ΔpilB ΔpilA) were constructed similarly using YZ690 (ΔpilA) as the parent.YZ690 through YZ1888 in Table pilBTC att::pilBTC), YZ1645 (ΔpilMNOPQ att::pilMNOPQ) and YZ1870 (ΔpilGHI att::pilGHI). Other strains similarly constructed are YZ1504 (ΔpilB ΔpilA att::pilBWA), YZ1505 (ΔpilB ΔpilA att::pilBWB), YZ1849 (ΔpilB ΔpilA att::pilB) and YZ1850 (ΔpilB ΔpilA att::pilB*) as well as YZ1512 (ΔpilB att::pilBWA), YZ1522 (ΔpilB att::pilB*), YZ1548 (ΔpilB att::pilBWB) and YZ1674 (ΔpilB att::pilBWB). The pilB merodiploid strain YZ1521 (ΔpilA att::pilB*) was constructed by transforming pWB572 into YZ690 (ΔpilA).The plasmids constructed with pWB425 as the vector were electroporated into the appropriate deletion strains to construct YZ1644 (ΔpilB stkA::tetR) and YZ1887 (pilB* difE::kanR) were constructed by transferring the stkA::tetR allele in LS1102 to DK10416 (ΔpilB) and the difE::kanR allele in SW501 to YZ1875 (pilB*) by genomic transformation41, respectively. pWB581 (ΔpilB) was used to replace pilBWT with ΔpilB in YZ641 (ΔdifD ΔdifG) to construct YZ1888 (ΔpilB ΔdifD ΔdifG).YZ1879 35. Genomic DNA from potential suppressor mutants was electroporated into the original parental strain and selected on plates with kanamycin and Congo red to examine the link of the EPS phenotype with the integrated plasmid41. Genomic DNA from suppressor mutants was cut with PstI, religation and transformation into E. coli XL1-Blue to recover the plasmid and pil mutations were identified by DNA sequencing.pWB559, pWB565 and pWB566 were mutagenized by propagation in NR9458, an 4) at 5 × 109 cells/ml. 5 µl of this cell suspension was spotted onto CYE plates with 0.4% agar and regular CYE plates with Calcofluor white (50 µg/ml) for S-motility and EPS analysis, respectively. Plates were incubated at 32 °C for 5 days before documentation under white light for S motility and 365 nm UV light for EPS production.Log phase cells grown in CYE were harvested and resuspended in MOPS buffer . The appropriate expression strains were grown at 35 °C to an OD600 of 0.5–0.6 in 1 liter of LB plus ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Protein expression was induced by addition of IPTG  to a final concentration of 0.1 mM, followed by incubation at 30 °C for 4–5 hrs. Please see SI Materials and Methods for more details for the purification.Plasmids pWB750, pWB751 and pWB752  were constructed and maintained in the 2, 0.5 mM EDTA, 1 mM β-ME and 10% glycerol) using a 5× stock buffer. Sypro Orange  from a 5,000× stock (Invitrogen) was diluted to 50× in deionized water and used at a 5× concentration in DSF assays. For examining the effects of ligand, adenosine 5′-triphosphate (AMP-PNP) (Sigma-Aldrich) was added to a final concentration of 0.4 mM. The experiment was carried out using a Bio-Rad CSX96 Real-Time System (Bio-Rad), starting at 25 °C with temperature increments of 0.5 °C to a final temperature of 99 °C. Samples were held at each temperature for 30 sec prior to measurement of fluorescence. Data were analyzed and Tm’s were obtained using Bio-Rad’s CFX Manager software. The data points starting at 45 °C were presented in this paper.Proteins were diluted to a final concentration of 8 μM in 1× stock buffer .Protein samples were adjusted to a final concentration of about 6 µM for far UV (200–260 nm) by diluting in storage buffer. For near UV (260–320 nm), proteins were concentrating using Amicon stirred cells to 230 µM and 170 µM for the experiment with ATP and ADP, respectively. CD spectra were generated on a Jasco J-815 Spectropolarimeter equipped with the Jasco PFD-425S temperature-control unit in a 1-mm path-length quartz cell at 25 °C. Each spectrum was from three accumulated scans with a 1 second response time at a scan speed of 100 nm per minute. Scans were performed with a bandwidth of 1 nm in far UV and at 0.5 nm increments in near UV. Data was recorded using Spectra Manager software (Jasco). For examining the effects of ligand, ATP and ADP (Sigma-Aldrich) was added at a final concentration of 0.1 mM. Data was plotted after background subtraction of identical scans of controls without the protein. ATP and ADP were used in this experiment because these max * [MANT-ATP]/(Kd + [MANT-ATP]) where ΔFmax is the maximum ΔF and Kd is the dissociation constant. Curve fitting and determination of Kd values were performed using XLfit (ID Business Solutions).Binding of MANT-ATP [2′-/3′-O-(N-methylanthraniloyl) adenosine-5′-triphosphate] (Invitrogen) was monitored by measurement of fluorescent intensity using an Infinite M200 microplate reader (Tecan). The excitation was set to 356 nm and fluorescence emission was recorded at 448 nm. Samples with MANT-ATP at specified concentrations (0.0012 to 10 μM) were incubated with or without the protein at 0.2 μM at room temperature for 2 hours in binding buffer  prior to fluorescence measurements. The difference in fluorescence (ΔF) with and without the protein at different MANT-ATP concentrations ([MANT-ATP]) was fitted to the binding isotherm ΔF = ΔFSupplemental Information


This work reports on the evolution law of helium bubbles in Hastelloy N alloy on post-irradiation annealing conditions. After helium ion irradiation at room temperature and subsequent annealing at 600 °C (1 h), the transmission electron microscopy (TEM) micrograph indicates the presence of helium bubbles with size of 2 nm in the depth range of 0–300 nm. As for the sample further annealed at 850 °C (5 h), on one hand, a “Denuded Zone” (0–38 nm) with rare helium bubbles forms due to the decreased helium concentration. On the other hand, the “Ripening Zone” (38–108 nm) and “Coalescence Zone” (108–350 nm) with huge differences in size and separation of helium bubbles, caused by different coarsening rates, are observed. The mechanisms of “Ostwald ripening” and “migration and coalescence”, experimentally proved in this work, may explain these observations. Nickel-based Hastelloy N alloy has been widely accepted as a promising structural material candidate for molten salt reactor (MSR) systems, due to their excellent corrosion resistance to fluoride salts and high-temperature mechanical properties ,2. HowevIn this work, the evolution law of helium bubbles in Hastelloy N alloy on post-irradiation annealing conditions has been investigated. Elastic recoil detection analysis (ERDA) was used to probe the diffusion of helium atoms under different annealing conditions. In addition, the evolution of the helium bubbles was characterized using transmission electron microscopy (TEM). A series of small and large bubbles distributed in different depths were observed when annealing at 850 °C. The presence of a distinct critical depth provides obvious evidence for the operation of different mechanisms related to the coarsening of helium bubbles. Moreover, the coarsening mechanisms (rate) driven by the “Ostwald ripening” and “migration and coalescence” with the evolution of bubble pressure are also discussed.+ ions at room temperature using a 400 kV ion implanter located at Beijing Normal University to an irradiation dose of 2 × 1016 ions/cm2. The irradiated samples were annealed at 600 °C for 1 h, and then some of them continued to be annealed at an elevated temperature of 850 °C for 5 h in a vacuum greater than 10−4 Pa.Hastelloy N alloy was provided by Haynes International Company with the chemical composition of Ni , 17.1 wt. % Mo, 7.1 wt. % Cr, and 4.2 wt. % Fe . The bulk material was firstly cut into 1 mm-thick sheets (6 mm × 8 mm), and they were then successively mechanically polished with silicon carbide paper and diamond polishing paste. After the vibratory polishing, these sheets were then ultrasonically cleaned with a solution of 50% acetone and 50% absolute ethyl alcohol. The prepared samples were irradiated with 30 keV HeThe ERDA technique was used to obtain the quantitative depth profiles of helium concentration in these samples before and after annealing. The systemic error of the amounts of helium determined by the ERDA experiments was within 5%. All ERD spectra were converted to helium concentration distribution by Alegria 1.2 code . The thenbubble) obtained by counting bubbles in each 25 nm width sub-region, damage depth profile (dpa), and ratio of vacancy to helium (vac./He) are also given in this figure. In the case of annealing at 600 °C (1 h), a high number density of nano-scale white dots with almost the same 2 nm size were observed in the region of 0–300 nm  . Therefon nickel ) and r aAccording to the “Ostwald ripening” mechanism, a decreased pressure within the bubble decreases the self-diffusion energy, which enhances the bubble migration in the matrix as well as in the bubble–matrix interface; thus, helium bubble coarsening rates will increase with decreasing pressure in bubble. As for the “migration and coalescence” mechanism, the coarsening rate will initially increase to a maximum and then decrease with decreasing pressure in bubble, since too high (too low) pressure within a bubble would increase the dissociation energy of vacancy (helium atom) dissociating from a bubble. Moreover, the calculation by P. J. Goodhew shows that the coarsening rate of “Ostwald ripening” is several orders of magnitude higher than that of “migration and coalescence” . On the In this work, helium ion irradiation of Hastelloy N alloy with subsequent annealing was performed to investigate the evolution law of helium bubbles using ERDA and TEM. The schematic of the coarsening mechanisms (rate) with the evolution of bubble pressure was given. On this basis, the presence of a “Ripening Zone” and a “Coalescence Zone” related to the helium bubbles after high temperature annealing were believed to be mainly due to coarsening driven by “Ostwald ripening” and “migration and coalescence”, respectively. Finally, the helium bubble coarsening mechanisms based on the calculation using growth rate equation was confirmed by the experimental work in this study.


Our simulations reveal that at room temperature AgI undergoes an adiabatic temperature shift of 38 K under a biaxial stress of 1 GPa. Likewise, Li3OCl displays a cooling capacity of 9 K under similar mechanical conditions although at a considerably higher temperature. We also show that ionic vacancies have a detrimental effect on the cooling performance of superionic thin films. Our findings should motivate experimental mechanocaloric searches in a wide variety of already known superionic materials.Solid-state cooling is an energy-efficient and scalable refrigeration technology that exploits the adiabatic variation of a crystalline order parameter under an external field . The mechanocaloric effect bears one of the greatest cooling potentials in terms of energy efficiency owing to its large available latent heat. Here we show that giant mechanocaloric effects occur in thin films of well-known families of fast-ion conductors, namely Li-rich (Li Mechanocaloric effects are a promising path towards solid-state cooling. Here the authors perform atomistic simulations on the well-known fast-ion conductor silver iodide and computationally predict a sizeable mechanocaloric effect under biaxial strain. Superelastic shape-memory alloys (SMA) displaying first-order martensitic transformations between a high-T martensitic and a low-T austenitic phase , are archetypal MC compounds2. For example, a giant adiabatic temperature shift of 25.5 K has been measured in NiTi wires upon a small tensile stress of 0.65 GPa3. Nevertheless, there are still several hurdles that need to be overcome in order to develop successful MC commercial applications. For instance, the first-order nature of the martensitic transformation involves concomitant hysteresis losses, which is detrimental for cooling efficiency4, and as the size of SMA shrinks towards the nanoscale the martensitic phase transformation may be suppressed due to overstabilisation of the high-T distorted phase5. Finding novel MC materials with sharp second-order phase transitions occurring near room temperature and which persist down to the nanoscale, therefore, may advance the field of solid-state cooling.Conventional cooling methods based on gas-compression cycles present a series of critical drawbacks including the use of environmental hazards and lack of scalability to micro sizes. Solid-state cooling represents an elegant solution to all these issues, with mechanocaloric (MC) effects possibly holding the greatest promise in terms of energy efficiency36, PbTiO37, and Bax1−CaxTiy1−ZryO3 solid solutions8 , can be synthesised as nano-sized materials and typically exhibit displacive second-order phase transitions9. However, current adiabatic temperature shifts, 4)2SO410), and the involved mechanical stresses appear to be unsuitably too large 2SO410). Recently, a giant MC effect has been predicted in fluorite-structured fast-ion conductors (FIC), typified by CaF2 and PbF2, which is comparable in magnitude to the benchmark adiabatic temperature shifts measured in SMA11. The MC effect disclosed in FIC may be understood in terms of stress-driven changes in ionic diffusivity, which in turn cause large variations in the entropy and dimensions of the material11. The originating superionic transition is of second-order type, in analogy to archetypal FE, and fluorite-structured FIC also can be synthesised as nanomaterials12. Unfortunately, the superionic transition temperatures in fluorite-structured FIC are far above ambient conditions , thus hindering the development of likely solid-state cooling applications.Ferroelectric compounds (FE) like, for instance, BaTiO3OCl) and type-I (AgI) compounds (following Hull’s notation14), some of them at room temperature. We use atomistic computer simulations based on force fields and density functional theory to estimate the isothermal entropy and adiabatic temperature shifts attained in FIC with biaxial stresses (σxx = σyy and σzz = 0).  In AgI thin films, we find a room-temperature adiabatic temperature shift of T = 300 K (~1 K6) and equals in magnitude the MC records set in SMA (~10 K20). Our findings in Li3OCl thin films, namely T = 1000 K, suggest that analogous Li-rich FIC with lower superionic transition temperatures  should display giant MC effects as well. Therefore, we argue that solid-state cooling could benefit immensely from the intensive research already undertaken on solid-state electrochemical batteries22.Here we demonstrate giant MC effects in other predominant families of FIC, namely Li-rich , calculated for unstrained CaF2 (type-II FIC14) in the absence and presence of ionic vacancies as a function of temperature (note that D increases by >80% in the system containing vacancies). In Fig. cv = 2.5% case, Ts is reduced by ~300 K almost independently of σ. This outcome can be rationalised in terms of a steady lowering of the energy barrier and creation energy of Frenkel pair defects , calculated in type-II FIC thin films with an arbitrary F− vacancy concentration of 2.5%. Large values of 30 JK−1 kg−1 and −30 K are obtained respectively at the highest temperature and tensile stress  renders a transition temperature of Ts = 1000 K at σ = 0 conditions. In what follows, we focus on tensile stresses (σ < 0) as in Li3OCl these favor ionic conductivity the most.We choose Limentclass2pt{minim+ diffusion coefficient and in-plane strain, 3OCl as a function of negative biaxial stress at a fixed temperature of 1000 K. Both quantities increase under applied tensile stress, and in the case of S = 16 J·K−1 kg−1 at the maximum tensile stress σaxm = −1 GPa. The accompanying adiabatic temperature change is 2 thin films considering identical  conditions and vacancy concentration . Actually, larger Cσ, is small and depends on temperature as ∝ T3, see Methods section).The largest contribution to the entropy of the superionic transformation can be assumed to be due to the configurational degrees of freedomβ, hexagonal P63mc) and zincblende  in which the I− anions arrange in a cubic bcc lattice and the Ag+ cations are mobile. In practice, AgI thin films can be synthesised either in the β or γ phase depending on the employed preparation method30. For reasons that will become clear later on, we investigate here the cubic γ phase consisting of two interlaced monoatomic fcc sublattices with fourfold ionic coordination  as we find the largest MC effect at such conditions.We select AgI as a representative member of the family of type-I FIC. At ambient conditions bulk AgI is found as a mixture of wurtzite , is not possible for distances larger than few angstroms at biaxial stresses higher than σc  ≈ 1, is reached very rapidly with the radial distance . As compared to ferroelectric materials, the estimated ΔT is several times larger in absolute value. In terms of mechanocaloric strength, AgI also shows great promise as it follows closely to shape-memory alloys (−120) and outperforms perovskite oxide materials .The giant room-temperature MC effect revealed in AgI thin films, which is originated by a mentclass2pt{minim11. In this context, the rich variety of superionic materials that have been already investigated with a focus on electrochemical applications grants solid-state cooling with vast new possibilities.Our findings should stimulate the development of new cooling devices based on FIC whose energy efficiency as compared to magnetocaloric and electrocaloric materials is very auspicious. In contrast to other mechanocaloric materials driven by first-order transitions, mechanical hysteresis losses and scalability limitations towards nanosizes should be absent in FIC. Mechanical stresses other than biaxial  also can be expected to produce similar mechanocaloric responses than reported here in FICN, P, T) simulations are performed with the LAMMPS code31. The pressure and temperature in the system are kept fluctuating around a set-point value by using thermostatting and barostatting techniques in which some dynamic variables are coupled to the particle velocities and simulation box dimensions. The interactions between atoms are modeled with rigid-ion Born-Mayer-Huggins potentials. Large simulation boxes, typically containing 6,144 atoms, are used in which periodic boundary conditions along the three Cartesian directions are applied. Newton’s equations of motion are integrated using the customary Verlet’s algorithm with a time-step length of 10−3 ps. A particle-particle particle-mesh k-space solver is used to compute long-range van der Waals and Coulomb interactions and forces beyond a cut-off distance of 12 Å at each time step.Molecular dynamics  can be found in the 2, Li3OCl, and AgI thin films at zero temperature  is the position of the migrating ion labelled as i at time t, t0 an arbitrary time origin, and The ionic diffusion coefficients are determined as:xx and yy and stresses σxx and σyy are identical. Consequently, the accompanying isothermal entropy changes can be estimated with the formula:V0(T) ≡ Lx,0(T) · Ly,0(T) · Lz,0(T) is the T-dependent volume of the crystal at equilibrium , Li represents the length of the simulation box along the i Cartesian direction, and the mechanical strain components are defined as Cσ is the heat capacity of the crystal calculated at fixed σ. In this study we assumed that Cσ ≈ Cσ. Further technical details on our calculations can be found in the Owing to the cubic symmetry of the thin films considered in this study, strains The data that support the findings of this study are available from the corresponding author (C.C.) upon reasonable request.Supplementary InformationPeer Review File


Carcharodon carcharias) are migratory, apex predators, which have undergone significant population declines across their range. Understanding their ecology is key to ensuring that management strategies are effective. Here, we carry out the first stable isotope analyses of free-swimming white sharks in South Africa. Biopsies were collected in Gansbaai  between February and July 2015. We used Stable Isotope Bayesian Ellipsis in R and traditional statistical analyses to quantify and compare isotopic niches of male and female sharks of two size classes, and analyse relationships between isotopic values and shark length. Our results reveal cryptic trophic differences between the sexes and life stages. Males, but not females, were inferred to feed in more offshore or westerly habitats as they grow larger, and only males exhibited evidence of an ontogenetic niche shift. Lack of relationship between δ13C, δ15N and female shark length may be caused by females exhibiting multiple migration and foraging strategies, and a greater propensity to travel further north. Sharks < 3 m had much wider, and more diverse niches than sharks > 3 m, drivers of which may include individual dietary specialisation and temporal factors. The differences in migratory and foraging behaviour between sexes, life stages, and individuals will affect their exposure to anthropogenic threats, and should be considered in management strategies.Demographic differences in resource use are key components of population and species ecology across the animal kingdom. White sharks ( These aggregations are not genetically distinct . Finn Larsen Ceta darts (4 × 0.9 cm) affixed to a biopsy pole were used to excise cores of tissue, comprising muscle and dermis, from the dorsal surface of free-swimming sharks. Samples were stored immediately in ethanol.Tissue biopsy samples were collected from white sharks between February and July 2015 within the designated white shark cage-diving area in Gansbaai, South Africa. Collection took place from either a 9 m research catamaran or a 14 m custom-built shark cage-diving catamaran, owned and operated by the Dyer Island Conservation Trust and Marine Dynamics Shark Tours. Sharks were brought close to the vessels using fish oil chum and a salmon head bait lure. Photographs were taken for individual identification based on distinguishing marks and DARWIN dorsal fin ID software  − 1] × 1000, where X is 13C or 15N and R is the corresponding ratio 13C/12C or 15N/14N, and Rstandard is the ratio of the international references PDB for carbon and AIR for nitrogen. Replicate analyses of internal lab standard alanine yielded standard deviations of 0.15‰ for δ15N and 0.09‰ for δ13C. δ13C and δ15N data were averaged between the two analytical runs and tested for outliers using the package ‘Outliers’ in R statistical software version 3.3.1., which was used for all analyses  and sex, respectively. Models specified a Gaussian distribution and identity link function, and all two-way interactions were included in the full models. Backward stepwise elimination of variables, using Akaike Information Criterion (AIC)  were used to assess the relationship between outlier-removed δ) Akaike , and vars Hennig .13C and δ15N were plotted in SIBER, and values of niche size produced from estimates of small sample size corrected standard ellipse areas (SEAc) and total area (TA) of convex hulls. Bayesian estimates of standard ellipse area were generated using 10,000 repetitions and the probabilities of each demographic group (“Group A”) being smaller than the other demographic groups in turn (“Group B”) were calculated and plotted with 50, 75 and 95% credible intervals. Layman metrics were computed for each group, providing values of nitrogen range (NR), carbon range (CR), mean distance to centroid (CD), mean nearest neighbour distance (MNND), and the standard deviation of MNND (SDMNND)  were identified, resulting in 24 samples being included in GLM analyses, and 22 included in SIBER analyses. δ13C and δ15N values were significantly related , but there was a significant interaction between the effects of shark sex and length on δ13C . There was no relationship between δ13C and female length , and the variances of the data also did not differ between males and females for either δ13C or δ15N . The pamk function revealed that paired δ13C and δ15N data split optimally into three clusters for the averaged data, heavily influenced by the inclusion of outliers  and reef manta ray  prey species (Smale Females did not exhibit the relationships between length and δes Smale , b. Femaes Smale , or the 13C values of kelp could also partially explain the variation in SIBER niches between juveniles and larger sharks as juveniles make comparatively more use of coastal habitat as opposed to the pelagic or tropical habitats utilised by larger individuals (Cliff et al. 15N values found in Christiansen et al. (While we found overlap between isotopic niches of all demographic groups, similar to other South African white shark diet studies, we also found evidence of expanded and diverse niches in juvenile sharks in comparison to those > 3 m (Cliff et al. n et al.  and thisOur results, combined with multifaceted evidence of individual and sexual variation in diet, movement, and foraging strategies in South Africa and globally, suggest that that sex and individual specialisation are key drivers in ecological variation in white sharks, which remain important through ontogeny (Estrada et al.


Measuring at ~30 nm, a fully customizable holliday junction DNA nanoconstruct, was designed to simultaneously carry three unmodified SiRNA strands for apoptosis gene knockout in cancer cells without any assistance from commercial transfection kits. In brief, a holliday junction structure was intelligently designed to present one arm with a cell targeting aptamer (AS1411) while the remaining three arms to carry different SiRNA strands by means of DNA/RNA duplex for inducing apoptosis in cancer cells. By carrying the three SiRNA strands  into triple negative breast MDA-MB-231 cancer cells, cell number had reduced by up to ~82% within 24 hours solely from one single administration of 32 picomoles. In the immunoblotting studies, up-elevation of phosphorylated p53 was observed for more than 8 hours while the three genes of interest were suppressed by nearly half by the 4-hour mark upon administration. Furthermore, we were able to demonstrate high cell selectivity of the nanoconstruct and did not exhibit usual morphological stress induced from liposomal-based transfection agents. To the best of the authors’ knowledge, this system represents the first of its kind in current literature utilizing a short and highly customizable holliday DNA junction to carry SiRNA for apoptosis studies. This is largely due to the wide choices available in innovative molecular/drug designs, ranging from nanoparticles6 to drug-antibody conjugates7. Yet not a single system can be truly beyond reproach9. Of the many novelties advocated in literature, delivering RNA interference (RNAi) remains a strong contender for treating diseases at a cellular level by means of repressing and shutting down disease-causing genetic anomalies13. While RNAi technology currently represents the forefront in gene suppression from an academic standpoint, it does suffer from some drawbacks as well. High degradation rate within the cytoplasmic environment14 as well as the requirement for high dosage16 had drawn much criticism from the scientific community. Chemically modified RNA may improve the circulation longevity15 but is highly susceptible towards immunogenic responses18. Hence, the plasmid derivatives in the form of ShRNA (short hairpin RNA) are often promoted in its place for the maintenance of interfering RNA levels19. Nevertheless, the main problem remains in the selection of the most appropriate delivery mechanism into the cell. The most common mode of deliverance is via liposomal-based technology but the issues of cytotoxicity21 as well as the lack in cell-specificity were serious enough to impede its development as a viable clinical option. While handling issues pertaining to cytotoxicity remains a tricky and daunting task, gaining high cell specificity is comparatively more straightforward. Many groups in the past had tried to covalently conjugate antibodies directly to liposomes24 but the problems of immunogenic responses towards the antibodies had plagued these hybrid systems right from the start26. This had subsequently regressed antibodies-liposome hybrids (coined “immunoliposomes”) to the role of useful in-vitro tools. In contrast, DNA aptamers, with its lower level of immunogenicity27 as well as being more economically viable compared to antibodies, are often proposed as another alternative for cell targeting.Some of the quintessential characteristics of any customizable therapeutics should be non-immunogenic, low toxicity as well as high tissue specificity31. Indeed, synergizing both aptamer and RNAi had already gained much footing in literature and reports had already shown considerable success in recent years34. Advantages of using aptamers over antibodies are that they are usually inexpensive and have a higher shelf life compared to antibodies35. They can be easily tailor-made through SELEX enrichment procedures and are more thermally stable. Compared to liposomal-based delivery36, aptamers do not require any additional preparation steps other than purification prior to administration to cells and they do not typically induce any of the cytotoxicity compared to liposomal delivery37.DNA aptamer are short strands of DNA that can readily self-hybridized with itself to present important tertiary structures. They can serve to bind to cell surface receptors and ultimately gaining entry into cell targets with high specificity38. These bioconjugated DNA/RNA nanocomplexes were then administered directly to the designated cell, and gene suppression was subsequently measured. However, the process of bioconjugation can be technically difficult in untrained hands, and RNA chimeras may suffer from potential immunogenic responses. Coupled by the fact that many of these systems only dealt with a single gene target, this may not be as efficient compared to multiple gene targets being singled out simultaneously. It was with these thoughts that we proposed this DNA/RNA nanoconstruct that was designed to simultaneously carry multiple copies of antisense RNA strands into cells. Borrowing from the concepts of the self-assembled holliday junction as demonstrated by Li et al. delivering fluorophores39 as well as complex DNA/RNA machinery by Lee et al.40, we assembled a four-armed nanoconstruct in a single pot fashion and an aptamer (AS1411) was selected to anneal towards one of the arms via a sequence specific ‘sticky’ end. The remaining three arms comprised of overhanging sequences that were complementary to its individual antisense RNA sequences and the respective RNA strands were then hybridized to these selective regions via DNA/RNA hybrid duplex.Much of the current research has reported the use of aptamers to deliver single antisense RNA (double stranded) and most often involved covalent conjugation of the ends of the DNA aptamer directly to the functional end of the RNA or as “chimeras”In short, all the strands were introduced into a single PCR tube and placed in a thermocycler and the temperature slowly reduced from 95 °C to 4 °C in order to self-assemble into its final holliday configuration as shown in Fig. et al.41. One other important consideration was the potential cleavage by cellular Ribonuclease H1 of the DNA/RNA duplex within the cell cytoplasm. To mediate this, the system was designed in such a way that the 5′ end for all three RNA strands (19 bases) was approximated less than 5 nm relative to the center of holliday junction. Molecular dynamics simulation on the cellular ribonuclease H1 interaction with DNA/RNA hybrid strand revealed that the binding process would require a surface area of 177 nm2 on site42. But it is essential that the DNA region to be free of any structured loops (such as hairpin loops etc) in order to accommodate the binding domain of the ribonuclease43, which would not be in our case due to the immediate flanking of the RNA’s 5′ end with DNA. The key recognition of the binding domain for Ribonuclease H1 to the width of the minor groove had been reported to be around 7.5 Å44 and is highly intolerant towards other dimensions. The holliday junction DNA right next to the 5′ RNA/DNA duplex antisense strand would distort and rigidize the backbone of the DNA/RNA which could offer resistance towards ribonuclease H1 cleavage. Moreover it was already well-established that structured RNA tends to cleave off significantly slower than unstructured RNA45. Hence, we postulated that the cross-like contorted nano-architecture could offer much steric hindrance towards the ribonuclease antisense and this would also help to reduce the overall processivity for the ribonuclease antisense to work on cleaving the DNA/RNA. It is important to note that if cleavage had indeed occurred for the DNA/RNA duplex, calculations from closest neighbor parameter would shown that the fragmented RNA oligomers were incapable of forming stable duplex with mRNA at 37 °C. It was therefore with these considerations that we proceeded with unmodified RNA strands rather than chemically modified species in order to reduce toxicity and facilitate for rapid clearance from the cellular system. The successful attainment of protein repression in later section of this manuscript had helped to reinforce these notions.Four strands of DNA were specially designed to host three separate antisense RNA strands while leaving a single arm for hybridization to an aptamer DNA see Fig. . We had 47. Nucleolin was highly expressed in cancer cells and they were often localized on the outer-membrane while their surface presentation was not typically found on non-cancer targets48. The rationale was that upon the complete assembly of the nanoconstruct, the aptamer would bind to the cell surface to facilitate for uptake  were mixed in equimolar concentration within a single PCR tube and were subjected to a melting temperature of 95 °C before allowing all oligomers to reassemble via complementary pairing with the slow gradual reduction of temperature to 4 °C. In order to validate the proper assembly of the full nanoconstruct, non-denaturing acrylamide gel electrophoresis was also performed  was subsequently performed to verify the sizing of the nanoconstruct. As shown in Fig. As observable in Fig. Using a holliday junction decorated with the AS1411 aptamer, a FITC tagged DNA sequence homologous to that of the antisense RNA belonging to the MDM2 was used to hybridized to the nanoconstruct and MDA-MB 231 cells were incubated for 2 hours. This was to examine if the cells were able to uptake nanoconstruct on the virtues of the AS1411 aptamer as selected. As shown in Fig. In a series of dose dependent studies, the full nanoconstruct carrying the three apoptosis inducing antisense RNA was purified from non-denaturing polyacrylamide gels and were added at 4, 8, 16 and 32 pmoles to MDA-MB 231 cells for a period of 24 hours. After the incubation period, cells were collected and the cell numbers were determined using the hemocytometer. Figure In order to further demonstrate the stringent cell-type selectiveness of this nanoconstruct, two other cell types were incubated with the apoptosis-inducing nanoconstruct at the dosage of 16 pmoles. As shown in Fig. Cell count for lipofectamine-based transfection was performed using the three single strand antisense RNA at 16 picomoles. As these antisense were unmodified, we did not envisage for any high efficiency from this delivery event and this was as shown in Fig. et al.54 by which that the maintenance of high p53 levels was deemed as a prerequisite for inducing cell apoptosis. However, this study could not be directed comparatively with that report as we had deliberately suppress three proteins in a simultaneous fashion although it is important to note that this was not the first time that this simultaneous repression of multiple protein target was attempted56. Nonetheless the general consensus was that multiple protein suppression in a concurrent fashion could indeed shortened the time-line required for apoptosis (24 hours)58 and our findings here were no different.As mentioned three antisense RNA protein were selected under special consideration that all the genes contribute towards cell death. AKT and its downstream MDM2 are proteins that regulate the expression of p53 and the suppression of these genes would upregulate phosphorylated p53 expression levels that would contribute towards cell apoptosis. On the other hand, survivin was another protein that inhibits caspase activity, thus contributing to the overall unnatural longevity of cancer cells. Hence, by suppressing all these three genes, it was in principle possible to induce rapid cell death. In Fig. Furthermore, in order to demonstrate that this nanoconstruct was easily programmable as a proof of concept, we decided to introduce three separate RNA antisense strand (8 picomoles) that would simultaneously suppressed the same metastatic Twist protein and we were able to obtain an even early repression at the 2 hour mark of up to ~77%  was purchased from Genetex, Survivin  was acquired from Novus Biologicals and AKT  was obtained from Cell Signalling. Anti-rabbit IgG, HRP-linked Antibody (#7074) was purchased from Cell Signalling as well. DMEM/F12 cell culture media, EGF (20 ng/ml), Insulin (10 ug/ml) was purchased from Invitrogen while Hydrocortisone (0.5 mg/ml) was from Sigma. 5% Horse Serum was acquired from HyClone Donor Equine Serum (#SH3007403) while Fetal Bovine Serum was purchased from Gibco2 incubator at 37 °C. MDA-MB-231 cells were cultured in DMEM-F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 90% RMPI 1640 medium, 10% fetal bovine serum and 1% penicillin-streptomycin was used to maintained AGS cells. Upon reaching 80% confluency, the cells were treated with trypsin after washing with PBS (3 times). The detached cells were centrifuged at 1000 rpm for 5 minutes. The pellets were then resuspended with growth medium at appropriate dilution, and cultured in a new 75 ml polystyrene cell culture flask.All cell cultures were incubated in 5% CO2 with DMEM and Nutrient Mixture F12 (Ham) (Invitrogen) with high glucose containing L-glutamine, pyridoxine hydrochloride, and HEPES buffer supplemented with 5% Donor Equine Serum (Hyclone), 20 ng/ml Epidermal Growth Factor, 0.5 mg/ml Hydrocortisone, 10 ug/ml Insulin, and 1% penicillin-streptomycin.The medium recipe for MCF-10A cells was adapted from Brugge’s lab. In brief, MCF-10A cells were grown at 37 °C incubator with 5% CO™ unless otherwise specified. To assemble the holliday junction nanoconstruct, the following sequences were selected to form a holliday junction with 4 overhanging ends that are complementary of the the individual RNA antisense strands and the respective aptamer sequence are also as listed below:All DNA and RNA were obtained from InvitrogenHolliday Junction 1 = 5′TTTGTGCAGCCAACCCTCCGTGTGTGTGCCATAGTGCATTGCGAGAGAGAG 3′Holliday Junction 2 = 5′ATACTATCAGATTTGTGGCTTTCCTTTGCATTCGGACTATGGCACACACAC 3′Holliday Junction 3 = 5′GCAGTGGATGAAGCCAGCCTTAAGGCCCGTGCTCACCGAATGCAAAGGAAA 3′GGGGGGGGGGGCTCTCTCTCGCAATGCTGAGCACGGGCCTTAA 3′Holliday Junction 4 = 5′59AS1411 (nucleolin) aptamer = 5′ GGTGGTGGTGGTTGTGGTGGTGGTGG CCCCCCCCCCC 3′CCCCCCCCCCC 3′60AGS cell specific aptamer = 5′ CGACCCGGCACAAACCCAGAACCATATACACGATCATTAGTCTCCTGGGCCG Three strands of antisense RNA was selected from well-cited sources in literature and was shown below:62AKT: 5′ GGAGGGUUGGCUGCACAAA 3′64MDM2: 5′ GCCACAAAUCUGAUAGUAU 3′65Survivin: 5′ GGCUGGCUUCAUCCACUGC 3′™) and 1 nmol for each strands  were collectively pooled to a PCR tube and annealing buffer  was added to bring the mixture to a final volume of 100 μl. The oligomer mixture was then mixed well before subjecting to the thermocycler  to 95 °C and held for 10 minutes. The temperature was then slowly reduced to 4 °C over 30 minutes before purification.To assembly the full nanoconstruct, all received oligomers (DNA and RNA) were firstly resolubilized in Rnase Free DEPC water (Invitrogeng for 10 mins. All purified nanoconstruct was quantified with UV-Vis Spectrophotometer (Eppendorf Biophotometer plus) at 260 and 280 nm. The “roadmap” of the nanoconstruct was as shown in Supplementary Figure The nanoconstruct was purified using an 8% native polyacrylamide gel in Tris/Borate/EDTA buffer. The gel was sectioned out according to the size of the fully assembled nanoconstruct (a lane was stained with EtBr as a guideline). The sections were crushed and transferred to Nanosep® MF centrifugal devices and spun at a 17949 × 4 in a 24-well plate and then transfected with the nanoconstruct at the nanoconstruct concentrations of 4, 8, 16 and 32 picomoles (plated out in quadruplicates) for 24 hours. The cells were washed three times with phosphate-buffered saline and resuspended with 0.05% Trypsin-EDTA for ~15 mins. After which, 10 μl of the cell suspension was loaded into a hemocytometer and counted according to the manufacturer’s protocol. Statistical analysis of differences was performed via a one-way ANOVA analysis whereby P value of ≤0.05 was taken as statistically significance.For all cell types, the cells were seeded at a density of 2 × 106 and co-incubated with the nanoconstruct. Total protein concentration was determined by bicinchoninic acid assay (Thermo Fisher). Whole cell lysates were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Milipore) using a wet transfer system (Hoefer) with a Tris-glycine buffer containing 20% methanol. The membrane was then blocked with 3% bovine albumin serum in Tris-buffered saline with 0.1% Tween-20 (TBST) for an hour room temperature and gently washed twice with TBST. The membrane was probed with antibodies against Akt , MDM2 (Genetex), phosphorlayted-p53 , and GADPH (Genetex) for ~16–18 hours at 4 °C and washed three times for ~10 mins with TBST. Afterwards, the blot was detected with HRP-linked Anti-rabbit IgG antibodies  for an hour at room temperature and followed by three washes with TBST for ~10 mins. The blot was developed using Immobilon Western Chemiluminescent HRP Substrate (Milipore).For immunoblotting, the cells were seeded at a density of 1 × 10The following staining protocol was adapted from Sir William Dunn School of Pathology. In brief, Uranyl acetate was dissolved in deionized water to prepare a 2% stain solution. The nanoconstruct was diluted by 10-folds in DEPC-treated water. Equal ratios of the stain solution and nanoconstruct were combined. The mixture loaded onto the grid for ~30 seconds and then absorbed with filter paper. Transmission electron microscopy was performed on Hitachi HT7700 platform at an acceleration voltage of 70 kV.Atomic force microscopy (AFM) images were acquired using Bruker Dimension Icon AFM system using an in-build AFM tapping mode. Scan area on the surfaces were of 0.3 μ m × 0.3 μ m and the scan speed was set at 0.5 hz with the integral and proportional gain set at automatic mode. Post image processing was performed with Gwyddion MacOS version 2.38.Particle size of the nanoconstruct was determined using a disposable plastic cuvette through the Zetasizer Nano  at room temperature using DEPC water as the solvent.6. After 2 hours, the cells were washed 3 times with PBS followed by the nominal fixation protocol with paraformaldehyde and stained with Alexa Fluor® 594 phalloidin before mounting on cover slips. The surfaces were then examined under Nikon® N-SIM super resolution microscopy using using an oil immersion objective lens CFI SR at laser wavelength of 488 nm and 561 nm for the visualization of the nanoconstruct carrying FITC and the cell membrane respectively. Image processing was done with NIS Elements Revolutionizes Imaging software .To evaluate the uptake of the nanoconstruct, the four arm holliday junctions with the AS1411 aptamer was assembled without antisense RNA but was replaced with a FITC-tagged complementary strand to holliday junction 2 (5′ GCCACAAATCTGATAGTAT 3′). The fluorescence labeled nanoconstruct (16 picomoles) was administrated to MDA-MB-231 cells at density of 1 × 10Supplementary Information


In addition, reQTLs are enriched for recent positive selection with an evolutionary trend towards enhanced immune response. Finally, we uncover reQTL effects in multiple GWAS loci and show a stronger enrichment for response than constant eQTLs in GWAS signals of several autoimmune diseases. This demonstrates the importance of infectious stimuli in modifying genetic predisposition to disease.The immune system plays a major role in human health and disease, and understanding genetic causes of interindividual variability of immune responses is vital. Here, we isolate monocytes from 134 genotyped individuals, stimulate these cells with three defined microbe-associated molecular patterns , and profile the transcriptomes at three time points. Mapping expression quantitative trait loci (eQTL), we identify 417 response eQTLs (reQTLs) with varying effects between conditions. We characterize the dynamics of genetic regulation on early and late immune response and observe an enrichment of reQTLs in distal  Insight into the genetic influence on the immune response is important for the understanding of interindividual variability in human pathologies. Here, the authors generate transcriptome data from human blood monocytes stimulated with various immune stimuli and provide a time-resolved response eQTL map. Given this central role in many human pathologies, it is crucial to understand the variability of immune responses at the population level and how this variability relates to disease susceptibility. Studying the genetic influence on immune response is complicated by the complexity of the immune system, which consists of many different cell types that respond to a plethora of signals, interact with each other and induce different effector functions under diverse kinetics11. However, given the complexity of the immune system, additional work is needed to illuminate the genetic influence on many aspects of the immune system. For instance, reQTLs of certain pattern recognition receptor (PRR) families such as NOD-like receptors using purified microbial ligands have not been studied yet, and thus far the dynamics of immune reQTLs have only been explored in LPS-treated cells9.An increasingly popular approach to identifying genetic factors influencing the interindividual variation in immune response is to map expression quantitative trait loci (eQTLs) —variants that associate with gene expression—and to identify so-called response eQTLs (reQTLs) where the eQTL effect differs between immune stimuli. Such genetic variants can impact the transcriptional response to infection, and also represent genetic effects that are modified by the infectious environment via gene-by-environment interactions. We and other groups have previously published reQTL studies of stimulated immune cells and demonstrated that the effects of a genetic variant on gene expression are highly context-specific and informative for disease6, we address these gaps by studying functional genetic variants in monocytes activated with microbial ligands for three different PRR families at two different time points. We identify context-specific reQTLs and describe their specificity for time point and treatment. In addition, we analyze differences in reQTLs and constant eQTLs in terms of their genetic architecture and contribution to explain GWAS loci. Finally, we describe reQTLs that shed light on the pathogenesis of immune-mediated diseases. Collectively, these results improve our understanding of the complexity of genetic regulation of the immune system. We provide a user-friendly access to our results via the ImmunPop QTL browser (http://immunpop.com/kim/eQTL).Building on our previous study of baseline and LPS-stimulated monocytes6, we have analyzed a subset of these data consisting of baseline and 90 min LPS-stimulated monocytes in this cohort.To examine the time course of innate immune responses, we first profiled gene expression in monocytes of five individuals using Human HT-12 v4 Expression BeadChips (Illumina) at six time points after stimulation with three prototypical microbial ligands: Lipopolysaccharide (LPS) was used to activate Toll-like receptor 4 (TLR4), muramyl-dipeptide (MDP) to stimulate Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), and 5′-triphosphate RNA (RNA) to activate retinoic acid-inducible gene I (RIG-I). Hierarchical clustering revealed early differentially expressed (DE) genes at 45 and 90 min after stimulation and late DE genes between 3 and 24 h  of 5%. We identified 717–1653 genes with an eQTL in each condition .In order to study genetic variation influencing gene expression levels, we performed eQTL mapping for all seven conditions, defining ion Fig. . The eQTβbaseline) vs stimulated  in a z-test, with reQTLs defined as having Bonferroni corrected p < 0.05 (see Methods section). This approach is highly consistent with a previously used method10 where differential expression is used as the quantitative trait . This indicates that reQTLs are relevant regulators of differential expression but additional regulatory mechanisms are involved in shaping the transcriptional response of reQTL genes. Altogether, our analysis of temporal reQTLs sheds light on mechanisms of the highly dynamic immune response, and the role of genetic variants in it.To obtain better insight into the dynamic link between reQTLs and differential expression upon immune stimulation, we classified reQTLs into those with early transient, late, and prolonged effects (see Methods section). We find that active reQTLs, reQTLs that are absent at baseline and active under stimulation, are more common and have higher effect sizes than suppressive reQTLs, reQTLs that lose their baseline eQTL effect under stimulation Fig. . Interesp > 0.05) . These ceQTLs displayed no change in regression slope across all conditions  Fig.  and gene05) Fig. . Functiotes Fig. . While r11, we detected a signal of increased positive selection in eQTLs, ceQTLs, and reQTLs using the integrated haplotype score15  and the singleton density score16 , comparing each eQTL class to a genome-wide null set of variants matched for minor allele frequency (MAF) and linkage disequilibrium (LD). Next, we examined the direction of the effect of the derived allele, dividing reQTLs into two groups  reQ17 method on summary statistics of 33 GWAS traits  with a disease-associated SNP listed in the GWAS catalog22 , including reQTL genes such as APOL2 potentially associated with glomerulosclerosis, PTGER4 with allergy, and PIP4K2A with acute lymphoblastic leukemia. These results do not exclude other possible mechanisms in other cell types or conditions, but the reQTL analysis discovers potential causal genes for individual GWAS loci with an effect that is potentially modified by infections.Given the central role of inflammation in many diseases, we examined reQTLs as a potential mechanism underlying genetic associations to complex diseases, discovered by genome-wide association studies (GWAS). First, we identified individual GWAS loci that are likely to share a causal variant with an reQTL in the same locus. We used the coloce19 Fig.  and the a20 Fig. , the eQTg22 Fig. , which m23. Interestingly, in seven out of eleven traits reQTLs had a significant enrichment, whereas ceQTLs were enriched in only three of these seven traits, and narcolepsy (NAR) was the only trait significantly enriched for ceQTLs but not for reQTLs. Most notably, systemic lupus erythematosus (SLE) GWAS signals24 were very strongly and significantly enriched among reQTLs with no enrichment in ceQTLs, suggesting that the innate immune response to pathogens may be a particularly important environmental modifier of genetic predisposition to SLE, while possibly playing a smaller role in the genetic architecture of e.g. psoriasis and type 1 diabetes. Even though fgwas analysis for multiple sclerosis (MS) did not show stronger enrichment of reQTLs over ceQTLs, the inflation of reQTLs in the QQ plot of MS advocates the importance of immune response genes in the etiology of MS , 100 ng/ml L18-MDP (Invivogen) or 200 ng in vitro transcribed 5′-ppp-dsRNA transfected with Lipofectamine 2000. These microbial ligands target three distinct pattern recognition receptor families and were chosen to study a broad spectrum of innate immune response in human monocyte. Differential expression analysis showed that early response genes are well captured at 90 min after stimulation followed by a “second” wave of late response genes that plateaued between 6 and 24 h after stimulation. Based on this pilot study, we profiled mRNA expression at 90 min and 6 h after stimulation in the larger eQTL cohort.l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin  and 10 ng/ml GM-CSF (ImmunoTools) was used to culture cells in 96-well round bottom wells at a density of 250,000 cells per well in 100 µl overnight. Cell viability after overnight incubation was > 85%. Cells of each volunteer were divided into subsets that were either left untreated or treated with 200 ng/ml ultrapure LPS from Escherichia coli (Invivogen), 100 ng/ml L18-MDP (Invivogen) or 200 ng in vitro transcribed 5′-ppp-dsRNA (IVT4) transfected with 0.5 µl Lipofectamine 2000 in a 50 µl reaction. Based on the pilot study described in Supplementary Fig. In total, 185 healthy male volunteers of German descent were recruited. The study was approved by the institutional review board of the University of Bonn and informed consent was obtained from all donors. All volunteers were between age 18 and 35 (mean 24). Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque density gradient centrifugation of heparinized blood. Monocytes were isolated by MACS using CD14-microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Cell purity was assessed by FACS analysis of cell-surface antigens with a FACS LSRII (BD Biosciences). Monocytes were stained with an antibody against CD14  and purity was ≥ 95%. RPMI 1640 (Biochrom) supplemented with 10% heat-inactivated FCS (Invitrogen), 1.5 mM After stimulation cells were lysed and RNA was extracted using the AllPrep 96 DNA/RNA Kit (Qiagen). RNA quantity was determined using NanoDrop (PeqLab) and quality was assessed for a subset of samples using a Bioanalyzer (Agilent Technologies).pdetection < 0.01 in at least 10 samples across all conditions were analyzed. Batch effects were removed using the R packages ComBat29 and sva30. Probes with an interindividual standard deviation > 5 were set to NA. Probes found to map to multiple locations in the human genome or to non-autosomal chromosomes were not used. In addition, probes with SNPs that showed an eQTL effect on the respective gene were excluded, resulting in 18,988 probes  for further statistical analyses.RNA was amplified and biotinylated using Illumina TotalPrep-96 RNA Amplification Kit (Life Technologies) and gene expression analysis was quantified using Human HT-12 v4 Expression BeadChips (Illumina) comprising 47,231 probes. Expression profiles were quantile normalized, and only probes which showed a pdetection across all conditions was used and differential expression  was computed using the linear modeling-based approach implemented in the Bioconductor limma package31. Genes differentially expressed in at least one condition were grouped into six distinct clusters corresponding to genes with similar response pattern using hierarchical clustering. Over representation of Gene Ontology terms in these clusters of differentially expressed genes were assessed using hypergeometric-based tests implemented in the R package GOstats32. Genes that were expressed in our monocyte data were used as background set in all enrichment analyses. Only enrichments significant at FDR of 0.05 are reported in Supplementary Data To determine the number of differentially expressed genes, the probe with the best Genomic DNA was extracted from 10 ml blood using Chemagic Magnetic Separation Module I (PerkinElmer Chemagen) according to the manufacturer’s instructions. DNA was quantified by NanoDrop (PeqLab).pHWE > 10–5, call rate > 98%, MAF > 5%), a total of 579,090 SNPs were available for analysis. Samples showing potential admixture within the multi-dimensional scaling (MDS) analysis were removed. All samples showed a call rate > 99%.Genotyping was conducted on the Illumina’s HumanOmniExpress BeadChips comprising 730,525 SNPs. After quality control (33 and imputed with IMPUTE234 in 5 Mb chunks against the 1000 genomes phase 1 v3 reference panel35. Sites with an information score of less than 0.8 or significant departure from Hardy–Weinberg equilibrium (p < 10−5) or MAF < 5% were excluded from further analysis. Genotype probabilities for all remaining sites were converted into dosage estimates.Genotypes were phased with SHAPEIT236. Significance of the most highly associated variant per gene was estimated by adaptive permutation with the setting “--permute 100 10000”. Permutation p-values obtained via beta approximation were used to access genome wide significance via Benjamini-Hochberg (FDR < 0.05). Downstream analyses were carried out in R. Network analysis of reQTL genes was performed using the STRING 10.0 database37 selecting only interactions that were either experimentally validated or originated from curated databases.As quantitative phenotypes, we used absolute expression values of untreated (baseline), LPS-treated (LPS), 5′-ppp-dsRNA (RNA), and MDP-treated (MDP) cells. Complete expression profiles of each of the seven conditions  were available for 134 donors. eQTL mapping was performed for SNPs located within 1 Mb of the gene expression probe using FastQTL9, we used Storey’s qvalue R package38. The π1 statistic considers the full distribution of association p-values (from 0 to 1) and computes their estimated π0, the proportion of eQTLs that are truly null based on their distribution. Replication is reported as the quantity π1 = 1−π0 that estimates the lower bound of the proportion of truly alternative eQTLs.We compared our results with two previous reQTL studies. For quantifying eQTL replication with a genome-wide study of monocyte eQTLs8 used a targeted approach (415-gene signature) to identify eQTLs after LPS, Flu or IFNβ treatment in dendritic cells. π0 could not be calculated using Lee et al. because less than 10% of eQTL genes in our data were represented in the 415 targeted genes, and thus replication was assessed by the proportion of our eQTLs with nominal significance (p < 0.05) in Lee et al.Lee et al.β) and its variance (σ2) of these eQTLs were calculated for all seven conditions using the linear model function summary(lm) in R. We then tested if the regression coefficient of an eQTL was significantly different between two conditions in a z-test:In each condition, we first determined the best eQTL per gene (lead eSNP). Regression coefficient . Previous reQTL studies10 have used differential expression as a quantitative trait to identify reQTLs (pdiff). We calculated pdiff for all reQTLs identified by β-comparison and used Spearman correlation as a measure of similarity.Resulting z-tests. A reQTL was treatment-specific if the Bonferroni-corrected p-value in the z-test was < 0.05. To detect time point specificity of reQTLs, for each treatment, we tested all significant reQTLs of one time point  vs the other time point (e.g. LPS6h) in a z-test. Time point-specific reQTLs were determined using Bonferroni-corrected p-values (p < 0.05). To compare reQTLs with eQTLs that are constitutively active (ceQTL), we defined ceQTLs as eQTLs with pbeta > 0.05 when testing each of the six stimulated conditions with the baseline condition.To detect treatment specificity of reQTLs, we tested all significant reQTLs of one treatment  vs the other two treatments of the same time point  in two separate p-value at each of the three time points or not . If a reQTL was shared between treatments, the treatment with the best p-value was used. This resulted in following groups: Transiently active (“0-1-0”), transiently suppressing (“1-0-1”), late active (“0-0-1”), late suppressing (“1-1-0”), prolonged active (“0-1-1”), and prolonged suppressing (“1-0-0”) reQTLs. The average of absolute eQTL-β and distribution of reQTL among these groups are shown in Fig. To study the dynamics of reQTLs, we encoded as a binary call whether reQTLs had a significant eQTL β and differential expression (DE) of the eQTL gene are congruent, DE between baseline and 90 min stimulation (Δ90 min-baseline) and DE between 90 min and 6 h stimulation (Δ6 h-90 min) were calculated using limma and significant Δ90 min-baseline (p < 0.01) was encoded in binary  whereas significant Δ6 h-90 min was encoded as “not significant” (0), “significant” (1), “significant, but opposite direction of Δ90 min-baseline” (2). To determine the proportion of reQTL genes with congruent DE, we quantified for transiently active/suppressing reQTLs the proportion of reQTL genes with significant Δ90 min-baseline and significant Δ6 h-90 min with opposite direction (“1-2”), for late active/suppressing reQTLs we quantified the proportion of reQTL genes with not significant Δ90 min-baseline and significant Δ6 h-90 min (“0-1”) and for prolonged active/suppressing reQTLs we quantified the proportion of reQTL genes with significant Δ90 min-baseline and either not significant Δ6 h-90 min (expression stays the same) or significant Δ6 h-90 min with same direction . To test if the proportion of reQTL genes with congruent DE was significantly enriched in each group , we quantified the proportions of the same DE code  in the remaining groups (late active/suppressing and prolonged active/suppressing) and tested the proportions using Fisher’s exact test.To further examine if eQTL-12 software to investigate the extent to which reQTLs and ceQTLs were enriched within specific annotation categories. Annotation information used by fgwas was derived from CADD variant consequence annotation39 (14 annotations) and monocyte-specific annotations from Ensembl Regulatory build40 (6 annotations). To identify the set of annotations that would best fit the model, we first tested each of the 20 annotations in a joint data set of reQTLs and ceQTLs including distance to TSS in the analysis. Sixteen annotations individually improved the model likelihood but as many of these annotations are correlated with one another we used a stepwise selection approach to identify a final best-fitting model that included 13 annotations asterisked in Fig. We used the fgwasFor each locus that contained at least one SNP with a posterior probability of association (PPA) > 0.3, we considered the SNP with the highest PPA from fgwas and tested the overlap of functional annotation sites of reQTL vs ceQTLs using Fisher’s exact test. To increase power of reQTLs/ceQTLs overlapping functional annotation sites, we mapped eQTLs using the mean of gene expression across all seven conditions. Fgwas steps were repeated as described above. Estimated enrichment parameters showed similar results and indicate the robustness of our analysis 16, which detects very recent changes in allele frequencies from contemporary genome sequences. Publicly available SNP level SDS scores calculated from the UK10K Project reflect allele frequency changes during the past ~2000–3000 years in modern Britons, who are closely related to the German population42. We therefore applied these SDS scores to our cohort.We used two metrics, iHS and SDS, which detect signals of positive selection. The integrated haplotype score (iHS) measures the degree of extended haplotype homozygosity of the putatively selected allele over that of the putatively neutral alleler2 > 0.8) with the best eQTL/ceQTL/reQTL SNP per gene. To assess significance, we then compared for each eQTL set the proportion of SNPs with |iHS| > 1.5 with the expected distribution obtained from re-sampling 10,000 sets of random SNPs matched for MAF and the number of SNPs in LD using the same parameters as described in Quach et al.11 using bins of MAF of 0.05 and LD bins of 0–2, 3–5, 6–10, 11–20, 21–50, and > 50 SNPs with r2 > 0.8). Similarly, for SDS, we compared the median of SDS scores of eQTLs/ceQTLs/reQTLs, to the expected distribution obtained from resampling 10,000 sets of random SNPs matched for MAF and LD patterns.For each statistic , we determined the strongest signal of selection of all SNPs in high LD  along with our eQTL data. A list of GWAS traits used in this analysis is provided in Supplementary Data p12 = 10−6. Coloc estimates posterior probability of association for either trait (PP0), association with gene expression (PP1), association with the trait (PP2), association with both phenotypes but distinct causal variants (PP3) and association with both phenotypes sharing the same causal variant (PP4). Regions with evidence for colocalization between gene expression and trait were defined as PP3 + PP4 ≥ 0.90 and PP4/PP3 ≥ 3 similar to what has been proposed by Guo et al.44 and are illustrated in Fig. Colocalization analysis was conducted using the R package colocp-value. This is expected to provide robust discovery even in highly condition-specific loci. Furthermore, we also ran coloc with eQTLs mapped using the mean of gene expression across all seven conditions, which is expected to improve power when the eQTL signal is present in many conditions. All coloc results with PP3 + PP4 ≥ 0.90 are reported in Supplementary Data As eQTL summary statistics in the coloc analysis, we used two approaches to maximize our discovery power. First, from each locus we used the summary statistics of the condition with the strongest r2 > 0.8) with the lead eSNP per gene. A full list of these GWAS reQTLs is provided in Supplementary Data To assess the overlap between reQTLs and trait-associated variants, we downloaded the NHGRI-EBI GWAS Catalog . A reported GWAS SNP was considered to coincide with an reQTL if the GWAS SNP was in high LD  suggesting that the enrichment is not simply due to the power of detection  under accession number E-MTAB-5631. In addition to results tables for all seven conditions provided in Supplementary Data http://immunpop.com/kim/eQTL), which provides multiple interactive visualization and data exploration features for eQTLs.Full summary statistics of the eQTL analysis and gene expression data are available in the ArrayExpress database (Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Peer Review File