Hydroxyl groups are the cornerstone species driving catalytic reactions on mineral nanoparticles of Earth’s crust, water, and atmosphere. Here we directly identify populations of these groups on ferrihydrite, a key yet misunderstood iron oxyhydroxide nanomineral in natural sciences. This is achieved by resolving an enigmatic set of vibrational spectroscopic signatures of reactive hydroxo groups and chemisorbed water molecules embedded in specific chemical environments. We assist these findings by exploring a vast array of configurations of computer-generated nanoparticles. We find that these groups are mainly disposed along rows at edges of sheets of iron octahedra. Molecular dynamics of nanoparticles as large as 10 nm show that the most reactive surface hydroxo groups are predominantly free, yet are hydrogen bond acceptors in an intricate network formed with less reactive groups. The resolved vibrational spectroscopic signatures open new possibilities for tracking catalytic reactions on ferrihydrite, directly from the unique viewpoint of its reactive hydroxyl groups. Ferrihydrite nanoparticles have many hydroxyl sites which can react with environmental contaminants and nutrients, but the surface structure of this common mineral is still not fully understood. Here, a combination of vibrational spectroscopy and molecular simulations identify hydroxyl groups exposed along rows at the edges of sheets of iron octahedra. Fh nanoparticles display large concentrations of reactive hydroxyl (OH) sites responsible for scavenging and transforming contaminants22 and nutrients24, in natural and industrial waters, and in terrestrial environments. Interactions with these OH groups can even alter the conversion pathways of Fh to other iron oxides30, and permanently occlude these compounds in the crystalline structure of these minerals. Still, our lack of knowledge on the composition and structure of Fh nanoparticle surfaces continues to challenge understanding reaction mechanisms that Fh hosts in nature and technology34.Ferrihydrite (Fh) is a puzzling nanosized mineral with a 50-year-old history of debates over its structure, composition and formation mechanisms3−O) underlying iron atoms  over Fh structure — Hiemstra38 explored an approach for rethinking the surface chemistry of Fh by identifying OH populations on crystallographic faces of idealised nanoparticles. The model envisions crystalline Fh nanoparticles composed of (1) a defect-free core consisting of the low OH-bearing structure of Michel et al6. (Fe5O8H), and (2) crystallographically-oriented surfaces of greater OH/H2O loadings (Fe5O8H + n H2O) but depleted in Fe2 octahedral and Fe3 tetrahedral sites (hydr)oxide minerals consist of oxo groups bound to one (−O), two (μ−O), or three oxides  involved in a broader network of hydrogen bonds 0.76 · 0.05 H2O , chemisorbed water molecules  and doubly-coordinated hydroxyl groups . Triply-coordinated μ3−OH sites were not detected in the expected spectral range where they occur in other crystalline iron (oxyhydr)oxide48 minerals  or bare Fe Lewis acid sites in acid-neutral and charge-neutral surfaces. We note that rows with greater populations of inter-site hydrogen bonds, such as in the mineral goethite (α-FeOOH)43, generate a band downshifted by 6 cm−1, and are not present in Fh. This difference is highly significant as these bands are phenomenally responsive to the slightest changes in hydrogen bond strength, and to the tune of ~150 cm−1 per pm change in O–H bond length44. This 3667 cm−1 band is therefore key evidence for a dominance of Fh −OH groups of relatively lower hydrogen bonded environments than on goethite.Our previous work showed that −OH groups responsible for the 3667 cm48, and can be appreciated by the loss of our 3667 cm−1 band and its shift to lower frequencies. These changes signal that new hydrogen bonds were formed between unreacted −OH groups and −OH2 sites along the rows we have previously mentioned. It is these chemisorbed water molecules that contribute to our second flagship band at ~3690 cm−1. The high O–H stretching frequency of these water molecules is from an entirely isolated O–H bond from the hydrogen bond network of the Fh surface, and we note that we have previously detected such species on iron (oxyhydr)oxide48 nanominerals. In this previous work, we showed that while a fraction is always resilient to outgassing under dry gases, water can be removed by prolonged exposure to ultra high vacuum or heat. We will come back to this point when looking at the thermal stability of Fh.To illustrate the sensitivity of −OH groups to their bonding environments, we altered their speciation by exposing Fh to HCl Fig. . The pro45. This suggests that, although generally of spheroidal morphology, Fh nanoparticles could have developed a minor basal face exposing neutrally charged and proton inactive μ−OH groups45. Our previous work on this face shows that these groups are embedded in a highly regular hydrogen bonding environment responsible for the narrow 3620 cm−1 band. The other μ−OH groups of the dominant spheroidal surface are, in contrast, not discernable as they are involved in a broad network of hydrogen bonds. We have arrived to the same conclusion through our work on crystalline iron (oxyhydr)oxides45, and will support this further in molecular simulations in the last section of this study.The band for μ−OH was only of small intensity, yet its appearance is of high value for understanding the surface chemistry of Fh, as it is a well-known signal for groups on the basal faces of iron (oxyhydr)oxides  of Fh to haematite  and −OH groups (3667 cm−1). These events produced a high temperature Fh core exposing the most heat-resistant core OH and surface μ-OH groups. Using a chemometric analysis49 we show that the concentration profile  and core OH (3395 cm−1) groups.We can validate these findings further by inspecting the thermal response of these spectral signatures at temperatures well below the conversion temperature  site. Drawing from the catalytic alumina literature40, where OH groups bound to tetrahedrally-coordinated Al3+ are well documented, we would have expected a band overlapping with that of the chemisorbed water. However, as this band shifted congruently with gradients in proton loadings oxide minerals  on Fh. As these spectroscopic signals also persist under atmospheric humidity50, we can explore interfacial reactions and phase transformation in nanometrically thick water films formed at Fh surfaces. In the following sections, we bridge these findings with new theoretical analyses and molecular simulations underscoring the importance of −OH and μ−OH groups on the surface chemistry of Fh nanoparticles.By resolving the complex vibrational spectroscopic signals of Fh, we here offer new possibilities for tracking the catalytic reactivity of Fh from the unique viewpoint of its OH groups. We can, of instance, begin resolving the binding of critically important gases , atomic displacements (0–4 Å) and Fe site vacancies (vFe). All broken Fe–O bonds were healed and protonation levels were set to −OH and μ-OH, while those of μ3−O(H) sites were set to levels predicted from the core structure. Finally, all nanoparticles were made charge-neutral by protonation of a required number of randomly selected −OH sites to −OH2. Inspection of 280 computer-generated nanoparticles reveals a dominance of −OH/−OH2 and μ−OH groups but only low densities of μ3−O(H) sites at Fh surfaces  densities align with our experimental results, and again suggest that the spherical Fh surface should be dominated by −OH/−OH2 and μ−OH groups.We support our experimental findings by identifying OH groups on simulated Fh nanoparticles of spheroidal morphology. Spheroidal nanoparticles, with no or little defined crystallographic faces, are not only more representative of real Fh but are also expected to expose greater proportions oxygens of lower Fe coordination number than on crystallographically flat surfaces. To explore this idea, nanoparticles were generated from a spherical cut of the crystalline structure of Michel et al.ements 0– Å and Fe−3 for ~2−3 nm particles11). In accordance with the work of Hiemstra37 on crystalline Fh nanoparticles, we find that depleting surface Fe2 and Fe3 sites is a highly effective means for reproducing experimental mass densities. A depletion depth of no more than 4 Å from the average positions of the top O atoms is needed to obtain such values. We can also justify introducing up to ~20% additional vacancies Fe2 and Fe3 vacancies  throughout the Fh core  and edge (~2.0 –OH/nm2) sites  from the surface, and retained full core Fe occupancies. This configuration was chosen to focus the discussion on the hydrogen bonding environment of OH groups of a hydroxylated Fh surface over a defect-free core. It thus emulates strongly dehydrated Fh surfaces, which were likely achieved in N2(g) dried samples studied experimentally.To understand how the distribution of surface OH groups impact inter-site interactions we turned to molecular dynamics simulations of single Fh nanoparticles with a surface depletion of Fe2 and Fe3 sites down to the first 4 Å. For these simulations, we removed all chemisorbed water molecules  to those involving surface OH groups.Our simulations retained the core crystallographic structure of Fh while mostly relaxing its hydroxylated surface Fig. . The sur1/2 of the available −OH (μ−OH···−OH) . The hydrogen bonding network also involves lateral μ−OH···OH−μ bonds taking place over both the dominant spherical surfaces and over the basal face. These interactions take proportionally more importance in the 2-nm particle, given the greater representation of the basal face  Fig.  and up tace Fig. . We alsoace Fig.  over the2, CH4, NOx, SOx), as well as photo(electro)chemical transformation of organics in nature and technology. As these spectroscopic signals also persist under atmospheric humidity47, this work also open new possibilities for exploring interfacial reactions and phase transformation in nanometrically thick water films at Fh surfaces. It may, additionally, be pertinent to investigate the possible roles that neutrally-charged basal faces play in the oriented aggregation of Fh in water. Future consideration of such possibilities, and especially based on our findings that hydrogen-bonded −OH and μ−OH groups dominate the surface speciation of Fh, represents some of the many anticipated implications that this work can have for understanding the surface chemistry of Fh nanoparticles in nature.By resolving the complex vibrational spectroscopic signals of Fh, this work offers new possibilities for tracking the catalytic reactivity of this nanomineral from the unique viewpoint of its OH groups. It immediately opens new possibilities for resolving the mechanisms through which Fh alters the fate of environmentally critical gases (e.g. CO2 (g) using the method of Schwertmann and Cornel56. To minimise atmospheric CO2 contamination, we degassed all deionised water (18 MΩ⋅cm) used for synthesis and washing procedures by boiling then purging with N2 (g) overnight. Material characterisation is reported in the Supplementary.We synthesized 6-line Fh under N2(g)-dried Fh particles by Fourier transform infrared spectroscopy. Following our previously established protocol43, we altered the protonation levels of Fh surfaces as described in the Supplementary Methods. Centrifuged pastes of equilibrated minerals were thereafter transferred onto an attenuated total reflectance cell and dried to a thin film under N2 (g) atmosphere. Spectra were collected with a Bruker Vertex 70/V Fourier transform infrared spectrometer, equipped with a DLaTGS.We collected vibrational spectra of N49 detailed in the Supplmentary Methods.We monitored the thermal stability of OH groups of Fh by temperature programmed desorption on dry samples by heating from 40 °C to 400 °C at a rate of 10 °C/min in a vacuum of <0.3 Pa. Dry Fh powders used for these experiments were pressed on a fine tungsten mesh  under a pressure of 5 tons, and inserted in a copper-heating shaft in direct contact with a K-type thermocouple. Transmission vibration spectra were collected in an optical chamber and water expelled from the sample was concomitantly detected by quadrupole mass spectrometry . The resulting spectra were analysed using a chemometric analysis6. The code then automatically modified the resulting nanoparticles with various combinations of the following strategies: (i) The surface depletion scheme was implemented by removing Fe2 and Fe3 sites within a preselected distance  from the average position of top layer O atoms; (ii) Atomic displacements of random orientation were applied to all atoms with distances of up to 5 Å of their crystallographic position; (iii) Vacancies were randomly applied to up to 20% of only Fe2 and Fe3  or all Fe atoms . Next, we healed all undercoordinated atoms created as follows: (i) Free O and Fe atoms were removed from the structure; (ii) Fe sites lacking up to three coordinating O atoms were healed by adding first neighbour O atoms at their crystallographic positions, while those lacking more than three coordinating O atoms were removed; (iii) Dangling −O and μ−O were saturated by H atoms but the protonation state of core OH−1, O2, O3, O4 sites as in Michel et al.6 were preserved as in the original crystallographic structure. Finally, charge neutrality was attained by ensured in all 280 Fh nanoparticles by protonating randomly selected −OH sites, which are the most proton-active sites of Fh.We wrote a code to automatically generate and objectively analyse atomic populations of 280 different Fh nanoparticles. The nanoparticles generated for this work were 1–10 nm-wide spheroids cut from the crystallographic structure of Michel et al.3−O and μ4−O) of all O sites. For the latter, we use a cut-off distance of 2.4 Å for the Fe−O bond. The programme also determined nanoparticle volumes from the total number of oxygen, using the relationship VO = 10.8 cm3/mol oxygen developed by Hiemstra and van Riemsdjik38. VO was then used to estimate mass densities. We found that this approach produced more reliable size-dependent mass densities than from volumes estimated by particle radius. For the same reason, we use the VO value to determine the total surface area of the particles, assuming a perfect sphere, to calculate all O(H) surface densities.All nanoparticle generation, treatment procedures and compositional analyses were performed using this code written in the computational environment of Matlab 9.1. The programme bookkeeps the identities of all Fe  and O atoms, as well as the identity of Fe sites to which all O are connected, and the coordination number 47(μ−OH)75) to 10 nm (Fe17058O21864OH3428(−OH)727(μ−OH)3291) in diameter in an empty 20 × 20 × 20 nm3 box. The protonation state of −OH and μ−OH sites was chosen based on previous actual bond valence arguments55, and charge neutrality was achieved by protonated randomly selected −OH groups. The resulting −OH2 sites were then removed from the surface, thus exposing Lewis acid Fe3+ sites. Full details of the simulation procedures are reported in the SI Appendix.We performed molecular dynamics of dehydrated Fh nanoparticles generated from our nanoparticle simulation code. We simulated neutrally-charged nanoparticles of 2 nm (FePeer Review FileSupplementary Information


Although Ag77Cu22(CHT)48 bears one unpaired shell-closing electron, the magnetic moment is found to be mainly localized at the S linker with magnetic isotropy, and the sulfur radicals were experimentally verified and found to be unstable after disassembly, demonstrating assembly-induced spin transfer. Besides, spin nanoparticles are found to couple and lose their paramagnetism at sufficiently short inter-nanoparticle distance, namely, the spin coupling depends on the inter-nanoparticle distance. However, it is not found that the spin coupling leads to the nanoparticle growth.Nanoparticle assembly paves the way for unanticipated properties and applications from the nanoscale to the macroscopic world. However, the study of such material systems is greatly inhibited due to the obscure compositions and structures of nanoparticles . The assembly of atomically precise nanoparticles is challenging, and such an assembly of nanoparticles with metal core sizes strictly larger than 1 nm has not been achieved yet. Here, we introduced an on-site synthesis-and-assembly strategy, and successfully obtained a straight-chain assembly structure consisting of Ag The assembly of atomically precise clusters into ordered superstructures enables new functional material designs. Here, the authors propose a strategy for linear arrangements of AgCu clusters and explore the consequent transfer and coupling of magnetic spins. The further assembly of nanoparticles enhances or even enriches the properties of sole metal nanoparticles and constitutes a novel type of promising material that is significant not only for fundamental research but also for practical applications17. Due to numerous atoms and complex structures as well as instability, it is generally difficult to assemble metal nanoparticles in a controlled and atomically precise way20. Even the controlled and atomically precise synthesis of metal nanoparticles has long been a target for the scientific community, and this target has only come into becoming a reality recently after many trials21. Atomically precise nanoparticle assemblies provide excellent opportunities for structure (composition)-property correlation and rational tailoring of their performances, thus attracting extensive interest in recent years32. Unfortunately, despite the successful assembly of small metal nanoclusters (NCs) with sizes less than 1 nm38, the assembly of atomically precise metal nanoparticles with metal core sizes strictly larger than 1 nm has not yet been reported due to the increasing challenge in synthesis and characterization . To resolve these challenging issues, the linker is one critical factor for consideration. A relatively short (simple) linker is preferred in simplifying assembly and facilitating characterization, and the most ideal linker in this context is a single atom (including charged and uncharged atoms). Cations have been widely applied in previous assemblies;17 however, anions such as S2– and N3– have rarely been employed as linkers, especially for the assembly of particles. S2– has a good affinity to metal atoms40 and it can be in-situ produced during the synthesis of thiolated metal nanoclusters41. It is possible that the on-site produced sulfur anion acts as a linker to stick the concurrently produced nanoparticles during the synthesis of nanoparticles , and, more importantly, revealed interesting spin transfer and coupling phenomena, which will be discussed below.Metal nanoparticles can be regarded as an assembly of metal atoms and surface-protecting ligands2– access to the metal surface. A kinetical control and thermodynamic selection strategy was applied to synthesize atomically precise bimetal nanoparticles42. Briefly, the synthesis and assembly processes are shown below: a freshly prepared solution of NaBH4 and PPh4Br was added dropwise to a dichloromethane suspension containing AgCHT and CuCl in the presence of triethylamine. After stirring for 6 h at 0 °C, a dark brown solution was obtained, which was rotary evaporated to give a dark solid. This solid was washed with methanol and then dissolved in a mixed solvent of dichloromethane/hexane for crystallization (assembly) (see Methods for details).Silver salt was employed as the major metal precursor due to its accessibility, and a second metal, copper salt, was added to strengthen the stability and enrich the properties of the metal nanoparticles. Cyclohexanethiol (CHTH) was employed as a protecting ligand due to its relative flexibility, which is beneficial for S77Cu22(CHT)48]2+, and the peak of 3+ charged cluster ion with one missing CHT ligand was located at m/z = 5040.6 Da. The other peaks can be rationally assigned to the fragments, among which two peaks are assigned to [Ag76Cu21(CHT)47 + S]3+ and [Ag76Cu21(CHT)48 + S]2+ 48]2+, which was also supported by additional EDX measurements 48, as illustrated in Fig. 19S18 for short). Gradient 3 is the inverted shape of gradient 1, which appears as a mutual mirror from the top view. Six Ag3S2 units constitute gradient 2, which sews up gradients 1 and 3. Ten sets of equivalent Ag-atom sites are found in the half shell via the measurement of the radial distances and marked by nine triangles and one Ag on the apex 30 shell bonds 2 or 3 Ag atoms in the Ag56S48 outer shell) except for the six metal atoms closed to the six vertexes  staple and five types of Ag4S4 (5 × 6) staples  in the crystal phase  in terms of the Ag–S–Ag angle  is elongated to 2.835 Å, which is obviously longer than the common Ag–S bond (2.3–2.7 Å)  in the outermost shell of Ag77Cu22, which has not been reported previously in silver nanoparticles (nanoclusters). Every three S atoms are assembled into a ring with one Ag atom at the center, leading to a ripple-like pattern with six rings  around the poles shows H···H interactions with three adjacent ligands  does not prefer the forming of HS–, too. IR spectrum further excludes the existence of HS– since the feature signal of HS– at ~2550 cm–1 was not found in the IR spectrum  in the shell. The metal core diameter of Ag77Cu22 is ca. 1.69 nm, and the interparticle distance along the chain is ca. 2.19 nm  is 2.422 Å  for the Ag77Cu22 nanoparticle is 49 (n* = NvA − M − z = 77 × 1 + 22 × 1 – 48 − 2 = 49)49, reminding one of its paramagnetism50, which was confirmed by experiments. For comparison, the paramagnetism of neutral Au25 was also investigated. As shown in Fig. 25 single crystals shows multiple peaks (red curve). When the large crystals are broken into microcrystals by ultrasonication, the refined peaks disappear, and broadband  appears, indicating the influence of the crystal orientation and the magnetic anisotropy of the Au25 crystal. The EPR measurement was conducted by automatically or manually rotating the single crystal sample  are well consistent with the experimental observations , and the collinearity of C3, the linker, and the z-axis is preserved when relaxed, see Supplementary Fig. 2– linker rather than the metal atoms (Ag or Cu). Only weak spin density is revealed on the surface or kernel atoms adjacent to S2–, and the magnetic moment of the linker S2– is almost 12 times larger than that of the Ag atom with the largest magnetic moment. The detailed spin distribution is given in Supplementary Table 25 is found to be mainly localized at the kernel Au atom , exchange interaction, etc. For Au25, the magnetic anisotropy can be attributed to the strong SOC of Au atoms since the noncollinear calculation reveals large magnetic anisotropy energy . The torque method was further applied for analysis57, and it was revealed that the positive uu (27.78 meV) and dd (27.96 meV) channels contribute more (97 μeV) than the negative ud + du (–55.65 meV) channel, leading to a positive MAE, which provides further support for the SOC mechanism for the magnetic anisotropy. For the Ag77Cu22 assembly, given the consideration of the major spin on S2–, the SOC mechanism is not applicable since the SOC parameter for S (288 cm–1) with a relatively small atomic number is much lower than that for Cu (1150 cm–1) or Ag (3540 cm–1)58, and, thus, the SOC effect is negligible; the exchange interaction can also be excluded given the large distances between S spins ; magnetic measurements using a Quantum Design MPMS3 SQUID magnetometer were used to also exclude the magnetic interaction since the Curie-Weiss temperature obtained by fitting variable temperature magnetic susceptibility was close to 0  are shown in Supplementary Figs. g-value (1.9790) was close to 2.0023  and the obtained effective moment μeff = 1.62 μB is also consistent with S = ½ per cluster molecule (1.73 μB), supporting the notion that the magnetism of Ag77Cu22 assembly originates from the unpaired shell electron of the Ag77Cu22 nanoparticle. Notably, the hyperfine splitting (fine structure) was not observed in the EPR spectra, which might be interpreted by the delocalization60 of the unpaired spin or the weak spin–nuclei interaction due to the extremely low abundance of magnetic 33S nuclei62. A natural question arises: why the magnetic moment mainly locates at S2–. It is possible that electron transfer occurs from linker S2– to the metal atom. Such electron transfer between sulfur and the nanoparticle core was revealed in our previous photoluminescence study63. The close linker (Ag–S distance) also provides support for such a phenomenon. A study of the projected density of states (PDOS) revealed that the linker S shows a magnetic moment of ~1 μB with half-occupied spin-down states of px/py orbitals, while the connected Ag exhibits a very small moment with partially occupied spin-up and spin-down states of 5s orbitals , DMPO was used to capture the radical. As shown in Supplementary Figs. 77Cu22 assembly, but no significant signals were detected for the case of Au25, confirming the presence of sulfur radicals in the Ag77Cu22 assembly and the absence of sulfur radicals in Au25, since that the sulfur radicals can be captured by DMPO and lead to the emergence of the EPR signal of the trap adducts as well as the decrease of the paramagnetism signal.The nominal shell-closing electron count  are connected directly via Au−Au interactions, and the interparticle distance is 1.3001 nm, which is significantly shorter than that in the x- and y-directions . To further probe the influence of the ligand on nanoparticle coupling, we conducted an EPR test for neutral Au25 protected by a stiffer ligand  or a more flexible ligand (CHT) than 2-phenylethanethiolate (PET). Note that the 2-phenylethanethiolates on the Au25 nanocluster are fully exchanged by naphthalenethiolates but not by cyclohexanethiolates, as identified by SCXC or mass spectra 180 180. The substitution numbers (Xs) were estimated from ESI-MS as follows: Au25-I, x = 2–4; Au25-II, x = 3–6; Au25-III, x = 5–9; Au25-IV, x = 11–15; Au25-V, x = 13–17  than n-butanethiolated Au2533. Taken together, these facts demonstrate that the inter-nanoparticle distance  is critical for spin coupling . It is worth noting that such spin coupling does not result in the growth of nanoparticles since no large nanoparticles (or dimers) were detected by ESI-MS, UV-Vis spectrometry, and TEM 48]2+ nanoparticles and linker S2– was obtained, and it was precisely characterized by using ESI-MS and SCXC. Interestingly, a rare Ag/Cu alloy shell was observed in the nanoparticles, and cyclic staples and a ripple-like thiolate distribution on the nanoparticles were, to the best of our knowledge, also reported for the first time. In particular, both calculations and experiments such as EPR and DMPO probing were used to reveal that the linker is sulfur radical with magnetic isotropy, which is formed by 2p electron transfer from S2– to the connected silver atoms and stabilized by the chain structure. The disassembly of the straight-chain structure in solution leads to the return of the electron to S2– and restoration of the paramagnetism of the Ag77Cu22 nanoparticles. However, the EPR intensity for Ag77Cu22 decreases with increasing time, which is ascribed to the spin coupling of Ag77Cu22 nanoparticles in solution. Further experiments reveal that the inter-nanoparticle distance is critical to the nanoparticle (nanocluster) spin coupling . Such an assembly-induced spin transfer and inter-nanoparticle distance-dependent spin coupling are not only interesting and surprising but also have important implications for the novel functional development of nanoparticles and the understanding of the shell-closing electron behavior of superatoms. It is expected that our work will extend a promising but still challenging field−the assembly of metal nanoparticles with atomic precision.In summary, we introduced an on-site synthesis-and-assembly strategy to solve the challenge of assembling atomically precise metal nanoparticles. A straight-chain structure composed of [Ag3, 99.99%), cuprous chloride , cyclohexanethiol , tetraphenylphosphonium bromide , naphthalenethiol , 2-phenylethanethiol (97%), and sodium borohydride  were purchased from Aladdin. 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Dojindo. Triethylamine (99%), ethanol (AR), dichloromethane (AR), hexane (AR), Toluene (AR), and ethanol (AR) were purchased from Sinopharm chemical reagent co., ltd.All chemicals were commercially available and used as received. Silver nitrate  and TPPB (42 mg) was added dropwise to the mixture, which showed a color change from yellow to dark brown. The reaction was then kept under continuous stirring for 6 h under ice-cold conditions in the absence of light. The mixture was rotary evaporated to dryness to give a dark solid, which was washed with methanol three times. The as-obtained precipitate was extracted using 15 ml of CH2Cl2, and the supernatant was aged for over a week at room temperature to form a green solution. Finally, a concentrated green solution (0.5 ml) was placed into a clean NMR tube layered with ca. 2 ml of hexane. Then, the tube was carefully capped and stored in a refrigerator at 4 °C. Black crystals were obtained after approximately 1 month.AgCHT complexes were prepared by mixing 120 mg of AgNO25 NCs were first prepared according to the previous literature:68 10 mg Au25 anions were dissolved in 8 ml CH2Cl2 and treated with 200 μl H2O2 (30%) for 20 min at room temperature, then the organic solution was extracted and purified using preparative thin-layer chromatography to obtain Au250 NCs. Six milligrams of neutral Au25 was dissolved in 300 μl of toluene and then reacted with different amounts of CHT at room temperature for 30 min or 60 min. The products were precipitated and washed with methanol. Au25(Nap)18 was synthesized at 70° by mixing neutral Au25 with excess Nap. The final product was purified using preparative thin-layer chromatography71.Neutral AuOptical absorption spectra were acquired in the wavelength range of 190–900 nm using a Shimadzu UV-2600 spectrophotometer. Electrospray ionization mass spectrometry (ESI-MS) was performed on a Waters Q-TOF mass spectrometer equipped with a Z-spray source. Single crystal X-ray diffraction data were collected on a Bruker D8 Venture X-ray diffractometer . The possible reasons for the high residual density are the inadequate absorption correction of silver which strongly absorbs X-rays or the uncertainty of the metal atoms (Ag or Cu), resulting in unreal residual Q peaks. The low C-C bond precision originates from the restraints on the hexyl ligands (S2 and S5) due to their varied configuration and weak diffraction intensity. X-band CW-EPR spectra were collected using a Bruker EMX plus 10/12 X-band EPR spectrometer equipped with an ER4119HS (TE011) high-Q cavity. Cryogenic temperatures were achieved using an Oxford Instruments ESR910 liquid helium flow cryostat and an Oxford Instruments ITC503 temperature controller. A programmable one-axis goniometer was used for the sample rotation. Transmission electron microscopy (TEM) images were recorded using a Tecnai G2 TF20 instrument . Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on an Autoflex Speed TOF/TOF mass spectrometer (Bruker) in positive ionization mode. Magnetization measurements were carried out for the NCs  as a function of temperature and field by using a SQUID magnetometer (Quantum Design MPMS3).72. The ion-electron interaction was described by the projector augmented wave (PAW) approach74. The generalized gradient approximation (GGA) in the scheme of the Perdew-Burke-Ernzerhof (PBE) functional was used to describe the exchange-correlation interaction76. The force and energy convergence criteria used for the electronic structure calculations were less than 0.02 eV/Å and 10–5 eV, respectively. Given the consideration of the strong correlation effect on the d orbitals of Au, Ag, and Cu, GGA + U calculations with effective U values of 1.5 eV (Au) and 1 eV (Ag and Cu) were performed by including a Hubbard U term78. The spin moment and MAE were calculated by taking into consideration of spin-orbital coupling and magnetic noncollinearity79. EPR simulations were carried out by assuming a distribution of randomly oriented S = 1/2 spin centers with a Lorentzian lineshape using the EasySpin toolbox working on the MatLab software platform.Ab initio calculations were performed based on spin-polarized density functional theory (DFT), as implemented in the Vienna Ab initio Simulation Package (VASP)Further information on research design is available in the Supplementary InformationReporting Summary


Thirteen patients completed treatment . Adverse events occurred in 12/13 patients. The most common G47Δ-related adverse events were fever, headache and vomiting. Secondary endpoint was the efficacy. Median overall survival was 7.3 (95%CI 6.2–15.2) months and the 1-year survival rate was 38.5%, both from the last G47∆ administration. Median progression-free survival was 8 (95%CI 7–34) days from the last G47∆ administration, mainly due to immediate enlargement of the contrast-enhanced area of the target lesion on MRI. Three patients survived >46 months. One complete response (low dose) and one partial response (set dose) were seen at 2 years. Based on biopsies, post-administration MRI features (injection site contrast-enhancement clearing and entire tumor enlargement) likely reflected tumor cell destruction via viral replication and lymphocyte infiltration towards tumor cells, the latter suggesting the mechanism for “immunoprogression” characteristic to this therapy. This study shows that G47Δ is safe for treating recurrent/progressive glioblastoma and warrants further clinical development.Here, we report the results of a phase I/II, single-arm study  assessing the safety (primary endpoint) of G47∆, a triple-mutated oncolytic herpes simplex virus type 1, in Japanese adults with recurrent/progressive glioblastoma despite radiation and temozolomide therapies. G47Δ was administered intratumorally at 3 × 10 G47Δ is a third-generation, triple-mutated oncolytic HSV-1 that has demonstrated anti-tumor efficacy in preclinical studies. Here the authors report the results of a phase I/II study of G47Δ in patients with recurrent or progressive glioblastoma. The standard-of-care treatment for primary glioblastoma has been chemoradiotherapy with 60 Gy total of fractionated irradiation combined with the alkylating agent, temozolomide, after maximum surgical resection. Tumor-treating fields (TTF), which delivers low intensity alternating electric fields to the tumor2, is currently included in the standard-of-care for newly diagnosed glioblastoma in the United States. The regimen using TTF has been reported to provide a median overall survival of 20.9 months2, but its recommendation level as a standard-of-care varies among countries outside the US. Glioblastoma invariably recurs and there is no effective treatment at recurrence. Meta-analysis data of phase II studies show a median survival of 5.0 months and a 1-year survival rate of only 14% for patients with glioblastoma after recurrence3. Therefore, to improve outcomes in patients with glioblastoma, new therapies that differ from conventional approaches need to be developed4.Glioblastoma, the most common and aggressive form of brain tumor in adults, has an extremely poor prognosis, with 5-year survival rates of <10% after standard treatment5. Commonly, viruses are genetically engineered to restrict virus replication to cancer cells, and infected cancer cells are destroyed directly by the virus in the course of viral replication. Herpes simplex virus type 1 (HSV-1) is especially suited for cancer therapy, because: (1) it infects most cell types, so can be applied to a variety of cancer types; (2) a relatively low multiplicity of infection is needed for total cell killing; therefore, high efficacy can be expected in clinical settings; (3) availability of anti-viral drugs means that therapy can be terminated, if necessary; (4) the large genome allows insertion of large and/or multiple transgenes; (5) circulating anti-HSV-1 antibody does not affect cell-to-cell spread of the virus; therefore, efficacy is not inhibited in seropositive patients and dosing can be repeated without diminishing efficacy6. G207 is one of the first oncolytic HSV-1 used in clinical trials7, with deletions in both copies of the γ34.5 gene and a lacZ insertion inactivating the ICP6 gene8. Intratumoral inoculations with G207 in a syngeneic mouse tumor model of N18 (neuroblastoma) revealed that, in addition to a direct oncolytic activity, the antitumor efficacy is augmented by induction of a systemic and specific antitumor immunity associated with a longstanding elevation of a cytotoxic T lymphocyte activity against tumor cells9. G207 has been shown to be safe in patients with malignant glioma, including children, in several clinical trials11. However, there seems to be room for improvement in efficacy with G207.Oncolytic virus therapy is an approach first conceptualized ~70 years ago, in which a replication-competent virus is used to treat cancer47 gene of the G207 genome12. The deletion of the overlapping US11 promoter results in placement of the late US11 gene under the control of the immediate-early α47 promoter, which leads to partial recovery of the deleted γ34.5 functions, and results in enhanced replication in tumor cells13. The main function of the α47 gene is to allow the virus to escape from immune surveillance by downregulating the major histocompatibility complex (MHC) class I expression in infected host cells through binding the α47 gene product to the transporter associated with antigen presentation14. The α47 deletion in G47∆ causes a further attenuation of the virus in normal cells, but enhances the stimulation of antitumor immune responses12. In fact, G47Δ demonstrated higher cytocidal activity and greater antitumor efficacy than G207 both in vitro and in vivo, while retaining high safety characteristics12. In addition to glioma, G47Δ has also shown efficacy in a wide range of solid tumors24. A phase 1 study was completed in patients with castration-resistant prostate cancer, in which 3 × 108 pfu (plaque-forming units) of G47Δ was injected into the prostate using a transrectal ultrasound-guided transperineal technique (UMIN000010463). Also, a phase 1 study was recently completed in patients with malignant pleural mesothelioma , in which 1 × 109 pfu of G47Δ was injected repeatedly into the pleural cavity (result unpublished), and another phase I trial is ongoing in patients with olfactory neuroblastoma . Further, G47Δ has been shown capable of killing cancer stem-like cells derived from glioblastoma patients and inhibiting their self-renewal28.G47Δ, a third-generation, triple-mutated oncolytic HSV-1, was created by introducing a further deletion within the αHere, we report the results of a phase I/II trial using G47∆ in Japanese patients with recurrent or progressive glioblastoma. This single-arm, dose-escalation study primarily aims to assess the safety of repeated stereotactic intratumoral G47Δ administrations at two dose levels. We follow patients enrolled in this study for a long period for any long-term G47∆-related adverse events in survivors. Secondary objectives are to assess the efficacy of G47Δ in terms of tumor shrinkage assessed by MRI, overall survival, and progression-free survival. We show that G47Δ is safe for treating glioblastoma and that further clinical development is warranted.9 pfu per dose , as the highest dose in this study. This decision was based mainly on the finding that all three patients in Cohort 2 showed increased frequency of convulsion. In addition, because G47∆ replicates within the tumor, the administered dose is amplified and the actual amount of G47∆ should vary widely among patients. Therefore, the conventional 3-fold dose-escalation scheme often used for chemotherapy was considered unsuitable for this study. Thus, 1 × 109 pfu per dose  was the highest dose tested in the phase I part, and this dose was used for the phase II part. Seven additional patients were enrolled in the phase II part and, in November 2014, the completion of this study was approved as the Phase II clinical trial (UMIN000015995) started.This phase I/II study was conducted from November 2009 to November 2014  at the University of Tokyo Hospital and the Institute of Medical Science Hospital of the University of Tokyo. According to the study protocol, it was required to allow a certain period of time between patients and between cohorts to ensure safety. This study consisted of a phase I part and a phase II part. The study protocol for the phase I part was a typical 3 × 3 scheme with a 3-fold dose-escalation , which corresponded to the low dose, whereas patients in Cohort 2 and the phase II part received G47Δ at a dose of 1 × 109 pfu , which corresponded to the set dose. For each administration, a total volume of 1 mL was equally divided and G47∆ was injected to two different sites (0.5 mL per site) of contrast-enhanced areas within the tumor in all patients.Accordingly, 13 patients who completed treatment between November 2009 and July 2014  were included in this study  and MGMT expression using biopsy specimens revealed two patients (15.4%) positive for IDH1 mutation, and seven patients (53.8%) showed positive immunostaining for MGMT.Patient demographics and clinical characteristics are shown in Table n = 8 [61.5%]), fever (n = 8 [61.5%]), and vomiting (n = 6 [46.2%]) (Table Regarding safety outcomes (primary endpoint), adverse events occurred in all three patients (100%) in the low dose group and nine of ten patients (90%) in the set dose group. The most common adverse events occurring up to 90 days after the last G47Δ administration included headache (n = 6 46.%) (Tablen = 6 46.%) (Tablen = 6 46.%)  from the initial surgery  was 30.5 95% CI 19.2–52.7) months. The median overall survival (OS) from the last G47Δ administration was 7.3 (95%CI 6.2–15.2) months  clearing of contrast enhancement at the injection site, and 2) enlargement of the entire enhanced lesion. These characteristic MRI features occurred in most patients and within 14 days (and mostly within 7 days) of the first G47∆ injection  of the 12 patients who died underwent autopsy. Autopsy brain tumor specimens showed viable glioblastoma cells on hematoxylin and eosin staining, and persistent intratumoral infiltration of CD4iod Fig. . HSV-1 i+ and CD8+ T lymphocyte counts and CD4/CD8 ratio at baseline and on days 30 and 60. Figure Seroconversion was observed for HSV-1 antibody (IgG) 1 week after G47Δ treatment in all four patients who were seronegative for HSV-1 (IgG < 2.0) before treatment. In the other 9 patients, IgG antibody titers tended to increase until 2–3 months after G47Δ administration and then decreased  adverse events. Adverse events clearly or likely related to G47∆ were all Grade ≤ 2. HSV-1 is neurotropic and the most common cause of sporadic viral encephalitis in humans, with severe manifestations and a high mortality rate. Therefore, the largest safety concern of oncolytic HSV-1 is naturally its potential to cause encephalitis, especially when injected into the brain. Although T-Vec , a second generation oncolytic HSV-1 that has mutations in the γ34.5 and α47 genes and expressing GM-CSF, has been approved as the first oncolytic virus drug in the US and Europe for malignant melanoma29, preclinical studies have shown that T-Vec (expressing mouse GM-CSF) exhibited toxicity when injected into the brain of BALB/c mice at 1 × 105 pfu/dose . Therefore, it should be stressed that G47∆ at a concentration of 1 × 109 pfu/mL and 1 mL/dose, higher than T-Vec used for melanoma patients in clinic (1 × 108 pfu/mL and up to 4 mL/dose), showed a favorable safety profile when used in the brain. G207, double mutated in the γ34.5 and ICP6 genes, has proved to be safe when used in the brain in several phase I clinical trials11, but preclinical studies have demonstrated that G47∆ exhibits much higher efficacy than G20715. Therefore, the achievement of both efficacy and safety likely resulted from the careful designing of the G47∆ genome to create mutations effectively in the three viral genes, γ34.5, ICP6 and α47.This study sought to primarily determine the safety of G47Δ in patients with recurrent or progressive glioblastoma. The treatment of repeated intratumoral injections with G47Δ, up to 1 × 107. The IDMC decisioned to set the second cohort dose (1 × 109 pfu/dose) of the phase I part as the highest dose of this study and to use this dose to proceed to the phase II part. Maximum tolerated dose (MTD) is generally defined as the highest dose of a drug that does not cause unacceptable side effects. Dose-limiting toxicity was not observed in this study, so, by definition, MTD was not determined in this study. One of the questions raised by the IDMC was whether the concept of MTD, which is generally equivalent to the optimal dose in the development of chemotherapy drugs, can be applied to development of oncolytic HSV-1. A 3 × 3 scheme has been used for oncolytic viruses30, but, at the time of this study, the IDMC raised a question regarding the significance of the 3-fold dose-escalation scheme. In fact, in preclinical studies, G47Δ was found to show a 100-fold difference in replication capacity depending on the cell type12, and it was deemed questionable whether the 3-fold dose difference in virus titer is significant. A recent trial with G207, the parental virus of G47∆, used a 10-fold dose-escalation scheme in children with malignant glioma31. DLT has not been seen in the clinical development of G207, and, although 3 × 109 pfu was the highest dose used in the first phase I trial for malignant glioma, this highest dose was not the dose used for subsequent clinical trials11.The study protocol for the phase I part was a 3 × 3 scheme, with a 3-fold dose escalation similar to the first G207 trial protocol for malignant glioma32. The adverse events observed with G47∆ in this study were mainly related to immune responses presumably to eliminate explosively replicating G47∆ and towards newly recognized tumor cells. A recombinant polio-rhinovirus chimera (PVSRIPO) was reported to have caused severe brain edema and nervous system disorders with peritumoral inflammation in glioblastoma patients, even causing death in one patient at 3.3 × 108 50% tissue-culture infectious doses, and all patients required steroid administration33. In contrast, G47∆ caused side effects to a much lesser extent and was generally well tolerated.The duration of median overall survival from the initial diagnosis (30.5 months) was longer than expected. Most patients enrolled in this study were relatively young (average 50 years old), possibly reflecting the relatively narrow eligibility criteria. However, a meeting by the Eligibility Assessment Committee took place before every enrollment, and all eligible patients were enrolled sequentially without exception and intentional selection. Post-hoc immunohistochemistry for IDH1 mutation (R132H) revealed that two patients (15.4%) were IDH1 mutated, but all three long-term survivors were IDH1 wild type. It has been reported that there is no difference in survival time after recurrence of glioblastoma between patients with and without IDH mutation+ and CD8+ T lymphocytes seemingly infiltrating into areas with remaining viable tumor cells rather than HSV-1 positive areas, although it is possible that infiltrating lymphocytes could be trafficking towards injected virus or virally-infected cells. Secondly, the enlarged enhanced lesions returned to their original size after 7 days of steroid administration in cases that received steroids. Thirdly, in Patient #5, the high accumulation of MET on PET images between the resected primary tumor and recurrent tumor site, which was observed soon after G47∆ administration, was also resolved after steroid administration. Therefore, the high accumulation observed on MET-PET likely reflected the infiltration of activated lymphocytes towards tumor cells. These findings support the idea that the enlargement of the enhanced lesion on MRI immediately after G47∆ administration is caused by a sudden influx of lymphocytic infiltrates towards the tumor cells. It has been reported that angiogenesis is stimulated by G47Δ in a mouse model34, and the rapid increase in contrast-enhancement may also reflect the virus-induced increase in vascular permeability35. This phenomenon is distinct from the pseudoprogression observed after radiation therapy in terms of both mechanism and timing, so we propose to refer to it as “immunoprogression”. Presumably, the infiltration of lymphocytes towards tumor cells occurs, because they come to newly recognize the tumor cells as non-self. Potentially, in the course of the host immune system eliminating G47∆, antigen-presenting cells process G47∆ bound to destroyed tumor cells and present tumor-associated antigens as non-self targets. It is still uncertain whether and how the emergent lymphocytic infiltrates are related to long-term specific antitumor immunity. MRI results from two patients (#3 and #4) showed that it likely takes 4–5 months for the antitumor immunity to have a therapeutic effect leading to tumor shrinkage. Thus, lymphocytes entering the tumor from a very early stage of G47∆ therapy may be an unknown subset of lymphocytes with previously unknown roles. Further investigations are necessary to confirm the relationship between “immunoprogression” and specific antitumor immunity.On MRI, all patients commonly showed a clearing of contrast enhancement at the injection site and an enlargement of the entire enhanced lesion almost immediately after the first G47∆ administration. This explosion crater-like phenomenon was a fresh observation characterizing the MRI features of glioblastoma treated with G47∆, and not previously reported with other oncolytic viruses, including G207. The histology of biopsy specimens of the G47∆ injection site obtained 5–14 days later suggests that the clearing on MRI reflects the destruction of tumor cells caused by intense viral replication. Regarding the enlargement of the contrast-enhanced lesion, several lines of evidence point to immune responses as the putative mechanism. Firstly, even for glioblastoma, this enlargement occurred too soon to be caused by tumor growth. The histology of biopsy specimens showed large numbers of CD436. In this study, therefore, we restricted the use of steroids as much as possible. Steroids were used in two patients for only 7 days in both for marked tumor swelling on MRI (Patient #4) and for persistent fever (Patient #5). In these cases, enlargement of the enhanced lesion and a remarkable high accumulation on MET-PET were resolved and the fever recovered immediately after steroid administration. These findings indicate that steroids can effectively counteract the side effects of G47∆ caused by immune responses. Patient #4 remains alive for >11 years after G47∆ treatment, suggesting that 7 days of steroid administration did not adversely affect treatment efficacy.Steroids have been shown to inhibit the induction of antitumor immunity and to have a negative effect on long-term therapeutic effects in oncolytic HSV-1 therapy37 when designing the subsequent phase II trial. Secondly, host immunity appears to significantly affect the elimination as well as the efficacy of the oncolytic virus42. Thirdly, because the extent of viral replication and the strength of immune responses vary among individuals, an effective method for oncolytic HSV-1 therapy may be to repeat the injections based on therapeutic responses. Finally, the long-term efficacy of G47Δ likely depends on the induction of specific antitumor immunity, which may take 4–5 months or more to become apparent; hence, oncolytic virus therapy should be applied at the earlier stage of the disease.Several important practical points have been learned from this first-in-human G47∆ study. Firstly, because the “immunoprogression” was commonly observed on MRI in most patients soon after G47∆ administration, conventional response criteria such as WHO and RECIST are not suitable for evaluating oncolytic virus therapy. Instead, this finding led to the formulation of oncolytic virus-specific response criteria referring to immune-related response criteria46. The results of preclinical data indicate that G47Δ decreases regulatory T cells while recruiting other lymphocytes47. Indeed, in a study using various syngeneic murine subcutaneous tumor models, G47Δ and systemic anti-CTLA-4 antibody administration enhanced antitumor activity with upregulation of gene signatures related to inflammation, lymphoid lineage, and T cell activation suggesting an increase in immune susceptibility47.One of the benefits of G47Δ is that it can be combined with other anti-cancer treatments. Given the favorable safety of G47Δ apparent in the present study, it is reasonable to consider that there are few concerns about increased adverse effects when G47Δ is used in combination with other treatments. In the clinic, it is likely that G47∆ will be used in addition to the Stupp protocol when G47∆ becomes widely available for malignant glioma patients. Some promising results have been reported on the therapeutic effects of combining G47Δ and immune checkpoint modulators9 pfu/dose for two doses within 14 days. Side effects were mainly caused by immune responses which, when necessary, could be effectively counteracted by a 7-day steroid administration. G47∆ caused immediate infiltration of lymphocytes that seemingly directed towards tumor cells, which was reflected on image studies with features characteristic to G47∆ therapy. Long-term survival (>46 months) was observed in 3 of 13 patients, which may be due to the delayed effect of G47∆ via antitumor immunity. The results justified and formed the basis of the phase II clinical trial in glioblastoma patients, including repeated G47∆ administrations into different coordinates of the tumor and adopting of response criteria specifically designed for oncolytic HSV-1.In summary, this study proved the safety of G47Δ when administered intratumorally at up to 1 × 107. The highest dose tested in the phase I part was to be further used in the phase II part. The primary objective of this study was to assess safety while efficacy assessment was the secondary objective. The study was registered at the UMIN-CTR Clinical Trial Registry (UMIN000002661) on October 23, 2009. The first patient was registered on November 6, 2009, and the last patient on June 30, 2014. Eligible patients were 18 years or older who had undergone prior surgery at the time of initial or recurrent disease with (i) a pathologically confirmed diagnosis of glioblastoma not responsive to radiation therapy, (ii) MRI-enhanced lesions ≥10 mm, (iii) Karnofsky Performance Scale (KPS) score >60%, (iv) no abnormalities that meet the exclusion criteria and normal major organ function. Patients could be enrolled regardless of prior chemotherapy treatment status. Concomitant usage of antitumor treatment other than G47∆ was not permitted during the 2-year observation period or until the patient is judged PD. Patients were excluded if G47∆ administration would be required to the ventricle (or via the ventricle), brainstem, or posterior fossa, or if any of the following were present: (i) multiple (≥2) glioblastoma lesions, (ii) subependymal and subarachnoid dissemination, (iii) allergy to anti-HSV drugs (acyclovir). A full listing of inclusion and exclusion criteria is included in Supplementary Table This was a phase I/II, single-arm study of G47Δ in Japanese patients with glioblastoma. This study consisted of a phase I part and a phase II part. The study protocol for the phase I part was a typical 3 × 3 scheme . Generated and purified G47∆ was resuspended in phosphate buffer saline (PBS) containing 10% glycerin, dispensed into cryotubes, and stored at −80 °C. A tube of clinical lot G47∆ was thawed prior to each administration and adjusted to 1 × 108 pfu, 1 × 109 pfu, and 3 × 109 pfu , respectively. After the second cohort, the IDMC decided not to proceed to the third cohort and to set the second cohort dose as the highest dose in this study, mainly because all three patients in the second cohort showed increased frequency of convulsions, which was later found to be coincidental. Further, because G47∆ replicates and amplifies after administration, the actual amount of G47∆ should vary widely among patients. Therefore, the IDMC decided to use 1 × 109 pfu/dose , as the set dose to proceed to the phase II part of the study. DLT was not observed in the phase I part, so this set dose was not MTD by definition.Three cohorts of three patients each were planned to determine the maximum dose of G47Δ (the phase I part). The 3 × 3 scheme is shown in Supplementary Fig. G47Δ was administered directly into the tumor by stereotactic surgery. Each cohort dose was diluted with 10% glycerin PBS to a total volume of 1 mL and, according to the study protocol, divided equally into 2–5 target sites within the tumor. G47∆, filled into a 1 mL syringe, was administered manually and slowly into the tumor at ~0.2 mL/min using the Biopsy/Injection Needle . After each injection, the biopsy/injection needle was kept in place for 5 min before being pulled out from the brain to avoid leakage. The second dose was given using the same burr hole and to exactly the same coordinates 5–14 days after the first dose.+/CD8+ count and ratio). The assessment of viral shedding was performed according to the study protocol. Quantitative PCR was used to detect G47∆ DNA in blood, saliva and urine collected on the day after the first administration of G47Δ, the day after the second administration, and 7 days after the second administration. In addition, viral culture was performed with blood samples.The primary endpoint was safety of G47Δ, assessed by the type, frequency, and severity of adverse events. The frequency of adverse events up to 90 days after the second dose of G47Δ was determined in all treated patients using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0. Relationship of adverse events to G47Δ was assessed on a 5-point scale. Patients also underwent laboratory testing for blood chemistry, coagulation, and lymphocyte fraction . The response rate was defined as the proportion of patients with either CR or PR among eligible patients with measurable disease.Secondary endpoints included tumor response in the 90-day observation period after G47Δ administration. Response was evaluated using WHO-Response Evaluation Criteria based on MRI examinationIn addition to MRI imaging, PET imaging with C11 methionine (MET) (MET-PET) was conducted in select patients as exploratory analyses. Frequent MET-PET imaging studies were performed only in Patient #5.IDH1 mutation status and MGMT expression was tested post-hoc by immunohistochemistry of biopsy specimens obtained immediately prior to the first G47∆ injection. Formalin-fixed, paraffin-embedded tissue sections of 3-μm thickness were deparaffinized in xylene and rehydrated in a series of graded alcohols. Antigen retrieval was performed with citrate buffer pH6 for CD4, CD8, and MGM and EDTA buffer pH9 for HSV.Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxidase for 5 min at room temperature. Then sections were incubated with the following primary antibodies: CD4 ; CD8 ; HSV ; IDH1 R132H ; MGMT  for 30 min at 37 °C, respectively. After washing, sections were treated with secondary antibody  for 30 min at 37 °C. To visualize the antigen-antibody complex, ImmPACT DAB substrate kit (Vector Laboratories) was used, and then sections were counterstained with hematoxylin. Supplementary Table http://www.med.or.jp/wma/); Ministerial ordinance on Good Clinical Practice (GCPs) ; Application for Class 1 Use Regulations Based on the Law Concerning the Ensuring Biological Diversity through the Regulatory of the Use of Genetically Modified Organisms; and the Guidelines for Clinical Research on Gene Therapy .This study protocol was approved by the Ministry of Health, Labour and Welfare of Japan (MHLW) on May 11, 2009 for the University of Tokyo Hospital and on March 22, 2012 for the Institute of Medical Science Hospital. This study was also reviewed and approved by the Gene Therapy Clinical Research Review Board of the participating institutions and conducted in compliance with the following ethical standards and regulations: World Medical Association Declaration of Helsinki  as the set dose to proceed to the phase II part of this study. Seven additional patients were treated  before the IDMC approved the completion of this study due to the start of Phase II clinical trial. Numerical data on the frequency of different adverse events were tabulated, but no statistical analyses were performed. Overall survival was calculated from the first surgery and from the last administration of G47∆ until the date of death or last follow-up. Progression-free survival was calculated from the date of the last administration of G47Δ until date of progression or period up to the earlier date of death from any cause. The Kaplan–Meier method was applied to describe the distribution of survival time. All data analyses were done with IBM SPSS Statistics version 22 software .Initially, three cohorts of three patients each  were included in determining the safe set dose (phase I part), with an additional 12 patients at the set dose or MTD (phase II part) to reach a target sample size of 21 patients (maximum 30 patients). After the second cohort of the phase I part (six patients), the IDMC decided to use 1 × 10Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


We have generated an 80 million-year Permian–Triassic δ34Sevap curve from the Staithes S-20 borehole, Yorkshire, England. The Staithes δ34Sevap record replicates the major features of the global curve, while confirming a new excursion at the Olenekian/Anisian boundary at ~ 247 million years ago. We incorporate the resultant δ34Sevap curve into a sulphur isotope box model. Our modelling approach reveals three significant pyrite burial events (i.e. PBEs) in the Triassic. In particular, it predicts a significant biogeochemical response across the EPME, resulting in a substantial increase in pyrite burial, possibly driven by Siberian Traps volcanism. Our model suggests that after ~ 10 million years pyrite burial achieves relative long-term stability until the latest Triassic.Despite the extensive use of sulphur isotope ratios (δ The EPME is associated with a reduction in marine species biodiversity on the order of 80–90%2, extinction amongst tetrapods, and a possible dieback of terrestrial vegetation3. Driven by volcanism from the Siberian Traps4, the EPME is intimately linked with increased CO2, CH4 and SO2 fluxes7, heightened global atmospheric and sea surface temperatures (SST)8, intensified chemical weathering9, ozone depletion10, a reduction in marine pH11 and an expansion of anoxic, and possibly euxinic, oceanic water masses13. It has been proposed that the Early Triassic represents a period of climatic, geochemical, and biological instability, delaying the recovery from the EPME17. Multiple SST changes18 likely coincided with major fluctuations in ocean chemistry expressed as excursions in the carbon and sulphur isotope geochemistry of marine carbonates and evaporites19, followed by conditions of relative stability in the Middle Triassic15.The Permian–Triassic interval has attracted much attention due to significant biological and geochemical events, including the end-Permian mass extinction (EPME)—the most catastrophic extinction event of the Phanerozoic20 and the Smithian/Spathian boundary17. These records lack temporal coverage and fail to capture long-term biogeochemical conditions for the Triassic at high resolutions. One exception is by Song et al.15, who compiled a δ34S record of carbonate-associated sulphate (CAS) from the late Permian to Middle Triassic from sections in south China. However, CAS is prone to diagenetic alteration22, with much of the isotopic heterogeneity of δ34SCAS records across the EPME attributed to post-depositional alteration23. Bernasconi et al.19 compiled a δ34Sevap record of sedimentary evaporites from the late Permian to Middle Triassic, including multiple sections across several countries in Europe. Although evaporites are less prone to diagenetic alteration19, their coverage in the sedimentary record is often sparse and not continuous, thus resulting in a lack of high-resolution δ34Sevap curves.Despite the biogeochemical significance of the Triassic, robust sulphur isotope data are sparse, with most studies focusing on specific, short periods of time, such as the EPME34Sevap curve from the Staithes S-20 borehole , Yorkshire, England .To address the lack of a single geographic and stratigraphic record, we have generated a high-resolution δA total of 364 individual evaporite samples  were collected at regular intervals. For gypsum and anhydrite, a drill was used to produce a fine powder for isotopic analysis, whilst for halite the sulphate was obtained through barium sulphate precipitation (see Supplementary for Methodology).34Sevap curve for the Permian and Triassic, consisting of ~ 1000 δ34Sevap results (see Supplementary); our new, continuous record from a single site adds 38% more data to the global curve. All results were double-checked for their age assignment against a standardised geological timescale25. Based upon trends and inflection points in the global δ34Sevap record, we correlated the Staithes S-20 curve to generate a more robust global δ34Sevap record of the late Permian–Late Triassic; especially the Middle and Late Triassic , and stratigraphic correlation with the composite δ34Sevap curve, a new rapid negative δ34Sevap excursion (on the order of 15‰) is recorded at the Olenekian/Anisian boundary (OAB) (~ 247 Ma). Following this, the δ34Sevap record exhibits an abrupt recovery to pre-excursion values of 29‰ at ~ 246 Ma.The composite late Permian—Early Triassic δity Fig. , interpr19. Accordingly, our δ34Sevap record exhibits a gradual and persistent decline from ~ 246 Ma in the early Anisian, before stabilising at ~ 236 Ma in the early Carnian , potentially suggesting the environmental changes during the CPE had little impact on the global sulphur cycle. This is of interest, as the CPE is associated with major carbon cycle perturbations, the emplacement of the Wrangellian LIP (large igneous province) and a mass extinction event (followed by biotic radiation)27. It is thus intriguing that our δ34Sevap record maintains relative stability across this time interval. Higher resolution δ34Sevap records spanning the CPE, accompanied by further biogeochemical modelling, are required to confirm the apparent disconnect between the carbon and sulphur cycles during the CPE.Interestingly, we see no evidence for a substantial change in δ34Sevap record also highlights the presence of a small positive δ34Sevap excursion (~ 4‰) prior to the Norian/Rhaetian boundary  into a sulphur isotope box model28 (see Supplementary). The model outputs predict three pyrite burial events (PBEs) during the time interval of this study, at ~ 251 Ma, ~ 246 Ma, and ~ 213 Ma  associated with microbial reduction of sulphate to sulphide  has been shown to vary according to a range of biological and environmental factors31. Recent biogeochemical modelling approaches suggest that variability in the δ34S of seawater sulphate during the Cenozoic can be accounted for by a shift in δ34S, reflecting a change in the locus of pyrite burial to deeper more oxygen-sparse water masses, rather than a simple change in pyrite burial rates32. Unfortunately, previous work19 did not consider a possible change in δ34S when interpreting variability in the δ34S of seawater sulphate observed for the Early Triassic.It should be noted however, that the fractionation factor . We explored a range of values for δ34S between −35 and −50‰ for the Early Triassic  in the presence of reduced iron. With the expansion of anoxia, pyrite formation may have occurred more readily within the water column41, heightening the magnitude of isotopic fractionation42. As suggested by our model results, this process would have sequestered isotopically light sulphur (32S) from the ocean reservoir, which would have contributed to the major positive δ34Sevap excursion in the Early Triassic . As before, it was necessary to test for the sensitivity of inferred pyrite burial rates to changes in δ34S, and we thus completed several sensitivity tests with a range of values between –25 and –40‰ for the time interval 249 to 247 Ma  has been demonstrated to correlate with sea level fluctuations30, and is of interest considering the OAB coincides with a general fall in eustatic sea level45  pyrite-rich shales from Early Triassic continental shelves) to weathering, (2) or extensive late Permian evaporite deposits (Zechstein). The sulphate released from pyrite oxidation and/or weathering of Permian Zechstein evaporites would be isotopically depleted , thus contributing to the negative δ34Sevap excursion at the OAB. This is in line with our model outputs, which suggest a reduction in pyrite burial to ~ −0.02 Tmol/year . Using either atmospheric oxygen and/or ferric iron as oxidants, the weathering of pyrite would yield sulphuric acid48, hence exacerbating weathering rates and contributing to the high 87Sr/86Sr values at the OAB49.Coincident with the temperature decrease is a general fall in eustatic sea level34Sevap values to earliest Triassic levels of 29‰ immediately after the OAB is concomitant with an increase in the pyrite burial flux to ~ 1.54 Tmol/year at ~ 246 Ma . We propose this reflects a recovery from the pyrite oxidation/evaporite weathering event responsible for causing negative δ34Sevap excursion at the OAB. In line with decreasing 87Sr/86Sr values in the early Anisian49, a relative decline in terrestrial weathering of sedimentary sulphides and evaporites would have reduced the flux of isotopically light sulphur into the ocean reservoir. In turn, this would have ensured rates of pyrite burial outpaced those of pyrite weathering, sequestering isotopically light sulphur from the seawater sulphate reservoir, facilitating a return to previous long-term δ34Sevap values . Again, sensitivity tests were performed with a range of δ34S values between −25 and −50‰, yielding estimates for pyrite burial between 1.67 and 1.15 Tmol/year, respectively (see Supplementary). As before, shifting δ34S to more negative values reduced the magnitude of the predicted increase in pyrite burial; nonetheless, we still consider it a noteworthy PBE.Global δ34Sevap excursion is currently unclear. A likely candidate is the emplacement of the Angayucham complex  at 214 ± 7 Ma53, which coincides with an oceanic warming event18, high CO2 concentrations54, and increasing humidity in Eastern Europe55 and the Alps56. Such environmental responses would have invigorated the hydrological cycle, thus increasing weathering and nutrient fluxes57, driving oceanic productivity in surface waters and oxygen consumption at depth in the water column. These environmental changes would have stimulated pyrite burial, and hence a positive δ34Sevap excursion. Tighter age constraint of the Angayucham Complex and additional δ34Sevap records over this time interval are necessary to ascertain their linkage. Why a δ34Sevap excursion is not present during a similar environmental event (CPE) is unclear and requires further investigation.The precise mechanism behind this δ2 emissions are potentially 14 times greater than peak emission rates during the EPME11 is a matter of grave concern. The environmental changes recorded in our δ34Sevap record and the EPME lasted on the order of 10 million years before the sulphur and carbon biogeochemical cycles became stabilised. Current anthropogenic emissions have already shown a measurable impact on marine ecosystems globally58, a reduction in the pH of surface waters59, a decline in oxygen concentration60, and an increase in ocean stratification61. Understanding the long-term record of global Earth system perturbations caused by an elevation in greenhouse gases will improve our understanding of marine anoxia, weathering and pyrite burial events in the geologic record.A direct comparison between LIP-induced environmental change in the geologic record and anthropogenic climate forcing is complex and ambitious. However, the fact that modern COSupplementary Information 1.Supplementary Information 2.