Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures. Filamentous cable bacteria conduct electrical currents over centimeter distances through fibers embedded in their cell envelope. Here, Boschker et al. show that the fibers consist of a conductive core containing nickel proteins that is surrounded by an insulating protein shell. The observation that electrical currents are transported along centimeter-long filaments4 extends the known length scale of biological electron transport by orders of magnitude, and suggests that biological evolution has resulted in an organic structure that is capable of highly efficient electron transport across centimeter scale distances4. Recent studies demonstrate that cable bacteria effectively harbor an internal electrical grid, which displays a unique topology and exceptional electrical properties8. The cell envelope of cable bacteria contains a distinctive network of parallel fibers (each ~50 nm diameter) that run along the whole length of the filament6. These fibers are embedded in a joint periplasmic space and remain continuous across cell-to-cell junctions5. Direct electrical measurements demonstrate that these periplasmic fibers are the conductive structures8. Additionally, the cell-to-cell junctions contain a conspicuous cartwheel structure that electrically interconnects the individual fibers to a central node, and in this way, the electrical network becomes redundant and fail-safe8. The electrogenic metabolism of cable bacteria necessitates that nano-ampere currents are efficiently conducted over centimeter scale distances through this network4, and in effect, the periplasmic fibers display extraordinary electrical properties for a biological material7. The estimated in vivo current density of ~106 A m−2 is comparable to that of household copper wiring, while the estimated conductivity of the fibers exceeds 20 S cm−1 and thus rivals that of doped synthetic conductive polymers7.Cable bacteria are multicellular microorganisms in the Desulfobulbaceae family that display a unique metabolism, in which electrical currents are channeled along a chain of more than 10,000 cellsBio-materials typically have an intrinsically low electrical conductivity, and so the availability of a bio-material with extraordinary electrical properties has great potential for new applications in bio-electronics. This prospect of technological application however requires a deeper understanding of the mechanism of electron transport as well as the structure and composition of the conductive fibers in cable bacteria. Yet at present, these aspects remain highly enigmatic. One important obstacle is that cable bacteria have a highly complex metabolism and life-style, which strongly hampers culturing and biomass collection. The inability to obtain sufficient cable bacteria biomass precludes the implementation of many traditional bulk analytical techniques that could shed light on the chemical composition of the electrical network. Here, we solved this problem by the application of high-resolution microscopy, spectroscopy, and chemical imaging methods to individual filaments of cable bacteria. This approach allows to elucidate the chemical structure and composition of the conductive fibers in cable bacteria.In this work, we show that the long-range electron transport in cable bacteria is crucially dependent on proteins containing a sulfur-ligated nickel group. This finding sets the conduction mechanism in cable bacteria apart from any other known form of biological electron transport, and demonstrates that efficient conduction is possible through centimeter-long protein structures.5, which contains the conductive fibers8. Previous studies have already elucidated the geometrical configuration of this fiber network8. Here, we applied high angle annular dark field–scanning transmission electron microscopy (HAADF-STEM) and subsequent tomography, which provided additional details of the fiber sheath architecture  and polysaccharide (1202 cm−1 and broad feature at 1115–1166 cm−1) (band assignment based on11). The detection of ester groups in the AFM-IR spectra  suggested that the polysaccharide contains acidic sugars9. A band at 3056 cm−1 likely represents aromatic C–H stretching for instance from aromatic amino acids. Cell junctions give similar AFM-IR spectra as central cell areas, although signals are higher in the junctions  provided a first insight into the biochemical composition of the fiber sheath Fig. , and indure Fig. , thus su16. After ~150 s of sputtering, the amino acid-derived signal levelled off, while counts of oxygen-rich fragments, including carbohydrate specific ions 17 peaked. In these samples, the fiber sheath forms a flattened hollow cylinder upon the supporting substrate, which has a total thickness of 117 ± 10 nm  analysis was applied in combination with in situ AFM calibration of the sputtering depth. This enabled us to map the nanometer-scale depth distribution of both organic and inorganic constituents within individual fiber sheaths  the conductive fibers are positioned in a regular, parallel pattern on the outside of the fiber sheath, (2) that the fibers consist of protein, and (3) that the fibers rest upon a basal sheath rich in polysaccharide. In bacteria with a Gram-negative cell envelope such as cable bacteria, the most likely source of the polysaccharide layer is the periplasmic peptidoglycan layer (see Supplementary Note  the cond−1) that have been seen previously3, but additionally revealed two prominent bands within the low-frequency region at 371 and 492 cm−1  Raman microscopy spectra obtained from living cable bacteria showed the resonance bands of cytochrome heme groups , the Amide I peak of protein (1672 cm−1), CH2 bending (1462 cm−1), and phenylalanine ring-deformation (1005 cm−1)19. Cross-sectional scans of intact filaments showed a unimodal signal for biomolecular signals generally present in microbial biomass . In contrast, the signal of the two low-frequency bands showed a bimodal maximum at the filament edges  and C–H (1451 and 2950 cm−1)19. Ratios between the background-corrected peak heights of the two low-frequency bands were similar in all Raman spectra recorded , suggesting that both bands originate from a single moiety.When air-dried intact cable bacteria were investigated with near infrared (NIR) laser (785 nm) Raman spectroscopy, the two low-frequency bands were again present, but the spectra also showed additional bands Fig. , charactges Fig. , which sges Fig. . This wages Fig. , which pm−1 Fig. , which m34S or 13C stable isotope tracers and investigated intact air-dried filaments with Raman spectroscopy as before. Labeling with 34S did not affect the cytochrome bands as expected, but resulted in a shift in both low-frequency bands toward lower wave numbers .To further examine the origin of the two low-frequency bands, we grew cable bacteria in sediments amended with ers Fig. . This su5, fiber sheaths contain ~4 times less biomass compared to intact filaments, thus explaining selective Ni enrichment. In contrast, Fe was partially removed by a factor of ~2, which is consistent with the loss of cytochrome bands in the Raman spectra after extraction  Fig. . Metals ion Fig. . Cu remaion Fig.  showed t58Ni, 60Ni, 61Ni, and 62Ni) showed a sharp subsurface peak, while the minor isotope 64Ni showed mass interference (likely from a low amount of 64Zn). Other transition metals had either low counts  at the same position as the Ni peak  within <0.5 nm, depth (Z) within <2 nm), otherwise these NiS-clusters would not form in the ToF-SIMS ion plume23. Finally, we detected two organic sulfur fragments (C2S2− and C2S2H−) that were specifically associated with the Ni3S3− peak  and functionally intact . This chemical resistance could be aided by protein disulfide bridges, which could be abundant as suggested by the high S content  could hence come from the disulfide bridges. Alternatively, these two organic fragments could derive from the Ni ligating group. Combined, our results demonstrate that the individual fibers are rich in Ni and S, and that Ni represents the metal in the sulfur-ligated group as detected by Raman analysis.High-resolution NanoSIMS analysis confirmed that the conductive fibers were Ni and S rich. Sern Fig.  with a seas Fig. . Furthertra Fig.  also sugeak Fig. . Fiber sent Fig. . The two4FeII(CN)6 resulted in a small increase in the green laser Raman signal of the two low-frequency bands, while oxidizing the fiber sheath with K3FeIII(CN)6 almost completely removed these signals 6 restored the Raman signal, suggesting that the Ni/S group is a reversible redox group. Intriguingly, the conductance of the fiber sheath was also reversibly affected by the redox state of the Ni/S group  higher conductance than oxidized fiber sheaths (independent of the direction of the oxidation/reduction step). Note that the Ni group is possibly not completely reduced, as measurements were done in air, and so the effect of oxidation state on conductance may actually be larger. The decrease of conductance upon oxidation is consistent with previous observations, showing that the conductance of the fiber sheath decreases in ambient air7. This suggests that the Ni/S group is oxidized upon exposure to oxygen, inducing a loss of conductivity.To verify whether the Ni/S group truly plays a role in long-distance electron transport, we studied the effect of redox state on Raman signals and conductance. Chemically reducing the fiber sheath with Kals Fig. . Such oxoup Fig. . ReducedAdditional experiments, in which Ni was partially removed from the fiber sheath through extraction with high EDTA concentrations, provided further support that the Ni/S group plays a crucial role in electron transport. EDTA is a known mobilizing agent for metals in biostructures. Raman signals from the SDS only treatment and the standard SDS + EDTA extraction were similar Fig.  suggesti18.By combining and integrating the various types of compositional data collected, we can construct a chemical model of the conductive fiber sheaths in cable bacteria Fig. . The fib8. The cross-sectional structure of the fibers therefore resembles a standard household electrical wire, with a conductive core surrounded by an electrically insulating shell layer. We speculate that this insulating layer prohibits that electrons go astray during long-range transport, thus avoiding radical formation and damage to the surrounding cell environment. However, the insulating outer layer also provokes questions on how electrons generated during electrogenic sulfur oxidation are uploaded onto or downloaded from the conductive core, i.e., how these electrons pass through the insulating layer of the fibers to reach the inner conductive core. Cable bacteria must contain a mechanism for this electron transport to and from the conductive fiber core, which may involve periplasmic cytochromes12 that are present in intact bacteria but are removed during fiber sheath extraction .ToF-SIMS analysis Fig. , E furth26. We analyzed a single, isolated fiber that had separated from a fiber sheath  and homogeneous , it could not fit both the modulus and phase data of the electric force (see Supplementary Note σc = 0 S cm−1), this resulted in an anomalously high relative permittivity εr = 11 ± 3, implying that the dielectric response of the fiber material would substantially exceed the typical values for common proteins (εr = 3–5)28, and would even surpass that of nucleic acids (εr = ~8)26. This is not congruent with our AFM-IR and ToF-SIMS data, which demonstrate that the fibers are made of protein. However, when we parameterized the fiber model to include a conductive protein core  surrounded by a non-conductive protein shell (εs = 3 conductivity σs = 0 S cm−1), we could fit both the modulus and phase data of the electric force, arriving at a shell thickness d = 12 ± 2 nm , which enables AFM-based electrostatic force detectionath Fig. , and intath Fig. C, D of tM, which ath Fig. . When ths εr = 3–28, and w29, though not enzymes with Ni centers. Nickel acts as a catalytic center in only nine enzymes, which are mostly involved in the metabolism of gases32, but not in electron transport.Our results indicate that conduction in cable bacteria occurs through proteins with Ni-dependent cofactors. The observation that Ni plays a crucial role in long-range biological conduction is remarkable, as biological electron transport typically involves Fe and Cu metalloproteins33. Clearly, we are dealing with a new type of Ni cofactor, and our data provide a first insight into the structure of this sulfur-ligated Ni-dependent cofactor. The low-frequency band at 371 cm−1 . Future studies should better resolve the molecular structure and Ni center coordination of this novel cofactor, and clarify its role in electron transport.The sulfur-ligated Ni group in the periplasmic fibers has a well-defined Raman signature, which does not resemble that of any of the known sulfur-ligated nickel enzymesm−1 Fig.  is most tra Fig. . Our sta40, as well as through micrometer-long protein appendages of metal-reducing bacteria from the genera Shewanella and Geobacter22. Our data suggest that the periplasmic fibers in cable bacteria consist of protein, with an outer layer of Ni deficient protein that is electrically insulating, and a central core of Ni-rich protein that is electrically conductive. Our results thus extend this known length scale of protein conduction from micrometers to centimeters. The periplasmic fibers in cable bacteria are continuous across cell–cell junctions, and hence, they provide one uninterrupted pathway for electron transport along these centimeter-long bacterial filaments5.While protein is generally considered to be an electrical insulator, recent work demonstrates that electrical currents can propagate efficiently through nanometer-thick protein films sandwiched between electrodesShewanella nanowires occurs through Fe-containing cytochromes20, up until recently, Geobacter nanowires were exclusively thought to consist of metal-free pilin protein, where pi–pi stacking of aromatic amino acids provides a conductive pathway13. This view of electron conduction in Geobacter nanowires has substantially changed in recent years, as high-resolution molecular analysis has revealed that these nanowires can be composed of stacks of OmcS or OmcZ multiheme cytochromes41. Together, our data suggest a mechanism of long-range electron transport in cable bacteria that is hitherto unknown to science. The involvement of Ni in conduction and the lack of cytochromes in the periplasmic fibers of cable bacteria imply a different mechanism of electron transport than in Shewanella and Geobacter nanowires.While conduction in 7, providing typical values of ~10 S cm−1 with a maximum up to 79 S cm−1, which are comparable to those of highly doped organic semiconductors. The core-shell model proposed here suggests that only the conductive core (26 nm diameter) channels the current, thus condensing the electron flow to within 27% of the total cross-sectional area of the 50 nm diameter fiber. This implies that the Ni-containing protein core has a typical conductivity of ~37 S cm−1, with a maximum up to 292 S cm−1. The conduction data for the standard SDS-EDTA extracted fiber sheaths in Fig. −1 and a maximum of 73 S cm−1. These conductivities are substantially higher than measured for the cytochrome based nanowires in Shewanella (1 S cm−1 20) and the OmcS type nanowires in Geobacter (0.02–0.04 S cm−1 22), but comparable to the more conductive OmcZ cytochrome nanowires from Geobacter (4–30 S cm−1 at pH 7 to 373 S cm−1 at pH 222). A clear challenge is to establish a solid physical and molecular foundation for these high protein conductivities of cable bacteria fiber cores.As electron conduction in cable bacteria needs to spans centimeters, the electron transport needs to be highly efficient, thus requiring organic structures with high conductivity. When it was discovered that the periplasmic fibers in cable bacteria were the current bearing structures, it was already noted that mean fiber conductivities were high42, and assuming an homogeneous distribution of Ni atoms throughout the conductive fiber core  and tunneling times are only ~5 ns . It has been argued that the high conductivity of OmcZ nanowires is primarily due to the enhanced pi/pi stacking of closely arranged heme groups22. The fast electron tunneling times of 50–90 ns suggests that the conductive fiber core must possess a supramolecular organization that supports highly efficient electron transport between Ni centers. This could involve particular configurations of aromatic amino acids, or maybe an unknown prosthetic group involved in Ni-ligation. Future studies should further elucidate the genetic and molecular basis of the highly efficient long-range electron transport in cable bacteria.The exact mechanism of electron transport in the periplasmic fibers of cable bacteria remains unclear, but our results indicate that the novel Ni cofactor is an essential component. Electron transport in metalloproteins critically depends on the distance between metal centers43). Enrichment cultures with sediment collected from a salt marsh creek bed  consistently developed thick (~4 μm diameter) filaments that were used for majority of the experiments (unless stated otherwise). To verify whether Raman spectra were similar for different groups of cable bacteria, filaments were retrieved from enrichment cultures using sediments from other environments: Mokbaai (The Netherlands), marine intertidal sediment providing thick filaments (~4 μm), Aarhus university pond (Denmark), a freshwater site with thin filaments (~1 μm); Yarra River, Australia, brackish estuarine sediment, generating thin filaments (~1 μm) that were also used for stable isotope labeling.Cable bacteria were cultured in natural sediments which were sieved, homogenized, repacked into PVC core liner tubes (diameter 40 mm) and incubated in aerated artificial seawater (ASW) at in situ salinity  of MilliQ water on a microscope cover slip, thus providing “intact cable bacteria filaments”. Fiber sheath samples were obtained by extraction of intact filaments in a droplet (~20 μl) of 1% (w/w) sodium dodecyl sulfate (SDS) for 10 min followed by six MilliQ washes. Subsequently, filaments were incubated for 10 min in a droplet (~20 μl) of 1 mM sodium ethylenediaminetetraacetate (EDTA), pH 8, and again six times washed in MilliQ2 covers for combined green laser and NIR laser Raman analysis. In addition, we also recorded green laser Raman spectra on living cable bacteria in sediment gradient slides3. Raman spectra were subjected to cosmic spike removal (CRR), averaged, and baseline corrected . The background spectrum—as recorded next to the filaments—was then subtracted.Raman spectra were recorded on several confocal microscopy systems equipped with different lasers: Renishaw InVia , Horiba LabRAM , and WITec confocal CRM alpha 300 Raman microscope (30 mW 532 nm green laser and 180 mW 785 nm NIR laser). Intact cable bacterium filaments and fiber sheaths were deposited on various substrates  for the green laser Raman analysis and Raman grade CaF−1 (1.3 to 4.7% laser power) was used to record IR spectra. A QCL laser (1500–1700 cm−1 at 10% laser power) was used to map the Amide I band maximum at 1642 cm−1. We recorded AFM images of samples before and after recording the IR spectra to ensure that we did not damage the fiber sheath during the collection of spectra. Contact mode AFM was used throughout with a spring constant k ~0.07–0.4 N m−1. Tips were supplied by Anasys and are custom made for the NanoIR2. Spectra from both the central cell area and the cell junctions were recorded. Spectra were smoothened with a Savitzky–Golay filter, averaged and the background as recorded next to the filaments was subtracted.Fiber sheaths were deposited on gold-coated silicon wafers, and AFM-IR spectra were recorded on a NanoIR2 instrument (Anasys). An OPO laser covering the IR range from 900 to 2235 and 2500 to 3600 cm25. Briefly, an AC voltage of amplitude 4 V and frequency 2 kHz was applied between a conductive AFM probe  of a Cypher S AFM . Fiber sheaths were deposited on highly oriented pyrolytic graphite (HOPG) substrate (μMash Inc.). The amplitude and phase of the 2ω harmonic of the cantilever oscillation were recorded in constant height mode at 0% RH (N2 flow) in SNAP mode. The oscillation amplitude was converted to a capacitance gradient25. The experimental noise was ~0.7 zF nm−1 for the amplitude and ~0.4° for the phase45.SDM was performed following the methods developed in28 to which we added a core-shell structure with a conductive core and a surrounding shell of thickness d. The fiber dimensions (major and minor semi-axis) were extracted from the deconvoluted topographic images. The accuracy of the elliptic cylinder model was assessed against a more realistic model that includes the small (±5 nm) surface topographic variations of the fiber. This analysis supports the assumption that electrical properties are homogeneous along the longitudinal axis of the fibers . The macroscopic cantilever contribution was modeled by a constant offset28. In the model, a sinusoidal voltage of frequency ω and amplitude vac is applied between the tip and the metallic substrate. The force acting on the tip (modulus and phase) was calculated by solving the built-in electric currents model, which employs the AC/DC electrostatic module of COMSOL Multiphysics 5.3 with software routines written in Matlab (Mathworks Inc.), as previously described45  using a Tecnai Osiris microscope (Thermo Fischer Scientific) operated at 200 kV. For electron tomography, projection images were acquired over a tilt range of ±50° with an increment of 2° and reconstructed using a simultaneous iterative reconstruction technique implemented in the Astra toolbox47. The EDX measurements were carried out using a ChemiSTEM system and analyzed using the Bruker ESPRIT software.Intact cable bacterium filaments or fiber sheaths were deposited on a Formvar-coated Au grid or Quantifoil Cu grid and excess water was removed by filter paper. For STEM-EDX, the grid with the filaments was first dried, whereas for the 3D tomography, the grid was immediately plunged in liquid ethane using a Vitrobot Mark 2 plunge freezer (Thermo Fischer Scientific). Afterwards, grids were transferred to liquid nitrogen (~77 K) and mounted into a Gatan cryo holder. Finally, the sample was freeze dried over a period of 8 h in vacuum at 77 K and subsequently allowed to heat to room temperature48 in the Elettra Sincrotrone . For the experiments, TwinMic was operated in scanning mode under a microprobe delivered by an Au zone plate diffractive optic with 600 μm diameter and an outermost zone of 50 nm. In this modality, a fast readout CCD camera produces absorption and phase contrast images, thus providing morphological information on the scanned areas. Simultaneously, LEXRF spectra can be acquired by eight silicon drift detectors places in front of the sample, providing the elemental distribution of excited elements. The excitation energy was chosen accordingly to the elements of interest. Here, an excitation energy of 2 keV was used to have optimal excitation of Si, Al, Mg, Fe, Ni, and Cu. The beam spot size was 570 nm. Firstly, samples were examined with a light microscope to select the regions of interest, then placed inside the TwinMic vacuum chamber operating at 10−6 mbar pressure. The acquisition time was typically 20 s per pixel. LEXRF maps were obtained by processing the LEXRF spectra with the PyMCA multiplatform software49.Intact cable bacteria and extracted fiber sheaths were deposited on Formvar-coated Au TEM grids and dried. X-ray microscopy and fluorescence experiments were carried out at the TwinMic Beamline+ ions (<500 eV) to increase the secondary ion yield without sputtering the material of the sample. Subsequently, the high-energy primary Cs+-ion beam (16 keV) was rastered over the sample surface while detecting secondary ions 12C−, 16O−, 12C14N−, 31P−, and 32S−. To ensure high lateral (~50 nm) and depth resolution, the primary ion current of 0.5 pA and dwelling time of 1 ms/pixel were used, and the same area  was imaged multiple times . The magnetic field strength and exact positions of the electron multiplier detectors were adjusted using an element standard . The obtained image data was processed with the Look@NanoSIMS software50.The elemental composition of the fiber sheaths was assessed by nano-scale secondary ion mass spectrometry  operated at Utrecht University. Upon extraction, fiber sheaths were transferred onto a polycarbonate filter  pre-coated with a 5-nm thick gold layer and air-dried in a desiccator for at least 24 h. Fields of view selected with scanning electron microscopy were first implanted with low-energy Cs56Fe+, 58Ni+, 60Ni+, 63Cu+, and 66Zn+ by NanoSIMS, with the following exceptions. Sputtering was done with the primary O−-ion beam  generated by the radio-frequency plasma source. No low-energy implantation was done with the O−-ion beam. Due to low signals, dwell time was increased to 5 ms per pixel to ensure reliable alignment of the imaged planes; a total of 168 frames were collected. When detecting zinc, isotope 66Zn was selected because the position of the detector measuring 63Cu was too close to the position required for the measurement of the more abundant isotope 64Zn.A similar approach was used for the detection of positive secondary ions 3+ analytical beam  and an Ar4000+ gas cluster ion beam  was interlaced to obtain depth profiles. The SurfaceLab software  was used for data analysis. Mass spectra were internally calibrated with C+, CH3+, C2H3+, C4H5+, and Au+ for positive mode and CH−, C2H−, C3H−, C4H−, and Au− for negative mode spectra and peaks were identified based on exact mass and isotopic composition for metal ions in combination with the Hybrid-SIMS analysis (see below). Lateral regions-of-interest were selected for the filaments and potential spots of sediment clay particles or minerals  or salt  were excluded.ToF-SIMS  analysis was performed on intact cable bacterium filaments and fiber sheaths to obtain high mass resolution spectra and detailed depth profiles of both organic fragments and metals. A bundle of well washed filaments was deposited on gold-coated silicon wafers. Using incident light microscopy, spots with free lying filaments were identified for ToF-SIMS analysis. The ToF-SIMS was operated in mass spectrometry mode with a Bi2 or 20 × 20 μm2  of the ToF-SIMS analytical area was imaged by AFM. AFM images before analysis and just after the Ni peak or the carbohydrate peak were recorded and aligned and subtracted in the Gwyddion software to obtain average sputtering depths for the fiber sheaths  compared to standard ToF-SIMS (~6000 for our samples), and therefore, it was used to identify fragment ions in both positive and in negative modes and to check peak purity. Disadvantages of the Orbitrap are that masses below ca. 50 amu cannot be assessed, which is a minor issue as ToF identification is generally sufficient here, and the detection limit is poorer because of a ca. 200 count background.Additionally, we analyzed fiber sheaths with a Hybrid-SIMS  operated with an ArMethanobacterium  and a 1:5 mixture of freshly precipitated NiS mineral particles in BSA. These samples were all spotted on gold-coated silicon wafers and analyzed by ToF-SIMS as described above in both positive and negative mode.To assist with the interpretation of the ToF-SIMS spectra and the identification of the polysaccharide layer, we analyzed additional samples as reference: bovine serum albumin (BSA), starch from potato, pectin from apple, peptidoglycan from 13C labeled glucose and 34S-labeled sulfate to sediments in which cable bacteria were grown. For the 13C-glucose experiment, 5 mg of glucose was added to each core  and mixed into the top ~3 cm of the core immediately after it was repacked. These cores were incubated submerged in an aerated aquarium. For the 34S-sulfate labeling experiment, ASW was prepared to a salinity of 20  and 20 mM 90 atom % 34S-sulfate. Before repacking, 25 ml sediment was centrifuged at 110 g for 5 min (supernatant discarded), re-suspended in 25 ml 34S-labeled ASW then repacked into 1 cm diameter cut off syringes. Once the sediment had consolidated, the syringes were topped up with 34S-labeled seawater (~5 cm water column) and a bubbler line was added to each core. Cores were regularly topped up with ultra-pure water to compensate for evaporation. For both labeling experiments a control with unlabeled substrates was treated identically. For 13C, filaments were collected for Raman imaging at 14 and 21 days after labeling. In the case of 34S, filaments were collected after 20 and 50 days for Raman imaging.Stable isotope labeling was performed by adding −1 was determined. For the Raman measurements, extracted fiber sheaths were pretreated for 10 min in 20 μl droplets of the reductant (10 mM K4FeII(CN)6) or oxidant (10 mM K3FeIII(CN)6) and enclosed in the same solute between a glass microscope slide and cover slip sealed with nail polish. At least 15 spectra from five individual fiber sheaths were recorded with a 532 nm green laser Raman microscope from Renishaw as described above. Subsequently, the effect of oxidation or reduction on the conductance of fiber sheaths was evaluated. To this end, individual fiber sheaths were stretched out on a microscope cover slip and a small spot of conductive water-based carbon paste (EM-Tec) was added as an electrode on both ends of the filament. After the carbon paste electrodes had dried, they were connected with conductive copper tape and additional carbon paste to a Palmsens 4 potentiostat and sealed with nail polish to make them water resistant. The effect of reduction or oxidation on filament conductance was determined by adding 2 μl of 10 mM K4FeII(CN)6) or 10 mM K3FeIII(CN)6 respectively to the fiber sheath area between the electrodes. As control we added a 2 μl droplet of MilliQ. The electrical current was measured directly before and after drop addition. Adding a second  2 μl droplet of oxidant or reductant typically did not result in a further change in conduction. We performed repeated oxidation, reduction and MilliQ cycles on single filaments. Occasionally unexpected, sharp drops in conduction were seen between treatments, suggesting the fiber sheath had been damaged during exchange of solutes. These suspect data were excluded from the analysis. Conductance measurements were performed at room temperature and current–voltage (IV) curves were recorded . The length (Δx) of the filament between the electrodes was measured with a Dino-lite microscope. Normalized current was calculated as Inorm = I × (Δx/Δxref) with ΔV = 0.1 V and Δxref = 0.3 mm (see7).Two types of experiments were performed to determine the role of the Ni/S group in the conduction of the fiber sheath. First, the effect of oxidation or reduction on the Raman signal of the two lower bands at 367 and 492 cm5.In a second set of experiments, Ni was removed selectively by extraction in 50 mM EDTA and effects on Raman signals and conduction were determined. For the Raman measurements, SDS only extracted fiber sheaths were treated for 10 or 40 min in 20 μl droplets of 1 or 50 mM EDTA and deposited after 5 MilliQ washes on a glass microscope slide. At least 15 spectra from five individual fiber sheaths were recorded with a 532 nm green laser as described above. Additionally, the conductance of ten individual extracted fiber sheaths that were either treated with the standard 10 min 1 mM EDTA or 10 min 50 mM EDTA were compared. Conductance was measured in terms of normalized current as described above. A 10 min extraction time was used for both treatments as this resulted in similar handling times. Fresh EDTA solutions were prepared daily from 0.5 M EDTA stock solution (pH 8) and filaments were treated in batches of five filaments to reduce the exposure to oxygen. Morphology of the fiber sheaths treated with 1 mM or 50 mM EDTA was studied by AFM on a Multimode 8 microscope  in the peak-force quantum nano mechanical modeFurther information on research design is available in the Supplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Data 1Supplementary Data 2Reporting Summary


The metabolic changes that occur in tumor microenvironment (TME) can influence not only the biological activity of tumor cells, which become more aggressive and auto sustained, but also the immune response against tumor cells, either producing ineffective responses or polarizing the response toward protumor activity. γδ T cells are a subset of T cells characterized by a plasticity that confers them the ability to differentiate towards different cell subsets according to the microenvironment conditions. On this basis, we here review the more recent studies focused on altered tumor metabolism and γδ T cells, considering their already known antitumor role and the possibility of manipulating their effector functions by in vitro and in vivo approaches. γδ T cells, thanks to their unique features, are themselves a valid alternative to overcome the limits associated with the use of conventional T cells, such as major histocompatibility complex (MHC) restriction, costimulatory signal and specific tumor-associated antigen recognition. Lipids, amino acids, hypoxia, prostaglandins and other metabolic changes inside the tumor microenvironment could reduce the efficacy of this important immune population and polarize γδ T cells toward IL17 producing cells that play a pro tumoral role. A deeper knowledge of this phenomenon could be helpful to formulate new immunotherapeutic approaches that target tumor metabolisms. It is well known that metabolic alterations related to cancer development are part of the “hallmarks of cancer” . Cancer To date, an increased number of studies focus on the role of tumor cell metabolism . The pre+ T cells and sustain their effector cytokine production and cytotoxic function. In contrast, memory and regulatory T cells are dependent upon FAO and OXPHOS pathways as naive T cells   .Tumors are characterized by the development of two types of hypoxia: chronic and cycling. Chronic hypoxia results from a persistent lack of oxygenation due to the inability of blood vessels to keep up with the growth rate of cancer cells, thus increasing the distance (>70 μm) between cells and blood vessels. Instead, cycling hypoxia, owing to unorganized vasculature, switches from episodes of oxygen reduction to re-oxygenation events .2] concentrations (<10 mmHg), compared to normal vascularized tissues  . FurtherSince our and other authors’ studies show that the TME alters the functions of γδ T cells ,38, ther


Glacial CDRs are positively correlated with air temperature, suggesting glacial chemical weathering yields are likely to increase in future. Our findings highlight that chemical weathering beneath glaciers is more intense than many other terrestrial systems and may become increasingly important for regional biogeochemical cycles.Physical erosion and chemical weathering rates beneath glaciers are expected to increase in a warming climate with enhanced melting but are poorly constrained. We present a global dataset of cations in meltwaters of 77 glaciers, including new data from 19 Asian glaciers. Our study shows that contemporary cation denudation rates (CDRs) beneath glaciers (2174 ± 977 Σ*meq Global glacial chemical denudation is one of the largest contributors to global elemental cycles and, amplified by climate warming, will significantly impact nutrient loads in downstream ecosystems. The highly efficient physical erosion beneath glaciers creates an abundance of reactive mineral surfaces14, and the resultant glacier flour is susceptible to rapid chemical weathering because of very high microparticle surface areas15 and amorphous mineral coatings18. Chemical weathering associated with glaciers (and ice sheets) may also be a CO2 sink or source via dissolution of bedrock minerals , thereby influencing carbon cycles and global climate23.Glaciers  and ice sheets play a substantial role in the global cycles of water27 because major cations  in meltwaters are predominantly derived from crustal mineral weathering 32. To date, glacial CDRs have been quantified at almost thirty basins with published values ranging from ~36 to 56 Σ*meq+ m−2 year−1 (Watson River in Greenland33) to ~4160 Σ*meq+ m−2 year−1 . Global mean CDR was estimated at 740 ± 830 Σ*meq+ m−2 year−1 for glaciers before 2000 40, which is almost two times higher than global continental mean CDR (~380–390 Σ*meq+ m−2 year−1)43. However, previous estimates of CDRs in glacial catchments are outdated and incomplete46. This study will update these estimates and identify the chemical and physical sampling needed for future progress.Chemical weathering rates can be estimated using cation yields derived from mineral weathering reactions 51.Here we present concentrations of major cations in meltwaters of the 19 glaciers within Asia. The new data is combined with a global analysis of CDRs in glacial environments to help elucidate the role of future climate-driven glacier retreat and meltwater export in regional chemical weathering elemental cycles. We propose that the subglacial chemical weathering rates are directly correlated to changes in climate and glacial runoff, and subglacial chemical weathering will become increasingly important for regional elemental cycles and weathering-climate feedbacks in response to accelerated global glacier (and ice sheet) mass loss in a warming environment2+ for 14 of the 19 glaciers, followed by Mg2+, Na+, and K+  containing 5465 samples generated in this study and published literature  after accounting for the uncertainty56.The range of cation concentrations for the 19 Asian glaciers (where data from 4 glaciers extended existing records) overlaps the majority of previously published data from 28 glaciers in Asia and from other glaciers worldwide  for K+, 0.90 to 1.42 (1.11) for Mg2+, and 0.94 to 1.32 (1.07) for Ca2+ during the morning hours with high cation concentration and low runoff, in comparison to from 0.73 to 0.94 (0.85) for Na+, 0.83 to 0.98 (0.90) for K+, 0.73 to 0.98 (0.86) for Mg2+, and 0.85 to 1.04 (0.93) for Ca2+ during the afternoon and evening hours with low cation concentration and high runoff. The daily amplitude of these ratios provides a template that sampling at ~13:00 h or ~21:00 h can capture the “most representative” meltwater  are preferentially weathered from glacial flour even if bulk bedrock is dominated by silicate minerals 13. Previous studies also indicate that Ca2+ and Mg2+ are primarily derived from carbonate weathering and Na+ and K+ from silicate weathering in glacial environments69. Our data set is consistent with this, reflected by higher contributions of Ca2+ and Mg2+ to total cation load from the 19 Asian glaciers (71–97%) and from previously published glaciers  and mean cation concentrations (apart from K+), despite the lowest specific discharge (0.43 m year−1) of mountain ranges  and Himalayas (6821 km2), also have high cation fluxes of 156 Gg year−1 and 271 Gg year−1, respectively, compared to all other Asian glaciers. The smaller glacier-covered mountain ranges of Hengduan (1395 km2) and Altai (179 km2) have the lowest cation fluxes of 14.9 Gg year−1 and 11.8 Gg year−1, respectively  we estimate a regional cation flux of 6305 ± 5843 Gg year−1 from Asian glaciers , is also the region with the highest cation flux (7796 ± 321 Gg year−1). The lowest cation flux of 37.0 ± 36.4 Gg year−1 is found in Scandinavia, one of glacial regions with least glacier cover  than the Himalayas (271 Gg year−1) even though glacial runoff is smaller 75, total cation export from the Greenland Ice Sheet (GIS) was estimated as ranging from 2158 to 2595 Gg year−1 (Table −1)76. Cation release from the Antarctic Ice Sheet (AIS) was estimated as 1209 to 5584 Gg year−1 based on mean cation concentrations from five outlet glaciers in the Antarctic and Subantarctic multiplied by total runoff (55–254 km3 year−1)78 The largest source of uncertainty is the scarcity of data on cation concentration from non-ice sheet glaciers outside of Asia 61. For example, daily mean Mg2+ concentration during 7–8 June is 9.4 times higher than during 8–9 August of 2013 at Dongkemadi Glacier in the Tanggula26. Monthly mean concentration of Mg2+ during July is 0.8 times lower than during an entire melt season (June to September) of 2013 at Urumqi Glacier No.1 in the Tianshan45.There is considerable regional variability and the data assembled here do not allow for any statistically valid interpretation about how glacier size, sampling location/season/duration, and sample size at basins with similar bedrock as well as diverse geology spatially influence the cation concentration. The timing and duration of sampling may result in large errors owing to the high fluctuation of seasonal cation concentrations and/or meltwater runoff(iii)81, but the distance of 80% of sampling sites is below 2 km from ice margin in the current global data set n = 593) is used from 24 non-ice sheet glaciers worldwide, the estimated glacier cation flux is ~64% of that using all data (n = 4641) from all non-ice sheet glaciers, which is lower but does not significantly change the conclusions of this study Geochemical data is currently not available for the large glaciers in Patagonia and Southern Andes Fig. . AlthougOur estimates contain substantial uncertainties, especially in the absence of catchment discharge-weighted mean cation concentration data from glaciers worldwide.Despite these reservations, our findings provide evidence that glaciers play a key role in catchment geochemical weathering and associated elemental mobilization.−1)83, and the high observed Ca2+ concentration (750 μeq L−1) is the main component of the very high CDR (3425 Σ*meq+ m−2 year−1)  and cation concentration are relatively low. Higher CDR from the Himalayas  is similar to Arctic Canada, Svalbard and Jan Mayen, Alaska, Western Canada and USA, Central Europe and Iceland, and in the intermediate range of regional CDRs  and regional  scales . The lowest regional glacial CDR of 285 ± 233 Σ*meq+ m−2 year−1 we estimated occurs in Greenland Periphery  is ~3 times higher than previous mean glacial CDR . Furthermore, glacial CDR is ~3 times higher than the mean cation yield  for Greenland Ice Sheet catchments 25, and is approaching two orders of magnitude higher than CDR when compared to the whole ice sheet  for the major world’s rivers 88 and ~6 times higher than the global river mean CDR (see above)43.Glacial cation denudation rates in glacial catchments appear to be globally elevated. Current CDR from glaciers globally  and mean annual precipitation  on both basin and regional scales  in the Tianshan, six glaciers  in the Qilian, two glaciers  in the Kunlun, one glacier (DKG) in the Tanggula, three glaciers  in the Pamir, two glaciers  in the Hengduan, and one glacier (RBG) in the Himalayan mountain ranges Figs. . This isn = 591) were taken every hour over a 1–4 day period in July–October of 2007 or April–August of 2008 to observe the diurnal process of cation concentrations and relevant drainage system in glacial environments  were sampled as close to the ice margin and river channels as possible in the proglacial area at KOG to characterize their mineral compositions  were determined by ion chromatography using a Dionex-600 fitted with an IonPac CS-12A-HC column, using 20 mM MSA eluent and a CSRS-ULTRA-II suppressor. The detection limits for all ions were below 0.85 μeq L−1 and the precision was better than ±1% for all measured ions. The comparative experiments on some samples indicated that the influence of the frozen-melt process after sampling on cation concentrations is negligible. The mineral compositions were determined by X-ray diffraction (D/Max-IIIB) using a copper butt, step-continuous scanning with 4°/min of speed, and tube 40 kV and 25 mA60.Glacier outflows from the 19 Asian glaciers were sampled manually at locations as close to ice margin as possible during fieldwork that is supported by the PRC Ministry of Science and Technology  were compiled from 62 glaciers in eleven glacial regions  and global glaciers  were assumed to be negligible92. Notably, glacier mass balance is the basis of the runoff model, and all variables involved are integrated over an entire glacier-covered area. The same input data, calibrated parameters for the mass balance model were utilized to drive the glacier runoff model, and the observations from 45 monitored glaciers were used to calibrate related parameters93, following the method of Radic and Hock90. To improve the accuracy of modeling results, the ASTER DEMs-derived geodetic glacier mass balance from Brun and others94 were used to further constrain the modeled results. In detail, the glacier mass balance and glacier runoff models are briefly illustrated below.Modeled annual glacial runoff for eight mountain ranges within Asia during 2000–2014 was taken from Wang90 was used in this study to model glacier mass balance for mountain ranges in High Mountain Asia during 1952–2014. In detail, the primary input data involved in this model includes monthly precipitation and air temperature. Glacier area-weighted specific mass balance (B) for the whole glacier in each mountain range was computed as a sum of specific mass balance of each elevation band on a glacier.bi and Si denote specific mass balance and glacier area, respectively, and subscripts (i) represent the number of elevation band on a glacier .The monthly-scale mass balance modelbi) of each elevation band on a glacier  was calculated asai denotes glacier surface ablation (negative), ci represents glacier mass accumulation (positive), and Ri refers to snowmelt refreezing (positive) at each elevation band.Monthly specific mass balance (ai) was calculated based on a degree-day model, in which snow/ice melt is considered as a linear correlation to monthly air temperature. Here, monthly ai (mm w.e.) was expressed asfsnow/ice denotes degree-day factor for snow/ice (mm w.e. d−1 °C−1), and Ti denotes monthly air temperature (°C) above the glacier surface.For debris-free glaciers, the glacier surface ablation  was calculated aski denotes the scale factor of each elevation band (i) which depends on the debris thickness.For debris-covered glaciers, the relationship between debris thickness and glacier surface ablationci (mm w.e.) was calculated asδm is a constant, Ti denotes air temperature at each elevation band, and Tsnow represents the threshold temperature. If Ti is below Tsnow, Pi is assumed to be snow; if not, Pi is regarded as liquid precipitation.Monthly glacier mass accumulation for each elevation band Ri,pot (cm) and air temperature Ta (°C) at each elevation band95, Ri,pot was calculated asRi) does not flow away until accumulated melt in a mass balance year exceeds the annual potential refreezing .Based on the relationship between annual potential refreezing Tlap, and Glap) were utilized. Tlap is a parameter similar to a ‘statistical lapse rate’ between the CRU TS altitude of grid cell with glacier location (hCRU) and the highest elevation of glacier (hmax), while Glap denotes glacier surface temperature mainly taking into account glacier surface difference, such as orientation and glacier surface climate environment. Monthly air temperature at each elevation band (Ti) was calculated asTCRU denotes the CRU TS monthly air temperature during the period of 1952–2014, and h represents the altitude of glacier elevation band.To interpolate the Climatic Research Unit Time-Series (CRU TS) monthly temperature at each elevation band, two temperature lapse rates  was assigned, while a precipitation gradient (dpre) was used to interpolate precipitation to each elevation band (the percentage of precipitation decreases with every 50 m decreases in elevation) from the highest to lowest elevation of the glacier. Thus, monthly precipitation at each elevation band was calculated asPCRU denotes CRU TS monthly grid precipitation for 1952–2014 at the cell of the glacier located.To interpolate the CRU TS monthly precipitation to 70, glacier runoff was calculated using a water balance approach. For individual glaciers in mountain range within High Mountain Asia during 1952–2014, annual glacier runoff (Qi) at each elevation band on a glacier was calculated as:ai denotes the melt of ice, firn and snow, as in Eq. , Pi is regarded as liquid precipitation. Ri refers to potential refreezing at each elevation band as in Eq.  the multiplication of regional discharge-weighted mean Na+, K+, Mg2+, and Ca2+ concentrations (as the mean cation concentrations for glaciers worldwide) by annual glacial runoff globally70 and then annual Na+, K+, Mg2+, and Ca2+ fluxes were summed (Table n = 593) from 24 glaciers worldwide in six non-ice sheet glacial regions  of the annual *Na+, *K+, *Mg2+, and *Ca2+ fluxes to the annual total Na+, K+, Mg2+, and Ca2+ fluxes which is calculated based on published data from 2 glaciers in Asia, more than 8 glaciers in Svalbard, 1 glacier in Central Europe and 1 outlet glacier in Greenland for each mountain range or glacial region  and global glaciers, the mean contributions were calculated based on the crustal-derived cation contributions at all non-ice sheet glaciers (apart from Kuannersuit Glacier) because data are not available in these six regions (Table To calculate the crustal CDRs we use the annual *NaSupplementary Information


Active deformation mechanisms have been identified and compared for two different deformation modes—uniaxial drawing and plane-strain compression. The initially amorphous PLLA was capable of significant deformation in both tension and plane-strain compression. In contrast, the samples of crystallized PLLA were found brittle in tensile, whereas they proved to be ductile and capable of high-strain deformation when deformed in plane-strain compression. The main deformation mechanism identified in amorphous PLLA was the orientation of chains due to plastic flow, followed by strain-induced crystallization occurring at the true strain above e = 0.5. The oriented chains in amorphous phase were then transformed into oriented mesophase and/or oriented crystals. An upper temperature limit for mesophase formation was found below Td = 90 °C. The amount of mesophase formed in this process did not exceed 5 wt.%. An additional mesophase fraction was generated at high strains from crystals damaged by severe deformation. After the formation of the crystalline phase, further deformation followed the mechanisms characteristic for the semicrystalline polymer. Interlamellar slip supported by crystallographic chain slip has been identified as the major deformation mechanism in semicrystalline PLLA. It was found that the contribution of crystallographic slip increased notably with the increase in the deformation temperature. The most probable active crystallographic slip systems were (010)[001], (100)[001] or (110)[001] slip systems operating along the chain direction. At high temperatures (Td = 115–140 °C), the α→β crystal transformation was additionally observed, leading to the formation of a small fraction of β crystals.The ability of PLLA, either amorphous or semicrystalline, to plastic deformation to large strain was investigated in a wide temperature range (T This polymer can be synthesized from monomers obtained from annually renewable natural resources, e.g., corn, and is biodegradable under industrial composting conditions , (100)[001], or (110)[001] slip systems, all operating along the chain direction [001].The deformation mechanism of amorphous samples compressed at a low temperature (Td < 90 °C, the crystal transformation α→β was also observed, but at high deformation temperature Td = 115–140 °C and high strains. This transformation produces only a small fraction of β crystals in plane-strain compression.Apart from the mesophase, which is produced at high strains from residues of the damaged or destroyed crystalline phase at T


This result fundamentallychallenges the commonly accepted views of polymorphic phase transitionmechanisms.Many crystallinematerials form polymorphs and undergo solid–solidtransitions between different forms as a function of temperature orpressure. However, there is still a poor understanding of the mechanismof transformation. Conclusions about the transformation process aretypically drawn by comparing the crystal structures before and afterthe conversion, but gaining detailed mechanistic knowledge is stronglyimpeded by the generally fast rate of these transitions. When thecrystal morphology does not change, it is assumed that crystallinityis maintained throughout the process. Here we report transformationbetween polymorphs of ZnCl Unfortunately, mechanistic understanding of the conversionpathways between polymorphs and the factors directing them is limitedby the generally fast kinetics of such processes.9 Many transformations,particularly those of first order, seem to require a reconstructionof the crystal lattice, while in single crystal to single crystal(SC–SC) transitions both polymorphs typically exhibit closestructural relationship,10 and it is assumedthat crystallinity is maintained throughout the process.Polymorphism, the abilityof a particular compound to exist intwo or more crystalline states, is widespread in nature11 and frequently restricts our ability to closelymonitor them. Only sufficiently slow processes can be followed closelywith a set of conventional complementary techniques and investigatedin detail. Herein, we present the shape preserving, polymorphic SC–SCphase transformation between polymorphs of ZnCl2(C2C2ImT)2  which is slow enough to unambiguouslydetermine an intermediate amorphous phase.Kineticsplays a significant role in phase transformations2 with 2 equiv of C2C2ImT in water followed by slow evaporation under ambient conditionsresulted in large, mm-sized, colorless single crystals , confirmed by single-crystalX-ray diffraction analysis (SCXRD) to be ZnCl2(C2C2ImT)2, a typical coordination compound (The reaction of ZnCl2(C2C2ImT)2 fromaqueous solution under dynamic vacuum .SCXRD unambiguously revealed the orange phase , here designated as form β,to be a polymorph of the colorless form (referred to as form α).PXRD data confirmed quantitative conversion of α to βupon grinding .Larger crystals of orangecolor could be obtained by rapid crystallizationof ZnCl2(C2C2ImT)2 featureZn(II) coordinated by two chloride anions and twothione ligands in, albeit differently, distorted tetrahedral coordinationenvironments. The conformations of the thione ligands and the ethylgroups within the ligands . In addition, the mutual thione orientation within a complex inα can be considered flared out in a butterfly shape, while inβ the thione ligands align almost orthogonal to one another energeticpreference for the β phase has been calculated .Forms α and β are not symmetryrelated; not even theheavy metal atom positions and no direct conversion pathways couldbe found for the conversion of one form into the other, suggestingthat the phase transition is not second order (continuous). WhileSC–SC transformations are well studied,15 All repeatedthermal cycles are identical with the second one and are also identicalwith the DSC cycles recorded for β. For virgin β samples,a melting point of 154 °C was recorded. The first derivativeof the α to β transition  indicates that the phase transition is first order. The endothermiccharacter suggests that both forms are thermodynamically stable underthe respective conditions, albeit it appears to be  irreversible,since only β crystallizes from the melt.To investigate the phase transitionfurther, particularly its order,differential scanning calorimetry (DSC) 2 was conMovie S1) that coincides with the loss of birefringence observed under polarization(T = 67 °C). Upon continued heating, the wavepropagates in reverse and the orange color change is observed at ∼80°C. The crystal gradually regains transparency and birefringenceuntil it displays a bright orange color at 87 °C. No detectablechange in morphology is observable until the melting process setsin at 97 °C .The rather broad solid–solid phase transition points toa kinetically slow process that is sufficiently slow to be followedby optical microscopy (OM) and laboratory X-ray diffraction 3. The OMSupporting Information Figure S12).The observation of a SC–amorphous–SC transitionissupported by temperature dependent SCXRD  do not show changes in the characteristic vibrationsduring the transition. Particularly, the Raman spectra show that thecomplex remains intact as the characteristic Zn–S and Zn–Clvibrations remain unaltered even in the molten state. The X-ray pairdistribution function (PDF) calculated for the intermediate stagepresented in Figure S4iv also confirmspreservation of the local coordination environment (as only one strongpeak can be observed at ∼2.3 Å covering both the Zn–Sand Zn–Cl contacts) and the absence of any long-range order.Slight color changes were observed past the amorphousstage. Onceat 87 °C, the crystal exhibited an intense orange color indicativeof β. After the transition, the unit cell of β could beeasily indexed. At 97 °C, the crystal starts to lose crystallinityas expected for the melting process. It is worth noting that bothIR and Raman spectroscopic measurements  and the significant decrease of thediffraction spots  which can be maintainedfor an extended time unless heated further. This raises the questionof how the crystals can retain their shape and surface detail in aSC–amorphous–SC transformation.Despite the fact that Figure S6). We hypothesize that freely rotating ethyl groupsand thione rings could act as a “glue” providing a rigidglassy state, holding the tetrahedra formed by S–Zn–Cl,and allowing slow recrystallization into β while maintainingthe morphology of the crystal. This would be supported by the wave-likepropagation of crystalline-to-amorphous and amorphous-to-crystallineobserved by microscopy.As noted earlier,the conversion of α to β requiresthe thione ethyl groups and the thiones themselves to reorient, whichwould require sufficient energy to give rotational freedom and allowtranslational freedom for the Zn complexes (see 2(C2C2ImT)2 complex provided kinetically slow transformationsbetween the polymorphs which could be followed, providing clear evidenceof a first order SC–amorphous–SC transformation withshape preservation. We believe this observation is more common thancurrent literature would suggest, and our finding may have more relevanceto the broader field and will stimulate deeper investigations on thesolid–solid phase transformations. Since this transition requiresessentially just a flexible ligand being able to adopt its shape inorder to support the backbone of the structure, we suspect that manyother purported SC–SC and, in a wider sense, solid–solidphase transformations may also proceed through a solid amorphous phase.In summary, what appearedto be just another example of a traditionalSC-to-SC shape preserving polymorphic transition of a simple coordinationcompound was revealed to be anything but traditional and quite intricate.The unique nature of the ZnCl


After this article  was publFigs The corresponding author stated that the original data underlying results reported in this article are no longer available.The corresponding author further explained that densitometric analysis was performed on scans of the original western blot films/plates, after which representative blot images were selected for the figures. In preparing figures, images were adjusted for brightness/contrast, and in some cases image fragments were rotated or re-ordered to present bands at a consistent orientation and layout across the panel. The corresponding author stated that all data in each panel originated from the same blot, and adjustments to brightness and contrast were applied to all data consistently in the assembled figures.PLOS ONE’s Editorial Board assessed S1 and S2 and stated that the methods are appropriate and the data support the findings of In regard to the concerns in The corresponding author provides here an alternative version of PLOS ONE’s Editorial Board assessed The corresponding author provides results of real-time quantitative PCR (RT-qPCR) for UCP2 mRNA  Files toPLOS ONE Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data and updated figures provided by the corresponding author.In light of the unresolved concerns in S1 FilepAKT/AKT western blotting analysis was performed following the protocol for gel electrophoresis and transfer described in  and usin(TIF)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File−ΔΔCt method [RT-q-PCR for UCP2 was performed using samples from 4 different animals for each experimental group . Total RNA was extracted from liver fragments processed according to the Tri-Reagent (Sigma-Aldrich) protocol. After extraction, 1μg of total RNA was processed with QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol to remove genomic DNA contamination and for the subsequent cDNA synthesis. The analyses were carried out on an Applied Biosystem 7500 Real-Time PCR System, using SYBR Green method (Life Technologies), following the procedures recommended by the manufacturer. Reactions were performed using forward and reverse primers designed using Primer Express software (Applied Biosystems). Each amplification mixture of 20 μl final volume contained 12 μl of real-time PCR Master Mix, 1 μl each of UCP2 or β-actin forward and reverse primers (10 μM), 2 μl of cDNA diluted 1:1 and 4 μl of nuclease-free water. Amplifications were performed with an initial step at 95°C for 1 min, followed by 40 cycles at 95°C for 15 s and 60°C for 40 s. A melting curve analysis of PCR products was performed from 60°C to 95°C in order to ensure gene specific amplification. UCP2 gene amplification was associated with β-actin as standard control. Changes in the UCP2 gene expression in the different samples were obtained in according to standard 2(TIF)Click here for additional data file.S4 File(XLSX)Click here for additional data file.


A review of the effects of vitamin C on the immune system and respiratory tract infections was recently published . We are Cerullo stated “According to Pauling, a daily vitamin C intake of 1,000 mg can reduce the incidence of colds by about 45% , 4. Howe“there was a statistically significant difference (P <0.05) between the two groups in the number of subjects who remained free of illness throughout the study period. Furthermore the subjects receiving the vitamin experienced approximately 30% fewer total days of disability (confined to the house or off work) than those receiving the placebo, and this difference was statistically highly significant (P <0.001)” (The large Anderson (1972) trial  did not ce P <0.0 between “the effects [of vitamin C] demonstrated might be explained equally well by a break in the double blind”  stated e blind” . However75 statedntially” .The Chalmers meta-analysis 1975) calculated that vitamin C shortens common cold duration just by 0.11 (0.24 SE) days 5 calcula, 13. ThiThe Dykes and Meier review 1975) , also co75 , alsoCerullo also writes “Vorilhon et al. , analyzeCerullo further writes “Kim et al.  carried “the most recent meta-analysis including 2,774 participants from seven clinical studies, underlined that current evidence is insufficient to sustain the efficacy of vitamin C supplementation in preventing or treating pneumonia, due to the small number of trials and very low quality of the existing results  meta-analysis  as follo results .”The Padhani review also contains significant errors. We described several of these including the inclusion and exclusion of trials and the statistical calculations .As to the effect of vitamin C on the mortality of ICU patients, Cerullo referred to the CITRIS-ALI trial as follows: “findings from the CITRIS-ALI study  showed a“not confined to the house”, “not off work”, and “not ill during the trial” because of common cold related symptoms were all about 8 percentage points lower in the vitamin C group than in the placebo group (Our meta-analysis on vitamin C and the common cold found that vitamin C did not decrease the average number of colds in general community trials . Howeverbo group Table 1.Vitamin C is known to cure scurvy, but it may also have an effect on other conditions , 21, 22.All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the original article, there was an error. “FTO is misspelled as HNRNPA2B1 on six occasions.”Abstract, Paragraph 1:A correction has been made to in vitro experiment.”“Premised on the expression of CBLL1, we also identified potential therapeutic agents for prostate cancer, and knockdown of FTO prominently inhibited prostate cells migration and invasion Materials and Methods, Cell Culture and Transfection, Paragraph 1:A correction has been made to “The two Small interfering RNAs (JTSBIO) sequences used to reduce FTO expression sequences used to reduce FTO expression were as follows: GCAGUGUAUCUGAGGAGCUCCAUAA UUAUGGAGCUCCUCAGAUACACUGC; CAGGCUGCACCUACAAGUACCUGAA UUCAGGUACUUGUAGGUGCAGCCUG.”Materials and Methods, RNA Extraction and Quantitative Real-Time PCR, Paragraph 1:A correction has been made to −ΔΔCT method was used to analyze the expression level of FTO normalized to GAPDH. The primer sequences are as follows: FTO  and GAPDH .”“RT-qPCR was performed using SYBR Premix EX Taq™ (Takara) and 2Results, Knockdown of HNRNPA2B1 Significantly Inhibited Prostate Cells Migration and Invasion, Paragraph 1:A correction has been made to in vitro experiments were performed. Transwell assay was performed to measure the migration ability of FTO-knockdown cells. The results indicated that the migration ability of cells in the FTO-knockdown group was lower than that in the NC group . The wound-healing assay was used to investigate the influence of FTO on the migration of prostate cancer cells. The results indicated that decreased FTO resulted in a decrease in the migration ability of prostate cancer cells . Transwell assay with Matrigel indicated that the invasion ability of prostate cancer cells was decreased due to the knockdown of FTO . The process of the results was summarized in Figure 9.”“Knockdown of FTO significantly inhibited prostate cancer cells migration and invasion to further explore the biological function of FTO in prostate cancer, a series of Discussion, Paragraph 7:A correction has been made to “In our study, we investigated the role of FTO in the proliferation of the prostate cancer cell. We found that FTO was down regulated in prostate cancers. Knockdown of FTO prominently inhibited prostate cells migration as well as invasion. So our study highlights that FTO is an oncogenic gene that promotes the progression of prostate cancer, and it is a potential novel therapeutic target for treatment of prostate cancer.”Discussion, Paragraph 8:A correction has been made to “In summary, our study carried out a multi-omics comprehensive analysis in prostate cancer to identify the m6A regulators. Further, premised on DNA methylation of m6A regulators, prostate cancers were divided into three different subgoups, which were associated with different disease-free survival. Subsequently, we found that Knockdown of FTO prominently inhibited prostate cells migration and invasion. Our study helps to explore the mechanism of prostate cancer and develop new strategies for personalized treatment of prostate cancer.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Unprecedented drought events occurred towards twentieth–twenty-first centuries as indicated by the July precipitation reconstruction. Positive AO phases in May were most pronounced during the second part of the first millennium, but also increased in frequency in the modern period of the twentieth–twenty-first centuries. Negative AO phases are associated with cold anomalies and show a remarkable decrease in the nineteenth century caused by a series of major volcanic eruptions. Our findings help explaining the increased frequency of Siberian forest fires over the past decades in Central Siberia consistent with a reduction of summer precipitation, triggered by a positive phase of the Arctic Oscillation in May.Rapid changes in the hydrological and temperature regimes over the past decades at the northern latitudes enhance significantly permafrost degradation accelerating carbon release, increase the frequency of drought events and extensive wildfires. However, the mechanisms and dynamics driving drought events and their influence on Siberian forests are currently the subject of numerous research activities. Newly developed and annually resolved stable carbon and oxygen isotope chronologies of larch tree-ring cellulose (δ Recent rapid changes in the hydrological and temperature regimes affect significantly permafrost degradation4 accelerating carbon release, increase frequency of drought events and extensive wildfires expanded over a large territory in Siberia8. However, the mechanisms leading to drought events in the permafrost region with available thawed water for Siberian forests remain unknown.Over the past two decades, heatwaves occur more often in the Eurasian north and particularly in the Siberian subarctic. Larch forests growing in these regions are highly sensitive to climatic changes due to severe environmental conditions10. Tree rings have been proven to be a valuable tool for climate reconstructions from the Eurasian subarctic back in time up to several millennia14. Tree-ring width chronologies from the Siberian subarctic record mainly June-July air temperature signal13. The variation of stable carbon isotopes in tree rings can provide complementary information about changes in precipitation19, sunshine duration20, vapor pressure deficit21 and cloud cover22, while oxygen isotopes can record information about atmospheric circulation patterns27.Siberian weather stations observation covers mainly the last 100 years, with the best quality data back to 1966 CE only. To obtain information about past climatic changes it is, therefore, necessary to consider indirect climatic archives like tree rings28 and particularly in Siberia29. A positive AO phase (AO+) indicates higher pressure at mid-latitudes, which brings higher than average temperatures to northern Eurasia, leading to enhanced greening conditions over large regions, which is confirmed by satellite-derived vegetation indices30. A study by Baltzer et al.31 showed a significant relationship between annual burned forest areas in Central Siberia and AO, while Kim et al.7 showed that positive Arctic Oscillation (AO) phases lead to an increasing fire activity in Asian regions. A high-pressure of the AO circulation was related to a high-temperature anomaly in late winter and spring during the past decade32.The Arctic Oscillation (AO) has an impact on winter temperature and precipitation patterns over Eurasia and North America28. Reconstructions of AO may, therefore, provide information on past temperature and precipitation patterns with annual and seasonal resolutions.Opposite to the AO+, a negative AO brings cold weather anomaly, storms, and wetter conditions at the high-latitude regionsTo apply any measure and mitigation strategies to decrease carbon release in Siberia under rapid temperature and hydrological changes, it is urgent to obtain information about fluctuation rates of climatic changes over the past. To reach this goal we built continuous annually-resolved stable carbon and oxygen isotope chronologies from larch tree rings and used multiple linear correlation analysis to test for the driving factors leading to precipitation changes in Siberian forests over the past 1494 years.13C and δ18O tree-ring cellulose (δ13Ccell and δ18Ocell) chronologies based on the 42 larch trees for the period from 516 to 2009 CE were developed , 1980 (+ 3.9σ) and 2009 CE (+ 3.8σ) .The increasing trend in δ18O values were recorded during the tenth and twenty-first centuries, with most extreme values in the years of 923 CE  and 2009 CE . Minimum δ18O values were recorded during the eleventh, twelfth and nineteenth centuries with the minimum value in 1822 CE  compared to the average  over the whole analyzed period (Supplementary Table Maximum δP < 0.001) were revealed between summer air temperature  and δ13Ccell , July (r = 0.44), averaged July–August (r = 0.54), JJA (r = 0.49) temperatures, respectively, while a negative significant correlation between δ13Ccell and July precipitation  was revealed , July–August (r = 0.39) and JJA (r = 0.35), respectively  for the common period 1969 to 2009 CE.Significant Pearson correlations , July precipitation reconstruction was computed for the period from 516 to 2009 CE  to 44.3 mm for the period 1800–2009 CE  was observed . A significant decrease of July precipitation from 60.4 mm for the period 516–1799 CE  for the period 1969–2009 CE and for the period  1948–2009 were found .25  phases in May Fig. a bring a25 Fig. b showed 25 Fig. b, while Starting from the twentieth century a clearly increasing trend of the AO+ towards twenty-first century is observed. Along with AO+ a rapid decrease in precipitation is recorded in the July precipitation reconstruction Fig. . A signiThe most pronounced negative phase of the AO was revealed during the past millennia Fig. , which cP < 0.01) computed for the common period of observation (1969–2009 CE) for both proxies.Spatial correlation analyses showed a positive significant correlation between reconstructed AO in May and CRU TS4.04 maximum May–July air temperature Fig. a and JulThe AO index in May correlated significantly with the May–July air temperature and shows a wide distribution from 80° N to 60° N, and from 95° E to 150° E 27 via precipitation patterns. For instance, reconstructed NAO25 based on 48 proxy data with annual resolution distributed around the Atlantic Ocean showed similarities to our AO May reconstruction. Our AO reconstructed data agreed with NAO25 mainly during Little Ice Age , emphasizing cold and dry conditions, while there was a disagreement for the recent decades. The Western part of Eurasian subarctic, which include Scandinavian regions, is strongly affected by internal variation and impact of North Atlantic Ocean, which brings wetter climate conditions during the recent period similar to the medieval time27.Oxygen isotopes in Siberian tree rings record not only a temperature signal21. Earlier warming in springs lead to earlier snowmelt1, developing a water shortage for trees by increasing the evapotranspiration in the Siberian subarctic21. Early warming caused by AO+ in May along with lack of precipitation and ground water availability (active soil layer at the top of 10 cm is still frozen) at the beginning of the growing season (late May–early June) in Taimyr Peninsula increases drought stress for larch trees. Recent severe fire periods were reported to occur early June–late July with a fire duration of 1.5 months38. Kharuk et al.5 showed that at high-latitude sites (> 70° N) extensive fires occurred in 1700–1999 CE based on fire scars and data of tree natality. This is in line with our July precipitation reconstruction, which indicates the development of drought, supporting the hypothesis that recent climatic warming will result in an increase of fire frequency at the Siberian subarctic. Furthermore, shifts of the growing period to earlier dates predicted by the process-based vegetation models39 will prolong the forest fire season.In the Siberian north, the Medieval Warm Period during the tenth century was warmer but wetter compared to the recent period derived from our results. This is opposite to the northeastern part of Siberia, where recent atmospheric drought events significantly increased compared to the past8 confirm that lack of water during modern dry summers as well as pre-growing season precipitation can lead to the development of drought for trees and increase the frequency of fires. A study by Kim et al.7 revealed extensive fires in inner-Asian region linked to preceding positive Arctic Oscillation over the recent decades. An increase of atmospheric drying over the Siberian north21 and fire activity over the past decades38 can lead to long-term ecological consequences, where the main trigger are changes in the atmospheric circulation processes.We have therefore clear evidence that AO+ phases further promote the frequency of forest fires and fire spreading in boreal forests. Recent studies by Kirdyanov et al.40 suggests a strong positive phase of AO in the twentieth century, which is in line with our study. Recent drought anomalies were recorded also in other regions, like in European Alps and Central Europe42, however, not so pronounced compared to the Siberian subarctic regions. This can be explained by the impact of NAO index43, which responds more to a regional component and variability, than to the hemispheric one.The increase in drought events may have started in the eighteenth century already, reaching a maximum and unprecedented values in twenty-first of century as shown with the July precipitation reconstruction. AO reconstruction based on the tree rings onlyOur findings are consistent with an increased frequency of Siberian forest fires over the past decades in Central Siberia, very likely caused by the ongoing reduction of summer precipitation and triggered by a positive phase of the Arctic Oscillation in May.Our study site is situated on the eastern part of the Taimyr Peninsula (TAY), where samples from living larch trees and rest of the stem wood on the ground surface were collected  . Larch trees growing at this site can reach an age of 600 years, which is represented by the generation that appeared at the turn of twelfth–thirteenth centuries and are found in fresh and flowing-wet habitats . A larger number of larch wood samples from preserved dead trees (n = 138) and subfossil wood (n = 27) was collected. Sampling was carried out at the present upper timberline in the Stow Ary-Mas (72° 28′ N) and at the present upper timberline at 200–300 m a.s.l. in the Kotuy River valley (70° 30′ N–71° 00′ N).The eastern part of the Taimyr Peninsula is represented by tundra forests, which include zonal lowland forest tundra, pre-tundra and sub-alpine and open forestshttp://climexp.knmi.nl) is the only one climate station located near the study site with available measurements from 1966 to 2009 CE. According to the Khatanga station data, the average annual temperature is − 12.8 °C. The warmest month is July with the maximum temperature plus 12.6 °C. The amount of precipitation is up to 280 mm per year. The vegetation period is rather short and does not exceed 85–90 days (early June-middle/end of August). Precipitation data from the local Khatanga weather station were used for the regression analysis and climate reconstruction.Khatanga weather station 46 is available from https://www.bio.mie-u.ac.jp/kankyo/shizen/lab1/AOindex.htm for the period from 1948 to 2020 CE. Year-round monthly anomaly and seasonal variability correlates significantly  from 1969 to 2009 CE46. The seasonal dataset was used for the climate analysis with newly developed stable carbon and oxygen isotope chronologies in tree rings for the common period 1948–2009 CE.The seasonal Arctic Oscillation (AO) index is the leading mode in empirical orthogonal function analysis of wintertime monthly mean sea level pressure anomalies and is characterized by a seesaw of atmospheric mass between middle (20–30° N) and high (60–90° N) latitudes. The AO is also referred to as the Northern Hemisphere annular mode (NAM). The summertime atmospheric pattern hints at the link between the winter and summer NAM patterns47. At least four sub-samples for the overlapping periods were used.Cross-dated 42 wood samples from larch trees with pointer years marked with steel needles were selected for the stable carbon and oxygen isotope analyses based on the following criteria: (i) similar age patterns; (ii) no missing rings; (iii) visually healthy trees without damages and scars. The average age of trees used for the analysis was 300 years. The first 50 years were not used for the analysis due to the possible juvenile period48. Each extracted tree-ring cellulose sample was homogenized, dried and weighted (0.2–0.3 mg) into the tin capsule for the 13C/12C analysis and into the silver capsule (0.5–0.8 mg) for the 18O/16O analysis. An isotope ratio mass spectrometer delta-S  linked to two elemental analyzers  via a variable open split interface  was used. The 13C/12C was determined by combustion and the 18O/16O by pyrolysis.Each annual tree-ring was split manually using a scalpel under the Leica stereo microscope . Each tree-ring was put in a filter bag with the identification number for the cellulose extraction according to the standard inter-laboratory protocol2 exclusion in helium. This system was linked to an IRMS .Samples for the periods 1110–1250, 1280–1630, 1670–1795 CE, were analyzed with a vario-PYRO cube  via thermal decomposition at 1450 °C and conversion to CO under O49.Both systems the EA-IRMS and the PYRO cube yielded very similar precisions (± 0.2‰) and values from two instruments were in high agreements13Ccell is necessary because the combustion of fossil fuels and biomass has resulted in a decrease of δ13C of the atmospheric CO2 over the last 150 years. By calculating the differences for each year to the pre-industrial value (1850 CE) for δ13C of atmospheric CO2 obtained from ice cores and direct atmospheric measurements at the Mauna Loa Observatory, Hawaii50 we subtracted these differences from the raw stable carbon isotope series from tree rings for each year. We did not apply any other corrections for the industrial period and back in time.Correction of δTo determine which climatic parameter or combination of climatic parameters impact stable carbon and oxygen isotopes in tree rings we applied multiple linear regression analyses.s)10 were computed. Calibration and validation statistics are illustrated with their 2.5 and 97.5 percentiles and the reconstruction is given with its 95%-confidence intervals.Statistical characteristics such as Pearson correlation coefficient (r), reduction of error (RE), coefficient of efficiency (CE), and Durbin–Watson statistics (DW), coefficient of synchronicity , while a and b are intercept variables.To reconstruct the climate back in time we applied a regression analysis, where climatic variables were the dependent variables, while stable isotope values were independenttion Eq. :1\docume13Ccell chronology .P < 0.01) between newly obtained annually-resolved climate reconstructions and climate parameters within 60°–80° N, 10°–150° E and a grid net 0.5°.Climate explorer  was used for computing spatial correlations CRU TS4.04 (The Arctic Oscillation index in May and July precipitation reconstructions were correlated with a field of averaged May–July air temperature and July precipitation for the common period of instrumental measurements from 1966 to 2009 CE.Relevant permits/permissions/licences were obtained: Wood sampling in the study site is complied with institutional and national guidelines and legislation. All methods were carried out in accordance with relevant guidelines and regulations.Supplementary Information.


Achalasia is a rare disorder of esophageal motility characterized by the absence of peristalsis and incomplete lower esophageal sphincter (LES) relaxation during swallowing. It usually presents with progressive severe dysphagia and its most effective treatment is surgical disruption of the LES with a Heller myotomy (HM) . ObesityHere we present a rare case of a 46-year-old patient with a Body Mass Index (BMI) of 47 kg/m2 and an intractable achalasia diagnosed by a high-resolution esophageal manometry (HRMi) and x-rays of upper digestive tract showing aperistalsis and dilation of the esophagus with failure of LES relaxation. Patient had already undergone two endoscopic pneumatic dilations over the 3 years prior to presentation with temporary efficacy.He underwent concomitant laparoscopic HM and LRYGB and 6 moths follow-up demonstrated a reduction of BMI to 34,7 kg/m2 and the remission of esophageal symptoms.  The patient was classified with an ASA (American Society of Anesthesiologists) score 3 and did not have any previous surgery. Operative time was of 115 minutes and no intraoperative complications were recorded. On post-operative day (POD) 3 the patients started a liquid diet; on POD 5 he was discharged. No early (<30 days) complication occurred.With the increasing spread of obesity worldwide, the combination of these two diseases may became more frequent and our report suggest thatsimultaneous surgical management of achalasia and obesity with a minimally invasive approach (Heller myotomy and RYGBP) is feasible with successful results for both weight loss and esophageal dysmotility.ESM 1(MP4 374118 kb)


Valonea tannin is a natural product readily extracted from acorn shells that has been suggested to have potential skin whitening properties. This study investigated the tyrosinase inhibition activity of extracted valonea tannin and the associated structure–function activity. Nuclear magnetic resonance spectroscopy and molecular weight analysis with gel permeation chromatography revealed that valonea tannin could be characterized as a hydrolysable tannin with galloyl, hexahydroxydiphenoyl and open formed-glucose moieties and an average molecular weight of 3042 ± 15 Da. Tyrosinase inhibition assays demonstrated that valonea tannin was 334 times more effective than gallic acid and 3.4 times more effective than tannic acid, which may relate to the larger molecular size. Kinetic studies of the inhibition reactions indicated that valonea tannin provided tyrosinase inhibition through mixed competitive–uncompetitive way. Stern–Volmer fitted fluorescence quenching analysis, isothermal titration calorimetry analysis and in silico molecule docking showed valonea tannin non-selectively bound to the surface of tyrosinase via hydrogen bonds and hydrophobic interactions. Inductively coupled plasma-optical emission spectroscopy and free radical scavenging assays indicated the valonea tannin had copper ion chelating and antioxidant ability, which may also contribute to inhibition activity. These results demonstrated the structure–function activity of valonea tannin as a highly effective natural tyrosinase inhibitor that may have commercial application in dermatological medicines or cosmetic products. Quercus ithaburensis subsp. macrolepis , a common tree throughout Eurasia, and can be readily extracted using hot water  was the tannin concentration and Ksv is the Stern–Volmer quenching constant. The Ksv was the slope calculated from the linear regression plot of F0/F against [Q]. Kq was the biomolecular quenching rate constant. τ0 was the average lifetime of the fluorophore in the absence of the tannin (τ0 = 10−8 s)  were taken for apparent binding constant calculation based on double log Stern–Volmer [a was the apparent binding constant that was obtained by the slope of log [(F0 − F)/F] versus log [Q] plots. Double log plotting needed restrict requisite otherwise false n values could be obtained [0/F against [Q]) and double log Stern–Volmer plot (log [(F0 − F)/F] versus log [Q]) are provided in As for the static quenching, the Fn–Volmer  Equationobtained . TherefoIsothermal Titration Calorimetry (ITC) analysis was conducted as previously described  but modiPeak interpretation, stoichiometry (n), the binding constant (K), change in enthalpy (ΔH) and change in entropy (ΔS) were calculated on Origin 7.0 software package , while changes in Gibbs free energy were calculated based on a previously report . A “two Agaricus bisporus  was obtained from RCSB Protein Databank. Then, tropolone and water molecules on tyrosinase were removed, followed by a polar hydrogen atoms addition, a missing atoms correction and a Gasteiger charges assignment [In silico molecule docking models were performed using AutoDock Vinasoftware  to provide an understanding of the mechanism of tyrosinase inhibition by valonea tannins. The crystallographic structure of the tyrosinase–tropolone complex from signment . Gallic signment .In accordance with Heitz’s report , the bli·+) as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH·). The DPPH· scavenging ability was analysed in accordance with Brand-Williams’s report [· solutions  were mixed with 50µL valonea tannin solutions at different concentrations , and placed in the dark for 30 min. After the reaction time, 300 µL of the mixed solution was transferred into a 96 well microplate reader, and the absorbance measured at 517 nm and corrected using methanol. This method was used in place of the valonea tannins as a negative control.The antioxidant activity of the valonea tannin was evaluated using scavenging assays with 3-ethylbenzthiazolin-6-sulfonic acid  with different concentrations  were mixed with 3.9 mL ABTS·+ solution. After 6 min reaction (at 25 °C), 300 µL of the mixed solution was transferred into a 96 well microplate and the absorbance at 734 nm was recorded, corrected with methanol. Methanol (0.1 mL) was used to replace the valonea tannins as a negative control.The ABTSescribed . Briefly· and ABTS·+ free radical scavenging rates were calculated by using Equation (7):1 was the absorbance of control, and A2 was the absorbance of sample.The DPPH50 was obtained using the linear regression of a valonea tannin concentration to free radical elimination rate plot. Following the method described above, IC50 values were also obtained for gallic acid, tannic acid and ascorbic acid for comparison.The IC2+-binding capacity of valonea tannin was measured using ICP-OES as previously described [2+ concentration measured. A 12.0 L/min plasma flow rate, 1.5 L/min auxiliary flow rate and 0.70 L/min nebulizer flow rates were provided during the analysis, while sample uptake and instrument stabilization delay were 15 s. Gallic acid and tannic acid were also used to react with copper sulphate and remnant Cu2+ were quantified following above method for comparison.The Cuescribed . ValoneaNon-parametric test  was performed to determine differences between the experimental samples (triplicated) using Minitab 18 . The model fitting results are all applied in Elucidation of the isolated valonea tannin revealed the tannin was composed of gallic acid, HHDP and glucose with an average molecular weight of 3042 ± 15 Da. Tyrosinase inhibition ability provided by valonea tannin was 334 times greater than that of gallic acid and 3.4 times higher than tannic acid. Mechanism studies suggested that the observed enzyme inhibition was driven by a combination of hydrogen bonding and hydrophobic interactions which involved non-selective binding to the surface of tyrosinase, resulting in a competitive and non-competitive mixed inhibition mechanism. Antioxidant activity and copper ion chelating ability of valonea tannin also contributed to tyrosinase inhibition. These results suggest that extracted valonea tannins may be of value as a whitening agent in cosmetics or dermatological medicines as an effective replacement for commercial tyrosinase inhibitors.