Gas stream cooling at 100 K is found to yield lower mosaicity than plunge cooling into liquid nitrogen at 77 K for several different protein crystals. Cryocooling for macromolecular crystallography is usually performed via plunging the crystal into a liquid cryogen or placing the crystal in a cold gas stream. These two approaches are compared here for the case of nitrogen cooling. The results show that gas stream cooling, which typically cools the crystal more slowly, yields lower mosaicity and, in some cases, a stronger anomalous signal relative to rapid plunge cooling. During plunging, moving the crystal slowly through the cold gas layer above the liquid surface can produce mosaicity similar to gas stream cooling. Annealing plunge cooled crystals by warming and recooling in the gas stream allows the mosaicity and anomalous signal to recover. For tetragonal thermolysin, the observed effects are less pronounced when the cryosolvent has smaller thermal contraction, under which conditions the protein structures from plunge cooled and gas stream cooled crystals are very similar. Finally, this work also demonstrates that the resolution dependence of the reflecting range is correlated with the cooling method, suggesting it may be a useful tool for discerning whether crystals are cooled too rapidly. The results support previous studies suggesting that slower cooling methods are less deleterious to crystal order, as long as ice formation is prevented and dehydration is limited. However, when macromolecular crystals are exposed to X-ray radiation, they accumulate free radicals that cause degradation of the diffraction data quality over time . The anomalous signal can also be affected. In our analysis below, we consider possible physical explanations for the observed differences, and for the similarity between protein structures from plunge cooled and gas stream cooled crystals. We also describe approaches for recognizing, limiting and recovering plunge cooling sourced crystal disorder.2.2.1.v/v) thermolysin from Bacillus thermophilicus (150 mg ml−1) in 45%(v/v) dimethyl sulfoxide (DMSO), and 50% DMSO [45%(v/v)] and 1 M ZnCl2, and the drop size was 6 µl , with protein catalog numbers as follows: thermolysin = P1512, lysozyme = L6876, proteinase K = P6556 and α-lactalbumin = L5385. All crystals were grown using hanging drop vapor diffusion in 24-well VDX plates  at room temperature . For tetragonal thermolysin, the drop composition was 50%. (2) Slow plunge cooling using a dewar of liquid nitrogen and moving slowly (∼3–15 s) through a 1–3 cm-thick cold gas layer into the liquid nitrogen below (the speed through the cold gas layer and entering the liquid is ∼0.1–1 cm s−1). (3) Normal plunge cooling using a dewar of liquid nitrogen and moving quickly through a 1–3 cm-thick cold gas layer into the liquid nitrogen below (the speed through the cold gas layer and entering the liquid is ∼100 cm s−1). Throughout, unless otherwise specified, protocol (3) was used for plunging. (4) Hyperquenching using a dewar of liquid nitrogen and a small fan to blow away the 1–3 cm-thick cold gas layer above the liquid surface . Methods (2)–(4) all employed hand plunging using a CryoWand (Hampton Research) with transfer to the goniometer for diffraction data collection using tongs.Crystals were placed on glass coverslips under a humid flow at ∼95% relative humidity (r.h.)  Crystals were unmounted directly from the gas stream into a vial containing 500 µl of the crystal cryobuffer, incubated in the vial for ∼1–3 min at RT, and remounted in the gas stream. Controlling the local environment of the crystal in this way improves the reproducibility of annealing compared with conventional annealing in which the cryostream is blocked for a few seconds with the crystal in place  at 50 kV, 0.8 mA. The beam divergence was 5.2 mrad (0.30°). The crystal-to-detector distances were 60–70 mm and the target resolution was set to 2.0 Å. Low-temperature  data were collected at 100 K using a nitrogen gas stream. RT data were collected without a capillary by mounting a crystal directly from the drop onto a micromesh (MiTeGen) and holding the crystal in humid flow at 98.5% r.h., 7.5 l min−1, through a 1.9 cm-diameter nozzle positioned about 1 cm from the crystal. In each case, a pre-experiment was conducted with 2 × 6  frames separated by 90°. The pre-experiment outputs estimates of cell parameters and mosaicities. In some cases, additional data were then collected and integrated in CrysAlisPro, yielding post-refined crystal parameters  in two orthogonal directions tangent to the Ewald sphere . e3 is the mosaicity in a direction perpendicular to e1 and S − S0, which is roughly the mosaicity in the scanning direction, where S and S0 are the scattered and incident X-ray vectors, respectively , which is the value reported by XDS as the mosaicity  and (1) above]. The crystals were similar in size (edges of 0.37–0.45 mm), and the data were collected with identical detector distances (70 mm), oscillation ranges (0.5°), target resolutions (1.4 Å) and exposure times (75 s per frame). The e3 mosaicities were 0.67 and 0.61° for the plunge and gas stream cooled methods, respectively. The data sets were of similar redundancy and quality (Table S1 of the supporting information). Both structures were determined via single-wavelength anomalous diffraction (SAD) in PHENIX .For structure determination, data were integrated and scaled in N,N-dimethylformamide)]. Crystals were plunge cooled [protocol (3)], gas stream cooled [protocol (1)] or annealed in-vial [protocol (5)]. The X-ray data were collected with identical detector distances (70 mm), oscillation ranges (0.5°), target resolutions (2.0 Å) and exposure times (30 s per frame). Data collection strategies were determined with CrysAlisPro assuming Friedel pairs were not equivalent with 10-fold (xylose and MPD) or 15-fold (DMF) anomalous redundancy . To assess the impact of redundancy on SAD efficiency, data sets were truncated at lower redundancy levels. Data were integrated and scaled in CrysAlisPro and merged using AIMLESS  from Phaser and the Rfree of the autobuilt model. The substructure search was also carried out in SHELX, in which case the data were initially analyzed in SHELXC. The substructure search for ten sulfur atoms was then carried out in SHELXD using the resolution limit of the anomalous signal suggested by SHELXC (using a cutoff of CC1/2-anom < 0.25), or 3.0 Å, whichever was lower resolution. The indicators used in this case were the plots of CCall versus CCweak, the number of tries to find a solution and the pattern of occupancies in the heavy atom sites found. Executions of SHELXD were judged to yield successful solutions if the distributions of CCall and CFOM were multimodal, if the plots of CCall versus CCweak indicated groups of tries with higher values separated from a base level group with lower values (i.e. in the upper right corner of the plot), and if the plot of occupancy versus heavy atom site number in decreasing occupancy of the top solution indicated some breaks rather than varying smoothly. In some cases, the top solution from SHELXD was used in SHELXE for calculation of phases, density modification and autobuilding of an initial polyalanine model. The correlation coefficient between the X-ray data and the autobuild trace  was used to assess the quality of the solution from SHELXE.For SAD efficiency studies, crystals of tetragonal thermolysin were used with one of three cryoprotective agents . Normal plunge cooling, on the other hand, shifts the plot upwards and increases the slope [Fig. 3b)]. We examined these plots for several different crystals, finding gas stream cooling biased the plots towards negative slopes whereas normal plunge cooling biased them towards positive slopes  and, within the domain model, the domain size (s)  than with plunge cooling. As with the mosaicity, the effects were more noticeable for greater contracting cryosolvents .We also examined the efficiency of SAD structure determination. In this case, the metrics are more dependent on the data collection procedure than for the mosaicity, but some trends are clear. Gas stream cooling yielded a stronger anomalous signal and SAD structures could be determined more efficiently ; see e3 versus d plots . In the case of DMF, annealing of plunge cooled crystals allows the domain structure to recover to a more highly ordered state than is achieved by crystals only subjected to gas stream cooling.The relationship between the annealing procedure and the domain structure was examined with 3.4.et al., 2018et al., 1987Fo − Fc electron density maps are very similar, with many blobs appearing nearly identical for the two structures (not shown). The highest peaks in the Fo − Fo electron density maps are for small shifts of bound metal ions probably associated with the slight non-isomorphism between the two crystals. Both structures have the same 33 residues built with alternative side chain conformations, whose occupancies are highly correlated, with the largest occupancy difference being 0.1 . This can be compared with occupancy shifts between RT and cryocooled structures, in which about 1/3 of alternative side chain conformations cause occupancy shifts greater than 0.2 . Cooling shifts the distribution, consistent with previously described modulation of side chain conformational distributions from cooling . Keeping the unit cells similar should reduce the effects of differential crystal packing interactions. The differences between the refined protein structures are subtle. The Cα root-mean-square deviation (RMSD) is 0.05 Å, and the hinge bending angle between the two structures is 0.2°. For reference, the Cα RMSD between two different RT structures soaked in different solutions (50% MPD versus 50% xylose) was 0.09 Å, and hinge bending angles between different thermolysin structures have been observed up to 5° , although gas stream cooling may be faster down to ∼250 K  and a larger temperature difference (∼1.1× greater for liquid N2 at 77 K versus gaseous N2 at 100 K)  was observed to reduce the mosaicity of thaumatin crystals by a factor of 2 over plunge cooling ]1/2, where R is the channel radius, et al., 2018Lcrit may therefore be related to the domain size after cooling. Faster cooling would reduce Δt, causing Lcrit and thus the domain size to be smaller, increasing mosaicity.Cooling-induced damage can in principle include both non-homogeneous and homogeneous processes , sample sizes (300–500 µm edges) and cold gas layer thicknesses (1–3 cm) caused most of the cooling during normal plunging to occur in the liquid rather than in the cold gas layer, in which case cold gas layer removal would have little effect on the cooling rate.Removal of the cold gas layer above the liquid surface [protocol (4)] can increase the cooling rate during plunging by limiting cooling in the gas phase above the liquid (Warkentin −1 [protocol (2)] reduced the mosaicities relative to normal plunging. At this speed, even for our relatively large samples, most of the cooling probably occurs in the cold gas layer , or transport of material out of the cell . A rough estimate of the required signal-to-noise ratio for successful structure determination via SAD with this relatively low Bijvoet ratio is ∼70  increase in the intercept of d Table 2, suggestAnnealing of plunge cooled crystals reduced the redundancy required for structure determination. This was particularly effective for DMF, in which case the plunged-then-annealed crystals had lower mosaicities than crystals just cooled in the gas stream (Table S4). All three plunged DMF soaked crystals that did not yield solutions produced solutions after annealing with redundancy levels of 10–15.e3 versus d  suggest that annealing increases the domain size and reduces unit-cell variation and/or domain orientational variation compared with the originally plunge cooled crystal. Interestingly, the one plunge cooled DMF crystal that yielded a structure solution had relatively high e3 values, but the lowest slope of e3 versus d, implying a larger domain size and pointing to damage processes involving reduced domain size as problematic for SAD structure determination.Plots of 4.3.2 is about 10× smaller than for gas stream cooling , perhaps complicating the comparison.The predicted cooling time for plunge cooling into liquid NRinger Chi-1 plot correlations – see supporting information) were for surface exposed side chains with multiple conformations, including two clusters of residues. The first cluster  includes residues that interact with the substrate and the active site zinc. Three of these residues are modeled with two conformations, but between the plunge cooled and gas stream cooled structures the occupancies differ by less than 0.1. The second cluster  forms a part of a turn at the end of a helix and includes an interaction with a bound cation. These three residues are modeled with multiple conformations, including a peptide flip of Lys182 away from the RT configuration, which is modeled with occupancies of 0.33 and 0.26 in the plunge cooled and gas stream cooled cases, respectively.The most dissimilar side chain electron densities  remains an open question.There is thus very little evidence in this particular case that faster cooling via plunging into liquid nitrogen changes conformational ensembles present at LT compared with gas stream cooling. Whether larger differences in cooling rate can appreciably modulate main chain and/or side chain conformations (et al., 2018et al., 2018et al., 2018It has been previously shown that structures determined at LT can be as similar to each other as structures determined at RT (Juers 4.4.et al., 2018For some crystals the thermal contraction of the cryosolvent is an important parameter that can be adjusted to reduce cooling induced disorder (Juers aet al., 2018Thermal contraction is also impacted by cryoprotectant concentration. For thermolysin, 30% MPD still yields ice-less diffraction but with about 4–5% solvent contraction in comparison to 8–9% contraction at 50% (Juers & Matthews, 2004et al., 2006For many crystals there will be a practical limit to slow cooling; taking ∼30 s to move DMF soaked crystals through a 3 cm-thick gas layer usually resulted in ice formation. There is thus a tension in which faster cooling is needed to limit ice formation, whereas slower cooling gives time for temperature-induced relaxations in crystal packing and solvent distribution to occur more evenly throughout the crystal, improving the quality of diffraction data. The lowest mosaicity will therefore probably be achieved using the slowest cooling rate that prevents the formation of ice. However, precise control of the cooling rate is challenging. Some options include adjusting the flow rate of the cryostream and controlling the speed with which the crystal moves through the cold gas layer above the liquid nitrogen (Warkentin In summary, the response of the protein and the crystal to cryocooling procedures inevitably involves more than a simple temperature change. Contraction, solvent flow and dehydration are also in play and can cause not only crystal damage but also non-isomorphism, making comparisons of structures to understand the effects of ligand binding or mutations more difficult. Attention to the composition of the cryosolution as well as possible dehydration effects occurring during mounting procedures will therefore improve both the reproducibility and quality of LT crystal structures.4.5.et al., 2007et al., 2000Plotting mosaicity versus resolution can provide information about the crystal order within the domain model. Smaller domains may indicate the crystal was cooled too quickly and could benefit from gas stream cooling, moving more slowly through the gas layer or annealing in the gas stream. If the beam divergence and wavelength dispersion are known, the domain size can be quantified (Juers 5.For the nine crystal/cryoprotectant combinations tested here, gas stream cooling at 100 K using the vial mounting approach yielded lower mosaicity than normal plunge cooling into liquid nitrogen at 77 K. For the subset of crystals tested, the anomalous signal from gas stream cooling was comparable to or stronger than that from plunge cooling. For plunge cooling, in some cases moving the crystal slowly through the cold gas layer above the liquid surface yielded lower mosaicity. Therefore, as long as ice formation is prevented and dehydration is limited, disorder induced from cooling appears to be minimized by cooling more slowly. Finally, considering the resolution dependence of the reflecting range should provide some guidance towards the optimal cooling rate.10.1107/S1600576719010318/ei5040sup1.pdfSupporting information file. DOI: 6n4wPDB reference: 6n4zPDB reference:


The photocurrent distributions are independent of electric field, indicating that photoexcited electrons and holes form excitons. Remarkably, these excitons can transport over hundreds of micrometers along the topological insulator (TI) nanoribbons before recombination at up to 40 K. The macroscopic transport distance, combined with short carrier lifetime obtained from transient photocurrent measurements, indicates an exciton diffusion coefficient at least 36 m2 s−1, which corresponds to a mobility of 6 × 104 m2 V−1 s−1 at 7 K and is four order of magnitude higher than the value reported for free carriers in TIs. The observation of highly dissipationless exciton transport implies the formation of superfluid-like exciton condensate at the surface of TIs.Excitons are spin integer particles that are predicted to condense into a coherent quantum state at sufficiently low temperature. Here by using photocurrent imaging we report experimental evidence of formation and efficient transport of non-equilibrium excitons in Bi x2-SbxSe3 nanoribbons, which can transport over hundreds of micrometres before recombination up to 40 K, further implying exciton condensation.Exciton condensation may emerge at room temperature in topological materials with strong Coulomb interactions and vanishing electron effective mass. Here, Hou et al. report the formation of excitons in Bi Dirac materials such as graphene and topological insulators (TIs) with strong Coulomb attraction and vanishing effective mass emerge as a new promising platform for achieving exciton condensate potentially at room temperature11. The gapless TI surface state is protected against backscattering and has a linear energy dispersion with massless fermions. Although free-fermions have been extensively studied in TIs, much less work is carried out to understand interacting systems13, where electron–electron interaction may lead to emerging quasi-particles. Photoexcited electrons and holes in TIs relax to the surface Dirac cones on sub-picosecond (ps) timescales, while further carrier recombination can be much slower, ranging from a few ps to over 400 ps19. This long-lived population inversion allows electrons and holes in the transient state to form pairs 10. The figure of merit for exciton formation in materials is EC is the Coulomb energy and EK is the electron kinetic energy. The linear dispersion of the TI surface state results in e is the electron charge, vF is the Fermi velocity20. The two-dimensional (2D) surface state of a three-dimensional (3D) TI, with a single non-degenerate Dirac cone, relatively low vF (compared to graphene) and reduced Tc exciton condensates11. In addition, the topological nature of the band structure may create exotic spin texture to the excitonic quantum state. The spin-momentum locking demands that the ground state of excitons must be a spin-triplet p-wave, which spontaneously breaks time reversal symmetry21.A variety of systems, including double quantum wellsirs Fig. . Because2, or PL peak intensity sharply increases with reduced peak widths at lower temperature5. More recently, exciton formation has been experimentally demonstrated in both graphene22 and TIs23. Evidence of superfluidic excitons has also been obtained by quantum Hall drag in bilayer graphene7. Photocurrent imaging is a powerful experimental technique that can be applied to visualize the transport of locally photoexcited charge carriers25. Compared to spatially resolved PL, it does not require materials to have strong light emission and is hence ideal to study TIs. Previous photocurrent studies of TIs have largely been on degenerately n-doped TIs, where photocurrent is weak with an external quantum efficiency (EQE) of <1% and photocurrent decays rapidly as the local photoexcitation moves away from the electrical contacts to the TIs28.Previous experimental evidence of exciton condensation in gapped semiconductors has been obtained from spatially resolved photoluminescence (PL) measurements, where PL images exhibit macroscopically ordered patternsHere, we apply scanning photocurrent microscopy (SPCM) in intrinsic 3D TIs to provide evidence on the formation of excitons and their transport of macroscopic distance.x2−SbxSe3 nanoribbons were grown by chemical vapour deposition (CVD)29, with x = 0.38 determined from energy-dispersive X-ray spectra. Sb doping significantly suppresses bulk conduction as evidenced by field-effect characteristics (detailed later). The experimental setup is shown in Fig. Ld) at various temperatures is determined by fitting photocurrent distributions with a hyperbolic function x0 is the excitation position and L is the length of the nanoribbon outside the channel  as high as 60% , and 4 orders of magnitude higher than the highest reported mobility in 3D TIs (μ ~ 1 m2 V−1 s−1)32. Note that though the electron backscattering at the surface of a 3D TI is forbidden, scattering into other angles is possible34, resulting in finite carrier mobility. Therefore, free carrier diffusion does not explain the observation. Other photocurrent generation mechanisms, such as thermoelectric, photo-Dember effects, inhomogeneous doping and photo-recycling, are also excluded  at 7 K and found that the in-channel photocurrent profiles remain largely independent of VSD  is close to the Dirac point evidenced by the ambipolar gate dependence . Photocurrent first increases slightly at negative Vg when EF is lowered closer to the Dirac point. But as Vg becomes more negative and tunes the TI from n-type to p-type  phase transition49, which predicts the phase transition of a 2D system into a superfluid under the KT transition temperature TKT, despite the absence of conventional long-range order in 2D. In this theory, the vortex excitations emerge and become closely bound for T < TKT, resulting in a coherent state that displays frictionless exciton transport50. For T > TKT, the vortices are unbound and the correlation length scales exponentially with T51Ld, is then expected to have a similar scaling and decrease exponentially at higher temperature. The TKT values estimated by the Hartree–Fock mean-field calculations are in good agreement with the experimental observation  was mixed with 20–35 mg of Sb powder  and placed in a small quartz tube at the centre of the tube furnace. Se pellets (250 mg)  were placed in another quartz tube upstream by a distance of 16 cm. A silicon substrate was placed 14 cm downstream from the centre of the furnace. The surface of the silicon substrate was coated with 10 nm of Au as a catalyst by electron beam evaporation. The temperature at the centre of the furnace was 680 °C, the Ar flow rate was 150 sccm (standard cubic centimetres per minute) and the growth time was 5 h. After that, the furnace was cooled down to room temperature over ~3 h. The growth yields both nanoribbons and nanoplates  devices were fabricated using a standard electron beam lithography process. Top metal contacts (10 nm Cr/290 nm Au or 10 nm Ti/290 nm Au) were made using an electron beam evaporator (CHA) or a sputterer (Lesker). A typical device is shown in Supplementary Fig. The CVD growth was carried out in a Lindberg Blue M tube furnace, following similar procedures as in previous workThe low temperature measurements were performed in a cryostat (Janis ST-500). Current–voltage curves were measured through a current preamplifier  and a NI data acquisition system. SPCM measurements were performed using a home-built setup based upon an Olympus microscope. Briefly, a 532 nm CW laser or a tuneable laser (NKT SuperK plus) was focused by a ×10 NA 0.3 objective lens to a diffraction limited spot with a size of ~3 μm and raster scanned on a planar nanoribbon device by a pair of mirrors mounted on galvanometers, while both reflectance and photocurrent were simultaneously recorded to produce 2D maps. The laser power was controlled by a set of neutral density (ND) filters and was measured by a power meter underneath the objective lens. Fast photoresponse measurements were performed using a pulsed laser , high-speed amplifiers (Femto DHPCA-100) and a digital oscilloscope. The results in Figs. −11 mbar via attached glovebox. ARPES data were collected at 70 K using a hemispherical Scienta R4000 electron analyser, 100 eV photon energy and a beam size of 50 μm2. A typical micro-ARPES spectrum of a Sb-doped Bi2Se3 nanoplate is shown in Supplementary Fig. Micro-ARPES experiments were performed at the Microscopic and Electronic Structure Observatory beamline 7.0.2 at the Advanced Light Source. Samples were removed from growth chamber, sealed in an argon gas environment and inserted into the micro-ARPES UHV end-station with a base pressure better than 5 × 10Supplementary InformationPeer Review File


The dispersion measured from the angular standard deviation of the Virtual Geomagnetic Poles (VGPs) was found to be close to, but smaller than the predictions of geomagnetic models. Together with further directions from the nearby Martinique Island, the 45 directions obtained within the Brunhes chron provide the most robust estimate of the statistical distribution of paleosecular variation (PSV) at this latitude. The sequence of directions shows episodes of high amplitude secular variation that are coeval with several geomagnetic events including the last reversal documented by five transitional directions. Finally, three lava flows have recorded a transitional behavior which could be link to two excursions, the Laguna del Sello (at ~340 ka) and the Pringle Falls (at ~210 ka) events.Paleomagnetic directional data were obtained from fourteen 0 to 2 Ma old lava flows at Basse-Terre Island . Five reversed polarity flows are consistent with their Matuyama age between 1.6–1.5 Ma and 875–790 ka while the ages of the other nine normal polarity units tie them to the Olduvai subchron and the Brunhes Chron. These directions have been combined with previous results obtained from Basse-Terre Island. The overall mean direction (D = −1.2°, I = 31.4°, α However, second-order but significant deviations emerge when computing the mean direction and/or when scrutinizing the dispersion of the virtual geomagnetic poles (VGP)5. Several paleosecular variation (PSV) models have been proposed to account for these observations10, but so far none has satisfactorily accounted for all features. This may be caused by a varying fidelity of paleomagnetic records despite careful selection of the data. We must also consider bias introduced when averaging unit vectors11.When averaged over a long enough time interval the Earth’s magnetic field is predicted to be mostly that of a geocentric axial dipole20 indicate that many lava flows belong to the Brunhes and Matuyama Chrons and therefore offer a promising potential for paleosecular variation studies. Carlut et al.13 previously studied 0–1 Ma old flows from the Basse-Terre island (Guadeloupe). These authors calculated a mean paleomagnetic pole in agreement with the geocentric axial dipole (GAD) field direction and inferred that no significant persistent second-order features were present at Basse-Terre over the last million years. Similarly, Tanty et al.21 found a mean paleomagnetic direction at Martinique island that is indistinguishable from the GAD field though the observed dispersion was higher than the PSV model predictions. In these two studies, slightly different mean inclinations were reported for the 700–400 ka and 400–0 ka periods, respectively, questioning the statistical significance of the offset21.Geochronological studies conducted in the Martinique and Basse-Terre islands20. Fourteen lava flows distributed over the whole Basse-Terre Island and with ages ranging between 1810 ka and 87 ka were sampled for paleomagnetism  was determined from both alternating field (a.f.) and thermal stepwise demagnetization diagrams using principal component analysisThe magnetic behavior of the samples from flow GU41 showed evidence for partial self-reversal, which appeared only during thermal demagnetization Fig. . In this25. Data quality can be evaluated from the α95 confidence angle that never exceeded 8°  was reached; component #1 in Table 1/2 component ranging from ~26 to ~98 mT. A second lower coercivity component (down to ~3 mT) was also present in most samples and a higher coercivity phase with B1/2 values from ~126 to ~199 mT was detected in the GU54 sample and to lesser extent in GU50 and GU55. These results are interpreted in terms of mixture of titanomagnetites with varying grain-sizes, Ti-content and/or cation deficiency28. A fraction of titanohematite could be responsible for the higher coercivity signal in GU50, GU55 and GU54. Note that this phase was not observed during the NRM measurements and therefore has a negligible contribution to the remanence. Finally, we measured the hysteresis parameters (saturation remanence (Mrs), saturation magnetization (Ms), coercivity of remanence (Hcr), and coercivity (Hc)). The Mrs/Ms versus Hcr/Hc ratios are plotted in Suppl. Mat. cr/Hc ratio larger than 6.2 indicative of a population of MD grains30.During high field acquisition experiments, most samples were saturated after exposure to a direct field of 200 mT. Unmixing of IRM curves by cumulative log-Gaussian (CLG) function2.4Ti0.6O4 (TM60) that reveals a lack of exsolution. Multiple cracks, typical of maghemitization were also observed in this sample.The iron oxides observed with a Scanning Electronic Microscope (SEM) vary in size from sub-microns within the crystal glassy parts up to ten microns Fig. . The mat37. Multiple antiparallel components of remanent magnetization have been identified during thermal demagnetization of the samples from the andesitic flow GU4114  could have generated a stable normal chemical remanent magnetization. However, the thermomagnetic experiments do not show any evidence for maghemite. Interestingly, some studies have highlighted self-reversal processes in andesitic and dacitic materials37 and proposed that they would be related to fine scale exsolution. However, in these cases, the antiparallel component was removed at about 300 °C, therefore at lower temperatures than in the present situation.This 875 ± 21 ka old lava flow erupted during the Matuyama chron. Therefore, we anticipate that the reverse high temperature component recorded the ambient field direction during cooling of lava. Alteration of an initial Ti-rich titanomagnetite fraction with reverse polarity to titanomaghemite during the Brunhes chron (similarly to Hoffman’s suggestion39) or as a two phases process by magnetostatic interaction or super-exchange coupling41. Such behaviors should be detected by performing continuous remanence measurements. We used the Triaxe vibrating magnetometer42 on small untreated and unoriented cylinders43 from GU41-05. We did not observe any spontaneous magnetization behavior, nor could we reproduce an antiparallel magnetization within the 480–520 °C temperature range as observed during the NRM thermal demagnetization. The very weak magnetization at this temperature (on the order of 10−2 Am−1) was at the threshold of the Triaxe sensitivity41 and could have thus hampered its detection. However, we rather believe that laboratory induced alteration during NRM thermal demagnetization yielded the growth of a phase with negative magnetic coupling. In contrast, this process remained negligible due to the very fast heating rate inherent to the Triaxe experiments. Several recent studies45 show that complex remanence behavior and self-reversals could be more common than previously thought in subaerial volcanic rocks, and especially in andesite, dacite and rhyolite. It is frequent however that reliable paleo-directions can be isolated at the highest temperature steps37.Alternatively, self-reversal could arise if the spontaneous magnetization of one phase reverses at a given temperature  and proximity to the 773 ka old Matuyama-Brunhes polarity reversal  Geochronology laboratory using the experimental procedure described in Ricci et al.20. The K-Ar age of 777 ± 15 ka confirmed that this flow was coeval with the last polarity reversal. Sample GU52 was collected on the eastern flank of Matéliane volcano close to GU54 (659 ± 11 ka13). It possibly belongs to the same volcanic episode and hence has been given an age of 660 ka. Uncertainties of ±50 ka were conservatively assigned to the ages of flows GU49 and GU52.Three of the fourteen new lava flows were not dated  when the present study was initiated. We took advantage of the numerous ages now available for Basse-Terre island47) while sites GU42, GU43, GU49, GU50, GU52, GU53, GU54 and GU55 with ages ranging from 777 to 84 ka belong to the normal polarity Brunhes period (<0.78 Ma).These new age estimates and the former ones show that the five reverse polarity flows are consistent with ages ranging from 1.6 to 1.5 Ma , and from 875 to 790 ka (for GU41 and GU40). Similarly, the normal polarity flow GU47 with an age of 1.810 ± 27 ka erupted during the Olduvai subchron  and GU42 (777 ka) directions with deviation angles of 30.4° and 26.3°, respectively, can both be considered as marginally transitional.Inclination data are scattered and, in some cases, strikingly different from the expected average GAD values of ±29.8° Fig. . Steeper95 = 3.3° obtained after combining the 39 individual non-transitional directions from Basse-Terre Island is indistinguishable from the expected GAD field direction . The mean direction  with a larger uncertainty (α95 = 6.4°) obtained for the thirteen lava flows of the present study is in poorer agreement with the expected geocentric axial dipole field direction and suggests that a larger flow number is required for adequate calculation of the time-averaged field, as previously mentioned by Tanty et al.21.The overall mean direction D = −1.2°, I = 31.4°, α21. We restricted this regional dataset to the Brunhes chron (<0.78 Ma) that contains the highest measurement density with 46 independent directions. The results are presented in Fig. 21. Two episodes with abnormal directions are found around 340 ka  and 206 ka . As mentioned by Carlut et al.13 these ages could be linked to the Pringle Falls event that has been recorded at several locations worldwide and dated between 200 and 220 ka51. The Pringle Falls event has been more recently observed in sediments from site 919 in the northern Atlantic Ocean52. Based on anomalous inclination values, several excursions have been proposed within the 205–225 ka interval that were interpreted as a 20 kyr long period of directional instability during the low paleointensity interval documented in the East Pacific Rise sea floor magnetization53 as well as in the Sint-2000 and Piso-150054 composite curves of relative paleointensity55.In order to integrate the Basse-Terre Island data into a more regional perspective we added the paleomagnetic directions of the individually dated flows from the nearby Martinique Islandhron <0.7 Ma that hron <0.7 Ma that et al.56 has a similar age, but there is no consensus about its existence, despite one transitional flow found in Argentina (Meseta del Lago Buenos Aires) and dated by Brown et al.57 at 341 ± 33 ka . We can neither completely rule out the possibility that the deviations found in Martinique were caused by displacement of large outcrops due to massive explosive events. More data are needed for better knowledge of the field instabilities during this period before inferring the existence of a geomagnetic event.In the present dataset, the 320 ka old large directional changes were only detected at Martinique. Excursion 9b discussed by Lund et al.10):th site from the geographic north pole, N the number of sites and Swi the within-site dispersion determined from Nsi samples.We estimated the scatter (Sb) of VGPs caused by the variations of the Earth’s magnetic field by subtracting the within site scatter from the total measured dispersion using the formula (e.g. Johnson 59) which is lower than values predicted by the G or C1 PSV models60 but closer to the trimmed scatter values (S’b) of TK0361  and thermal stepwise demagnetization techniques in the magnetic shielded room of the Institut de Physique du Globe de Paris. Natural Remanent Magnetization was measured using Agico JR-5 and JR-6 spinner magnetometers. Eleven a.f. demagnetization steps were systematically performed at 04, 08, 12, 16, 20, 25, 30, 40, 50, 60 and 70 mT. Thermal demagnetization was carried out at 14 temperature steps  and two additional heating steps (595 and 610 °C) were used for eleven samples.3 sample powders from each flow using an Agico KLY-3 equipped with a CS-3 furnace. Heating-cooling runs were performed in air atmosphere from 20 °C to 630 °C to detect mineralogical changes. Hysteresis loops and high-resolution acquisition curves of Isothermal Remanent Magnetization (IRM) were conducted for one representative sample from each flow using an Alternating Gradient Force Magnetometer  in a 1 Tesla maximum field. The IRM acquisition curves were unmixed using the CLG function28 in order to isolate magnetic components with different coercivities.Measurements of low field thermomagnetic susceptibility (κ) were conducted on 1 cmFinally, microscopic observation and semi-quantitative chemical data were collected on thin sections from sites GU41, GU44, GU48, GU52 and GU53 Fig.  using a All paleomagnetic data and SEM analyses are available upon request to the authors.61, C18). Nevertheless, the significant number of data , allows us to consider these values as a reference for Guadeloupe Island.The paleomagnetic directional dataset for Basse-Terre Island  now includes 44 distinct directions from lava flows covering the last 2 Myr, of which 39 were dated by K-Ar technique. The overall mean direction (D = −1.2° and I = 31.4°) is indistinguishable from that of the geocentric axial dipole . The VGPs dispersion (Sb) of 10.2° and 9.6°, for the all Basse-Terre flows and only those restricted to the Brunhes chron, respectively, is lower than in PSV models (e.g. TK0349 with one or more excursions52 including the Pringle Falls event49. This period may be representative of the “bundle” behavior suggested by Lund et al.56 characterized by a rapid succession of excursions with intervening intervals of large amplitude secular variation. The Martinique and Guadeloupe records indicate that this event was likely pronounced in the Caribbean.In a regional perspective, the PSV records from Guadeloupe and Martinique islands indicate an episode of high amplitude secular variation at around 205 ka that we link to the extended period of low dipole intensity during the 205–225 ka interval64.Finally, we identified five flows that were erupted during the Matuyama-Brunhes transition. The weighted mean age obtained for the last reversal in the Caribbean is 777 ± 4 ka, which, considering the uncertainties, is fully compatible with the age estimate for the midpoint of the reversal at 773 ka derived from the sedimentary sequencesDataset 1&2


In contrast, in Trial B which 20 volunteers completed, there was no significant effect of the inulin on stool frequency . However, many subjects in Trial B had a stool frequency >5 per week also for the placebo period, in breach of the inclusion criteria. Combining the data of 16 low stool frequency subjects from Trials A and B showed a significant effect of inulin to increase stool frequency from 4.1 to 5.0 per week (p = 0.032). Regarding secondary outcomes, stool consistency was significantly softer with inulin treatment compared to placebo periods, it increased 0.29 on the Bristol stool scale (p = 0.008) when data from all subjects of Trials A and B were combined. No other differences in bowel habit parameters due to inulin consumption were significant. None of the differences in specific bacterial abundance, alpha or beta diversity were significant, however the trends were in directions consistent with published studies on other types of inulin. We conclude that 10 g per day of native chicory inulin can increase stool frequency in subjects with low stool frequency.Inulin is a soluble dietary fibre, also classified as a prebiotic, extracted from chicory roots. The present study aimed to determine the effect of consumption of native chicory inulin on the stool frequency of middle-aged to older adults (40–75 years old) with uncomfortably but not clinically relevant low stool frequency, specified as two to four days without bowel movements per week. Two randomised, double blind, placebo-controlled crossover trials were conducted using similar protocols in differing populations. Trial A was conducted in Amsterdam, The Netherlands and subsequently Trial B was conducted in Newcastle, United Kingdom. Both trials involved supplementation for 5 weeks with 10 g per day of inulin or placebo, a washout period of 2 weeks, and then crossed over to receive the other treatment. In Trial B, faecal gut microbiota composition was assessed using 16S rRNA gene sequencing. In Trial A, which 10 volunteers completed, the stool frequency was significantly increased to an average 4.9 ± 0.23 (SEM) times per week during inulin periods versus 3.6 ± 0.25 in the periods with placebo .•Directions of changes in secondary outcomes were generally consistent with expectations for prebiotic effects of inulin.•Secondary outcomes included quality of life symptoms, stool consistency and composition of the faecal microbiota.•Among secondary outcomes, only softening of stool consistency reached significance within the present study. Both the average and median age was 62 years, ranging from 51 to 74 years.Inclusion criteria: Inclusion criteria for both trials were both healthy men and women aged between 40 and 75 years inclusive with self-reported low stool frequency, i.e. 2 days or more per week without successful bowel movements.Exclusion criteria: Volunteers were excluded from the study if they had a clinical diagnosis of constipation as defined by the Rome criteria  from chicory roots. Frutafit® inulin has been confirmed as a safe ingredient through its formal review of Sensus’ GRAS documentation . Compliance to the recommended diet was confirmed verbally at each post randomisation study visit.2.4At the start of the first 5-week treatment period, volunteers were randomly assigned to either inulin treatment or placebo. In Trial A, volunteers were randomised into the two groups by the study team. In Trial B, randomisation was stratified by gender using a computer-generated semi-sequential multiple-coded approach , where eThis provided two specific benefits, compared with a simple random allocation of each volunteer into one of the two groups: a) If an adverse event required unblinding of one volunteer, this would not automatically unblind all the other volunteers as well; b) Even with relatively low volunteer numbers in one of the genders, the probability of substantially different numbers of subjects allocated to the two treatments within a gender was much lower than if simple random allocation was used . Study sachets were prepared by Sensus BV in pre-packed boxes pre-labelled with the subject numbers and study phase , as well as scores from 1 to 4  for intestinal symptoms  as previously described . In Tria2.6Volunteers from Trial B were asked to provide a stool sample prior to commencing the inulin or placebo treatment and again after 5 weeks supplementation, and after the washout period immediately prior to starting the second arm of the study; this was repeated upon completing the cross-over  four st, and a 516S rRNA gene pipeline incorporated alignment-based and phylogenetic approaches to maximise resolution of data. Paired reads were demultiplexed based on the unique molecular barcodes and then merged using the USEARCH v7.0.1090 , allowin2.7p-values for the 6 secondary outcomes (other than the primary outcome of change in stool frequency) resulting in α = 0.008, rather than the 0.05 for the primary outcome, The analysis and visualisation of bacterial communities was conducted using R  were added up into domain scores  and changes in scores between weeks 1 and 5 were calculated. As data were not numeric, a non-parametric Wilcoxon signed rank test was then conducted to compare the change from baseline scores between treatments.Data recorded in the bowel habit diaries during weeks 3 and 5 of each treatment period were transformed into difference data (by subtracting the average of the recorded week 3 and 5 scores during the placebo period from the average week 3 and 5 scores for the corresponding inulin period) in order to express the effect of the treatment as two numerical values for each volunteer. The overall trial average difference for each volunteer was also calculated, using the combined average of weeks 3 and 5 for the difference in outcome between the two treatments. These difference data were tested for  distribution before being analysed for the statistical difference from zero using a one sample T-test. Additionally, for data that were recorded in a comparable way in both Trial A and Trial B , the overall trial average difference for each volunteer were analysed together, combining all volunteers in both trials. The values were analysed using ANOVA . This analysis distinguished between volunteers who during week 1 of their placebo period met the criterion of ≥ two days without bowel movements per week i.e. ‘low initial stool frequency’, and those who did not meet this criterion, thereby had less than two days without bowel movements i.e. ‘high initial stool frequency’. Due to the run-in dosing schedule in week 1 and anticipated increasing effect with inulin treatment over time, the bowel habit data recorded during week 1 were not used otherwise. The ‘low initial stool frequency’ criterion was met by 10 volunteers from Trial A and 6 from Trial B. The model factors were initial frequency (either low or high), and site , nested within initial frequency, to determine the mean and standard deviation and significance corresponding to the hypotheses: (1) that the overall effect differs from 0; (2) whether the outcome differed among volunteers with low or high initial frequency ; and (3) whether the outcome was affected by site among volunteers with low initial frequency. Where the outcome showed a significant interaction with the initial frequency, an additional one-sample T-test was used to analyse the data from volunteers with low initial frequency only. A Bonferroni correction was applied to the  using R . The phy using R . The Krulgorithm . The efflgorithm . All p-v33.1p = 0.04) and for the average trial score (p = 0.01). These changes correspond to an increase in frequency of 1.3 bowel movements per week; thus bowel movements increased from an average ± standard error of mean (SEM) of 3.6 ± 0.25 to 4.9 ± 0.23 per week (p = 0.08), for flatulence at week 5 (p = 0.07) and averaged (p = 0.06), and for abdominal cramping for the average trial score (p = 0.06). Data tables can be found in The results of the bowel habit analysis for Trial A are presented in per week a. No cha3.2p = 0.36)In Trial B there was no effect on stool frequency outcome when calculated as average of all volunteers . HoweverThe recorded values for both primary and secondary outcomes are shown in 3.3Interactions between inulin versus placebo treatment with low or high initial stool frequency in subjects from Trials A and B were analysed for those outcomes in the bowel habit diaries or questionnaires where identical scales were used. The number of volunteers included in the combination of trials was 30. Histograms produced in the Minitab output were visually analysed and visible outliers were removed from the analysis. Analyses reported in this section are data with outliers omitted and the number of volunteers included in each analysis along with analysis outcomes can be found in p = 0.025), and the low frequency subjects showed an increase in stool frequency of 0.7 per week compared to placebo. In addition, there was an increase in stool type implying softer stool consistency of 0.28 points on the Bristol stool chart when data of both trials were combined, irrespective of initial stool frequency (p = 0.008). When the alpha level for this secondary outcome was corrected for multiple comparisons , this was significantly different. There was a non-significant increase in cramping in subjects with a low initial frequency from both Trials A and B (p = 0.08). There were no other significant differences for subjects in Trial B with either low or high initial stool frequency (data not shown).When the trial data for stool frequency for all subjects in Trial A and B were combined, stool frequency was not significantly different between inulin and placebo treatments. However, there was an interaction with initial frequency of defecation  between the inulin and placebo at pre or post each arm of the intervention. Volunteers had a mean of 33% and 32% for Bacteroidetes, and 55% and 56% for Firmicutes prior to consuming the placebo or inulin, respectively  and Bifidobacterium (p = 0.33), which were not present in the placebo arm of the study, and a larger increase in Faecalibacterium (p = 0.61), following inulin consumption (genus level (p > 0.05) in either the placebo or inulin consumption arm of the study. We also compared the differences between pre and post inulin and placebo periods in the change from pre to post (effect size), however, none of the effects were significant . For these volunteers all remaining samples were removed from Inulin and Placebo arms periods of the study, giving a final sample size of 20 for Inulin and Placebo. Compositional analysis showed that Bacteroidetes and Firmicutes were the dominant phyla at baseline in the stool microbiota and following both arms of the intervention, with no significant differences (ectively . At genugranulum . Followisumption . Followip = 0.73). There was a small increase in the number of OTUs following the inulin intervention (p = 0.42), which was mirrored by a small decline in OTUs following placebo consumption, suggesting inulin supplementation may be responsible for this increase. (p = 0.53) or inulin (p = 0.25) arms of the trial  and Unweighted Unifrac  , Unweighted Unifrac  .To assess β-diversity, weighted and unweighted Unifrac were conducted and visualised using principle co-ordinate analysis (PCoA). There was no significant difference in β-diversity between pre and post placebo demonstrated by the Weighted Unifrac (p > 0.05).We investigated the impact of initial stool frequency on gut microbiota composition, as determined using the inclusion criteria; low frequency: ‘≥ two days without bowel movements per week during week 1 of the placebo period', and high frequency: '< two days without bowel movements'. There were no significant changes in any taxa abundance or overall gut microbiota composition between low and high stool frequency volunteers within or between the inulin and placebo periods prior to or following the five week interventions (p = 0.004) and gender (p = 0.006) with microbiota profiles, accounting for 87% and 11% of the overall variability, respectively. Time point and treatment period were not significantly associated with the microbiota profiles (p = 0.920) and (p = 0.922), respectively, explaining only 0.3% and 0.1% of the overall variability, respectively. This corroborates the previous findings that each volunteer has a unique bacterial community, which is stable over time despite inulin intervention.Combining the sequence data from all volunteers at all time points we found significant associations between volunteers  reduction (−0.89 ± 0.75) in the frequency of defecation. This may be explained if volunteers pass fewer larger stools rather than several smaller ones, an outcome which has been previously shown upon inulin consumption . In futuBifidobacteria, but also with Bilophila, Anaerostipes and Clostridium in constipated subjects amongst others  a finding also reported after consuming 12 g of Orafti® inulin per day for 4 weeks (The only significant secondary outcome of the trial was the impact of 10 g of native inulin per day on stool consistency in the subjects. Results from Trial A and Trial B separately showed no statistically significant change in stool consistency. However, when data from both trial sites were combined, stool consistency increased by 0.29 points on the Bristol stool chart ( 4 weeks .We conclude that 10 g native chicory inulin improved stool frequency and stool consistency in otherwise healthy middle-aged adults with unsatisfactory low stool frequency. Inulin consumption did not modulate taxa abundance or overall gut microbiota composition significantly, however the directions of observed trends were consistent with published data on other types of inulin. Importantly there were no indications of potentially negative effects on gastrointestinal symptoms in these subjects.


The pooled ORs  of GST M1 and T1 were 1.186 and 1.107, respectively. In addition, subgroup analyses according to the type of SNHL and ethnicity showed no relationship between GST M1 and T1 polymorphisms and the susceptibility to acquired SNHL. Our results suggest that no significant relationship was found between GST M1 and T1 polymorphisms and the susceptibility to acquired SNHL.Acquired sensorineural hearing loss (SNHL), including age-related hearing loss (ARHL), noise-induced hearing loss (NIHL), drug-induced hearing loss (DIHL) and sudden sensorineural hearing loss (SSHL), is one of the most common sensory deficits in humans. Several studies have reported that antioxidant gene glutathione s-transferase M1 and T1 (GST M1 and T1) polymorphisms have a close relationship with the susceptibility to acquired SNHL, but other articles have reported opposite results. This meta-analysis aims to identify whether an association exists between GST M1 and T1 polymorphisms and the susceptibility to acquired SNHL. Seventeen independent studies containing 1749 cases and 2018 controls were included. According to the I Approximately 360 million people worldwide suffer from this health problem2. People with acquired SNHL exhibit decreased hearing sensitivity and a decline in speech intelligibility, which can lead to serious difficulties in an individual’s communication and social interactions and consequently reduce life expectancy4. Despite the high prevalence and serious effects of acquired SNHL, few therapeutic methods have been found to be clinically effective5.Acquired sensorineural hearing loss (SNHL), including age-related hearing loss (ARHL), noise-induced hearing loss (NIHL), drug-induced hearing loss (DIHL) and sudden sensorineural hearing loss (SSHL), is one of the most common sensory deficits in humans in modern society10. Therefore, exploring these genetic differences and then exploiting them may enable the development of individual prevention strategies for SNHL.The susceptibility to acquired SNHL among individuals is diverse. Some individuals are more susceptible to acquired SNHL, while others are not. Several studies have suggested that this individual difference in susceptibility to acquired SNHL is mostly due to the different genetic backgrounds of individuals, especially genetic polymorphisms that affect the expression of some functional proteins or enzymes14. Researchers have successfully alleviated several kinds of SNHL with the application of antioxidants in animal experiments15. Glutathione s-transferase (GST) encodes a system of antioxidative enzymes that have been demonstrated to play an important role in antioxidative protection in cochlear cells18. Among the GST subclasses, GST T1 and M1 are genetically deleted (null genotype) in a high percentage in humans. Approximately 30–50% of individuals have a null genotype for GST M1, depending on their race19, and 25–40% carry the null genotype of GST T120. Rabinnowitz et al. once suggested that individuals with the null genotypes of GST M1 or GST T1 are more susceptible to oxidative stress damage and are possibly more susceptible to NIHL21.Oxidative stress has been proven to be the most important molecular mechanism in the pathogenesis of acquired SNHL25, and others have reported conflicting results34. Meta-analysis is an effective way to address this type of contradiction. Therefore, we performed this meta-analysis to identify whether a close association exists between GST M1 and T1 polymorphisms and the susceptibility to acquired SNHL and whether GST M1 and T1 polymorphisms can serve as predictive factors for the susceptibility to acquired SNHL.Many studies have attempted to correlate mutant genotypes of GST to the susceptibility to acquired SNHL. Some have demonstrated a close relationship between GST M1 or T1 polymorphisms and the susceptibility to SNHLThe literature selection process is shown in Fig. 2 values for GST M1 and T1 were 50.9% and 64.3%, respectively, which are shown in Fig. 2 values were ≥30%, so we used a random-effects model to calculate the pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). The pooled ORs  of GST M1 and T1 were 1.186 and 1.107, respectively.The ITo address heterogeneity, we performed a subgroup analysis according to the acquired SNHL type and ethnicity. For GST M1, no heterogeneity was observed in the DIHL and the SSHL subgroups. However, relatively strong heterogeneity was observed in the ARHL and NIHL subgroups  most of the previous studies were single-center studies with small sample sizes; and (2) most of the previous studies focused on the associations of GST M1 and T1 polymorphisms with only one type of acquired SNHL and neglected the fact that oxidative stress is the most important and common molecular mechanism of acquired SNHL. Based on the above theoretical basis, this meta-analysis included more studies on oxidative stress-related acquired SNHL types and larger sample sizes to further identify the association of GST M1 and T1 polymorphisms with the susceptibility to acquired SNHL. According to our results, neither the collective nor the subgroup analyses suggested an association between GST M1 and T1 polymorphisms and susceptibility to acquired SNHL.Oxidative stress is the most important and common molecular mechanism of acquired SNHLet al. in 2014. The study concluded that GSTM1polymorphisms, but not GST T1 polymorphisms, are related to noise-induced hearing loss. There are at least two major differences between Zhou’s study and our study. (1) Zhou et al. evaluated the association of GST M1 and T1 polymorphisms only with NIHL. Five studies were included in their meta-analysis, with a total of (GST M1 and T1) 914/909 cases with NIHL and 885/876 controls. However, the purpose of our study was to identify the possible relationship between GST M1 and T1 polymorphisms and acquired SNHL, which includes three other kinds of SNHL in addition to NIHL, with a total of 1749 cases and 2018 controls included in our meta-analysis. (2) Zhou et al. concluded that GST M1 polymorphisms are related to NIHL, but in our study, we found opposite results, although the included studies and samples in the NIHL subgroup in our study were the same as those included in their study. The different results can be attributed to the different models used in the two studies. In Zhou’s study, the fixed-effects model was applied to calculate the pooled effect size even though significant heterogeneity (51%) was observed among the studies. Such a method is worth discussing.A meta-analysis regarding the association between GST M1 and T1 polymorphisms and NIHL was performed by Zhou Heterogeneity is a major problem that affects the reliability of the pooled effect size in meta-analysis. In our meta-analysis, heterogeneity was observed for both GSTM1 and GST T1. The results of the subgroup analysis according to the type of acquired SNHL and ethnicity showed that heterogeneity was much smaller in some subgroups, but it was strong in other groups, suggesting that some other factors besides the acquired SNHL type and ethnicity served as sources of heterogeneity. We also performed a meta-regression analysis  Several studies that possibly met the inclusion criteria did not include primary data, so the ORs with their 95% CIs could not be calculated. We attempted to contact the authors for more information, but we received no response. The results may be influenced by these missing studies. (2) Although 17 independent studies containing a total of 1749 cases with acquired SNHL and 2018 controls were included in this meta-analysis, the sample size is still limited, especially in the process of subgroup analysis. For the DIHL subgroup, most studies contained only 10–20 samples, and for the SSHL subgroup, only 2 articles met the inclusion criteria.To our knowledge, this is the first meta-analysis to focus on the association of GST M1 and T1 polymorphisms with the susceptibility to acquired SNHL. The results of our meta-analysis suggested that GST M1 and T1 polymorphisms may not serve as susceptibility factors for acquired SNHL. Considering the limitations of our meta-analysis, further prospective studies with large sample size and additional studies  are needed to validate study findings.A comprehensive literature search was performed in the following databases: (1) PubMed; (2) Web of Science; (3) EMBASE; (4) OVID; (5) CNKI Chinese database and (6) Wanfang Chinese database. The MeSH and free terms were all included in our search terms, which are listed as follows: “Glutathione s-transferase”, “Glutathione transferase”, “hearing impairment”, “hearing loss”, “ototoxicity” and “deafness”. Our search logic in the PubMed database is listed as follows: “((((“hearing”[MeSH Terms] OR “hearing”[All Fields]) OR  OR “inner ear”[All Fields] OR “cochlea”[All Fields] OR “cochlea”[MeSH Terms])) OR ototoxicity[All Fields]) OR (“audiology”[MeSH Terms] OR “audiology”[All Fields])) AND (((“glutathione transferase”[MeSH Terms] OR (“glutathione”[All Fields] AND “transferase”[All Fields]) OR “glutathione transferase”[All Fields] OR “glutathione s transferase”[All Fields]) OR (“Glutathione Transferase”[Mesh] AND “Glutathione S-Transferase pi”[Mesh] AND “glutathione S-transferase T1”[Supplementary Concept] AND “glutathione S-transferase M1”[Supplementary Concept])) OR (“glutathione transferase”[MeSH Terms] OR (“glutathione”[All Fields] AND “transferase”[All Fields]) OR “glutathione transferase”[All Fields])) AND “humans”[MeSH Terms]”.th, 2018. We also manually checked all articles listed in the reference lists of the retrieved literature.All studies that we searched were published before November 20Studies that met the following criteria were included: (1) independent studies investigating the relationship between GST M1 and T1 polymorphisms and the susceptibility to acquired SNHL and (2) studies including sufficient and definite original data (the genotype frequencies of GST M1 and T1 in the case and control groups) that could be used to calculate the OR with its 95% CI of each genotype. When duplicate publications were found, the data in the latest publication were used.The data in the included studies were extracted by two investigators independently using the same “Data Extraction Form”. The information extracted from the included studies is listed as follows: first author’s name, publication year, country of origin, ethnicity, genotype detection methods, the type of SNHL, and the number of cases and controls. The quality of each included study was evaluated using the Newcastle-Ottawa scale (NOS). The studies with an NOS score ≥7 were considered high-quality studies. All disagreements in the process of study selection, data extraction and quality assessment were discussed and resolved by consensus.2 test. For a value of I2 < 30% and p > 0.1, a fixed-effects model was used to calculate the pooled ORs; otherwise, a random-effects model was used for a value of I2 ≥ 30%.Woolf’s method was applied to estimate the 95% CIs. We considered that there was statistical significance when the overall 95% CI did not include 1 and the p-value transformed from the Z score was less than 0.05. In addition, a subgroup analysis was performed according to the type of acquired SNHL and ethnicity. Sensitivity analysis was used to evaluate the stability of the pooled effect size. Publication bias was assessed by Egger’s test. Publication bias was considered for a p-value < 0.05 or if the 95% CI did not contain 0. All statistical analyses were performed using the Stata 13.1 software.The association of GST M1 and T1 polymorphisms with acquired SNHL susceptibility was evaluated by the pooled OR and 95% CI.Statistical heterogeneity among the studies was measured with the IResults of meta-regression considering sample size, publication year and NOS scores of the included studies.