The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time. For several decades, it has been known that extracellular matrix (ECM) molecules can provide topographical cues and guidance for neuronal growth. Proteoglycans (PGs) are a major component of the ECM in several tissues in our body and have been shown to influence nerve growth in many systems from the developing central nervous system ,2 to theOne of the main areas of interest within our research group is the biology and degeneration and regeneration of the intervertebral disc. Degeneration of the disc is a common phenomenon, which is of interest clinically because of its association with one of the largest health burdens in the Western world, that of back pain –12. PrevPGs, with their considerable hydrophilic capacity, are fundamental to the main function of the disc, that of weight bearing in the spine and it is these molecules that are the first to alter when discs degenerate . AggrecaAlthough PGs make up 13–61% of the dry weight in the disc  and the The present study highlights the importance of evaluating the composition of commercially available preparations. We had set out to determine if decorin and biglycan might influence nerve growth in model systems used previously in our laboratory to study the influence of aggrecan ,31,32, b2/ml) that was then allowed to dry. Strips of Whatman filter paper, soaked in solutions of ‘decorin’ ranging from 10 to 1000 µg/ml or ‘biglycan’ ranging from 2 to 500 µg/ml diluted in PBS with 10% rhodamine-B, were blotted on to the nitrocellulose and allowed to dry. The filter paper was removed and the remainder of the wells were coated with type I collagen from rat tail  at a concentration of 100 µg/ml in PBS. The DRG-seeded Petri dishes were maintained in explant culture medium (Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 supplemented with progesterone, insulin, sodium selenite, phosphocreatine, transferrin and sodium pyruvate) containing 15 ng/ml nerve growth factor  at 39°C for 72 h prior to fixation in 4% paraformaldehyde. Neurite growth was monitored by phase-contrast and fluorescent microscopy and measured on captured images as described previously [Dorsal root ganglia (DRGs) were obtained as previously described and used as a source of sensory neurons . Brieflyeviously . A minimBoth ‘decorin’ and ‘biglycan’ were obtained from Sigma–Aldrich and had been prepared from bovine articular cartilage. Certificate analyses of these products stated that protein levels had been analysed  in the l/ml, cleaves unsulfated chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate from the core protein and chondroitinase ABC  cleaves all of these in addition to dermatan sulfate (DS). Filter paper strips previously blotted with 500 µg/ml ‘decorin’ or ‘biglycan’ were incubated with the enzymes at 37°C for 2 h.To investigate whether the GAG chains present on the SLRPs inhibit nerve growth, different enzymes were used to selectively remove specific GAG chains from ‘decorin’ and ‘biglycan’. Chondroitinase AC , which was used at concentrations ranging from 2 to 250 mUAggrecan was prepared using previously described methods ,34 and wWestern blotting was carried out as previously described  and repePGs were deglycosylated prior to Western blotting. To remove chondroitin sulfate (CS) and DS chains, the commercially sourced ‘decorin’ and ‘biglycan’ and purified human aggrecan were digested with chondroitinase ABC or chondroitinase AC (both at 250 mU/ml in Tris acetate buffer) or left undigested for 2 h at 37°C. In addition, they were digested with chondroitinase ABC (200 mU/ml) and keratanase (100 mU/ml) and keratanase II (2 mU/ml) in Tris acetate buffer to ensure removal of any CS, DS and keratan sulfate (KS) GAG chains. The enzymatically digested or undigested PGs were mixed with lithium dodecyl sulfate (LDS) PAGE application buffer (4×) and 500 mM dithiothreitol (10×) then heated for 30 min at 70°C, cooled and either 10 µg or 25 µg were loaded into each well and separated on 4–12% Bis-Tris gradient gels for 35 min at a constant voltage of 200 V  under reducing conditions.Protocol 1, the separated PGs (which had been digested with chondroitinase AC or ABC) were transferred to nitrocellulose membranes (0.22 µm) using NuPAGE transfer buffer plus 10% methanol at a constant voltage of 30 V for 1 h. The membranes were blocked for 1 h with 5% skimmed milk/0.1% Tween 20 in 50 mM Tris-HCl and 0.15 M NaCl, pH 7.2 (TBS). Primary mouse monoclonal antibodies against GAGs  at the concentrations provided in Two protocols for transferring to the nitrocellulose membranes and visualization were used. For Protocol 2, following separation via electrophoresis as described above, the commercial SLRPs  were transferred to nitrocellulose membranes using an iBlot system (Program 3) and associated transfer stacks (Invitrogen). These membranes were then probed for decorin, biglycan, aggrecan, fibromodulin, lumican and KS using the previously described primary antibodies (In tibodies . The pri4HCO3/acetonitrile (1:1), reduced with 10 mM dithiothreitol in 0.1 M NH4HCO3 at 56°C for 45 min followed by alkylation of cysteine residues by incubation with 55 mM iodoacetamide in 0.1 M NH4HCO3 for 30 min at room temperature. Prior to MS analysis, the protein digests were cleaned up and concentrated using C18 Ziptips according to the manufacturer’s recommendations (Millipore). Peptides were eluted from the Ziptips on to a stainless steel MALDI target plate with α-cyano-4-hydroxycinnamic acid (CHCA) matrix at 3 mg/ml ) and allowed to dry. Close external peptide standards were used to calibrate the instrument in both MS and MS/MS mode. Both MS and MS/MS analysis of the peptides was performed using a 4800 MALDI TOF/TOF (AB Sciex). The mass spectrometer was operated under the control of 4000 Series Explorer software (V3.5.2 Applied Biosystems). Peak lists of MS and MS/MS spectra were also generated with the 4000 Series Explorer software and the following parameters were used after selective labelling of monoisotopic mass peaks: MS peak lists: S/N threshold 3, Savitzky–Golay smoothing with 3 points across peak (FWHM), no baseline correction, MS/MS peak lists: S/N threshold 10, Savitzky–Golay with 7 points across peak (FWHM) and no baseline correction. Automated database searches were run using GPS Explorer software . Using MASCOT as the search engine, the Swissprot database was explored using the following parameters: precursor ion mass tolerance of 0.15 Da, fragment ion mass tolerance of 0.15 Da, mammalian taxonomy and finally, carbamidomethyl modification of cysteines and oxidation of methionine residues were allowed as variable modifications.Two different batches of the commercially sourced ‘decorin’ and one batch of ‘biglycan’ were enzymatically digested for removal of CS, DS and KS as described for Western blotting, before being separated on 12.5% SDS-PAGE gel for MS. Bovine nasal cartilage was treated similarly and used as a positive control. Following Coomassie Blue staining of the 12.5% gel, protein slices, spanning the entirety of each lane were excised and digested overnight at 37°C with trypsin , according to the method described by Shevchenko et al. . BrieflyP<0.05.The non-parametric statistical test, Kruskal–Wallis with post-hoc analysis (Dunn’s multiple comparisons test), was used to determine any differences in neurite growth on different substrata. Statistical significance was considered at P=0.0012) and 10 and 1000 µg/ml (P<0.0001). No significant difference on DRG neurite growth was observed between ‘decorin’ concentrations of 500 and 1000 µg/ml.Substrate choice experiments showed that ‘decorin’ inhibited DRG neurite growth in a dose-dependent manner A. SignifP=0.0013). In contrast, treatment with chondroitinase AC (which cleaves CS GAGs only) did not reverse the inhibitory effects of ‘decorin’ on DRG neurite growth  and when it was increased from 2 to 500 µg/ml (P=0.0007). No significant difference on DRG neurite growth was observed between ‘biglycan’ concentrations of 100 and 500 µg/ml. Again, the effect on DRG neurite growth of the CS and DS GAG chain components of the ‘biglycan’ preparation were assessed by using ‘biglycan’ at 500 µg/ml (as previously used to assess ‘decorin’) followed by either chondroitinase ABC or chondroitinase AC (both at 250 mU/ml). As can be seen in The experiments were repeated using ‘biglycan’ instead of ‘decorin’ as the test substrate and a similar dose-dependent pattern of DRG neurite growth inhibition was observed A. That iWestern blot analysis of the ‘decorin’, ‘biglycan’ and aggrecan revealed the presence of KS, in all three PGs with clear 5D4-positivity A. AlthouWhen the membrane was probed for 4-sulfated CS using the antibody 2B6 (after chondroitinase ABC pre-treatment), additional bands were seen at approximately 45 kDa in both ‘biglycan’ and ‘decorin’ B. This iMass spectrometric analysis of gel slices excised from sample lanes containing the two batches of ‘decorin’ preparations  resultedSimilar to the ‘decorin’ (2nd batch) preparation, MS analysis of the gel slices excised from the ‘biglycan’ sample lane resulted in the identification of significant peptides matching to biglycan, fibromodulin, aggrecan and decorin .Subsequent to the unexpected MS findings, further Western blot analyses were carried out on the PG preparations. Membranes were probed for biglycan and decorin and although ‘biglycan’ contained biglycan and ‘decorin’ contained decorin, the presence of biglycan in ‘decorin’ and decorin in ‘biglycan’ . AlthougFurther Western blot analysis of ‘biglycan’, both with and without chondroitinase ABC pre-treatment and undigested purified aggrecan as a positive control, clearly showed the presence of fibromodulin in ‘biglycan’ but none in the aggrecan preparation D. AdditiMolecules that contain GAG chains have previously been shown to influence neuronal growth with those containing CS in particular being implicated in neurite inhibition . The aimWestern blot analysis of ‘decorin’, ‘biglycan’ and aggrecan with varying degrees of glycosylation revealed the presence of KS chains on all three PG preparations. Although KS was expected to be present within the aggrecan molecule, it should not have been present in either of the SLRP preparations. In all three PG analyses, the Western blot bands appeared diffuse A, reflecThe combination of MS and Western blotting used in the present study has demonstrated that commercial preparations of ‘biglycan’ and ‘decorin’ bought and used in studies in our laboratory actually contained many contaminating substances, at least in these specific batches. The molecules we concentrated on validating were other PG family members identified via MS. Hence, the ‘decorin’ also contained biglycan, fibromodulin and aggrecan, while the ‘biglycan’ also contained fibromodulin, aggrecan and decorin, at least. This renders these preparations useless for the original purpose of our study which was to unravel the contributions that different members of the PG family, known to occur in the human intervertebral disc, may make to controlling neurite extension and nerve growth.The impact of our findings may be huge, not in the manner we originally intended but, rather in the amount of time, money and samples that have been wasted. Most importantly, however, it may affect the possibility of erroneous information entering the scientific arena. Concern about the purity and quality of commercial chemicals and biochemicals has previously been expressed by other researchers. For example, D’Hondt et al.  highlighThere are many sophisticated analytical techniques in the scientific world and it is hoped that companies will use these to provide more accurate details of the purity and constituents of their reagents and chemicals. Inherently there will be a cost to the consumer, but on balance, the reduction in time and funds spent on investigating the effects of ‘impure’ molecules within the scientific community, possibly resulting in inaccurate conclusions and ‘flawed’ studies, will offset this cost. Without this, the effect of contaminants within reagents may alter or confound the observed effect of the molecule being investigated; potentially leading to erroneous interpretation of data such as in the elucidation of mechanisms of action or the contribution a particular molecule may play in key biological processes.


A.For an algebra  BCI-algebras forms a quasivariety which is not a variety, see Arai et al. , Chajda nd Iséki  and Iséknd Iséki . It was nd Iséki  that forOur observation is that similar results hold for quasivarieties The symbol The symbol A defined by The symbol The symbol Further, we will consider the following conditions:A. Moreover, the following identities  wi wiThe sy(i)For any algebra Θ on it,  and 6)  \documenΘ on it, .(ii) does not(iii) and 6)  6) implyFor the conditions –8) the  the 8) t(i)If (ii)If ams  and  holds, b(iii)If atisfies  and 6),,\documenatisfies  since \d=b≠1 nor  since \dintegral commutative residuated poset is an ordered quintuple The following lemma is well known:An interesting example of an algebra mentclass2pt{minim(i)(ii)(iii)atisfies  and 6)..\documenIf (i)The following are equivalent: (ii)The following are equivalent: (iii)atisfies  since \dLemma mentclass2pt{minim-algebra if the relation defined by  ar ar\documrding to  and (7).atisfies  (cf. Chaatisfies ):9\documa et al. . The algy  and and\docuy  then  holds.If convex if The congruence (i)If (ii)If (iii)If The relations defined by  and 2)  2) satis(i)If (ii)This follows from (i).(iii)If algebra with induced order if the relation defined by  of Lemma Theorem If Next we present two conditions under which the property of If rder and  holds, td of g of Let  that if  holds annvex and  to hold.rding to , there erding to . Now \dosumption , transittive and  to hold.rding to , there erding to . Now \doThat assumption  in TheorIf ′{a} and  does notIn an analogous way as it was already done for Theorem If rder and  holds thIf d of According to Lemma rding to  there exrding to  there ex(i) and 7)  7) imply(ii) and 8)  8) implyThe following implications hold:(i)We have (ii)We have Let (i)If the relation defined by  is antisatisfies  and eithatisfies  or 8), , \docume(ii)If atisfies  and 6)  \documenatisfies  or 8), , \documeThe following implications hold:(i)This follows from Lemma (ii)This follows from Lemmata (i)(cf. Traczyk and Zarȩbski (ii)If The following implications hold:These follow from Lemmata


We demonstrate the creation of a vector beam by tailoring geometric phase of left- and right- circularly polarized beams. Such a vector beam with a uniform phase has not been demonstrated before because a vortex phase remains in the beam. We focus on vortex phase cancellation to generate vector beams in terahertz regions, and measure the geometric phase of the beam and its spatial distribution of polarization. We conduct proof-of-principle experiments for producing a vector beam with radial polarization and uniform phase at 0.36 THz. We determine the vortex phase of the vector beam to be below 4%, thus highlighting the extendibility and availability of the proposed concept to the super broadband spectral region from ultraviolet to terahertz. The extended range of our proposed techniques could lead to breakthroughs in the fields of microscopy, chiral nano-materials, and quantum information science. Pure vector beams, which form the basis of vector beams, such as radially and azimuthally polarized beams12, produce a longitudinal electric field13 and an optical Möbius strip14. Researchers have also shown that by adjusting the intensity distribution of a pure vector beam with radial polarization, one can control the three-dimensional distribution of the electric field15. Moreover, the ability to focus the electric field by a pure vector beam enables a beam spot to be sharpened to sizes smaller than the diffraction limit – an ability that cannot be achieved with linearly polarized beams13. Because of these optical properties, pure vector beams are expected to enable super-resolution in microscopy and laser processing16. However, the super-resolution property of pure vector beams is destroyed by the geometric phase induced by cyclic polarization changes19.Vector beams, which tailor spatial distribution of polarization and phase, have the potential to lead to cutting-edge developments in the fields laser physics21. Propagation of a beam containing a radially polarized optical vortex is different from that of a radially polarized beam12. Such a radially polarized optical vortex shows complex polarization dynamics because its polarization and phase are spatially distributed in three-dimensional free space. For this reason, vortex phase cancellation by use of an optical vortex with opposite vortex phase has been proposed as a method of creating pure vector beams22. This approach, however, is limited to experimental setups with complex optical alignments and low optical material throughput using polarization elements. To be best of our knowledge, radial polarization converters, which enable conversion to be the pure vector beams, have been proposed for use in a micro-structured photoconductive antenna23, a cone lens24, laser resonances1, liquid crystal modulators12, sub-wavelength structures17, nonlinear crystals25, wave plates of form birefringence fabricated by three-dimensional printing26, and meta-materials27 in several spectral regions from visible to terahertz. Our group also illustrated an achromatic axially-symmetric wave plate based on internal reflections and a generation of vector beams in the visible28, middle infrared18, and terahertz29 spectral regions. To verify details of the pure vector beam generated by use of those radial polarization converters, one must verify that the generated beam removes the vortex phase that can result from geometric phase. Although the vortex phase must be reduced in applications of pure vector beams, few studies have rigorously determined the spatial distribution of phase and its polarization of generated vector beams23, having observed only intensity distributions transmitted through a polarizer. The radial polarization converters used in these studies, moreover, are limited to narrow wavelength bands because broadband vortex phase cancellation has not been previously demonstrated.When a generated beam is radially polarized, but its phase is spirally distributed as vortex phase, the beam is referred to as a radially polarized optical vortex30 and quantum communications32 using pure vector beams, which are expected to represent a breakthrough for surpassing the diffraction limit, high-density transmission of signals, and generating longitudinal electric fields by light. We focus on the proof-of-principle demonstration of a terahertz (THz) pure vector beam by tailoring the geometric phase. In our demonstration, we also show that our concept is not limited to the THz region only and enables us to extend this approach to different spectral regions.In this paper, we specifically show a pure vector beam that eliminates the vortex phase by tailoring geometric phase at 0.36 THz. Our proposed concept of vortex phase cancellation considers symmetry of the polarization of the light and asymmetry of the polarization converter. The asymmetry of the radial polarization converter enables one to remove vortex phase in generated vector beam. Geometric phase, which is induced by the spatial distribution of polarization in the vector beam, determines whether the generated vector beam has vortex phase. For this reason, we also propose single shot determination of polarization distribution in the generated vector beam. Our work provides insights that could lead to new frontiers in highly important scientific fields involving singular opticsWe theoretically investigate the spatial distribution of polarization and vortex phase of vector beams. The key concept for reduction of vortex phase in vector beams is the cancellation of vortex phase by tailoring left- and right-hand circularly polarized optical vortices. This cancellation is achieved from the relationship between symmetry of polarized light and a rotational asymmetry of a polarization converter see Fig. . In the 33. The changes of the generated beam’s polarization state are sketched on a Poincaré sphere  of high-density polyethylene (HDPE) and n = 1.44  polytetrafluoroethylene (PTFE).  We estimate that total internal reflection produces achromatic phase retardance in the frequency region from 0.1 to 1.6 THz , Ey(θ)]T of the vector beam via Jones calculus. The Jones vectors of Ex(θ) (red solid line) and Ey(θ) (blue dashed line) are shown in Fig. Ex(0°), Ey(0°)]T = T, T = T, T = T, and T = T. In addition to the spatial distribution of the polarization, the phase of the vector beam is uniformly distributed as shown in Fig. 34.The range of our proposed approach extends to all spectral regions from ultraviolet to terahertz. Table “B” Fig. . These o(0°)]T = , 0T, [ExIn conclusion, we have proposed and demonstrated a pure vector beam without vortex phase by tailoring the geometric phase of the beam. This paper reports the first demonstration of a pure vector beam with radial polarization achieved by the concept for the reduction of vortex phase. We also proposed a continuous body extended to the segmented non-axially-symmetric half-wave plate. Our technique enables the vector beam to be manipulated in singular optics. Our proposed concept can also be used in conjunction with generic technologies in other different fields, such as super resolution imaging, material science, multiplexing quantum information science, high energy physics, and even astronomy.n(λ) and β denote the refractive index of the material and the slope angle of a rhomb, respectively. Under the proposed conditions, β is a constant, while n(λ) shows dependence on wavelength λ. An output beam generates the phase retardance of ϕ by total internal reflections35.Internal Fresnel reflections are an example of fundamental optical phenomena that result in phase retardance of ϕ = π, is required for a generation of a pure vector beam with radial polarization and a uniform phase. A fast axis of the non-axially-symmetric half-wave plate is rotated with azimuth of θ/2 as a function of angle θ. In this experiment, we fabricated the non-axially-symmetric half-wave plate with four Fresnel rhomb segments. T as shown in Fig. When a uniform beam with linear polarization is incident onto a non-axially-symmetric half-wave plate that is acting as a radial polarization convertor see Fig. , the polEin = 0, T as showd = 30 mm and focus length f = 100 mm), and the non-axially-symmetric half-wave plate with four segments. Figure Figure s0(θ), s1(θ), s2(θ), and s3(θ) along the angle θ on the input vector beam converted are modulated by the axially-symmetric wave plate. After passing through a wire-grid polarizer, the intensity distribution of the vector beam varied as a function of the angle θ can be expressed asI(θ) contains full Stokes parameters from sA0(θ) to sA3(θ) given in angular frequency terms — bias, sin 2θ, cos 4θ, and sin 4θ. To isolate each term in frequency space, we introduce Fourier transform method, which is commonly used for fringe pattern analysis in optical metrology40. Using Fourier transform method, Fourier spectrum of P(k) is expressed ask denotes angular frequency; C0(k), C2(k), and C4(k) represent Fourier spectra; a superscript asterisk “*” represents a complex conjugate. By individually extracting the C0(k), C2(k), and C4(k) components in Fourier space, the spatial distribution of Stokes parameters sA0(θ) − sA3(θ) are determined as38 T, and T, where ε is ellipticity and θ is azimuth, respectively. The surface area Ω of a spherical triangle is expressed as C is a domain of definition (for more information, refer Supplementary Note To evaluate the phase distribution of the generated vector beam, we introduce geometric phase using the spatial distribution of Stokes parameters. The geometric phase was first suggested by Berry and Pancharatnamsupplementaryvectorbeammagnitude


The high strain rate during the Charpy impact was found to enhance the strain hardening capability of the UFG Cu due to the suppression of dynamic dislocation recovery. The crack in the CG Cu was blunted by dislocation-slip mediated plastic deformation, while the cracks in the UFG Cu were formed at grain boundaries and triple junctions due to their limited plasticity. Near the crack surfaces the elongated grains in ECAP-2 sample were refined by recrystallization, while equi-axed grains in the ECAP-16 sample grew larger.Nanostructured (NS) and ultrafine-grained (UFG) materials have high strength and relatively low ductility. Their toughness has not been comprehensively investigated. Here we report the Charpy impact behavior and the corresponding microstructural evolutions in UFG Cu with equi-axed and elongated grains which were prepared by equal channel angular pressing (ECAP) for 2 and 16 passes at room temperature. It is found that their impact toughness (48 J/cm It is usually closely related with plasticity: higher plasticity leads to higher toughness. Strength and toughness do not follow simple reciprocal relationship although there is usually a trade-off between strength and ductility. For structural materials, it is ideal to simultaneously possess both high strength for carrying more load, and high toughness for avoiding catastrophic failure. Charpy impact test is the most common technique for evaluating the impact toughness of materials under high strain rates ~03 s−11. et al.2, of an UFG Al–11%Si alloy to the equal channel angular pressing (ECAP) process, which broke the large aluminum brittle dendrites and inter-dendritic networks. Stolyarov et al., found that the impact toughness of NS Ti increased with decreasing temperature, which is opposite to the trend in CG metals and alloys6Toughness is an important mechanical property for ultrafine grained (UFG) and nanostructured (NS) materials to be applied in many structural applications8911121314These literature results generally show that grain refinement can improve the impact toughness. However, compared with other mechanical properties such as strength and ductility, there has been a lacking of systematic study and understanding on the impact toughness of UFG and NS metals and alloys. In the present paper, we report a systematic investigation on the Charpy impact behavior of bulk UFG Cu with two different grain structures, elongated and equi-axed, produced by ECAP for 2 passes and 16 passes, respectively. For comparison, same investigation was also performed on the CG Cu counterpart.The initial CG Cu after annealing was found to have fully recrystallized homogeneous microstructure with equi-axed coarse grains of about 55 μm . After E1718Uni-axial tensile engineering stress-strain curves are shown in 21222425Ak could be obtained directly from the experiments and the impact toughness ak could be calculated via normalizing Ak by the cross-section area. Ak and ak of the CG and UFG Cu. One can see that the Ak and ak of the UFG Cu  are almost as high as those of CG Cu , and ECAP-2 and ECAP-16 Cu samples have comparable Ak and ak. The impact energy is proportional to the area of the impact load-displacement curve. Therefore, although the area in stage II of the UFG Cu is larger than that of CG Cu, the overall area of the UFG Cu is still a little smaller than that of the CG Cu because of the smaller area in stage III of the UFG Cu than that of the CG Cu.The absorbed impact energy Comparing the tensile and impact curves, one can see that there is little strain hardening stage for the UFG Cu subject to the quasi-static tension. However, under impact loading the UFG Cu have strain hardening capability that is comparable with that of the CG Cu. It has been reported that high strain rates can improve the strain hardening capability in UFG/NS materials272829303134The experiment also shows that UFG Cu is less resistant to crack propagation  can be described by:where Ak is impact energy, Va is impact affected volume, l is crack length measured in SEM images of UFG materials in d is sample thickness (3 mm) and w is assumed width of impact affected zone (500 μm as corresponding to the EBSD maps). Then the temperature rise can be calculated as:where et al.ρ = 8.97 × 103 kg m−3 and Cv = 394 J (kg K)−1. The crack length l of ECAP-2 Cu sample is about 2.72 mm and impact energy Ak is 4.4 J, so the temperature rise is calculated as ΔT = 274 K. As for ECAP-16 Cu, l is about 2.64 mm and Ak is 4.4 J, so ΔT = 282 K. About 290 °C can be reached for both samples, which is high enough for recrystallization process, as reported by Zhang et al.From Mishra et al.It is noted that the temperature rises in ECAP-2 and ECAP-16 samples are close. However, their microstructures near the crack edges after the impact tests are very different. Grain growth was found in ECAP-16 Cu while grain refinement caused by recrystallization was detected in ECAP-2 Cu. These differences of microstructural evolutions during impact were caused by their different initial grain structures. The ECAP-2 Cu has dislocation cell structures. Heating during impaction causes recrystallization at the small-angle GBs or region with high-density of dislocations. In comparison, the ECAP-16 Cu sample has equi-axed recrystallized grains, impact heating directly caused grain growth without nucleation. Liu As shown in 492, which is almost comparable with that of the CG Cu samples: 55 J/cm2. Compared with the tensile curves under quasi-static strain rate, high strain rate was found to enhance the strain hardening capability of the UFG Cu due to the suppression of dislocation dynamic recovery. EBSD mapping revealed that the CG Cu sample underwent large plastic deformation mediated by dislocation slip in near-crack region, which produced elongated grains and subgrain structure, while the UFG Cu samples formed cracks at the GBs and triple junctions due to limited plasticity and dislocation activity. Along the crack, recrystallized refined grains in the ECAP-2 Cu and large grown grains in the ECAP-16 Cu were found although the temperature rises were close for both samples. The higher thermal stability of the ECAP-2 Cu than ECAP-16 Cu was resulted from both low GB fractions and low-angle GBs.In this work, UFG Cu samples were processed by ECAP process for 2 and16 passes, producing elongated grain structure with low-angle GBs and equi-axed grain structure with high-angle GBs, respectively. Both the UFG Cu samples have comparable impact toughness of about 48 J/cm3 were machined by electrical discharge. These square bars were processed by ECAP using a die having a channel angle (both exterior and interior) of 90°. Samples were pressed to 2 passes and 16 passes through route BC (route B for 2 passes) where the bar was rotated in the same sense by 90° between each pass with 0.4 mm/s velocity at room temperatureAs-received pure Cu (99.99%) was annealed at 500 °C for 2 h in a vacuum furnace to produce a CG initial structure with an average grain size of about 55 μm. Square bars with a dimension of 20 × 20 × 80 mm−3 s−1. The dog-bone shaped tensile specimens were machined out along the longitudinal direction after ECAP. The gauge dimension is 2 × 1 × 10 mm3 and sample surface was polished by 1 μm diamond suspension before tests. The impact toughness was measured by means of Charpy impact tests using a 25 J pendulum instrumented Zwick HIT 50P. The longitudinal direction of the specimens is parallel to the ECAP extrusion direction with dimensions of 3 × 4 × 27 mm3. A 60° “V” groove was machined in the middle and the notch depth is 1 mm. To exclude the influence from microstructural heterogeneity of the ECAPed billets, the impact specimens were strictly cut from the central parts of the ECAP billets. The Charpy impact specimen dimensions in this experiment conform to DIN 50 115 standard. At least three successful measurements were used to evaluate the impact toughness for each testing condition. The average impact strain rate is about 1.3 × 103 as calculated the displacement of “V” groove versus impact time.Quasi-static uniaxial tensile tests were conducted at room temperature at a strain rate of 1 × 10+ ion-milling at 4 kV at temperatures less than 40 °C to get perforation. Moreover, Electron Backscattered Diffraction (EBSD) analysis was performed at the surface areas neighboring fracture section after impact tests. Sample surface was grinded to mirror-like condition and then electro-polished using 2.1 V ac in 85% H3PO4 + 15% deionized water to remove micro-scratches and relax strained sample surface. EBSD scanning was completed using Zeiss Auriga crossbeam microscope equipped with an Oxford EBSD detector working at 20 KV. Step size chosen for ECAPed samples was 25 nm to obtain accurate resolution for obtaining UFG microstructures. The fracture surfaces of the Charpy impact specimens were also examined by scanning electron microscopy (SEM) integrated in Zeiss Auriga.Microstructure was characterized using a Philips CM12 transmission electron microscope (TEM) operated at 100 kV. Diffraction patterns were also obtained by selected area electron diffraction (SAED). TEM specimens were cut from the plane parallel to the longitudinal direction of ECAPed bar. These specimen sheets were first grinded step by step using abrasive papers (400 #~1500 #) to <100 μm in thickness and punched to wafers with 3 mm in diameter, then prepared using ArHow to cite this article: Liang, N. et al. Effect of grain structure on Charpy impact behavior of copper. Sci. Rep.7, 44783; doi: 10.1038/srep44783 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


One important type of defect in 2D layered materials is known as rotational stacking fault (RSF), but the coexistence of multiple RSFs with different rotational angles was not directly observed in freestanding 2D MoS2 before. In this report, we demonstrate the coexistence of three RSFs with three different rotational angles in a freestanding bilayer MoS2 sheet as directly observed using an aberration-corrected transmission electron microscope (TEM). Our analyses show that these RSFs originate from cracks and dislocations within the bilayer MoS2. First-principles calculations indicate that RSFs with different rotational angles change the electronic structures of bilayer MoS2 and produce two new symmetries in their bandgaps and offset crystal momentums. Therefore, employing RSFs and their coexistence is a promising route in defect engineering of MoS2 to fabricate suitable devices for electronics, optoelectronics, and energy conversion.Electronic properties of two-dimensional (2D) MoS By having a nonzero bandgap, 2D MoS2 is more suitable as a semiconducting channel for electronics22, optoelectronics16, spintronics16, energy conversion17, piezotronics9, and photonics21, compared to graphene. Significantly, crystal defects easily occur in 2D MoS2, whose electronic structures can be modulated using many defects such as dislocations19, grain boundaries19, point vacancies6, antisite defects5, edges4, and dopants14. Hence, the properties of 2D MoS2 can be tailored via defect engineering to develop various functional devices19.Single- and few-layer molybdenum disulfide  are a type of fundamental defects that are widely present in layered materials such as overlapped or folded single- and few-layer 2D MoS2 sheet, the thinnest 2D MoS2 crystal in which RSFs can exist. These RSFs were observed using an aberration-corrected (AC) transmission electron microscope (TEM) with a low accelerating voltage of 80 kV for its electron beam. Their atomic structures were directly imaged and then compared with high-resolution TEM (HRTEM) simulations, which indicated that they originated from a crack and two dislocations in one component layer of the bilayer sheet. Their rotational angles were resolved as 27.80°, 20.27° and 14.60°, respectively. Furthermore, first-principles calculations were utilized to examine their electronic structures and those of RSFs with other rotational angles. The calculations indicate that two RSFs possess nearly symmetrical bandgaps and purely symmetrical offset crystal momentums when their rotational angles are symmetrical about 30°. That is, Eg (x) ≈ Eg (60° − x) and p (x) = p (60° − x), where Eg, x and p denote the bandgap, rotational angle and offset crystal momentum of RSF, respectively. The two symmetries can be utilized to engineer the electronic structure of 2D MoS2, and the coexistence of different RSFs can bring different electronic structures into a MoS2 sheet. This work introduces new opportunities to develop electronic, optoelectronic and energy-conversion devices from 2D MoS2.In this contribution we report the first direct observation of three RSFs with different rotational angles coexisting in a freestanding bilayer MoS2 sheets in this work were synthesized by annealing MoO2 microplates in sulfur vapor and then transferred onto TEM grids with poly (methyl methacrylate)-mediated nanotransfer printing. These procedures are exactly identical to those reported2. The AC-TEM characterization for HRTEM was performed by FEI Titan 80–300 with a spherical aberration (Cs) corrector for the objective lens at the accelerating voltage of 80 kV. The spherical aberration was set to be negative, which can give high contrast and low noise40 (see more details in the Methods section of Supplementary information).The bilayer MoS2 sheet with the standard AB stacking and without any RSFs. The atomic structure of the theoretical model . Figure 2 sheet contains three different Moiré patterns. The FFT pattern of the HRTEM image of the sheet contains twenty-four Defects can be produced in intact MoS2 layer without any cracks. Comparing Fig. 2 sheet with the three Moiré patterns. Since this uncracked layer is exclusively due to Hexagon 1, the HRTEM image of the second component layer of the bilayer MoS2 sheet can be reconstructed from Hexagons 2 to 4 47 (see more details in the Methods section of Supplementary information). In the first-principles calculations, the band structure of a standard MoS2 monolayer was firstly tested by the conventional Perdew, Burke and Ernzerhof (PBE) functional45 and the hybrid Heyd-Scuseria-Ernzerhof functional (HSE06)49 methods, which yielded bandgap values of 1.78 and 2.25 eV, respectively. The value of 1.78 eV is in good agreement with previous calculation results  by PBE52. Thus, we focused on the PBE band structures in this work. Besides, the influence of spin-orbit coupling in MoS2 is reported to be very small, because the Mo and S elements are not heavy enough54. Thus, we did not consider the spin-orbit coupling in our calculations.To further understand the effects of these RSFs, we performed first-principles calculations to explore their influences on the electronic structure of bilayer MoS2  ≈ Eg (60° − x) and p (x) = p (60° − x). Eg, x and p denote the bandgap, rotational angle and offset crystal momentum of RSF, respectively. The two symmetries were not reported before our work, because some works considered the atoms in the two component layers of RSFs of bilayer MoS2 to have nearly random relative distributions34 and the others constructed the RSF unit cells with different rotation axes35. In our calculation, only the rotational axes passing through the centers of hexagons of the two component layers were used to construct the unit cells  have similar structures: their sizes and numbers of atoms are identical, and the distributions of the spatial positions of their Mo and S atoms are similar to each other. The similarity of the atomic positions is described as follows: if we leave the bottom MoS2 layers immobile while rotating the upper layers by the x and 60° − x angles respectively, we observe a 180° polarity reversal in the upper layers, such as those shown in Fig. x and 60° − x angles are centrosymmetric to each other. This symmetry of the atomic structures consequentially results in the symmetries of the corresponding electronic structures, because atomic structures are the origins of electronic structures55. However, bandgaps are also affected by the accurate spatial positions of atoms in atomic structures55. Thus, since the atoms of the upper layer of one of the two RSFs in the aforementioned angles have spatial positions that are not exactly the same as but only centrosymmetric to those of the other RSF, the bandgap trend is not purely symmetrical. On the other hand, the fact that a pure symmetry about 30° is observed in the offset crystal momentums suggests that offset crystal momentums are not affected by the accurate spatial positions of atoms but depend on only the symmetry of the atomic structures.The symmetries of the bandgaps and offset crystal momentums occur because the unit cells of two RSFs with two different rotational angels symmetrical about 30° -dependent changes in the bandgap (Eg) and offset crystal momentum (p). The changes reveal symmetries about the RSF angle of 30°, Eg (x) ≈ Eg (60° − x) and p (x) = p (60° − x), due to similarities in atomic structures for specific pairs of RSF angles. Our work shows that by engineering specific RSFs into 2D MoS2 bilayers, the MoS2 electronic structures can be tailored to meet the specifications of particular devices for electronics, optoelectronics, and photovoltaics.In summary, three RSFs with different rotational angles have been found to coexist in a freestanding bilayer MoSSupplementary Information


Southern rice black-streaked dwarf virus’’  [1]. The data include fluorescence quenching spectrum, Stern–Volmer quenching constants, and binding parameters. In this article, a more comprehensive data interpretation and analysis is explained.The data present binding constants between ferulic acid derivatives and the Coat Protein (P10) by fluorescence titration in this article, which is hosted in the research article entitled “Interaction Research on an Antiviral Molecule that Targets the Coat Protein of  Specifications TableValue of the data•The data serve as a background for the affinity of P10 with ferulic acid derivatives by fluorescence titration.•The data present a method for interaction research of molecule and target protein.•Southern rice black-streaked dwarf virus compounds.The data provide a new insight for further design novel and potent anti-1The dataset of this article provide information on the interaction of 33 ferulic acid derivatives with P10 protein by fluorescence titration. The fluorescence quenching spectrum for compounds F28, F29, F30 are shown in 2Escherichia coliP10 protein was purified from At 275 nm excitation wavelength, the fluorescence quenching spectra were recorded at 298 K from 290 nm to 450 nm when the excitation and emission band slit widths were both 5 nm. Fluorescence quenching can be dynamic or static. Such type of fluorescence quenching can be verified through the Stern–Volmer equation, as shown in Eq. 10 M−1 S−1. When Kq > 2×1010 M−1 S−1, the main reason for the fluorescence quenching is static quenching KA and binding sites n of the complex can be calculated using Eq. The maximum quenching constant of the quencher on the biomacromolecule is about 2×10


The QT interval on the human electrocardiogram is normally in the order of 450 ms, and reflects the summated durations of action potential (AP) depolarization and repolarization of ventricular myocytes. Both prolongation and shortening in the QT interval have been associated with ventricular tachy-arrhythmias, which predispose affected individuals to sudden cardiac death. In this article, the molecular determinants of the AP duration and the causes of long and short QT syndromes (LQTS and SQTS) are explored. This is followed by a review of the recent advances on their arrhythmogenic mechanisms involving reentry and/or triggered activity based on experiments conducted in mouse models. Established and novel clinical risk markers based on the QT interval for the prediction of arrhythmic risk and cardiovascular mortality are presented here. It is concluded by a discussion on strategies for the future rational design of anti-arrhythmic agents. Both syndromes can arise congenitally from ion channel mutations, or can have acquired causes. In this article, the ionic basis of the QT interval is examined, summarizing recent advances into the electrophysiological mechanisms of arrhythmogenesis of both LQTS and SQTS.Long and short QT syndromes (LQTS and SQTS) are primary electrical disorders of the heart that predispose the affected individuals to sudden cardiac death 1.1c) interval by Bazett's formula is 440 ms for males and 460 ms for females. The latest European Society of Cardiology guideline produced in 2015 suggests upper and lower limits of 480 ms and 360 ms, respectively, for both males and females The QT interval of the human electrocardiogram (ECG) is a marker of the duration of the cellular action potential (AP) 1.2+ channels with rapid activation and inactivation kinetics. Phase 1 involves rapid repolarization mediated by the fast and slow transient outward K+ currents, Ito,f and Ito,s, respectively. Phase 3 is maintained by competing inward currents mediated by the voltage-gated L-type Ca2 + channel  and Na+-Ca2 + exchanger (INCX), and outward currents mediated by the voltage-gated delayed rectifier K+ channels (IK) + efflux due to a large potential difference between the membrane potential and the K+ equilibrium potential. Phase 4 is the resting membrane potential at − 80 and − 64 mV IK1 with contribution from the weak inward rectifying ATP-dependent K+ channels  Generation of the ventricular APs is dependent upon voltage-gated conductances, and AP durations are determined by the balance between inward and outward currents. An AP has five phases: fast upstroke (phase 0) followed by a spike (phase 1) and plateau (phase 2) morphology, and further repolarization (phase 3), where the transmembrane voltage returns to the resting membrane potential (phase 4) . Phase 01.3Long QT syndromes (LQTS) is characterized by an abnormally long QT interval of ≥ 450 ms on the ECG. The first hereditary long QT syndrome was discovered by Jervell and Lange-Nielsen (JLN) in 1957 + channels are responsible for LQTS types 1 (KCNQ1), 2 (KCNH2), 5 (KCNE1), 6 (KCNE2), 7 (KCNJ2) and 13 (KCNJ5). By contrast, gain-of-function mutations in Na+ channel subunits lead to LQTS types 3 (SCN5A) and 10 (SCN4B), and in the L-type Ca2 + channel produces LQT type 8 . Mutations in supporting proteins are responsible for the LQT type 4 (ANKB), 9 (CAV3), 11 (AKAP9) and 12 (SNTA1) phenotypes. Recent studies have implicated calmodulin mutations in patients suffering from a LQTS with previously unidentified genetic causes + current  mediated by neuronal Na+ channel isoforms LQTS is caused by a decrease in repolarizing currents or an increase in depolarizing currents, with either congenital or acquired causes. Today, thirteen genetic LQTS subtypes have been identified thus far. Loss-of-function mutations in the different types of K2 + current available Na+ channel reactivation during the repolarizing phase, leading to the development of early afterdepolarizations and subsequent triggered activity  is characterized by an abnormally short QT interval of < 350 ms on the ECG. It predisposes affected individuals to an increased risk of atrial and ventricular arrhythmias, in particular ventricular fibrillation, and is therefore an important cause of sudden cardiac death The mechanism of arrhythmogenesis in SQTS is less well-understood than that of LQTS. Recent work in mice demonstrated shortening in ERP in concert with APD 1.5cd) peak – Tend, reflecting increased TDR peak – Tend)/QT ratio Pre-clinical models have been useful for the studying the mechanisms of cardiac arrhythmogenesis and provide a platform for testing the arrhythmogenic potential of drugs peak–Tend)/QRS and (Tpeak–Tend)/(QT × QRS), have recently been put forward by Tse, but these remain to be validated clinically. Tse's indices are based on the observations that both conduction and repolarization abnormalities are important in arrhythmogenesis and Tpeak–Tend was a significant predictor of SCD even after adjusting for, inter alia, QTc and QRS durations d) reflecting CV dispersion, yielding QRSd × (Tpeak–Tend)/QRS, and QRSd × (Tpeak–Tend)/(QRS × QT) d/QRS could be a surrogate marker of CV dispersion coefficient based on the standard deviation of the mean CV However, none of the above repolarization markers takes into account action potential conduction, yet λ, which incorporates both processes, is an important determinant of arrhythmic risk 1.62 + transients, or suppressing after depolarization phenomena  insertion. There is therefore a need to develop more effective agents for anti-arrhythmic therapy. A better understanding of the mechanisms of arrhythmogenesis would allow rational drug design that aims to reverse the electrophysiological abnormalities in question. Application of pre-clinical results to clinical medicine could result in effective translation for the benefit of patients, which is illustrated by the following two examples that demonstrate important proofs-of-concept. Firstly, hypokalaemia modelling LQTS produces AP prolongation, reduced ERP, reduced λ, increased TDR, increased APD restitution slopes and increased amplitude of APD alternans. Gap junction inhibition using heptanol normalized ERP and therefore λ without correcting for the remaining repolarization abnormalities henomena . Novel aNone declared.


This has been proposed as one potential pathway to mitigate the effects of greenhouse gas release. Most of our current understanding of reaction mechanisms is based on hand specimen and laboratory-scale analyses. Linking laboratory-scale observations to field scale processes remains challenging. Here we present the first geophysical characterization of serpentinite carbonation across scales ranging from km to sub-mm by combining aeromagnetic observations, outcrop- and thin section-scale magnetic mapping. At all scales, magnetic anomalies coherently change across reaction fronts separating assemblages indicative of incipient, intermittent, and final reaction progress. The abundance of magnetic minerals correlates with reaction progress, causing amplitude and wavelength variations in associated magnetic anomalies. This correlation represents a foundation for characterizing the extent and degree of in situ ultramafic rock carbonation in space and time.Peridotite carbonation represents a critical step within the long-term carbon cycle by sequestering volatile CO Peridotite carbonation plays an important role in the carbon cycle. Here, the authors present a geophysical characterization of serpentinite carbonation from km to mm scale and confirm that the abundance of magnetic minerals provides a strong correlation with the overall carbonation reaction process. The uptake of atmospheric and hydrospheric carbon during ultramafic rock carbonation particularly represents a natural analog to geologic carbon sequestration and is considered as one potential pathway to offset anthropogenic CO2 emissions into the Earth’s atmosphere7. Natural carbonation of ophiolite—alpine-type ultramafic rocks forms alteration assemblages known as ophicarbonate, soapstone, and listvenite. These different carbonation products differ in the composition of secondary sheet silicate phases and the abundance of carbonate and thus their bulk rock CO2 content. Listvenite is predominantly composed of carbonate and quartz and represents a desirable product during in situ CO2 sequestration in ultramafic formations. While natural ultramafic rock carbonation may take place over long time scales, its efficiency has yet to be proven on human time scales. Carbonation reaction parameters are extensively investigated by laboratory-scale hydrothermal experiments11, thermodynamic modeling12. and natural analog studies19. However, a scheme that can delineate the carbonation reaction progress in situ has not yet been fully explored and upscaling of reaction parameters from small scale, controlled laboratory experiments to large scale, complex natural processes remains challenging22. Along the reaction path of hydrothermal alteration of ultramafic rock, silicate mineral replacement reactions concomitantly release Fe for incorporation into secondary oxide, sulfide, and carbonate phases24. Of particular interest is the production and consumption of magnetite during reaction of ultramafic rock with hydrothermal fluids due to its strong influence on bulk rock magnetic properties25. If coherently observable at multiple scales, we propose that changes in rock magnetic properties related to peridotite serpentinization and subsequent carbonation can be linked to distinct steps along the reaction path, and hence that the reaction progress can be monitored by field magnetometry.Peridotite serpentinization and carbonation play important roles in facilitating large-scale cycling of volatiles between the atmosphere, hydrosphere, and lithosphereIn this study, we investigate magnetic anomaly changes related to natural serpentinite carbonation using regional, outcrop-, and thin section-scale magnetometry coupled with microtextural analysis of mineral replacement reactions. The results show that the magnetic character of distinct alteration product assemblages changes in response to the stability of magnetic carrier phases. Progressive serpentinite carbonation is characterized by a transient increase in the magnetic field strength during intermittent carbonation. The final alteration product is almost devoid of magnetic carrier phases and thus characterized by a very weak magnetic field strength. These findings indicate that magnetic field measurements can be used to detect carbonation fronts in the field and to monitor reaction progress in space and time.Widespread and near perfect exposure of naturally carbonated serpentinite at the Linnajavri Ultramafic Complex (LUC) in the Upper Allochthon (Köli nappe) of the Norwegian Caledonides represents an excellent natural laboratory to study the effects of ultramafic rock carbonation on changes in geophysical properties at the field scale Fig. . The LUC2-bearing fluid resulted in the formation of distinct zones of minor serpentine-magnesite , extensive talc-magnesite  and quartz-carbonate  assemblages from completely serpentinized peridotite during the Caledonian orogeny  and listvenite (higher aCO2)  Fig. 12, 13, 2ore Fig. . MagnetiMg (XMg = Mg/(Mg + Fe) ≈ 0.93), whereas the euhedral magnesite rim is devoid of magnetite inclusions and distinctly enriched in Fe (XMg ≈ 0.86) , chlorite, albite, tourmaline, and sometimes sulfide phases Fig. . Magneti86) Fig. 13.Fig. 5We conducted a multi-scale geophysical investigation across the carbonation fronts by integrating aeromagnetic data (~10 km), total magnetic field and magnetic susceptibility surveys at outcrops (10−100 m), and magnetic mapping of thin sections (µm) from drill core samples acquired along the survey lines. The crustal-scale aeromagnetic field data of the LUC region were obtained by the Norwegian Geological Survey’s 1991 DRAGON aeromagnetic survey using a Scintrex MEP410 cesium magnetometer with an average survey altitude of 60 m and line spacing of 200 m.30. Magnetic susceptibility measurements were conducted along the magnetometry transects using a Terra–TK04 susceptometer  anomalies in contrast to the surrounding metasedimentary units, which exhibit only weak field values and minimal amplitude variations. The locations of high amplitude, short-wavelength anomalies coincide with the distribution of mapped ultramafic complexes Fig. . Outcropter Fig. . In contChanges in total field magnetic anomaly amplitudes over measurement profiles originate from the combination of both natural and induced remanent magnetization (NRM) of the rock formation, whereas changes in magnetic susceptibility values represent the abundance and chemical composition of magnetic carriers. In the serpentinite and soapstone samples magnetite represents the only magnetic carrier phase, while magnetite is absent in listvenite or present in only minor amounts with occasional additional iron sulfide phases  Fig. . ChangesaCO2, magnetic anomaly amplitudes are high in serpentinite, and even higher in soapstone, but minimal in listvenite with little variations in amplitude and wavelength  field measurements to assess the distribution of magnetic carriers within the samples at fine spatial scale. After full alternating-field demagnetization of the samples, we imparted ARMs with a bias field of 100 μΤ and a peak alternating field of 260 mT to activate magnetic sources in the low to high coercivity range and assess the capacity of the sample to acquire magnetization.Magnetic field mapping at sub millimeter-scale spatial resolution on representative thin sections from serpentinite, soapstone, and two listvenite samples acquired from two different localities at the LUC were conducted using a scanning superconducting quantum interference device (SQUID) microscope at the Massachusetts Institute of Technology Paleomagnetism Laboratory. The instrument’s magnetic field sensitivity is ~0.01 nT and measures the vertical component of the magnetic field in a rectangular grid of positions above thin sections26. CO2 mass fractions of each sample used for thin section preparation were measured as ~2.35 wt%, ~14.9 wt%, and ~29.3 wt% for serpentinite, soapstone, and listvenite, respectively,  NRM magnetic field values in serpentinite, even higher (~9.4 µT) in the soapstone and almost none (except for a few grains of 1.8–3.0 µT per measured sample) in the listvenite Fig. . Petrogr24. As a result, total magnetic field intensity is expected to increase along the isothermal carbonation reaction path, while the absolute magnetic field intensity value depends on the amount of magnetite formed and thus on alteration temperature. In the serpentinite and soapstone samples, magnetite is the only mineral phase contributing to the magnetic signal, whereas listvenite sometimes contains additional sulfide minerals  )23. For the dominant silicate phases involved in ultramafic rock carbonation, preferential uptake of Fe is in the order: olivine > antigorite > talc34. Alteration of peridotite to form serpentinite and soapstone is therefore accompanied by release of Fe that is not partitioned into secondary serpentine and talc but available for magnetite formation:Variations in the magnetic signal strength of the different alteration assemblages can be linked to the stability of magnetic carrier minerals at the relevant alteration conditions. Thermodynamic models and hydrothermal experiments predict an increasing abundance of magnetite during the alteration sequence from peridotite serpentinization, intermittently formed soapstone, to listvenitete) Fig. . Magnetite) Fig. , 932 andMg ≈ 0.95; chlorite XMg ≈ 0.92) and carbonate phases (magnesite XMg ≈ 0.93) in soapstone relative to serpentinite (antigorite XMg ≈ 0.94) is thus consistent with the thermodynamic prediction and reflected by magnetic field intensity variations in outcrop and thin section-scale magnetic mapping across serpentinite-soapstone interfaces , carbonate (as siderite component), and sulfide  phases predominantly incorporate Fe2+, while talc and chlorite may take up small amounts of Fe3+. At the high fluid CO2 activities required to stabilize the listvenite assemblage, siderite forms at the expense of magnetite thereby effectively reducing the released ferric Fe 36, providing charge balance and sulfur for the formation of sulfide phases in the listvenite  and alteration fluid 2 mass fractions from serpentinite (~2.35 wt%) to soapstone (~14.9 wt%) and to listvenite (~29.3 wt%) demonstrates that measureable geophysical signals are associated with changes in mineralogy during the carbonation sequence  loss (or gain) using a self-calibrated correction for Si Kα1, Na Kα1, Ti Kα1, K Kα1, Fe Kα1. Interference corrections were applied to Fe for interference by Mn, and to Mn for interference by Cr47. Results are the average of three points and detection limits ranged from 0.006 wt% for Si Kα1 to 0.008 wt% for Al Kα1 to 0.009 wt% for Na Kα1 to 0.012 wt% for Ti Kα1 to 0.028 wt% for Ni Kα1. Oxygen was calculated by cation stoichiometry and included in the matrix correction. The elemental maps were processed using Probe Software’s CalcImage application. The matrix correction method was ZAF and the mass absorption coefficients data set was LINEMU Henke  < 10KeV/CITZMU >10KeV. The ZAF algorithm utilized was Armstrong/Love Scott48.Quantitative elemental maps were acquired on a JEOL 8530 F electron microprobe equipped with 5 tunable wavelength dispersive spectrometers. Operating conditions were 40° takeoff angle, and a beam energy of 15 keV. The beam current was 20 nA for calibration and map acquisition. The beam diameter was 2 µm. Dwell time was 40 ms per pixel with a pixel dimension of 2 × 2 µm. Elements were acquired using analyzing crystals LiFH for Ti Kα1, Cr Kα1, Mn Kα1, LiF for Fe Kα1, Ni Kα1, PETJ for Ca Kα1, K Kα1, and TAP for Mg Kα1, Si Kα1, Al Kα1, and Na Kα1. The standards were an assortment of synthetic and natural minerals and metals. The counting time was 20 s on peak for all elements, and Mean Atomic Number background corrects were used throughout2 and FeO were performed by Actlabs Laboratories Ltd., using the lithium metaborate/tetraborate fusion ICP Whole Rock and the trace element ICP/MS packages.Whole-rock geochemical analyses including CO2 sample gas are measured by infrared absorption after decomposing 0.2 g of sample material in a resistance furnace in a pure nitrogen environment at 1000 °C, using an ELTRA CW–800 (www.actlabs.com).Samples are mixed with a flux of lithium metaborate and lithium tetraborate and fused in an induction furnace. The melt is immediately poured into a solution of 5% nitric acid containing an internal standard, and mixed continuously until completely dissolved (~30 min). The samples are run for major oxides and selected trace elements on a combination simultaneous/sequential Thermo Jarrell–Ash ENVIRO II ICP or a Varian Vista 735 ICP. Calibration is performed using 7 prepared USGS and CANMET certified reference materials. One of the 7 standards is used during the analysis for every group of ten samples. FeO is determined through titration, using a cold acid digestion of ammonium metavanadate, and hydrofluoric acid in an open system. Ferrous ammonium sulphate is added after digestion and potassium dichromate is the titrating agent. Weight fractions of dry COAll the data generated or analyzed during this study are included in this published article (and its Supplementary Information files). Samples and data used in this study are available through MAPLES  at Department of Geology and Geophysics, Texas A&M University via email contact (masako.tominaga@tamu.edu).Supplementary Information


Here, white and brown deposits cap chemically distinct sediments with varying hydrothermal influence. All sediments contain abundant genes for autotrophic carbon fixation used in the Calvin-Benson-Bassham (CBB) and reverse tricaboxylic acid (rTCA) cycles. Both forms of RuBisCO, together with ATP citrate lyase genes in the rTCA cycle, increase with distance from the active hydrothermal centres and decrease with sediment depth. Clustering of RuBisCO Form II with a highly prevalent Zetaproteobacteria 16S rRNA gene density infers that iron-oxidizing bacteria contribute significantly to the sediment CBB cycle gene content. Three clusters form from different microbial guilds, each one encompassing one gene involved in CO2 fixation, aside from sulfate reduction. Our study suggests that the microbially mediated CBB cycle drives carbon fixation in the Spathi Bay sediments that are characterized by diffuse hydrothermal activity, high CO2, As emissions and chemically reduced fluids. This study highlights the breadth of conditions influencing the biogeochemistry in shallow CO2-rich hydrothermal systems and the importance of coupling highly specific process indicators to elucidate the complexity of carbon cycling in these ecosystems.The seafloor sediments of Spathi Bay, Milos Island, Greece, are part of the largest arsenic-CO Hydrothermal activity generally occurs at divergent and convergent boundaries such as subduction and seafloor spreading margins, fault zones, and in submarine serpentinization systems1. Marine shallow-water hydrothermal vents (SHV) in particular are often linked to arc volcanism, for instance on the Hellenic Volcanic Arc (HVA), Milos Islands (Greece), offshore of Kueishan Island (Taiwan), and Tutum Bay (Papua New Guinea)2. Because of the elevated temperature and extreme pH conditions, hydrothermal fluids are often rich in dissolved sulphur, iron, and other reducing inorganic chemical species  normally not present in appreciable amounts in low temperature marine settings4. Milos Island, an emergent volcano on the HVA in the Aegean Sea, bound by shallow hydrothermal submarine venting on its bays6 . The high CO2 emissions5 are a major potential source to fuel inorganic carbon fixation and drive in situ organic matter production in the sediment. While the rTCA cycle may drive carbon fixation in deep-sea hydrothermal systems rather than the Calvin Cycle12, the same cannot be assumed for shallow submarine settings. Indeed, there is a dearth of information on carbon fixation in shallow submarine settings, especially regarding the genetic quantification of specific taxa or functional genes, such as those involved in carbon fixation pathways. Also, owing to geothermal activity, exposure to light, and fluids enriched in As, shallow hydrothermal systems are generally considered extreme environments where both photosynthetic and chemosynthetic microbial processes7 occur simultaneously. Taken together, the Milos SHV offers an ideal opportunity to investigate the predominant modes of carbon fixation and carbon utilization in the extreme, As-influenced SHV sediments. Besides lending insight to the main drivers behind the biogeochemical cycling of carbon in such systems, a study of the shallow hydrothermal environment also helps to define how these settings differ from the more intensely studied deep-sea hydrothermal areas12.Shallow submarine hydrothermal ecosystems occur in the photic zone at water depths less than 200 m below sea level2 uptake, we also measured δ13Corg, δ13CDIC and performed lipid biomarker analyses to investigate biological actors other than microorganisms in the system.Our aim here was therefore to investigate the mode of carbon fixation in the shallow submarine hydrothermal environment of Spathi Bay, Milos Island. Specifically, we focused on coastal, hydrothermally-affected and unaffected sediment as a function of depth and distance from the hydrothermal centre using a suite of biogeochemical techniques: microbial community profiling using quantitative polymerase chain reaction (qPCR) and lipid biomarker studies, as well as bulk geochemical analyses. We identified and quantified key carbon fixation genes from samples collected in three different habitats in Spathi Bay. Two habitats were capped either by white or brown deposits and one was a deposit-free sand reference Fig. . To evalZetaproteobacteria 16S rRNA genes. Areas in the direct vicinity of the white (amorphous silica and sulphur-rich) and brown (manganese and iron oxide-rich) deposits have a lower abundance of genes involved in carbon fixation relative to the reference sediment. Irrespective of depth or habitat, qPCR analyses indicate that the Calvin Cycle is the main CO2 fixation pathway mediated by microorganisms in this shallow hydrothermal setting, with the predominance of cbbm genes quantified over aclB genes (rTCA cycle). The δ13Corg and δ13CDIC composition of the sediment was found to be consistent with autotrophic carbon fixation processes. While CO2 consumption in the sediment is driven by both the hydrothermal CO2 gradient and microbial C-fixation, lipid biomarker analysis further revealed the influence of both plant and microbially derived organic matter in the sediments.Our results show a high spatial heterogeneity in carbon-fixing gene distribution related to the abundance of archaea, bacteria and X-ray diffraction (XRD) and Raman spectroscopy analysis revealed quartz, birnessite (Mn oxides), graphite and hematite (FeI(II)(oxyhydr)oxides) as ubiquitous minerals in the sediment, regardless of depth and hydrothermal influence Tables  and S2. The Raman data coupled to principal component analysis (PCA) indicates that the three habitats were distinct in mineralogical and chemical composition Fig. . Additio13Corg of all white- and brown-capped sediments revealed small variations ranging between −18‰ and −20‰ .The bulk carbon isotopic composition δ20‰ Fig. . The δ13ely Fig. . The rescbbL and cbbM ; aclB (ATP citrate lyase β subunit in the rTCA cycle); pmoA (particulate methane monooxygenase); mmoX (soluble methane monooxygenase); mcrA  and dsrA  (see SI for qPCR bias).Quantification of targeted specific genes was performed by qPCR on two sediment cores per habitat (core 1 and 2), i.e., white-capped, brown-capped and reference sediment. The quantified genes were bacterial, archaeal and zetaproteobacterial 16S rRNA; Zetaproteobacteria, were not detected after 4–6 cm depth in core 1 collected from the white-capped sediments.16S rDNA specific to cbbM genes were not detected between 2–4 cm and 4–6 cm in replicate cores sampled from the white-capped sediments.mmoX genes for the aerobic methanotrophic Alphaproteobacteria were not detected at 6–8 cm and in the deepest section of core 1 collected from the sediments capped with the white deposits.pmoA genes for the aerobic methanotrophic Gammaproteobacteria were not detected in the uppermost part of the reference material (core 1), in the white-capped sediments between 8–12 cm (core 1), and between 10–14 cm (core 2).mcrA genes were not detected in the white capped sediments below 8 cm (core 1) and between 10–14 cm (core 2).Targeted genes were quantified in all cores and depths Figs  and S3 wZetaproteobacteria community appeared to comprise a large part of the bacterial 16S rRNA gene pool, especially in the brown-capped sediments  and lower amounts associated with Form I (cbbL)  Figs  and S2. nts Figs  and S2. nts Figs  and 4.iso-C15:0 and anteiso-C15:0 fatty acids, were more distributed near the surface of the brown-capped and the reference sediment and less abundant in the white–capped sediments  had the highest distribution, which generally decreased downcore at each site. Saturated fatty acids were found at all sites and at all depths, but the unsaturated C16:1 cis-9 fatty acid was only present in surface layers at the white and brown-capped sites. Both saturated and unsaturated fatty acids showed an increasing distribution trend across the different habitats, i.e. going from the white to the brown-capped to the reference sediments   test indicated that habitat  generally played an important role in the amount of gene quantified by qPCR (5) Table . Indeed,ts Table , which sts Table . The samts Table  and S8 , available to chemoautotrophs. Regardless of depth or habitat, qPCR analysis suggests that the main CO2 fixation pathway is the microbially-mediated Calvin Cycle . Gaseous emissions from the white-capped sediments at this site contain up to 90% COcle Figs . RuBisCOell Figs   of the sediment mineralogy  and goethite (FeO(OH)), which are known to strongly regulate porewater trace metals and metalloid concentration20. Across sediments, the arsenic concentration in the porewater decreases from high concentration in the white-capped sediments to low concentration in the reference sediment  also show, that the arsenic concentration increases with depth in the sediment covered by a white patch4. Arsenic appears not to be a major selector of major carbon fixation genes that dominate along the sampled transect, indicated by the homogeneity of similar dominant carbon fixation genes in the different habitats, despite the heterogeneity of arsenic concentrations. However, the systematic increase in the most abundant carbon fixation pathway genes from the hydrothermal centres to the reference sediments, while arsenic content declines simultaneously in the same direction, suggests that arsenic may be a contributing factor to the decrease in hostile habitable conditions in this direction. This proposition is supported by our observation that the hydrothermal centres are generally the most hostile habitats in this environment.Dissolved iron concentrations were low in the porewater of all hydrothermally-affected sediments. In light of the sediment mineralogy, this might indicate Fe removal in the form of Fe-sulphides  in the white-capped sediments Table  and mainnt Table . In the nt Table  sedimentnt Table . Similar21 [and references therein], arsenic species prevent DNA replication, ATP formation or phospholipids and phosphoprotein synthesis. The arsenic gradient in the sediment, in addition to the hydrothermal activity, therefore, offers one possible explanation for why the white-capped sediment is less inhabited than the brown-capped sediment  (data not schown). We hypothesise that the hydrothermal activity and gradient directly affects microbial abundance in the surface sediment as has been seen by previous studies18. This would also explain why the microbial abundance of the 16S rRNA genes and the bacterially derived lipids are most abundant in surface sediments compared to deeper samples, and in the reference sediments relative to the hydrothermally affected sediments.MANOVA Tables , 7 and 8Zetaproteobacteria and RuBisCO form II (cbbM) genes, which is consistent with studies in Hawaiian and Greek hydrothermal settings24. Moreover, while both cbbL and cbbM genes have been detected in the genome of Marinoprofundus ferooxydans25, factor analysis revealed a cluster for only the cbbM gene with the Zetaproteobacteria in the Spathi samples. This suggests that the Zetaproteobacteria contribute significantly to the abundance of the cbbM genes in Milos’s sediments probably due to the high CO2 content of this environment. The factor analysis also grouped together the archaeal 16S rRNA genes with the aclB gene, suggesting that archaea likely contribute the most to the aclB gene content , mmoX, pmoA  and mcrA  genes . Furthermore, the entire metagenomics dataset, comprising ∼4000 assembled functional genes, indicates that oxygenic photosynthesis is not an important process in carbon fixation in this environment (data not shown).Statistical analysis Fig.  revealedent Fig. . This cones Fig. . RuBisCOmcrA and dsrA gene . This correlation, however, was low in the brown-capped sediments. These relationships highlight that some form of anaerobic methanotrophy coupled to sulphate reduction may be expressed in the white-capped sediments and in the reference sediments, although the cluster analysis did not support this conclusion. Further evidence for microbial sulphate-reduction is provided by the prevalence of Fe sulphide as pyrite and marcasite minerals mainly in the white-capped sediments  and the mcrA (anaerobic methane cycling) genes highlight the close connection between the methane producers and consumers, as well as the possible involvement of anaerobic bacteria methane oxidation via nitrite reduction by internally producing O2 in anoxic environments35.In the white-capped sediments and in the reference sediments, a significant correlation exists between the ts Table . HoweverdsrA gene is the sole gene not clustered with others and it also does not group with any autotrophic carbon fixation pathway genes  of the sedimentary organic matter is plant-derived. Indeed, the ratio of cholesterol to β-sitosterol, being 0.54, 0.40 and 0.52 in the top layers of the white, brown and reference sediments, respectively, indicates that some organic matter in the marine sediments may be of higher plant origin  which could explain some of the more enriched C16:0 fatty acid δ13C values. However, microorganisms using the rTCA cycle may also produce fatty acids of similar isotopic composition.The sulphate reduction nes Fig. . The reagin Fig. . Cholestbay Fig. 40,41. SeZetaproterobacteria population similar to the one suggested for the Spathi Bay sediments by our study, proposes that chemoautotrophy may account for up to 65% of total organic carbon production in SHV fields compared to photosynthetic processes44. Relative importance of chemoautotrohy for primary production in a light exposed marine shallow hydrothermal system further revealed that chemoautotrophic processes were promoted by microorganisms containing anteiso-C15:0, C15:0, and iso-C16:0 fatty acids. Related bacteria-specific anteiso-C15:0 and iso-C15:0 fatty acids identified in the sediments at Spathi Bay show typical CBB cycle δ13C signatures of up to −22.6‰. The values fall within the range of our bulk sediment δ13Corg measurements, indicating that chemoautotrophs that produce the anteiso-C15:0 and C15:0 fatty acids, together with microbes with the more generic fatty acids that expressed δ13C values of −19.4 to −19.9‰, contribute the most to carbon fixation via the CBB cycle in the Spathi Bay sediments. Given the bulk range for δ13Corg in the sediments, we suggest that lineages that produce predominantly fatty acids with δ13C values lower than −20‰, contribute the least to sediment organic carbon production. Moreover, because seagrass is expected to enrich heavy 13C in plant fatty acids by a factor of two relative to the bulk sediment δ13C values recorded in this study45, we caution that while good preservation of plant organic matter is likely taking place in the sediment, organic matter contribution by plants to the total sediment production pool is likely negligible. The δ13C values of the ubiquitous C16:1 cis-9 fatty acids in the white– and brown–capped sediments, show a significant difference of 14‰. The diffuse CO2 emissions in the white and brown sediments alone insufficiently explain this disparity. The δ13C values of the C16:1 cis-9 fatty acids are more depleted in the brown-capped sediments than the reference sediment, which is expected to have the lowest CO2 concentration. The more likely explanation for this discrepancy is the influence of in situ carbon fixation by organisms using the rTCA cycle.A recent study from the SHV fields of Dominica Island that support a prominent In situ production of fresh organic compounds therefore, likely provides available substrates for the heterotrophic and chemotrophic microbial community, such as sulphate-reducers. The Spathi Bay sediments contain the genetic capacity to enable both autotrophy via the CBB and rTCA cycles and heterotrophy with sulphate-reducers, which are not grouped with any of the autotrophic targeted genes . This, in addition to the fatty acid C15:0 cbbM gene), is the main driver of CO2 fixation within the microbial community in the sediments. This seems to be especially true for the Zetaproteobacteria for which the specific 16S rRNA gene abundance coincides with the cbbM gene content. The qPCR data show that different microbial guilds are grouped into three clusters, each one encompassing one gene involved in CO2 fixation  with the exception of the sulphate-reducers. The sulphate-reducers are not associated with any other quantified genes, highlighting their role in the heterotrophic part of the carbon cycle. Sedimentary organic carbon appears not only to be derived from in situ production, but also from extraneous sources, for instance seagrass fields further away. Our study suggests that the microbially-mediated CBB cycle drives carbon fixation in the Spathi Bay sediments of the Milos SHV characterized by diffuse, high CO2 emissions and high As and reduced species exudation. This study highlights the breadth of conditions influencing the biogeochemistry of SHVs and the importance of coupling different methods of highly specific process indicators to elucidate the complexity of the carbon cycle in shallow CO2-rich hydrothermal systems.The data also indicate that hydrothermal active and non-active sediments of Spathi Bay form a complex system where the carbon cycle is driven by both biotic and abiotic reactions, as well as by autotrophic and heterotrophic metabolic activities. The CBB cycle, and especially RuBisCO form II . The sediment cores were sliced every 2 cm. Push core 1 collected in the brown-capped deposit was sliced every 4 cm using a disposable sterile plastic spatula and then stored on dry ice. Each section was divided into two: one for genetic survey and the other for corresponding geochemistry. These samples were stored in 50 ml Falcon tubes on dry ice, until subsequent storage at −80 °C in the laboratory. Porewater was extracted from the chemistry replicate cores using rhizones connected to depressurized exetainer® bottles. Exetainers for DIC concentration and isotopic analysis were stored upside down at 4 °C until analysis. After porewater collection, one push core per habitat was transferred into the glove bag and sliced aseptically every 2 cm for further bulk δ13C isotopic analysis. Samples were placed in 50 ml Falcon tubes and stored under N2 in an anoxic bag, frozen and shipped on dried ice. At the laboratory, they were frozen at −20 °C.Shallow water hydrothermal sediments were collected at Spathi Bay, 36°40′N; 24°31′E Fig. , southeasit Fig. . Local ssit Fig. . Large aλ = 1.5406 Å). Samples were run between 5–80° (2θ) in step sizes of 0.017° in continuous scanning mode while rotating samples. The top, middle and bottom portions of cores were used to define depth mineralogical-profiles. Samples were dried at 60 °C overnight and powdered in an agate mortar before analysis.Powder X-Ray Diffraction (PXRD), using a PANalytical X’pert diffractometer equipped with an X’celerator silicon-strip detector was used to analyze the mineralogical composition of sediments. The instrument was run at 45 kV and 40 mA using Ni-filtered Cu-Kα radiation  source. A confocal Olympus BX41 microscope was combined to the instrument. The laser beam, with a power at the sample surface at 8 mW, was focused through an 80x objective with a long-working distance of 8mm. The analysed spot size was around 1 µm. The spectral resolution was ~0.4 per cm/pixel and the accuracy of the instrument was controlled by repeated use of a silicon wafer calibration standard. The data collection and spectral baseline correction were done with the LabSpec 5 software.Raman spectroscopy was done with a confocal laser Raman spectrometer , equipped with a multichannel air-cooled charge-coupled device detector as previously described in Chi Fru Porewater concentrations of major and trace elements were measured using Inductively Coupled Plasma-Atomic Emission Spectrophotometry  at Stockholm University (details are given in SI methods).13CDIC (Dissolved Inorganic Carbon) and δ13Corg (organic carbon), were analysed respectively using a GasBench II coupled to a Delta V Plus mass spectrometer (ThermoFinnigan) following the set-up described in Torres et al.48 with the exception that a chilled autosampler was not used and a EA-IRMS . Details about the procedure are in the SI (SI methods).Carbon isotopic analysis: δZetaproteobacteria, cbbL, cbbM, aclB, pmoA, mmoX, mcrA and dsrA genes , following the manufacturer’s recommendations. Samples contained 5 µL of DNA (1 ng.µL−1), specific primer set at appropriate concentrations and annealing temperatures , two copies of the pMMO-encoding pmoA protein per cell52, one for the sulphate-reducing bacteria dsrA gene53. From the sparse data available for the number of specific genes of mcrA, mmoX, cbbL, cbbM, aclB per genome, especially in prokaryotes inhabiting the shallow hydrothermal system, together with the remaining genes, we assumed one copy per genome.DNA extraction was performed in duplicates from ~0.25 g of sediment, using the Mo Bio PowerSoil DNA kit  according to the manufacturer’s instructions. The 16S rRNA gene abundance of archaea, bacteria, es Table , were ques Table , in 35 ces Table . The qPC2 blowdown system. The lipid extracts were then re-dissolved and eluted over a glass pipette filled with activated Copper powder to remove sulphur and subsequently split into 3 fractions over a silica column using hexane, hexane/DCM (1:1 v/v) and DCM/MeOH (1:1 v/v). The last eluting fraction was methylated using the method of Ichihara et al.54 and silylated by adding 20 µl of pyridine and 20 µl of BSTFA  and placed in an oven at 60 °C for 25 min.Sediment samples were frozen and freeze dried and then ultrasonically extracted using a mixture of dichloromethane and methanol , repeated 5 times. Extracts were combined and gently evaporated to dryness using a N−1 followed by a ramp of 4 °C min−1 until 320 °C where it was held for 20 min. The total run time was 60 minutes. MS operating conditions were an ion source temperature of 200 °C and 70 eV ionization energy55. All peaks were background subtracted and identified by comparing to commercially available or published mass spectra.Lipid extracts were analysed using a Shimadzu GCMS-QP2010 Ultra gas chromatography mass spectrometer (GC/MS) with an AOC- 20i auto sampler. Samples were injected in splitless mode onto a Zebron ZB-5HT Inferno GC column (30 m × 0.25 mm × 0.25 μm) using helium as carrier gas. The GC oven temperature was programmed to increase from 60 °C to 180 °C at a ramp of 20 °C min13C values using gas chromatography isotope ratio mass spectrometry (GC-IRMS) using a Thermo Delta V Plus mass spectrometer connected to a Trace Ultra GC, GC Isolink II and Conflo IV. Helium was used as the carrier gas and lipid extracts were injected in PTV mode onto a Zebron ZB-5HT Inferno GC column (30 m × 0.25 mm × 0.25 μm)56. The GC oven had an initial temperature of 100 °C then ramping at 20 °C min−1 up to 250 °C and 5 °C min−1 up to 340 °C, with a hold time of 18 min. Individual compounds were identified by comparing GC-IRMS chromatograms with corresponding GC/MS chromatograms.Lipid samples were analysed for δ57 using varimax (variance maximizing) with Kaiser Normalization58, was used to quantitatively unravel variability in gene content, between and within the habitats. Factor analysis plot was generated to describe the relationships between genes quantified by qPCR irrespective of the habitat. The 3D factor analysis was made to describe the relationships between each push core and genes quantified by qPCR. Three MANOVA  tested the relationship of the gene quantification between three independent variables: 1) between replicate push cores, 2) habitat, i.e., white- or brown-capped sediments and reference sediments and 3) depth. Multivariate tests of significance were run together with a Tukey’s post hoc tests. The results for which the P values are below 0.05 were considered statistically significant. Statistical analyses were performed in SPSS59. Principal Component Analysis (PCA) were made using the Raman data in order to find any correlation between habitat and depth. The PCA was performed using XLSTAT60, a statistic tool appended to Microsoft Excel.Factor analysisThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary information