Micromagnetic simulations with PMA and different second order anisotropies at the opposite Fe/MgO interfaces qualitatively reproduce the observed broken symmetry spin reorientation transition. Our findings open the possibilities to develop multistate epitaxial spintronics based on competing magnetic anisotropies.The observation of perpendicular magnetic anisotropy (PMA) at MgO/Fe interfaces boosted the development of spintronic devices based on ultrathin ferromagnetic layers. Yet, magnetization reversal in the standard magnetic tunnel junctions (MTJs) with competing PMA and in-plane anisotropies remains unclear. Here we report on the field induced nonvolatile broken symmetry magnetization reorientation transition from the in-plane to the perpendicular (out of plane) state at temperatures below 50 K. The samples were 10 nm thick Fe in MgO/Fe(100)/MgO as stacking components of V/MgO/Fe/MgO/Fe/Co double barrier MTJs with an area of 20 × 20  The bottleneck to have PMA is the control of the magnetic anisotropy which is characterized by an effective anisotropy constant (Keff) that has a volume contribution Kv and two surface or interface contributions Ks4. As a result, it can be described as t is the ferromagnetic layer thickness. The volume contributions arise from magnetocrystalline anisotropy, magnetoelastic anisotropy and shape anisotropy. The latter contribution induces commonly an in-plane magnetized configuration in magnetic thin films. However, in ultrathin layers several angstroms thick, the surface contribution to anisotropy can exceed the volume shape anisotropy contribution leading to the PMA. Surface contributions are linked to roughness and interface alloy, strain and mainly to the broken symmetry at the interface or at the surface of the magnetic layers. The constant Ks1 of the first order surface anisotropy energy per unit area may range between Ks1 ≈ 1 × 10−3 J/m2 in ultrathin Co, Fe and Ni films4 and Ks1 ≈ 3 − 4 × 10−3 J/m2 at CoFeB/Pt interfaces5.Magnetic films with the magnetization oriented perpendicular to the film plane are currently the best candidates for magnetic storage devices with respect to the challenge to decrease the bit size. The phenomenon of having a preferential magnetization perpendicular to film plane is usually referred to as Perpendicular Magnetic Anisotropy (PMA)6, low spin torque switching currents8 and record small lateral sizes9. Those features are critical for the progress towards spin transfer torque based magnetic random access memories. It appears that MgO/Fe interfaces show PMA substantially exceeding the values reported for the prototype Co/Ni(111) system10.The recent trends in spintronics using MTJs also take advantage of PMA to provide large tunneling magnetoresistance (TMR), enhanced thermal stabilityd-orbitals and O p-orbitals has been suggested as a main source of PMA at MgO/Fe interfaces12. First-principles calculations give Ks1 ranging between 1.5 × 10−3 J/m2 and 1.8 × 10−3 J/m2 12 in a reasonable agreement with experiments14. According to its interfacial nature, the PMA intensity varies strongly with the magnetic layer thickness. This means a great challenge to increase the critical thickness of the spin reorientation transition6 above a few nm, without the need of the permanent application of an external magnetic field stimulus15. By changing the normal metal (NM) in NM/Fe/MgO 14, the transition between out of plane and in-plane anisotropy remains between 4 and 6 Fe monolayers. Reducing the bulk magnetization through bulk Fe doping by V or Cr impurities reduces the easy-plane demagnetizing energy and slightly changes the critical thickness at which reorientation occurs16. Finally, decreasing the temperature down to 5 K, leads to the PMA and saturated magnetization enhancement. This has a rather limited impact on the critical thickness for the spontaneous out of plane magnetization alignment18.The spin-orbit interaction (SOI) emerging from the reduced interfacial symmetry of the Fe 20. According to our discussion, due to the shape anisotropy, such soft FM layers remain in-plane magnetized at room temperature and zero magnetic field. However, a ferromagnetic resonance analysis pointed out the presence of a PMA contribution to the total anisotropy21 even for in-plane magnetized FM electrodes. As a result sketched in Fig. 22. Therefore, for a film thickness below some critical value  the out of plane magnetic state becomes the ground state as sketched in Fig. Seminal MTJs grown by Molecular Beam Epitaxy (MBE) have in-plane anisotropy and typically incorporate about 10 nm thick Fe or FeCo soft ferromagnetic layers (FM) separated by MgO barriers from the magnetically hard layersThe main concept of the ferromagnetic system with competing in-plane and out of plane anisotropies is sketched in Fig. Sample growth and measurement techniques are explained in the Methods section. Figure Hext = 0) to the out of plane direction at about Hext = −1 kOe. In the range of the explored perpendicular magnetic fields (<3 kOe) and low temperatures (T < 50 K) this magnetization switch (from in-plane to out of plane direction and backwards) takes place only in the negative field direction as shown in Fig. Our main experimental finding has been observed at low temperatures below 50 K and is represented in Fig. For the experiments with out of plain field one unavoidably has some small (in our case ≤3 degrees) deviation of the applied field from a strictly perpendicular direction. We cannot therefore exclude an in-plane component of the external field to be present during such experiments. We have tried to verify a possible explanation of the transition as due to in-plane magnetization rotation induced by the above mentioned misalignment . With this aim we have studied the tunneling resistance under a full in-plane magnetic field rotation of the soft layer magnetization keeping the hard layer fixed. The experimental results  and a MTJ in series through the following expression:G1 is the low bias conductance of the V/MgO/Fe junction, G2 is the low bias conductance of the MTJs in the perpendicular state, p is the effective spin polarization and ϑ the angle between the magnetizations of the ferromagnets. From the room temperature out of plane and in-plane resistance vs. field measurements shown in Fig. ϑ = 0), anti-parallel (ϑ = π) and perpendicular (ϑ = π/2) states. The knowledge of those conductances provides p = 0.65 at 300 K and p = 0.68 at 10 K.The resistance values in three well defined magnetic states of the soft layer allow an evaluation of the effective spin polarization of the FM electrodes at corresponding temperatures. Following a simplified modelp values underestimate the effective spin polarization because they were obtained without consideration of the additional shunting effect coming from the coherent tunneling between V and top Fe across the two MgO barriers. This effect is particularly important for majority spins whose coherence length in Fe is known to be larger than 10 nm (see Ref.24). The above mentioned effect enhances the AP conductivity and reduces the expected large TMR. Moreover, we mention that the evaluated G1,2 conductances increase with temperature proving high quality and pinhole free barriers.The estimated decrease of spin polarization with increasing temperature could be attributed to thermally excited magnons. We also note that the obtained 24 also observed indirectly an accumulated disorder in the epitaxial double barrier magnetic tunnel junctions through a substantial difference in the two MgO barrier transparencies with the same nominal thickness.On the other hand, we find the second MgO barrier (between the 10 nm Fe and the Fe/Co hard electrode) to be about 4 times more transparent than the bottom MgO layer (i.e. Ks2 anisotropy) with competing anisotropies under applied perpendicular magnetic field. A perpendicular magnetization anisotropy has been introduced using two qualitatively different ways sketched in Fig. M1) uses a single step PMA variation within the first atomic layer. The concentration of Ks1 in the first layer only corresponds to the vacuum/Fe/vacuum case discussed in ref.12. The second (model M2) approach involves three slightly different versions labeled (M2i–iii) which allow the different PMA variation in steps of a half lattice period a/2 (an Fe atomic layer). The model M2i with an oscillatory decay of PMA inside Fe is the closest to the numerical predictions for the MgO/Fe/MgO case12.The details on simulations are explained in the Methods section. We first discuss the models used to simulate the magnetization reversal of the symmetric soft Fe layer  and 30% for the second one. The third layer, following DFT results12, is assigned an uniaxial in-plane anisotropy of roughly 10% in volume energy of the first layer of Fe atoms. Finally 5% of Ks1 is assigned to the 4th layer. Generally, those percentages approximately follow the DFT results12 concentrating Ks1 within the first four atomic Fe layers and approximating the surface anisotropy variation with the predicted Friedel-like decay oscillating between PMA and in-plane magnetic anisotropy (IMA). Other versions of the M2 model (M2ii and M2iii) modify the Friedel-type PMA decay towards a more monotonous PMA variation. Below we describe our main experimental findings and compare the results with simulations within the above stated approaches M1 and M2.The model Using the above introduced models we have carried out zero temperature micromagnetic simulations of the MgO/Fe/MgO structure shown in Fig. M1 and M2 involving three different PMA energy distributions keeping the total PMA energy fixed. The introduction of the Friedel-like PMA variation12 (model M2i) softens the reorientation transition with respect to the M2ii–iii models. Although the M1 PMA distribution also softens the reorientation transition, it reduces the non-volatility of the zero field magnetic state, in contrast to the experiment.Figure Ks1 = 5 × 10−3 J/m2. On the one side, to reproduce the observed magnetization flip between the near in-plane and the near out of plane magnetizations, modeling has to use Ks1 values not exceeding approximately 6.4 × 10−3 J/m2. These PMA values are somewhat bigger than those provided by DFT12 calculations. For the surface anisotropy exceeding 6.4 × 10−3 J/m2 a reorientation transition tends to take place directly between two nearly perpendicular magnetization states. This means without locking the magnetization in the intermediate, close to in-plane state. For confirmation see inset of Fig. The inset to Fig. Our experimental results point towards a relatively weak coupling between the MgO/Fe/MgO layer and the hard Fe/Co electrode. Indeed, simulations show that the in-plane to out of plane spin reorientation transition could be affected by either ferromagnetic or antiferromagnetic coupling between free and sensing electrodes  layers could be a specific feature of ferromagnetic films with competing PMA and IMA. That is to say with thickness few times exceeding the critical one where a perpendicular magnetic state becomes the ground state. We used a hard ferromagnetic layer to sense the effect. Nevertheless one may anticipate that a reorientation transition could be also detected by measuring the anisotropic magnetoresistance at sufficiently low temperatures through the application of magnetic fields along two different (in-plane or out of plane) directions and with the current directed within the film plane.The observed reorientation transition shows two remarkable properties at low temperature: non-volatility and an asymmetric magnetization response to the out of plane external field. Simulations taking into account PMA have reproduced the experimentally observed non-volatility. However the presence of the magnetization asymmetry was not reproduced in the case of symmetric MgO/Fe/MgO structures. The magnetization asymmetry points towards some net field acting perpendicularly to the interface direction. This breaking symmetry could originate from differences in the crystalline disorder at the two MgO/Fe interfaces and will be discussed separately below.Despite the general qualitative agreement between experiment and simulations, a certain number of open questions remain yet to be answered. Our simulations show that the magnetization flip occurs at fields around 1–2 kOe with the magnetization after the flip being not fully perpendicular to the interface Fig. . The swi25, because the latest numerical studies minimize the effect of Fe-O p-d hybridization on the PMA26.Several factors, not present in the simulations, could be responsible for such differences. Among them are the (i) presence of defects, (ii) a modified interfacial saturation magnetization and/or (iii) the presence of finite temperature in the experiments, among others. Some reduction of the lateral size of the simulated structure affects competing anisotropies leading to the suppression of the robustness in the simulation results. Our discussion is centered mainly on physical effects and omits the possible influence of chemical bonding on PMA27 well beyond those simple three relative magnetization states observed and discussed above for the uncoupled MgO/Fe/MgO.Let us focus on the possible influence of the electrodes coupling on the results. If the hard FM layer was magnetostatically and/or weakly exchange coupled to the soft Fe layer under study, this would give rise to a more complex magnetization reversal  needed for the spin reorientation transition approach towards those obtained by DFT, once we reduced the saturation magnetization at both interfaces  × 10−3 J/m2 to (5–6) × 10−3 J/m2 at the MgO/FeCo interface. Moreover, the PMA for the individual Fe atoms deposited on MgO(100) thin films could increase Ks1 in the same order of magnitude33. So far the PMA has been investigated in about 1–2 nm thick ferromagnetic layers because only ultrathin magnetically soft layers provided conditions for the room temperature operation of MTJs with PMA34.An additional discrepancy in the PMA values could be the lattice mismatch (stress) providing interfacial electric fields30 which should be different at two interfaces with different structural disorder. From the other side, we have already discussed that from the crystallographic point of view, the quality of the bottom and top MgO barrier should not be similar. The reason is that when growing MgO on bottom V(001), the V-(001)-MgO(001) lattice mismatch is 2%, smaller than the Fe(001)-MgO(001) mismatch (4%) implicated when growing the top MgO barrier on middle Fe. As a consequence, the plastic relaxation limit thickness of MgO will be smaller when grown on Fe than on V35. Both barriers have nominal 2 nm thickness, well below the relaxation limit, so dislocations clearly occur but the dislocation density in the top barrier, grown on Fe, will be larger. This has direct consequences on symmetry filtering, the defects promoting additional tunneling channels and therefore increasing the top barrier transparency.Below we discuss in more detail the possible origin of the observed perpendicular magnetization switching asymmetry. The two possible scenarios sketched in Fig. Ks2 could show up differently at two distinctly disordered interfaces. Already back in 1994 Dieny and Vedyayev showed analytically36 that spatial fluctuations of the film thickness with Ks1 being constant, and period of the fluctuations lower than the exchange length of the ferromagnet may lead to a higher Ks2 cos4 (Θ) contribution to the PMA in addition to the Ks1 cos2 (Θ) term. Here Θ is the angle between magnetization and perpendicular to the interface axis. Such possibility has been corroborated by a recent report28 showing that magnetization reversal could be substantially modified for opposite Ks1 and Ks2 signs.As a consequence, a higher (e.g. second) order interfacial uniaxial anisotropy Ks2 surface anisotropy term on the magnetization reversal. Figure Ks1 and Ks2 have opposite signs. The Ks2 anisotropy has been introduced only at the upper Fe/MgO interface, more disordered by the growth history. For the negative Ks2 values exceeding few times Ks1, we observed a strong asymmetry in the hysteresis cycle magnetization switch from the near in-plane to the near out of plane direction which resembles the experiment.Within these lines, we have carried out detailed simulations in order to verify the possible influence of the Hcp and the second jump (negative in-plane to out of plane) coercive field Hcn vary with increasing Ks2/Ks1 ratio. The last (maximum negative) switching field value is taken as just exceeding the maximum negative applied field. This is because the in-plane/out of plane transition does not take place in that case (within the field range used) and magnetization just returns along the same trajectory, representing a strong asymmetry in field magnetization response. This is qualitatively similar to the one observed experimentally. Although the asymmetry in spin reorientation transition appears only for Ks2/Ks1 > 5, the critical condition for the relation between the corresponding energy contributions is around 0.2–0.4. This is because the reorientation transition takes place at angles about Θ ≈ 1.37 rad where the relation between angular dependent factors is Ks2 along the four (instead of the single) interfacial cells does not alter substantially the simulation results.Figure Eper in addition to the well-established perpendicular interfacial electric field Eper. Since the Rashba field BR is proportional to the cross product of electron momentum k and electric field E, and these depend on the local interface disorder, each of the Fe/MgO interfaces could generate locally different Rashba field components perpendicular to the interface BRz. Indeed, in the real growth conditions, the interfacial defects concentrations and their type should be different for the bottom (MgO/Fe) and the upper (Fe/MgO) interfaces. One therefore could envisage on average a difference in the perpendicular components of the corresponding Rashba fields as sketched in the left part of Fig. One could also speculate on the above mentioned perpendicular component of the Rashba field induced by the oxygen defects inside the MgO and/or lattice mismatch at the Fe/MgO interface. Both factors could give rise to a local in-plane component of the electric field 31, could be a key property in the design and fabrication of new types of spintronic and superconducting spintronic devices with multilevel characteristics. The effects we observed in micron size magnetic tunnel junctions are a consequence of competing in-plane and out-of-plane anisotropies. Therefore, probing similar magnetization spin reorientation experiments on large (millimeter size) unpatterned films would require tunning of the Fe layer thickness in order to find other conditions for the competing in-plane and out-of-plane anisotropies. Once such conditions are found for the extended films, using a vector magnetometer allowing preparatory in-plane saturation of magnetization would benefit, in our view, clear observation of the out of plane spin reorientation transition.An abrupt magnetic field induced transition between in-plane and out of plane magnetization states has been observed and investigated experimentally through magnetoresistance and by simulations in MgO/Fe/MgO layers with competing anisotropies. At sufficiently low temperatures (below 50 K), the out of plane magnetization state becomes asymmetric in field and non-volatile, most probably due to the difference in disorder between the two Fe interfaces. The presence of three different remanent magnetic states in V/MgO/Fe/MgO/Fe/Co MTJs, potentially controlled by the electric fieldin-situ RHEED and ex-situ AFM experiments. Moreover, after the growth of the first barrier it has been annealed for atomic-level flattening, as proved by in-situ RHEED. This insures model quality MgO/Fe/MgO interfaces. The MTJs were patterned by UV photolithography and Ar etching to an area of 20 × 20 μm2. The magnetic state of the Fe layer has been probed through TMR measurements, as it is interfaced via two MgO barriers by a magnetically hard (10 nm thick Fe and 20 nm thick Co) layer on one side and by a 40 nm thick normal metal Vanadium layer on the other. The full layer sequence is V(40 nm)/MgO(2 nm)/Fe(10 nm)/MgO(2 nm)/Fe(10 nm)/Co(20 nm), represented in Fig. −11 mbar). Details of the sample growth can be found in ref.20. The DC resistive measurements have been carried out at low bias (5 mV) at temperatures down to 5 K  using a JANIS He3 cryostat equipped with a home-made superconducting vector magnet. The room temperature DC resistive measurements have been carried out in zero bias limit in a less shielded system which accounts for the apparently higher measurement noise in that case. Details of the main low temperature experimental setup were published previously24.The basic system under investigation, MgO/Fe(10 nm)/MgO, represents a 10 nm thick Fe(100) soft layer interfaced by two 2 nm thick MgO(100) layers. The Fe layer between the two MgO barriers is continuous, as checked by 37. The left part of the sketch shown in Fig. Msat = 1700 × 103 A/m, exchange stiffness Aexch = 21 × 10−12 J/m, damping α = 0.02 and cubic anisotropy Kc = 4.8 × 104 J/m3. Interfacial layers with PMA include the surface anisotropy first order term Ks1 and (separately discussed) a second order term Ks2 anisotropy. The results have been confirmed to be independent on whether the MgO cells were represented as vacuum or as a weakly (10−7) diamagnetic material. The parameters used for Cobalt are: Msat = 1400 × 103 A/m, Aexch = 30 × 10−12 J/m and α = 0.02. To approach the extensive in-plane dimension (in comparison with the thickness), the in-plane size of the simulated sample is set to 50 × 50 nm2 with periodic boundary conditions. The space was discretized in 16 × 16 × 147 cells. This discretization has been chosen because it allows us to introduce in the micromagnetic problem the different models of interface anisotropies.The magnetic field dependence of the magnetization has been simulated at T = 0 K by using MuMax3 codeSupplementary material


HTRA1 rs11200638 and ARMS2 rs10490924 gene polymorphisms with response to intravitreal ranibizumab therapy among neovascular AMD (nAMD) subjects in Malaysia was determined in this study, followed by the expression of HTRA1 and ARMS2 genes.The association of HTRA1 rs11200638 at P = 0.018  and ARMS2 rs10490924 at P < 0.001 . An association was also observed in response to ranibizumab for both SNPs in a logistic regression analysis (P < 0.001). The mRNA levels in the HTRA1 variant between responder and non-responder groups were significantly different for the homozygous non-risk GG genotype (P = 0.032).Both single nucleotide polymorphisms (SNPs) recorded a significant association between nAMD and controls with HTRA1 rs11200638 and ARMS2 rs10490924 gene polymorphisms are associated with nAMD among Malaysians. Both gene polymorphisms were also correlated with response to intravitreal ranibizumab therapy based on visual and anatomical outcomes especially the HTRA1 rs11200638 variant.The  Age-related maculopathy susceptibility 2 (ARMS2 or LOC387715) and high-temperature requirement A serine peptidase 1 (HTRA1) genes are among the susceptible genes that are well studied in most populations with contradictory findings [Age-related macular degeneration (AMD) is a complex multifactorial disease and a common cause of visual impairment among elderly populations worldwide . The neofindings –8.ARMS2 and HTRA1 genes are located nearby on the 10q26 chromosome in a strong linkage disequilibrium which could be the main reason for these inconsistent results as most genetic association studies have insufficient data on distinguishing these two genes with nAMD. The rs11200638 polymorphism at the promoter region of HTRA1 was suggested to be related to overexpression of the gene among nAMD patients with the minor A allele, which was related to a significantly lower expression of HTRA1 mRNA compared with the major G allele [HTRA1 rs11200638 and ARMS2 rs10490924 mRNA levels among AMD [The G allele , 10. HowHTRA1 encodes a heat shock serine protease and regulates the transforming growth factor-β (TGF-β) which is involved in regulating angiogenesis and extracellular matrix deposition; thus, the overexpression of HTRA1 among nAMD was suggested to be due to the inhibition of TGF-β [ARMS2 gene was shown to encode a function in the mitochondrial outer membrane of the retina; therefore, the rs10490924 polymorphism in the exon 1 region of the ARMS2 gene, which causes a change of alanine to serine amino acid (A69S), may be a genetic factor leading to nAMD by affecting the function of the retinal mitochondria [of TGF-β . The ARMchondria .VEGF) is also involved in nAMD development due to formation of angiogenesis and vascular permeability that results in fluid leakage across the blood vessels [VEGF with an intravitreal injection of anti-VEGF agents such as bevacizumab [CFH, VEGFA, ARMS2 and HTRA1 genes might be involved in this heterogeneous response [Vascular endothelial growth factor . Male subjects were recorded with significantly greater frequency (P = 0.019) in both nAMD (59.3%) and controls (72.4%) compared to female subjects in both groups.The subjects’ socio-demographic characteristics are presented in Table P = 0.002) whereby nAMD had a higher frequency of hypertensive subjects (62.1%) compared to controls (44.1%). Smoking was also observed to be significantly associated with nAMD compared to controls (P < 0.001).A comparison between ethnicities revealed that Chinese subjects were highest in the nAMD group (60.7%); however, among the controls, Malay subjects were reported highest (41.4%). The co-morbidities that reported a significant difference between nAMD and control was hypertension  except for the nAMD group in HTRA1 rs11200638 (P < 0.05). Both gene polymorphisms showed a significant difference for genotypes and alleles when compared between the two subject groups with a recorded OR = 2.44, CI = 1.75–3.42 (P < 0.001) for the ARMS2 rs10490924 polymorphism and OR = 1.52, CI = 1.07–2.15 (P = 0.018) for the HTRA1 rs11200638 polymorphism , suggesting the nearly identical association test and odd ratios.Genotyping was performed for both ARMS2 rs10490924 and HTRA1 rs11200638 showed a significantly worse mean BCVA and mean CRT at 6 months post-treatment with mean BCVA 0.62 ± 0.28 logMAR (P = 0.046) and 0.61 ± 0.29 logMAR (P = 0.044), respectively, and mean CRT 400.1 ± 127.90 μM (P < 0.001) and 387.5 ± 129.89 μM (P = 0.008), respectively. These findings were supported by the mean changes of BCVA and CRT from the baseline, which also reported significantly worse changes (P < 0.05) at 6 months post-treatment for both homozygous risk genotypes in both polymorphisms. In addition, male nAMD subjects were significantly frequent carrying the risk genotype compared to females for both ARMS2 rs10490924  and HTRA1 rs11200638 .Further genetic association analysis was performed between the genotypes of both gene polymorphism with BCVA and CRT of nAMD patients at baseline and 3 and 6 months post-treatment with intravitreal ranibizumab therapy (Table P = 0.001) and worse mean CRT  at baseline compared to non-responders. However, at 6 months post-treatment, non-responders recorded a worse mean BCVA  and worse mean CRT  compared to responders. The mean changes in both BCVA and CRT at 3 and 6 months post-treatment from baseline among responders and non-responders were also significantly different (P < 0.05). No significant differences (P > 0.05) were observed between age and smoking status between the two groups . For HTRA1 rs11200638, the mean BCVA at 3 months post-treatment recorded a significantly worse BCVA among non-responders which carried the non-risk genotype . A significant association was also observed in a regression analysis between the two response groups and genotypes for each polymorphism with the ARMS2 rs10490924 using a co-dominant model (P < 0.001) and HTRA1 rs11200638 using a recessive model (P < 0.001)  Table .Table 5VHTRA1 gene showed a significant difference between non-responders and responders for the homozygous non-risk GG genotype with the responders having a 15-fold higher expression of the HTRA1 gene compared to non-responders (P = 0.032). No significant difference was observed for the ARMS2 expression levels between the two response groups  and functional (BCVA) aspects in correlation with  studies .ARMS2 rs10490924 and HTRA1 rs11200638 polymorphisms, suggesting that the variable response to treatment might be affected by changes in the 10q26 chromosome biomarkers [ARMS2 rs10490924 and HTRA1 rs11200638 polymorphism was associated with a poor response to ranibizumab based on changes in BCVA [HTRA1 rs11200638 alleles in the present study were not consistent with HWE among the nAMD groups which reasons could be due to HTRA1 rs11200638 as being strongly associated with risk of nAMD among Asian populations [A significant association was reported after a logistic regression analysis in the present study between the two treatment response groups and the omarkers , 23. The in BCVA , 37–39 a in BCVA . Howeverulations . The nAMARMS2 rs10490924 TT genotype (P = 0.009) compared to non-responders with the normal genotype that have a smaller mean change in CRT. This suggests that subjects carrying the homozygous risk ARMS2 rs10490924 TT genotype are more likely to not respond to ranibizumab treatment compared to those with normal genotypes. These findings are consistent with previous studies such as those in Korea [ARMS2 rs10490924 with ranibizumab response was not observed [CFH rs1061170 and ARMS2 rs10490924 in Europeans did not generalise to the non-European populations [CFH, C2 and complement factor B were contradictory with findings in India. Furthermore, the variants in ARMS2 and HTRA1 genes were found to be associated with increased risk of AMD in the INDEYE study, but not in the Western populations, which further suggests the effect of ethnic differences in AMD polymorphism studies [Higher mean changes in CRT was observed at 6 months post-treatment among non-responders with homozygous risk in Korea , Australin Korea  and Polain Korea . Howeverobserved , 23, eveobserved  and CATTobserved . The undulations . Finding studies .HTRA1 rs11200638 genotypes also showed a significant difference, but only on mean BCVA at 3 months post-treatment with normal GG genotype (0.90 ± 0.20 logMAR) having the worse mean BCVA compared to risk AA genotype . This contradicts the ARMS2 rs10490924 findings, as the non-responders with the worst visual acuity were among the subjects carrying the risk genotype of ARMS2 rs10490924. This could be because the mean baseline BCVA was initially worse among subjects with HTRA1 rs11200638 GG genotypes. However, the risk AA genotypes reported a higher loss of BCVA from the baseline compared to the normal GG genotype which did not lose further vision at 6 months post-treatment.Similarly, the non-responder group between It is suggested that the response to treatment could also be affected by the baseline vision, due to a phenomenon known as the ceiling effect, whereby subjects with a worse baseline BCVA were less likely to lose further vision and subjects with better BCVA were less likely to improve , 45. ThiARMS2 and HTRA1 genes, it remains unclear which variants are at higher risk of nAMD pathogenesis; thus, the correlation of ARMS2 and HTRA1 variants to ranibizumab treatment response remains unclear. However, studies are suggesting that the risk allele might lead to changes in transcriptional activity and, thus, affect the proteins normal activity resulting in a differing response to the ranibizumab treatment [Due to the strong linkage disequilibrium among the reatment .ARMS2 rs10490924 and HTRA1 rs11200638 polymorphisms in response to ranibizumab and the strong linkage disequilibrium among the two variants, we further investigated the mRNA levels of both variants among the two treatment response groups. A significant association was only observed in the mRNA expression of HTRA1 rs11200638 GG genotype with the non-responders having the highest HTRA1 expression compared to the responders. The high expression of HTRA1 GG genotype was also reported by Yang et al. [HTRA1. The overexpression of HTRA1 has been related to the alteration of Bruch’s membrane, resulting in the invasion of the extracellular matrix with choroid capillaries and inhibition of the angiogenesis and extracellular matrix deposition regulator (TGF-β) [HTRA1 genes resulting to the poor response to the ranibizumab treatment.In the present study, since we observed significant associations between ARMS2 and HTRA1 genes due to the high risk of rs10490924 and rs11200638 polymorphisms with development of nAMD and their significant association with response to ranibizumab therapy such as reported in China [The present study focused on a targeted SNP analysis of in China , Korea [in China  and Spaiin China . The SNPin China  and Tabain China  in the sARMS2 and HTRA1 genes which do not exclude the possibility of other variants in the two genes or in other high-risk genes  or even a combined effect of the polymorphisms that might play a role in the heterogenous response to ranibizumab therapy. Future studies could also sequence other rare variants in the high-risk genes and this might provide a more depth information on pharmacogenetic associations of nAMD.Despite the findings reported in this study, there are limitations that should be taken into consideration. The study design itself is a limitation as this is an unmatched comparative case-control study. A matched case-control study is advantageous in controlling cofounders that could alter the exposure and outcome relationship in an analysis. However, the present study with an unmatched case-control could better examine the impact of the cofounders and further provide evidence on significant risk factors related to nAMD. The lack of CNV subtypes is one of the major limitations as performing analysis based on the CNV subtypes  could provide more specific information on which CNV is related with the different response to ranibizumab. A short follow-up period of 6 months post-treatment is also a limitation due to time constraint in the present study. Although the small sample size is a significant sample size for a two-centre study in a Malaysian population, future studies could be conducted in a larger sample size in multiple centres around Malaysia to confirm the pharmacogenetic findings. Furthermore, the variable criteria in grouping the responders/good response and non-responders/poor response to the ranibizumab treatment could also be one of the factors affecting the pharmacogenetic results, as there are contradictory findings in previous studies. The present study only focused on two variants of ARMS2 rs10490924 and HTRA1 rs11200638 polymorphisms in response to the ranibizumab treatment among nAMD subjects. An overexpression of mRNA in the HTRA1 GG genotype was also reported and could contribute to the non-responders’ reaction to ranibizumab. Despite the reported data, future studies are needed to confirm the findings with a larger sample size and more stringent criteria which accounts for the limitations of the present study.A significant association was observed between This study was approved by Universiti Putra Malaysia (UPM), Universiti Kebangsaan Malaysia (UKM) ethics committee and Medical Research Ethics Committee (MREC), Ministry of Health, Malaysia (MOH). This study was registered with the National Medical Research Registry with the registration number NMRR-14-1176-21475.A prospective cohort study was conducted from September 2014 to February 2016 in ophthalmology clinics at two tertiary centres, Universiti Kebangsaan Malaysia Medical Centre (UKMMC) and Hospital Selayang. A total of 145 nAMD subjects were selected as case subjects from a total of 158 nAMD subjects, as the remaining 13 subjects failed at follow-up. Informed consent was obtained from all the subjects prior to starting the study.All subjects underwent comprehensive ophthalmologic examinations, including BCVA test by Snellen chart, fluorescein angiography (FA) and central retinal thickness analysis using optical coherence tomography (OCT). The subjects were then diagnosed as nAMD by ophthalmologists at each centre based on the clinical examinations performed. None of the subjects were previously treated for nAMD.All subjects were aged above 50 years old with confirmed presence of exudation involving macula and a follow-up appointment of 6 months after the first injection of ranibizumab . They were eligible for this study. Subjects who do not fulfil the mentioned inclusion criteria and had a CNV secondary to other causes, such as inflammatory diseases, trauma, hereditary diseases, pathologic myopia, angioid streaks, other retinal diseases other than AMD and eyes previously submitted to posterior vitrectomy were excluded from this study.Control subjects with no previous history of nAMD, absence of other retinal disorders except mild cataract, no visual impairment and no family history of nAMD were recruited in both centres during routine ophthalmic examination. Subjects with severe cataracts were excluded from control subjects.Socio-demographic factors, medical history, smoking history and the complications of nAMD were retrieved from the medical records of the subjects. Each nAMD subject received 0.5 mg/0.05 ml ranibizumab per intravitreal injection following the treat and extend regimen and was followed up for 6 months. The BCVA and central retinal thickness (CRT) at baseline and 6 months were compared and evaluated with regard to the treatment. Those with an improvement of three lines or greater in the Snellen chart and/or a resolution of more than 100 μM decrease in CRT at 6 months post-treatment compared to baseline were regarded as responders, while the non-responders had no improvements.HTRA1 rs11200638 and ARMS2 rs10490924 gene polymorphisms were among the significantly associated variants with response to ranibizumab therapy in nAMD and, thus, were selected as a candidate SNP in the present study. Furthermore, the SNP was selected based on the functional significance, location and minor allele frequency of more than 10%. HTRA1 rs11200638 and ARMS2 rs10490924 have a minor allele frequency of 0.241 and 0.255, respectively, as reported by the National Center of Biotechnology Information (NCBI).Based on the obtained literature reviews, HTRA1 rs11200638 and ARMS2 rs10490924 gene polymorphisms was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR amplification was performed in a thermocycler  with a total volume of 25 μl for each polymorphism, including the following primers: forward: 5′-ATGCCACCCACAACAACTTT-3′ and reverse: 5′-CGCGTCCTTCAAACTAATGG-3′ for HTRA1 rs11200638 polymorphism and forward: 5′-TACCCAGGACCGATGGTAAC-3′ and reverse: 5′- GAGGAAGGCTGAATTGCCTA-3′ for ARMS2 rs10490924 polymorphism. Genomic DNA amplification was performed for 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C (HTRA1 rs11200638), 52 °C (ARMS2 rs10490924) for 30 s and extension at 72 °C for 45 s, followed by the final extension at 72 °C for 5 min.Peripheral blood was collected from all the subjects and the genomic DNA was extracted using a QIAamp DNA blood mini kit  and stored at − 20 °C for further analysis. The genotyping of the HTRA1 rs11200638 (385-bp fragment) and ARMS2 rs10490924 (449-bp fragment) was then digested with the EagI (rs11200638) and PvuII (rs10490924) restriction enzymes  at 37 °C for 3 h. The digested products were separated on 2% agarose gels and visualised using the UV alpha imager . Random samples were chosen to validate the results, and identical results were obtained.The amplified product of HTRA1 and ARMS2 genes. Total RNA was isolated from whole blood samples using the Tempus Spin RNA Isolation Reagent Kit . The cDNA synthesis was then conducted using the QuantiTect Reverse Transcription Kit . Following the synthesis of cDNA, quantitative real-time PCR was performed with the QuantiNova SYBR Green PCR Kit  using the MiniOpticon Real-time PCR System (Bio-rad Hungary Ltd) to evaluate the level of gene expression for the HTRA1 and ARMS2 genes. The primers for HTRA1 expression assay were forward: 5′-CGGAAGATGGACTGATCGTGAC-3 and reverse: 5′-GGTGATGGCTTTTCCTTTGGC-3′ and the primers for ARMS2 expression assay were forward: 5′-GATGGCAAGTCTGTCCTCCT-3′ and reverse: 5′-TTGCTGCAGTGTGGATGATAG-3′. The housekeeping gene, β-actin, was used as an endogenous control in the normalisation of each sample. The samples were prepared in triplicate in a final volume of 20 μl with real-time PCR conditions at 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 51 °C for 1 min and a florescence measurement. The relative gene expression was determined by a ∆∆Ct calculation.From the 145 nAMD subjects recruited, 20 responders and 20 non-responders to ranibizumab treatment were randomly selected to evaluate the mRNA expression of the t test and one-way analysis of variance (ANOVA), and skewed continuous variables were compared using the Mann-Whitney U test and Kruskal-Wallis test whereas categorical variables were compared using the chi-square test. Furthermore, a logistic regression analysis was performed to determine the association between genetic variants of HTRA1 rs11200638 and ARMS2 rs10490924 to ranibizumab treatment. All statistical analyses were performed using SPSS version 21.0 , and P < 0.05 was considered to be statistically significant.The logarithm of the minimal angle of resolution (logMAR) was derived from the BCVA recorded in the Snellen eye examination chart for further statistical analysis. The Hardy-Weinberg equilibrium and linkage disequilibrium were verified using Haploview software. Data on normal continuous variables was compared using Student’s


Schurz (Synthese 164:201–234,  Let us call abductive inferences of the former kind logic of scientific inquiry can be fruitful. However, since theoretical concepts are intimately connected to empirical phenomena via dispositions , creative abduction is intended as an inference method for generating hypotheses featuring new theoretical concepts on the basis of empirical phenomena. Most philosophers of science are quite sceptical about whether a general approach toward such a In this paper we take up Schurz’  proposalThe paper is structured as follows: In Section x has a disposition D depends on whether certain test conditions T lead to a specific reaction R. For an object x to be soluble in water, for example, it is required that x dissolves at some time t if put into water at t:T and R are empirical concepts, while the dispositional concept D is a not directly observable theoretical concept. Note that Eq. D on the basis of T and R, except that the dispositional term is not relativized to t. What distinguishes the characterization of a disposition D(x) as provided in Eq. D(x) with D in Eq. D might be interpreted as x is soluble in water at some point in time t) is that Eq. T and R alone. It is a well-known fact that the only non-conservative (or creative) import of Eq. t an object x satisfies the test conditions and brings about the corresponding reaction, then x will do so at any time t:In this section we present Schurz’  approachentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaD via Eq. D since for each regularity among test-reaction pairs a distinct disposition has to be postulated. Things become more interesting once we focus on regularities among several dispositions D1,...,Dn, each characterized by a corresponding test-reaction pair consisting of Ti and Ri (with 1 ≤ i ≤ n). Now assume that we found strict pairwise empirical correlations between all of these dispositions D1,...,Dn, meaning thatcrossed uniformity assumption. Given n test-reaction pairs for n dispositions D1,...,Dn, we get n2 such crossed uniformity assumptions (Schurz n test-reaction pairs:n2 to n. In this sense such a reduction can be understood as unificatory. The abductive inference consists in the introduction of D1) and that some objects have the disposition to produce electricity when moved along a wire (D2), meaning that they introduced the two theoretical concepts D1 and D2 on the basis of Eq. D1 and D2 are correlated A and B are correlated and neither A causes B nor B causes A, then A and B are effects of a common cause C.If two properties (CCP) demands that every correlation among any pair of properties not standing in direct causal dependence to each other has to be explained by the existence of an independent common cause. In this sense (CCP) provides a way of causally unifying observed regularities. In the case of pairwise empirically correlated dispositions such as D1,...,Dn above, (CCP) supports a realist interpretation of the unifying higher-level disposition D1,...,Dn is explained by postulating a common cause In the next section we take up the idea of combining creative abduction and principles of causation by modeling cases of successful creative abduction in a Bayes net framework. Though Bayes nets can be causally interpreted, one does not have to subscribe to a realist interpretation when employing this particular framework to model creative abduction. While the realist gets a justification for creative abductive inferences on the basis of a causal interpretation, the instrumentalist can still use the Bayes net framework without a causal interpretation as a tool for justifying abductive inferences in terms of unificatory power. In this paper we prefer to stay neutral on the realist vs. instrumentalist question. As we will show, modeling creative abduction Bayesian style comes with a couple of advantages regardless of the answer to that question.V of random variables X1,...,Xn, a set E of directed edges (→) connecting some of these variables, and a probability distribution P over V. A triple 〈V,E,P〉 is a Bayes net if and only if it conforms to the Markov factorization (Pearl Par(Xi) is the set of Xi’s parents in the Bayes net’s graph G = 〈V,E〉, i.e., the set of all Xj ∈V for which Xj→Xi holds. Whenever the probability distribution P of a triple 〈V,E,P〉 factors according to Eq. P from the graph G = 〈V,E〉. Every Xi ∈V has, for example, to be independent of every Xj that is not connected to Xi via a path Xi→...→Xj conditional on Par(Xi). In the causal interpretation, the arrows (→) of a Bayes net’s graph stand for direct cause-effect relationships. It is well-known that (CCP) is a consequence of assuming the causally interpretated Markov factorization. Note that Schurz ((CCP) in support for a realist interpretation of creative abduction.We start this section by briefly introducing the basics of the Bayes net formalism. Bayes nets allow for modeling and graphically representing the paths over which probabilistic information spreads between variables. A Bayes net consists of a set on Pearl , p. 166t Schurz , 2016 onD1,...,Dn and the abduced higher-level disposition by a variable Di (with 1 ≤ i ≤ n) is represented by a variable Ei which stands for an inductive generalization of instances of test-reaction conditions such as  ∧ Ri) ∧ ... ∧  ∧ Ri). The dependence of each lower-level disposition Di on its corresponding evidence Ei is represented the same way as the dependence of a hypothesis on its evidence is typically modeled in the Bayesian framework: For each pair Di,Ei we draw an arrow Di→Ei. Since the creative abductive step is conducted by applying (CCP) in Schurz’  > P(Di) if i≠j.  > P(Ei).Each From 1., 2., and 3. it follows that P(Ei|Ej) > P(Ei) if i≠j.Probability flow between dispositions Conditions 1., 2., and 3. are necessary conditions for successful creative abduction: They guarantee pairwise correlations among lower-level dispositions that have to be inductively inferred on the basis of observed evidence and build the basis for introducing the higher-level disposition n2 empirical law statements establishing pairwise empirical correlations among n lower-level dispositions to n higher-level dispositional statements. In the Bayes net setting, pairwise empirical correlations between n lower-level dispositions D1,...,Dn consist in P(Di|Dj) > P(Di), where 1 ≤ i≠j ≤ n. Similarly, for the dependencies among pairs of evidential variables there are Like in Schurz’  originaln + 1 probabilistic statements in conditions 1., 2. and 3.: n statements of the form P(Ei|Di) > P(Ei) (with 1 ≤ i ≤ n), n statements of the form i ≤ n), and 1 statement u intended to capture the intuitions about unification outlined above. Given n correlated lower-level dispositions, u(n) measures the ratio between x(n) empirical statements to be unified and y(n) unifying theoretical statements. In order to shift the neutral case to 0, we subtract 1 from this ratio: ∞), where a negative value means that the theoretical description is more costly than simply listing the empirical statements, 0 means that there is no gain but also no cost in providing a theoretical description, and a positive value means that the theoretical description provides unification.5It follows from the Markov factorization Eq.  that the Schurz’  approachD1,...,Dn in the 2n + 1 statements used for unifying the correlations among the evidence E1,...,En and explain these correlations directly by n statements of the form i ≤ n) and 1 statement D1,...,Dn might be practically necessary to find a more general higher-level disposition A comparison of the unificatory power of both, the original and the Bayes net approach, is provided in Fig. unconditional empirical correlations. However, many more empirical correlations are possible in the Bayesian setting. If the evidential base is strictly correlated  and i,j ≤ n are extreme), then it follows from Eq. Ei,Ej (with i≠j) are independent conditional on any set of other evidential variables. Thus, the unconditional dependence statements in Eq. E1,...,En in this setting. However, if some correlations among pieces of evidence cannot be screened off by some non-empty set of other evidential variables, then also many conditional empirical dependencies may hold among pairs of evidential variables. In particular, there can be up to P(Ei|Y) > P(Ei|Z), where Z ⊂Y and Y are sets of evidential variables different from Ei.  and producing electricity when being moved along a wire (D2) had been established by experimenting with lodestone. It is inferred by abductive inference that this correlation is brought about by the higher-order disposition of generating an electromagnetic field (D1). It follows from our model together with conditions 1. and 2. that this increases the probability that this object’s having disposition D1. Hence, the probability for D2 is increased. Thus, observing that the object has disposition D1 predicts that P(D2|D1) > P(D2) applies to it as well. Note that this prediction is use-novel since only lodestone was used in building the theoretical model.Let us illustrate how creative abduction in a Bayes net model allows for generating use-novel predictionsD1 and D2 are empirically correlated, it seems to be commonly accepted that one can use evidence for one of these dispositions to confirm the presence of the other disposition. If, for example, one finds that an object attracts iron (E1), then one tends to accept this as evidence that it has the disposition of producing electricity when being moved along a wire (D2) as well. So E1 can be understood as a test for whether an object has disposition D2. This can be justified by help of our model as follows: Once the model’s structure E1 increases the probability for the presence of D1 which, in turn, by conditions 1. and 2. increases the probability of the presence of D2 as well, also the probability for D2’s presence will be increased. Thus, P(D2|E1) > P(D2) applies to our object and, according to Bayesian confirmation theory, E1 confirms D2.(CCP), has several problems. In this sense, expanding the account by switching to the Bayes net framework seems to allow for increased applicability.Given two dispositions H1 and H2 can often account for some evidence E equally well. So, just considering E, it is underdetermined which hypothesis one should choose. One approach to this problem consists in employing indirect evidence E′ . Furthermore, E′ confirms H3.iiE′ confirms also Hence: iiiE′ confirms also H2. (with i and ii)Hence: However, Okasha ((CCC):This problem arises due to the fact that two different theories or hypotheses , Okasha  has note, Okasha  solution(CCC)A entails B and C confirms B, then C also confirms A.If (SCC):And the underlying principle which grants the inference of iii is the so-called special consequence condition (SCC)A entails B and C confirms A, then C also confirms B.If (CCC) and (SCC), were already discussed by Hempel : A confirms A.Hence, by A ∧ B entails A.Trivially also (CCC): A confirms A ∧ B.Hence, by (SCC): A confirms B.But then, again by Clearly, this problem does not show up for the (comparative and) quantitative notion of confirmation. If we take, for example, the positive relevance notion of confirmation, then for some A,B,C it is well possible that Pr(A|C) ≤ Pr(A) (C is not positively relevant for A) though Pr(A|B) > Pr(A) (B is positively relevant for A) and Pr(B|C) > Pr(B) (C is positively relevant for B). The question arises, how then Laudan and Leplin’s  > P(H2) holds due to conditions 1., 2., and 3.: The mentioned theorem of Dardashti et al.  = P(H1) holds. This is a direct consequence of the Markov factorization  to be explained or (ii) do not screen off all non-intersecting sets of lower-level dispositions (or pieces of evidence) from each other. If (i) were the case, then This paper was about modeling successful cases of creative abduction on the basis of empirically correlated dispositions within a Bayes net framework. After introducing Schurz’  strict aIn Section This paper was about modeling successful instances of creative abduction and about which interesting conclusions one can draw from a Bayes net representation. An issue that has not been tackled is the epistemic question of how exactly theoretical concepts should be abduced on the basis of empirical data. If dispositions can be adequately represented by continuous variables, then this seems to open the door for a fruitful application of much more sophisticated search procedures from the literature on causal discovery.


Respirable dust samples were collected in several key locations of eight underground coal mines in central and northern Appalachia. In total, there were 76 unique sampling events . Here, we present data from each event describing particle size and mineralogy class distributions across the ∼100–10,000nm size range, which were determined using SEM-EDX; and estimated mass concentrations of potentially bioaccessible and total acid-soluble metals and trace elements, which were determined using sequential digestions with digestate analysis by ICP-MS. Discussion of this dataset is included in a companion research article “Beyond conventional metrics: Comprehensive characterization of respirable coal mine dust” Sarver et al., 2019. Each sample represents a unique sampling event . 22.1A total of 76 sets of respirable dust samples were collected in eight underground coal mines in mid-central , northern , and south-central Appalachia . The samples were collected in five key locations: intake airway (I), just outby of the primary production area  or along the mantrip track; feeder (F), near the feeder breaker or along the main conveyor belt; production (P), just downwind of an active continuous miner or near the midface of a longwall section (except for Mine 4); roof bolter (B), just downwind of an active bolter; and return airway (R), just outby of the primary production area .50 cut size of about 4 μm at the sampling flow rate of 1.7 L/min. Each sample set represents a unique sampling event, during which multiple replicate samples were collected simultaneously, over a continuous 2–4 hr period. One sample from each set is included in the analysis described here. These samples were collected directly onto 37-mm polycarbonate filters .A detailed description of the mines and sampling protocol was previously reported Although the PC filters used in this study are expected to have very high overall collection efficiencies for the particle size range studied here . This approach served to minimize interference between particles for elemental analysis, but does assume that the distribution of non-aggregated particles on the sample filters is representative of the overall particle distribution. For each particle, two main types of data were collected: (1) dimensions, and (2) the elemental spectra. The dimensions were used to determine particle projected area diameters. The spectral peak heights (or their resulting atomic ratios) were used to classify particles by their mineralogy. Classification criteria for supramicron particles into five defined mineralogy classes  were previously described The classification criteria were adapted  and veri•2 (12.67 μm × 10.96 μm) and the frames were spaced 1 mm apart.Initially, the SEM stage was moved to the center frame . Analysis began at the center frame and then proceeded through subsequent frames respectively . Further, more frames were analyzed beyond the 45 shown in The manual submicron particle analysis proceeded as follows:Following completion of both the sub- and supramicron particle analysis, the resulting datasets were merged to allow description of particle distributions across the entire size range . This wa2.3Following is a detailed description of the method used to prepare and analyze respirable coal mine dust samples to determine mass concentrations of potentially bioaccessible and total acid-soluble metals and trace elements. The method involved two sequential digestions of each dust sample, the first in a simulated lung fluid (SLF) and the second in a strong acid solution. It is noted that the term “total acid-soluble” used here refers to the total mass concentration of each element that could be dissolved via both digestions. Because the strong acid digestion did not employ hydrofluoric acid (HF), it is also noted that some elemental concentrations  could be underestimated.•Each filter remnant was weighed to establish a pre-weight prior to dust removal.•Each filter was then placed into a glass digestion tube and rinsed with 18 MΩ water. Enough water was added to fully submerse the filter. The tubes were then capped and sonicated for 1 hr, followed by centrifuging for 10 min (@ 3000 rpm) to settle the dust. Tubes were then uncapped and water was evaporated in a clean oven (@110 °C).•Dry filters were re-weighed to determine recovered dust mass. For the purpose of estimating elemental concentrations in the current study a–c, any Next, SLF, which is sometimes called “Gamble's solution”, was prepared per •To prepare 1 L of SLF, the ingredients shown in •The solution was then placed in a water bath (constant 37 °C), and the pH was adjusted to 7.4 using trace-metal grade HCl.•The SLF solution was added to each digestion tube containing dry dust, as well as tubes prepared as matrix and blank samples. The SLF solution volume was determined using a 1/50,000 solid  to SLF liquid ratio per •The tubes were capped and placed in the sonication bath for 24 hours (@ constant 37 °C), and then centrifuged for 10 min (@ 3000 rpm).•A 5 mL aliquot of the liquid was taken by syringe using a PTFE filter (0.1 μm pore size), to trap any remaining dust particles, and then the SLF digestate was added to an ICP tube and acidified to 2% (by volume) HNO3 using trace-metal grade acid.D7439-14Then, a method modified from ASTM •3 (prepared with 18 MΩ water).The PTFE filter used to trap dust from the SLF sample was placed back into the tube used for the SLF digestion. The filter and tube walls were then washed by pipetting a solution of 10% HNO•Under a fume hood, the tubes were positioned in a hot block , covered with watch glass, and heated until completely dry.•Then 1.25 mL of concentrated HCl was added to each tube, and the tubes were again covered and placed back into the hot block for 15 min, followed by 5 min of cooling.•3.The above step was then repeated with 1.25 mL of concentrated HNO•Each tube was then diluted to a final volume of 25 mL with 18 MΩ water, taking care to wash down the sides of the tube and watch glass, and capped and shaken.•3 using trace-metal grade acid.A 5 mL aliquot of the liquid was taken by syringe using a PTFE filter (0.1 μm pore size), to trap any remaining dust particles, and then the strong acid digestate was added to an ICP tube and acidified to 2% (by volume) HNOFirst, dust was recovered from each PC filter remnant :•For each ICP run, at least 5 blank PC filters were prepared using both SLF and strong acid digestion procedures to allow for blank corrections. The SLF and strong acid solutions were also analyzed to allow matrix corrections.•ICP results (μg/L in the digestate solutions) were corrected and then transformed into dry dust concentrations (μg/g) using the dust mass recovered from each filter. The concentration determined from the SLF digestate is regarded as potentially bioaccessible; and the sum of the concentration from the SLF and strong acid digestates is regarded as total acid-soluble concentration. It is noted that, due to relatively low sample masses for the current dataset, results in •The elements that were measured by ICP-MS and reported here are listed in Finally, digestates from the SLF and strong acid digestions were analyzed by ICP-MS using a Thermo Electron X Series instrument :


Tomato yellow leaf curl virus (TYLCV), this system can detect one copy per microlitre of virus in a pure isolate of extracted DNA and can readily identify an infected plant with a healthy appearance. This system hence provides a highly sensitive and stable DNA diagnosis. This visual method has a potential for disease diagnosis and prognostication in the field based on advantages of simplicity, high speed, portability and sensitivity.A visual DNA diagnosis with a rapid and simple procedure has been developed on integrating recombinase polymerase amplification (RPA) and a gold nanoparticle (AuNP) probe. The entire process is implemented in only one tube with no precision instrument and requires in total 20 min to amplify a DNA fragment with RPA and to discriminate a DNA fragment with an AuNP probe. The result in various colors is directly observable with the naked eye. Through discovering a small DNA fragment of  Many molecular biotechnological approaches have been widely applied to diagnose plant disease including immunology-based and nucleic-acid-based methods, which investigate pathogen proteins and nucleic acids extracted from plant materials and can substitute for the observation and cultivation of pathogens2. Immunology-based methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow devices (LF), are useful to detect the pathogen antigens and have been developed into portable equipment. As the specificity of these methods has relied on monoclonal antibodies, the sensitivity generally varies with the species3. Nucleic-acid-based methods involve mainly a polymerase chain reaction (PCR), which first amplifies a specific gene or DNA fragment for subsequent detection with gel electrophoresis. Basic and advanced PCR methods are at present widely used to detect plant pathogens because of their great sensitivity5. Nucleic-acid-based methods have greater accuracy, specificity and sensitivity than methods based on immunology, but they rely strongly on expertise and instruments in the laboratory, which becomes a limitation for their portable application in the field7. To overcome these limitations, prominent and portable isothermal nucleic-acid amplification techniques and biosensors provide a solution to the drawbacks of their counterparts, respectively.Visual observations of disease symptoms in plant tissues contribute towards the diagnosis of disease, but this approach typically requires an experienced pathologist and is unreliable when symptoms appear at an early stage of infection. Early detection of plant pathogens is important in the management of plant disease, especially for plant quarantine at a country border and seedling cultivation in a greenhouse10, and even using human body heat11. The reaction is complete in 20–40 min but the duration of detection limit is as little as 5 min. Among those approaches, RPA has the advantages of being simple and convenient to perform at a low temperature and for a short period while still possessing the characteristics of exponential amplification efficiency and highly sensitive detection12. The applications of RPA in the diagnosis of plant pathogens have been reported, including Tomato yellow leaf curl virus (TYLCV)20. The RPA amplicons are unsuitable for detection with agarose gel electrophoresis and should be purified with PCR cleanup columns. Specially designed probes, enzymes, lateral flow strips and further processing are involved in the detection of RPA amplicons21. A simple and effective detection of RPA amplicons is the next issue in the development of rapid and sensitive diagnostic techniques.Isothermal amplification techniques have recently been employed for genotyping, disease diagnosis, whole genome amplification, miniaturization and automation of bioanalytical applications based on their isothermal properties, simple procedures and excellent amplification capabilities. Recombinase polymerase amplification (RPA) can work satisfactorily at constant temperatures ranging from 25 to 45 °C7. A colorimetric detection of a AuNP probe recognized with amplicons is implemented mostly with a lateral flow strip23 and AuNP aggregation26. Although no additional equipment is required to detect AuNP aggregation with the naked eye, there are still shortcomings such as accuracy, sensitivity and quantitative interpretation. A novel approach can enhance the reliability of colorimetric detection of AuNP aggregation in qualitative and quantitative analysis with the naked eye27, which can facilitate an expansion of the applications of a AuNP probe in DNA detection.Innovative and portable DNA-based biosensors have been used as diagnostic tools in recent years; they exhibit rapid and sensitive characteristics and can allow qualitative and quantitative analysis of pathogens with label-free techniques. For reasons of simplicity and convenience, a gold nanoparticle (AuNP)-labeled DNA probe as an optical DNA sensor is the best method for DNA detectionSolanum plants, and cause serious economic losses28. The performance of our approach is compared directly with conventional bench-top methods. This rapid and simple system can be employed in qualitative and quantitative analysis of pathogens and broad applications in disease diagnosis and prognostication.With the aim of accelerating the reaction and simplifying the procedure, we have integrated RPA with an optical AuNP probe to detect pathogen DNA with the naked eye. All reaction conditions in the procedure of a visual DNA diagnostic technique have been completely optimized under a modest requirement of equipment. In this work TYLCV was chosen as a model pathogen because it can infect tomato and genus Bemisia tabaci biotype B) for one week. The viral DNA was extracted from leaves of TYLCV-infected tomato plants using a Gene-SpinTM GenomicDNA Isolation Kit, Protech Technology Enterprise Co., Taipei, Taiwan). The plasmid DNA was extracted from liquid-cultured Escherichia coli  carrying TYLCV isolate 82-2-1 and 57-2 clones.Healthy tomatoes were planted in an insect-free net house and then placed with TYLCV-viruliferous whitefly 30. According to the consensus of multiple sequences, the TYLCV- specific primer set 1/2R (5′-GGATTAGAGGCATGAGTACA-3′/5′-GGATTAGAGGCATGAGTACATGCCATATA-3′) was designed  was eventually validated with gel electrophoresis on agarose gel (1.5%).The nucleotide sequences of 56 Taiwan TYLCV isolates adapted from Tsai ®, TOPO® TA Cloning® Kit; Thermo Fisher Scientific Inc., MA, USA) and sequenced . The highly conserved sequence of conventional PCR amplicons derived from separate samples and TYLCV isolates was discovered and designed to be the probe DNA conjugated with AuNP . The AuNP probe was prepared according to the literature procedure27. The concentration of the AuNP probe was growth-regulated to 50 nM with pure water and stored at 4 °C for ensuing use. For colorimetric assay, the AuNP probe  was added into DNA solution (100 μL) with sodium chloride . Artificial complementary ssDNA solution was examined at varied concentrations of from 50 pM to 10 μM. The admixture of the DNA sample and the AuNP probe was incubated at 95 °C for 5 min. Hydroxylamine hydrochloride  and hydrogen tetrachloroaurate (III)  were subsequently added and mixed with the solution. After approximately 30 s, the AuNP was grown and visualized with various colors. UV/visible spectral analysis of the growth AuNP solution was implemented with a spectrophotometer .The AuNP solution (particle size 13 nm) was purchased  according to the manufacturer’s recommendations with some modifications. RPA reactions were performed in total volume 20 μL containing isolated DNA (5 ng) and primer set (480 nM) and then implemented at 39 °C for 30 min. To establish the optimal conditions for DNA detection, RPA reactions were executed at successively 33, 35, 37 and 39 °C for 5, 10, 20 and 30 min. To test the sensitivity, various DNA concentrations of TYLCV isolate 82-2-1 were applied in the RPA reaction from 109 to 1 copies per μL in an appropriate order, and were then incubated at 33 °C for 10 min. RPA amplicons (4 μL) were validated with gel electrophoresis on agarose gel (1.5%). RPA amplicons (10 μL) were used in a AuNP colorimetric assay. For visual DNA diagnosis, a AuNP probe  was added into RPA amplicon (10 μL) with blending and then incubated at 95 °C for 5 min. Subsequently, H2NOH  and HAuCl4  were added and mixed. After approximately 30 s, the AuNP was grown and visualized with various colors. The image and UV/visible spectra of the growth AuNP solution were finally recorded.The recombinase polymerase amplification (RPA) reaction was achieved using a basic kit . The correlation between amount of amplicons and template concentration was determined with linear regression using Microsoft Excel 2016.All gel images were acquired with a digital gel image system  and recorded in JPEG format. The gel images were processed with cropping, automatic adjustment of brightness and contrast, and quantification of electrophoretic bands using Fiji 32. This approach achieves saving of time and effort, with modest requirement of equipment and expertise in DNA amplification using RPA.To clarify the entire concept of visual TYLCV DNA diagnosis in one tube, the procedure is described with a schematic illustration Fig. . Apart f2NOH and HAuCl4 (1 μL) were individually added and blended to trigger the growth of AuNP. At 95 °C, the amplified DNA fragments were denatured and transformed into ssDNA. Because a DNA probe conjugated with AuNP hybrids only complementary ssDNA and develops a double helix, a AuNP probe can discriminate complementary ssDNA from mismatched ssDNA. Moreover, the amount and sequence similarity of cDNA would affect the growth, size and shape of AuNP; the sizes and shapes of AuNP have varied colors and UV/visible spectra33. The color variation of growth AuNP can hence be regarded as a semi-quantitative interpretation of the cDNA concentration27. This feature shows that the detection of DNA relying on the AuNP probe can be accomplished with the naked eye. RPA integrated with a AuNP probe can fulfill a simple and convenient DNA diagnosis. In the future, this system could be applied to diagnosis of disease in the field.In the second step, AuNP probe (3.5 μL) was added directly to the RPA reaction (10 μL) with rigorous blending. For the purpose of highly stringent hybridization between a AuNP probe and a TYLCV-specific DNA fragment, the admixture was incubated at 95 °C for 5 min. Immediately afterward, HThe design of an identified amplicon and AuNP probe is a priority in visual DNA diagnosis. To fulfill the requirement of RPA reactions, the newly TYLCV-identified primer set was redesigned and carried a smaller length, greater sensitivity and greater accuracy than the conventional primer set  to hybridize with a short AuNP probe (20 nt). The increased length of cDNA meets the requirement of DNA identification. A longer cDNA seems to generate a wide range of color variation of growth AuNP solutions, suitable for visual observation because of an improved linear distribution of colors and cDNA concentrations.To clarify the effectiveness and sensitivity of the AuNP-probe detected target amplicon, cDNA of varied concentration was successively hybridized with a AuNP probe. The color of the growth AuNP altered from pink to other colors in a sequence,–dark blue, navy blue, indigo, purple, purple-red, mauve, which characterize cDNA concentrations 10, 5, 1, 0.5, 0.1 μM, 50 nM, respectively. A concentration less than 10 nM became an indistinct color; even a precipitate appeared at less than 0.5-nM reaction Fig. . Colorim® Basic Kit) work at a constant temperature (37–39 °C) for 20–40 min based on the manufacturer’s recommendations. The yield of RPA amplicons generated at 39 °C for 40 min is more than that of conventional PCR 11. The reaction can be detected in only 5 min32. To save time and effort, we studied the best conditions of RPA reactions to detect TYLCV. RPA reactions were executed successively at 33, 35, 37 and 39 °C for 5, 10, 20 and 30 min. As the result of gel electrophoresis, RPA amplicons had a maximum yield at 37 °C and a minimum yield at 33 °C for 30 min. The effect of varied duration indicates that a low limit of detection was generated at 10 min  and indistinct (5 min) detectability of RPA reactions were both purple. The color of a AuNP probe detected in various RPA reactions is similar to various concentrations of artificial complementary ssDNA Fig. . These rR2 = 0.9438) and RPA (R2 = 0.8909). RPA (1 copy/μL) was clearly one million times as sensitive as conventional PCR (106 copies/μL)  Fig. .Figure 43 copies/μL), purple (104–105 copies/μL), blue-violet (106–107 copies/μL) and navy blue (108–109 copies/μL) . To clarify accurately the relation between color and template concentration, an absorption ratio R (A570/A530) of growth AuNP was estimated. The relation between absorption ratio R and template concentration showed a linear distribution (R2 = 0.9239)  to colors in a sequence, such as mauve (1–10μL) Fig. . The absμL) Fig.  is a phe39) Fig. . It is sR (A570/A530), respectively  definitely indicated that sample 1 (R = 0.95) carried 10 copies/μL (R = 0.94) of TYLCV; the virus amount of sample 2 (R = 1.55) was more than 109 copies/μL (R = 1.31). Samples with a large amount of virus were expected to show a rich color, i.e. blue or blue-violet, and a large absorption ratio R (>1), except sample 7 that had an extremely large absorption ratio possibly due to the precipitation of AuNP. Moreover, reference isolate 57-2 with single nucleotide polymorphism (SNP) . This phenomenon was revealed by Fang et al.27, but the diagnostic result retains discrimination. In general, the diagnostic results of the colorimetric assay and the absorption ratio were consistent; visual DNA diagnosis can hence be applied to TYLCV identification.The nucleic-acid extraction of pathogens is invariably accompanied with a large amount of host genomic DNA (gDNA), because the nucleic-acid concentration of a pathogen is diluted with many host gDNA, which would interfere with the DNA diagnosis. For this reason, the reliability of visual DNA diagnosis was verified by means of infected tomato plants with various symptoms. Seven infected samples were diagnosed with conventional PCR  Fig.  still shi.e. mauve, purple, blue-violet, navy blue. The entire procedure consumed only 20 min and a modest requirement of equipment. According to the sensitivity test of this visual TYLCV DNA diagnosis, the results showed that this system is more rapid and more sensitive than conventional PCR and qPCR. In particular, 1 copy/μL of the virus and an infected plant with a healthy appearance can still be identified. The colors of the reactions can process the qualitative and quantitative analysis of virus. This system can clearly detect the virus in infected tomato plants even with an interference from plant genomic DNA in accordance with the reliability assessment. Based on the advantages of the above description, this system can be applied directly to monitoring health in the field. As the procedure and reagents of this system are simple and convenient, it could be combined with a microfluidic system to accomplish a multiplex or singleplex diagnosis.We developed a visual DNA diagnosis with integrated RPA and a AuNP probe. This work demonstrates that this excellent system can amplify and detect DNA in a rapid and simple procedure and maintain a high sensitivity. DNA amplified with RPA at a low temperature for less duration achieves saving of time and effort. RPA amplicons discriminated with a biosensor AuNP probe form a visible solution of varied color, Supplementary information


Corallinales, Rhodophyta) that are photosynthesizers, calcifiers, and ecosystem engineers and therefore represent important targets for ocean acidification (OA) research. Here, we exposed live rhodoliths to near-future OA conditions to investigate responses in their photosynthetic capacity, calcium carbonate production, and associated microbiome using carbon uptake, decalcification assays, and whole genome shotgun sequencing metagenomic analysis, respectively. The results from our live rhodolith assays were compared to similar manipulations on dead rhodolith  biofilms and water column microbial communities, thereby enabling the assessment of host-microbiome interaction under climate-driven environmental perturbations.Life in the ocean will increasingly have to contend with a complex matrix of concurrent shifts in environmental properties that impact their physiology and control their life histories. Rhodoliths are coralline red algae  contains supplementary material, which is available to authorized users. Increasing ocean CO2 uptake results in the elevation of partial pressure of CO2 (pCO2), lower pH levels, and lower carbonate saturation of seawater, all of which control the calcification process ; cell wall and capsule (p = 4.03 × 10− 6); iron acquisition and metabolism (p = 4.24 × 10− 2); membrane transport , motility and chemotaxis  and stress response (p = 4.60 × 10− 2).The dead rhodolith microbiome displayed higher mean relative frequencies assigned to metabolic biofilm formation when compared to seawater and live rhodoliths, including carbohydrates , RNA  and sulfur , as well as in the photosynthesis  and respiration .In comparison with dead rhodolith and seawater, live rhodolith metagenomes under both ambient air and high COpCO2. The inorganic carbon rates  were higher in rhodoliths under high pCO2 treatment, but organic content (live tissue) was lower, suggesting that the excess energy from photosynthesis went towards deposition of CaCO3 rather than growth.The results of this experimental study showed that the microbial community structure in the seawater and the dead rhodoliths biofilm changed during the course of the experiment, while the live rhodolith microbiome remained stable. Further, the algae photosynthetic capacity increased after exposure to high pCO2, as indicated by a higher Pmax, compared with observations for rhodoliths under ambient air treatment. Photosynthetic efficiency (α) was unaltered regardless of pCO2. A significant increase in sequences associated with photosynthetic activity was observed among live rhodolith metagenomes compared to water column and dead rhodolith biofilm. Accordingly, the proportion of sequences assigned to photosynthesis metabolism was 24.31% higher under elevated CO2 compared to the ambient air live rhodolith metagenomes. Photosynthesis draws carbon (CO2 or bicarbonate) from total CI [2. Calcification is an energy-demanding process and photosynthesis can provide the energy needed to support the precipitation of carbonate skeletons. In our study, the inorganic carbon rates  were higher in the rhodolith under high pCO2 treatment compared to rhodolith under ambient air treatment, but organic content was lower. Therefore, it is possible that more inorganic carbon was allocated to deposition of CaCO3, but at the cost of growth.Consistently elevated rates of carbon uptake during photosynthesis were observed in rhodoliths under high total CI , whereaspCO2, where two closely related species in the same genus presented disparate responses [2 availability.The research on OA in mesocosms poses challenges due to the constraints in fully reproducing field conditions in the laboratory. Coralline algae have shown strong species-specific responses to elevated esponses , or variesponses . Howeveresponses . Our res2 for photosynthesis, which, in turn, releases the carbonate (CO32−) used in algal calcification [pCO2 confirms the control the algae exert on the boundary layer. As rhodoliths precipitate CaCO3 in their cell walls, the increase in pH shifts the equilibrium toward an increase in CO32− concentration [3. Thus, the positive effect of photosynthesis on calcification to a certain extent could offset CaCO3 dissolution in calcifying algae in response to increased pCO2 [Photosynthesis rates and the surrounding concentration of inorganic carbon are related to calcification rates in coralline algae. Similarly, calcification can stimulate algal photosynthesis, if it acts as a carbon concentration mechanism, as proposed previously . Brieflyfication . Photosyfication , 48 and ntration , 50, whised pCO2 –51. In tpCO2 [2 concentrations [2 and temperatures are elevated, the photosynthesis-calcification metabolic processes in calcifying macroalgae might become uncoupled [Previous studies have demonstrated a parabolic response in coralline algae physiology to pH and pCO2 , 18, 23.trations , 44, 52.trations , 54, in ncoupled . HoweverpCO2 conditions, resembling a healthy holobiont.Rhodolith function is intimately linked to the composition and structure of their associated microbiome. Due to their diverse metabolic traits, microorganisms play important roles in macroalgae physiology, as suggested for instance, in the biomineralization process . When copCO2 could impair host physiology disrupting beneficial host-microbiome interactions  over geologic times . Whole goN, 118.3246oW), Catalina Island, California, USA in April 2014. The morphologic features of Catalina Island rhodoliths are consistent with the species Sporolithon australe [Rhodoliths were collected at a depth of 10–12 m at Avalon harbor  or predicted future (high pCO2) conditions, in duplicate mesocosms. Approximately 500 g of live and dead rhodoliths were placed in separated 18.5 L acrylic mesocosm tanks: tanks 1A and 1B for live rhodolith, ambient air treatment; tanks 2A and 2B for dead rhodolith, high CO2 treatment; tanks 3A and 3B for live rhodolith, high CO2 treatment; tanks 4A and 4B for dead rhodolith, ambient air treatment . Every two mesocosms were connected to one of four 400 L seawater holding tanks (n = 2 mesocosms per large holding tank) in a recirculating system. The large holding tanks were used to maintain a stable pCO2 levels during the experiment, which is not possible to achieve by injecting the CO2 into the smaller mesocosm [The experimental setup was performed, with few modifications, following Brown . Brieflymesocosm , 54. It mesocosm , we poinmesocosm , 63 and pCO2 levels within each large holding tank were adjusted to either ambient air (ca 500 μatm) by bubbling ambient air  by bubbling a certified CO2 mixture  into the large holding tanks. The predicted increases in seawater pCO2  are based on models for the open-ocean where values are expected to track atmospheric concentrations, and therefore they may not apply to coastal ecosystems where these values are much more dynamic  and total inorganic carbon (CI) [I and AT could then be entered into the CO2Sys software (http://cdiac.ornl.gov/ftp/co2sys/) to calculate pH and pCO2 at a constant temperature and salinity [Seawater ia coast ); or higc . Along c  . The titsalinity  (Additiot air ca 00 μatm b− 2 s− 1), according to previous descriptions of the conditions within Catalina Island rhodoliths beds [Temperature in the holding tanks was maintained at 15 °C and controlled using aquarium chillers. Light above the mesocosms was provided by full spectrum fluorescent bulbs set on a 12:12 cycle, with light levels set at ~ 250–300 photons  treatments were measured separately over four consecutive days (for both duplicates mesocosms tanks A and B). On each day, 500 mL BOD bottles were filled with seawater taken from the mesocosm being measured, sealed and stored in the dark within a temperature-controlled room, set to 15 °C. A separate water sample was taken from the large holding tanks to determine starting CI for each treatment. Concurrently, three separate rhodolith specimens (~ 0.7 g wet weight) were taken from the mesocosm being evaluated. Each rhodolith specimen was patted dry, weighed and placed into a BOD bottle. These bottles were then placed upright in separate water baths within the temperature-controlled room and photosynthetic carbon uptake by the algae was measured under six irradiances ) for one hour each. Light was provided by full-spectrum compact fluorescent bulbs hung directly above the bottles and light levels were adjusted either by moving the bulb closer to the bottles, or through window tinting of different shading properties until reaching the desired irradiance as measured with a photometer. To reduce the formation of boundary layers, magnetic stirring bars were placed into each bottle. After one hour, the rhodoliths were removed and the CI within the bottles was measured using potentiometric titration as described above. Carbon uptake was calculated as the difference between CI at the end of one hour versus the treatment’s starting CI and standardized to the weight of the rhodolith. The rhodolith specimens were transferred to new bottles and the irradiance was increased to the next level. For each mesocosm, the carbon uptake at each light level was plotted and fit as in Platt [max) and the photosynthetic efficiency (α) of each treatment were calculated.To determine the effects of elevated in Platt . From th2 diameter rhodolith individual) sampled at the beginning and end of the experiment, under both ambient air and high CO2 conditions, were weighed in triplicate and decalcified using formic acid. The rhodoliths were oven dried and weighed at the start and end of the decalcifying process, the mass difference after the removal of calcium carbonate skeleton was considered the proportion of flesh tissue in each sample. The amount of inorganic carbon  was calculated as the total weight minus weight of organic material. The percentage (%) of inorganic material was calculated as . Student’s T-test was used to compare the proportion of tissue in the rhodoliths at the start (day 1) and end (day 40) of the experiment.Calcium carbonate loss was calculated on live rhodoliths only, by comparing the proportion of live tissue present on a subset of rhodoliths at the start of the experiment (day 1) and again at the end (day 40), according to Price . To condn = 4) were collected and filtered using Sterivex filters (0.22 μm), the holding tanks are independent of each other. Microbial DNA extraction was performed using a modified column purification protocol  with proteinase K , followed by incubation at 55 °C with gentle agitation overnight. By the end of the experiment (day 40), approximately 60–80 L of water from each large holding tank were collected and processed through tangential flow filters (TFFs). The filtrate was concentrated to around 500 ml on a 100 kDa TFF, and then passed through 0.22 μm Sterivex filters  using a 60 ml syringe.At day 0, day1, and day 7 of experiment two 4 L seawater samples from each large holding tank (2 diameter) from each duplicate mesocosm (A and B) under both pCO2 levels (ambient air and high CO2) for the live rhodolith treatment; and one mesocosm (A) of both pCO2 levels (ambient air and high CO2) for the dead rhodolith treatment. The samples were collected at day 1 and day 40, i.e., the beginning and end of the experiment. All rhodolith samples were macerated with liquid nitrogen using sterile technique. The resulting slurry was processed using CTAB buffer with 100 mM of EDTA and the PowerSoil® DNA Isolation Kit . A purification column was used to recover high-molecular-weight DNA as described by [Rhodolith gDNA extraction was performed using multiple whole rhodolith individuals (~ 1 cmribed by .n = 16). Rhodolith holobiont libraries were prepared using Accel-NGS 2S DNA Library Kit for Illumina platform (Swift Biosciences) (n = 12). The two library preparation kits were used because the NGS 2S kit provides better recovery from samples with low DNA concentrations. Variations between the two kits occur at the extreme GC range only (application note from Swift Biosciences). All metagenomic libraries were sequenced on an Illumina MiSeq (MiSeq Reagent Kit v3) at San Diego State University.Water metagenomic libraries were prepared using a XT Nextera DNA sample Preparation Kit (Illumina)  was conducted using the R statistical software package to compare the functional grouping based on the SEED subsystems (level 1) [Statistical analysis was performed using the Statistical Analysis of Metagenomic Profiles (STAMP v.2.0.0) software . For mullevel 1)  of all 2-Curtis similarities, using either the statistical package PRIMER 6  or R. Comparisons over time of percentage changes in the relative abundance of taxa/functions in any treatment were calculated as (Δf / fd1) × 100, where Δf = fd40 – fd1 (relative abundance in day 40 – relative abundance in day 1).Multidimensional scaling (MDS) plots were used with the annotated metagenome data to visualize the similarities between the rhodolith microbiome and the microbial profile of seawater in terms of taxonomy structure. Taxonomy structure was determined by comparing the relative abundances of 15 higher-rank bacterial classes, eukaryotic classes and archaeal families, in separate analysis. Groupings depicted by the MDS were based on the BrayChanges in bacterial communities between day 1 and day 40 of the experiment were calculated based on Bray-Curtis similarities among each of the rhodolith thallus treatments (i.e. live and dead rhodoliths), and each of the two seawater locations (i.e. water column over the live and dead rhodoliths). Prior to further testing, all Bray-Curtis similarity data were examined for normality by visual examination of probability plots, with the data plotted against a normal distribution , and forAll metagenomes analyzed here are available through MG-RAST under the project “Rhodolith_Calcareous Algae under Ocean Acidification” following the accession numbers as listed in Additional file Additional file 1:General features of metagenomes. (XLS 45 kb)Additional file 2;Taxonomic similarities of Eukarya class (a) and Archaea family (b) in live rhodoliths, dead rhodolith biofilms and seawater metagenomes during the experiment. (EPS 3233 kb)Additional file 3:Relative abundance of bacterial families in Rhodolith (live and dead) metagenomes (1% > hits contribution). (EPS 4059 kb)Additional file 4:2 in days 1 and 40. (b) Metabolic profile of seawater tanks for both live treatment (left column) and dead treatment (right column) under ambient air and high CO2 in days 1 and 40. (EPS 7580 kb)Functional profile. (a) Rhodolith metabolic profile for both live algae (left column) and dead algae (right column) under ambient air and high COAdditional file 5:Average water chemistry in treatment tanks. (XLS 28 kb)