In the past few decades, Poly(vinylidene fluoride)/Polymethylmethacrylate (PVDF/PMMA) binary blend has attracted substantial attention in the scientific community due to possible intriguing mechanical, optical and ferroelectric properties that are closely related to its multiple crystal structures/phases. However, the effect of PMMA phase on the polymorphism of PVDF, especially the relationship between miscibility and polymorphism, remains an open question and is not yet fully understood. In this work, three series of particle blends with varied levels of miscibility between PVDF and PMMA were prepared via seeded emulsion polymerization: PVDF–PMMA core–shell particle (PVDF@PMMA) with high miscibility; PVDF/PMMA latex blend with modest miscibility; and PVDF@c–PMMA (crosslinked PMMA) core–shell particle with negligible miscibility. The difference in miscibility, and the corresponding morphology and polymorphism were systematically studied to correlate the PMMA/PVDF miscibility with PVDF polymorphism. It is of interest to observe that the formation of polar β/γ phase during melt crystallization could be governed in two ways: dipole–dipole interaction and fast crystallization. For PVDF@PMMA and PVDF/PMMA systems, in which fast crystallization was unlikely triggered, higher content of β/γ phase, and intense suppression of crystallization temperature and capacity were observed in PVDF@PMMA, because high miscibility favored a higher intensity of overall dipole–dipole interaction and a longer interaction time. For PVDF@c–PMMA system, after a complete coverage of PVDF seeds by PMMA shells, nearly pure β/γ phase was obtained owing to the fast homogeneous nucleation. This is the first report that high miscibility between PVDF and PMMA could favor the formation of β/γ phase. These semicrystalline-amorphous binary blends not only show miscibility on molecular level at all composition in the amorphous region, giving rise to a rich library of phase morphologies characteristics, but also combine advantageous physical properties from each individual polymer ,2,3, namlication , and, inlication . Besideslication ,7. There3GT3G′). The only difference between them is either parallel (γ) or anti-parallel (ε) chain arrangement [PVDF is a typical semicrystalline polymer exhibiting five crystal polymorphs, namely, α, β, γ, δ, and ε phases ,9. As a angement . It is rangement . Differeangement . Howeverangement . For melangement . Since aangement  and confangement ,17. In aangement ,19, wherangement . In addiangement . Given tIn this work, we focused on the intercorrelation between the PMMA/PVDF miscibility and the change of PVDF polymorphism. Inspired by our previous work on PVDF@Polystyrene core–shell composite particles prepared via seeded emulsion polymerization ,22, here® Latex 32, was kindly provided by Arkema Inc.  Methyl methacrylate (MMA) and 1,4-butanediol diacrylate (BDDA) in analytically pure grade were purchased from Tianjin Reagent Corporation . Prior to use, the monomer MMA was distilled under reduced pressure (30 mmHg) at elevated temperatures (60 °C) to remove inhibitors. The crosslinking agent BDDA was used without any further purification. Potassium persulfate (K2S2O8) and sodium dodecyl sulfate (SDS) were purchased from Tianjin Kermel Chemical Reagent , and used without further purification. Poly(vinylidene fluoride) (PVDF) latex, Kynar2 before polymerization, and fluxed during the entire polymerization. The monomer was added in one batch to swell the PVDF latex particles at room temperature for 40 min.The core–shell PVDF@PMMA colloidal particles were synthesized via seeded emulsion polymerization using PVDF latex particles as seeds. All polymerization reactions were carried out in a 250 mL four-necked round-bottom flask equipped with a condenser, a mechanical stirrer, a thermometer and an inlet for nitrogen. A determined amount of PVDF latex was introduced into the flask, and deionized (DI) water was added to make the total volume about 100 mL. The mixture dispersion was further ultrasonicated  for 40 min to avoid any colloidal particle agglomeration. Then, nitrogen was purged into the dispersion for 15 min to remove O2S2O8 relative to the amount of MMA monomer in 5 mL aqueous solution was added into the mixture. The polymerization time was varied from 0.5 to 6 h in order to obtain core–shell particles with different PMMA compositions. In this work, the feed ratio of MMA and PVDF was set at 4 to 1 in weight for making PMMA@PVDF particles. For PVDF@c–PMMA particles, a 4 wt % of BDDA relative to the amount of MMA monomer was added simultaneously with MMA to crosslink the grown PMMA shell during emulsion polymerization. The reaction mixture was then heated to 75 °C (or 65 °C) at a heating rate of 2 °C/min under a constant 300 rpm stirring. After an additional 15 min equilibration time, a 0.6 wt % of KThe obtained particles were purified by distillation below 40 °C under reduced pressure to remove unreacted monomer. An aliquot of sample was taken for morphology imaging and calculation of MMA conversion. Because the original PVDF weight was known before polymerization, the gained weight must come from the grown PMMA shell given that the MMA content in the condenser is negligible relative to the liquid phase.2S2O8 aqueous solution was added to initiate polymerization. An emulsion with translucent bluish color was seen in 1 h. Then, another aqueous solution, containing 0.6 wt % K2S2O8 of the MMA monomer, was added while 6.34 g of MMA was added dropwise at an adding rate of 2 g/min. After finishing feeding, the polymerization reaction was kept for another 2 h. The morphology and size distribution of PMMA latex particles are given in Neat PMMA latex particles were prepared using a similar way as the one discussed above. A 0.25 wt % of SDS relative to the amount of water was added into 130 mL of DI water in a 250 mL four-necked round-bottom flask. The solution was stirred at room temperature and nitrogen purging was maintained throughout the entire polymerization. After 15 min, 1.58 g of MMA monomer was transferred into the flask, followed by heating the mixture to 75 °C. Afterward, 10 mL 0.6 wt % Kwt/wt, respectively. To achieve uniform dispersion, the mixture was kept stirred, followed by ultrasonication for 1 h. Next, the PVDF latex and the prepared PMMA latex were blended together at room temperature, and the weight ratios of PVDF to PMMA latex particles were set at 70/30, 60/40 and 50/50 After mixing the above latexes, the resultant micro-particles were washed with DI water (3 × 20 mL), and collected by centrifugation. The collected micro-particles were dried overnight under reduced pressure at 40 °C. The particle morphology was imaged using a Hitachi Japan S-4800 scanning electron microscope . To make sample conductive, a thin layer of gold (10–30 Å) was sputter-coated under vacuum. The transmission electron microscope (TEM) images were collected by a JEOL Japan JEM-2010 FEF  transmission electron microscope. A drop of diluted latex solution was cast on 400-mesh carbon-coated copper grids and dried in the fume hood. The average size and size distribution of the core–shell particles were determined using a Malvern Zeta Sizer Nano 90 dynamic light scattering (DLS) particle size analyzer .Tcs) with about 7 mg load. Dry nitrogen was used as carrier gas. The DSC samples were heated from room temperature to 200 °C at a heating rate of 10 °C/min. The DSC cooling thermograms were obtained by annealing the samples for 10 min at 200 °C and cooling at a rate of −10 °C/min from 200 to 0 °C.Thermal analysis was carried out using a Diamond differential scanning calorimeter . A modulated DSC mode was used to measure the crystallization temperatures  was obtained under crossed polarizers using a Zeiss Axioskop 40 . The spherulite morphology of the above-mentioned samples were examined with a hot stage. The samples were melted at 200 °C for 10 min, then isothermally crystallized at about 145 °C for 20 min.Wide-angle X-ray diffraction (WAXD) was collected with a Bruker D8 Focus XRD diffractometer, using Cu Kα radiation  in the range of 5–50°, with a scanning step rate of 4°/min).−1. The melt-recrystallized samples after DSC test were ground, and pressed into pellets with KBr powder.Fourier transform infrared spectroscopy (FTIR) was conducted on a Bruker Vector 22 FTIR Spectrometer  in the spectral range of 4000–400 cmc ~ 50%), PMMA might form numerous nucleation/growth sites all over the seed surface. This was essentially different from the growth of polystyrene, which can only form a bulge on PVDF surface because of poor compatibility [The morphology evolution of the PVDF@PMMA core–shell nanoparticles during polymerization was studied by sampling aliquots at different time intervals. As shown in tibility ,22. Withtibility ,24. TherIn addition, it is noted that neither uncoated PVDF seeds nor isolated PMMA particle was identified in the final core–shell product, as convinced from the DLS results that the particles with a relatively narrow PDI value were finally obtained. This indicated that nano-sized PVDF latex efficiently acted as primary seeds to enable surface growth of PMMA via emulsion polymerization, presumably due to its large-area surface availability as well as great compatibility to MMA monomer and polymer. It was also corroborated by theoretical and experimental results reported in the past literature ,26,27,28Tc of PVDF gradually decreased as increasing PMMA content in both PVDF@PMMA and PVDF/PMMA samples, indicating a significant slow-down of crystal growth rate. It was usually attributed to the suppressed PVDF segments mobility caused by the intermolecular interactions between the carbonyl group of PMMA and the hydrogen of PVDF [Tc of PVDF in PVDF@PMMA decreased from 133 to 100 °C as the PMMA composition increased from 0 to 30 wt %. Nonetheless, it only decreased to 119 °C in PVDF/PMMA. Further increasing the PMMA content (up to 40 wt %), the exothermic peak disappeared in PVDF@PMMA while a relatively strong peak can still be identified in the normal blend . The crystallization peak in PVDF/PMMA remained discernable until the PMMA content was higher than 50 wt %. Similar effect was also observed during the first heating process, as shown in Tm) of PVDF gradually decreased from 160 to 149 °C when the weight ratio of PVDF@PMMA decreased to 60/40, while only little change in Tm (159 °C) were traced in PVDF/PMMA. The above DSC evidence suggested a stronger intermolecular interaction between PVDF and PMMA in PVDF@PMMA during crystallization. Note that the particular core–shell morphology in PVDF@PMMA featured an enormously large PMMA–PVDF interfacial area and pre-interwoven polymer chains at interface. This presumably led to an enhanced inter-diffusion thereafter starting from the interfacial area during the first heating. In other words, when temperature increased above both Tg of PVDF and PMMA, the inter-diffusion was so efficient in PVDF@PMMA that a much greater extent of PMMA–PVDF chain intermixing at finer length scale could be resulted. Therefore, PVDF and PMMA polymer chains were more intimately interwoven together, leaving more carbonyl groups on PMMA and more hydrogens on PVDF readily interacting with each other during cooling and recrystallization. In contrast, physically isolated PMMA and PVDF phases stayed in their latex particles in normal blend sample with very limited contact area, which means that it must be more difficult for normal latex blend to reach the same length scale of chain intermixing as that in the core–shell sample at a given heating time. Coarse scale of chain intermixing would somewhat make carbonyl groups and hydrogens less accessible to each other. As a result, the crystallization of some PVDF chains might be unaffected by PMMA, and hence a weaker crystallization suppression was observed in the normal blends.The non-isothermal crystallization behaviors of PVDF@PMMA, PVDF@c–PMMA and PVDF/PMMA samples with varied PMMA contents were studied by DSC analysis, as shown in  of PVDF ,29. CompTc was observed within 60–65 °C in well confined nanodroplets and PVDF@PS composite latex from our previous work [In contrast, the PVDF@c–PMMA exhibited totally distinct crystallization behavior. From the first cooling curve see , there wous work ,17; therTm = 168 °C) only appeared on the first heating curve. This indicated that in PVDF@PMMA, the inter-diffusion and chain mixing quickly proceeded in the amorphous regions at relatively low temperature (<Tm) during the first heating. The PVDF crystals were surrounded by perfectly mixed PMMA–PVDF composite phase, and the melting temperature was suppressed during the first heating. Contrarily, in PVDF/PMMA 60/40, besides the main melting peak equal to neat PVDF (centered at 160 °C) another distinct endothermic peak centered close to 90 °C was observed in first heating curve, which has previously been ascribed to melting of some small, imperfect PVDF crystallites [Tm during the first heating. After the first heating (T > Tm), PVDF and PMMA in normal blend had better mixing and interfacial area than they were before. During the second heating, the Tm of PVDF started to be affected by PMMA. Therefore, PVDF@PMMA could reach to equal chain intermixing more efficiently than PVDF/PMMA, Meanwhile, the same melting and crystallization temperature  for PVDF@PMMA 80/20 and PVDF/PMMA 60/40 revealed that less PMMA in PVDF@PMMA could provide same influence on the crystallization kinetics, referring to the better mixing of PMMA due to larger interfacial area.To further understand the difference, the heating–cooling cycle data for selected composition are shown in tallites ,30. DuriKeeping in line with the re-crystallization behavior, the melting behavior of PVDF@c–PMMA 60/40 was also distinctive. Compared with the previous two miscible series, three major differences should be addressed. First, the melting point of PVDF was not much influenced in the first heating scan C, indicaTc, as seen in previous DSC results  along with several relatively weak peaks for TTT conformation  were observed, indicating the formation of large amount of α phase [−1) with two more peaks from amorphous PMMA , their WAXD profiles looked very differently. The intensity of 100α and 020α peaks decreased dramatically, and 110α peak shifted to high angle. From the deconvolution curves, it is clear that both the diffraction peaks of α  and β phase (110/200 β) were detected, suggesting the coexistence of α and β crystals. Comparing the WAXD pattern of neat PVDF and PVDF@PMMA 80/20, we assume that only α phase was formed in neat PVDF. The weak signal of the TTT conformation in FTIR may result from external force-induced local conformation change during the grinding process for making the testing pellet. This is not unusual, since similar a phenomenon was also observed when examining the cutting surface of PVDF resin using attenuated total reflection (ATR) mode. Thus, the observed TTT conformation was mainly artifact. With increasing PMMA content in PVDF@PMMA, characteristic IR peaks attributed to PMMA [−1) became stronger, as marked by dot line in −1) or became extremely weak  and indistinguishable . It is worth to mention that an IR absorption peak centered at 600 cm−1 became noticeable when the PMMA content was higher than 30 wt %, and it shifted to 590 cm−1 in the PVDF@PMMA 50/50 sample after re-crystallization. This new peak can neither be indexed to any peak associated with amorphous PMMA, nor to any forms of PVDF crystal structures. At 50/50 weight ratio, only amorphous blend was obtained as confirmed by WAXD. Thus, we speculate that it may be related to the amorphous PVDF, given the fact that this peak is also visible in PVDF melts [−1), two peaks associated with TTT conformation of β or γ phase , and a weak amorphous PVDF peak (600 cm−1), were seen  together with relatively weak peaks from TTT conformation , indicating a combination of major α phase and very minor β/γ polar phase. The deconvoluted diffraction peaks corresponding to 110/200β and 021γ (2θ = 20.8°) are well documented in −1 β/γ peak in FTIR became more intense, and the 110/200β and 021γ diffraction peak could be well identified from the original WAXD profile. The induced β/γ phase primarily originated from the confined crystallization of PVDF that was fully covered by c–PMMA shell. An extra homogeneous crystallization peak at 62 °C, together with a normal crystallization peak at 133 °C, was clearly seen in DSC. With further increasing the PMMA content, almost all peaks assigned to TGTG’ conformation disappeared in FTIR. Meanwhile, only the diffraction peaks of β/γ phase could be seen in the original WAXD profile, indicating that nearly pure polar PVDF phase was achieved in each core–shell particle. Here, although there may be some contribution from the polar confined environment, since polar substrate also could induce the β/γ phase, it was not the main reason because fast homogenous nucleation would limit the influence of dipole–dipole interaction.Intriguingly, because the diffusion of PMMA was prohibited by the crosslinked network, the polymorphous behavior of PVDF@c–PMMA was totally different from the other two samples. From the FTIR spectrum of PVDF@c–PMMA 77/23, we observed some strong absorption peaks from TGTG’ conformation  (P(VDF–TrFE–CTFE)) [It is worth stressing that those PVDF@c–PMMA particles might be promising as novel ferroelectric polymers in electrical energy storage. Compared to our previous work , i.e., PE–CTFE)) .Based on the previous results and discussions, a schematic is given in For PVDF/PMMA, the diffusion of PMMA to PVDF was triggered after the latex going through ultrasonic mixing procedure, the pre-mixed part before melting was fairly limited .Tc of PVDF shifted to lower temperature and the crystallization peak gradually vanished with a decreased peak intensity as the PMMA content increased. Meanwhile, higher content of β phase with lower crystallization capacity was obtained, indicating that the crystallization of PVDF was significantly influenced by PMMA because of the intermolecular interactions. Moreover, at the same PMMA content, PVDF in PVDF@PMMA blends exhibited higher percentage of β phase and lower crystallization capacity than in PVDF/PMMA. In other words, the crystallization behavior of PVDF was more affected by PMMA in PVDF@PMMA. We attributed this to a better PMMA/PVDF chain intermixing or miscibility in PVDF@PMMA, as also evidenced by a more pronounced suppression of crystallization and a larger interchain spacing between neighboring PVDF chains. To the best of our knowledge, this is the first report that high miscibility between PVDF and PMMA could favor the formation of polar β/γ phase. On the contrary, the crystallization behavior of PVDF in PVDF@c–PMMA is much different from the cases in the other two blends, due to a crosslinked and immobile PMMA phase. When PVDF seeds were not fully wrapped by PMMA, the Tc and the crystal polymorphism were slightly changed, which mainly originated from the dipole–dipole interactions between PVDF and isolated PMMA domains. After a full PMMA coverage was achieved, the nanoscale confinement led to an exclusive homogeneous crystallization, resulting in the formation of β/γ phase. Moreover, the PMMA shell would break large ferroelectric domains into nano-domains, resulting in novel relaxor ferroelectric behavior with high dielectric constant and low hysteresis, which is desirable for various applications such as energy storage, electrocaloric cooling, electrostrition, and so on.By employing emulsion polymerization of MMA in the presence of PVDF latex seeds, three series of latex blends featuring different levels of miscibility between PVDF and PMMA were designed and prepared. Thereafter, the crystallization behavior of PVDF in these blends was systematically studied. In miscible systems (PVDF@PMMA and PVDF/PMMA), the


CSN1S1, CSN1S2, CSN2, and CSN3) and making a preliminary assessment of their effect on the gene expression (on the mRNA and protein levels) and milk composition traits in selected horse breeds. Altogether, 23 polymorphisms (21 described previously SNPs and two novel InDels) were found in the studied sequences, the majority of which are common in various horse breeds. Statistical analysis revealed that some are putatively associated with gene expression or milk composition — for example, the c.-2047_-2048insAT polymorphism (CSN1S1) turns out to be related to the total milk protein content in Polish Primitive Horse (p < 0.05), whereas c.-2105C>G SNP (CSN2) is related to beta-casein relative mRNA level and milk lactose concentration in the Polish Coldblood Horse breed (p < 0.05). We have also found significant effects of horse breed and lactation time-point on gene expression and mare’s milk composition. Our study indicates that the 5’-regulatory regions of genes encoding casein proteins are interesting targets for functional studies of their expression and the composition traits of mare’s milk.Genes encoding casein proteins are important candidates for milk composition traits in mammals. In the case of the domestic horse, our knowledge of casein genes is limited mainly to coding sequence variants. This study involved screening for polymorphism in 5’-flanking regions of four genes encoding equine caseins (The online version of this article (10.1007/s13353-018-0473-2) contains supplementary material, which is available to authorized users. Lama glama), the camel (Camelus dromadarius), and the domestic horse   because these remain the main sources of milk consumed by humans throughout the world. Years of intense investigations have revealed the presence of numerous polymorphic variants of casein genes and proteins. Some of these have turned out to be associated with different milk composition traits and important physicochemical properties  casein , and which results in frequent exon skipping events. The best-documented experiment regarding alternative splicing forms in equine caseins is that of Lenasi et al. , who dems1 (CSN1S1), αs2 (CSN1S2), and β (CSN2) caseins is forming the micelles — macromolecular structures responsible for the transfer of calcium to the newborn. κ-casein (CSN3) is thought to be the factor that stabilizes the micelles. Moreover, investigations of human breast milk have indicated that CSN3 may play an important protective function against Helicobacter pylori infections in infants , the number of investigations into equine casein genes and proteins continues to increase. This is related to the several important roles attributed to casein proteins. The most recognizable biological function of the αCSN1S2 polymorphic variants A and B (caused by a 1.3 kb deletion spanning two coding exons) may have an effect on the alpha-s2 casein concentration in equine milk . The major aim of our experiment was molecular characterization of equine casein loci including screening for polymorphisms in their 5’-flanking regions and assessment of their expression levels. We also determined the potential effects of found 5’-flanking variants on both — gene expression and mare’s milk composition. Finally, we assessed the changes in equine casein gene expression (on the mRNA and milk protein levels) between various horse breeds and at three lactation time-points .n = 8), Polish Coldblood Horse , Polish Warmblood Horse , Arabian , Thoroughbred , Welsh Pony , Shetland Pony , Haflinger , Percheron , Fiording , Silesian , and Hucul . The samples were derived from the collection of the Horse Genetic Markers Laboratory at Poznań University of Life Sciences , where they were previously used for parentage analysis. For each gene studied, the two overlapping fragments of the 5’-flanking region were PCR amplified. The obtained amplicons harbored 1115 bp, 1036 bp, 1113 bp, and 1137 bp of the CSN1S1, CSN1S2, CSN2, and CSN3 genes, respectively. PCR primers were designed using Primer3 software , for which the gene expression and milk composition traits measurements were carried out.All animal procedures in this study were approved by the National Commission for Ethics of Animal Experimentation, Local Ethics Committee for Animal Research .n = 20), PWH (n = 27), and PCH (n = 27). The animals were derived from the four Polish national horse studs , where they were kept in congenial environmental conditions. The milking procedure was carried out in the morning (7–9 a.m.), providing visual contact between mothers and foals. To obtain the best characterization of changes in gene expression (and milk composition) during lactation, each mare was milked three times , yielding a total of 222 milk samples. The collected milk was partly (15 ml) frozen in liquid nitrogen for relative transcript level analysis, and the remaining part was stored at − 20 °C for the milk composition studies.Milk samples (100 ml each) were collected manually from 74 mares representing three horse breeds: PPH  and four internal control genes  were determined. The methodology of selecting the reference genes was described in our previous paper , following to the methodology described in detail in our previously published article . Afterwards, the fat layer was removed and 3 ml of the solubilized sample was diluted (1:3 proportion) with a solution containing guanidine hydrochloride and solvent A . All samples were then filtered through a nylon filter. All four caseins concentrations were measured using an Agilent 1100 Series RP-HPLC . The separation was carried out at ambient temperatures on a Jupiter column 5μ C18 300A . The peaks obtained in this manner were compared with proper bovine casein protein standards .The concentrations of the major components of the milk  were measured using an automated infrared analysis with a Milkoscan FT2 instrument . The four milk casein protein concentrations were assessed by high-performance liquid chromatography (HPLC), using the methodology described in our recent study of equine alpha-s2 casein variants A and B , followed by the Tukey–Kramer adjustment. Genotype groups containing less than five horses (15 measurements) were excluded from the association study. All the statistical analysis was carried out using the SAS 9.3 package .The restricted maximum likelihood (REML) method was used to estimate the unknown variance components. Initially, the importance of each factor included in the statistical model was examined using the Kruskall–Wallis and Friedman tests. A model encompassing the fixed effect of breed and sampling time  as a repeated-measure factor was used in order to verify the potential effect of the casein gene polymorphisms on their relative transcript abundances (measured in milk somatic cells), the milk’s content of particular caseins, and the milk’s composition . Hypotheses were tested with the CSN3 gene sequence. The majority of these kappa-casein gene variants were widely distributed across the horse breeds , all the detected variants were present in at least one of the three breeds  for which the gene expression and milk composition analyses were carried out. However, many of these ultimately had to be removed from the association studies on account of their very low frequencies.In total, 23 polymorphic sites, 21 SNPs  and two novel InDels, were detected in the 5’-flanking regions of the four equine casein genes. Over one third of these sites (8) were found within the CSN3 and one of the three CSN1S2 polymorphisms. However, in several cases, the full LD was detected even between variants located in the most distant genes (CSN3 and CSN1S1)  chromosomal region (ECA3). We have therefore checked for potential linkage disequilibrium (LD) between the investigated SNPs using HaploView software  revealed the highest amount of alpha-caseins (CSN1S1 and CSN1S2) in PCH, while the CSN2 and CSN3 proteins were most abundant in the PPH breed (Table p < 0.01) between weeks 5 and 10 and remained stable until the last time-point (week 15). The concentration of the CSN2 protein was almost constant over the whole experiment range, whereas the concentration of CSN3 showed a significant decrease between weeks 5 and 15 (p < 0.05)  showed various tendencies for each casein concentrations. The amount of CSN1S1 in milk increased significantly  than did the ins/ins and ins/del genotypes (1.83 g/100 mL), p < 0.05. The heterozygous GC genotype in the c.-2105C>G SNP of the CSN2 gene was associated with an increased mean beta-casein relative transcript level and lower milk lactose content, compared to the CC homozygotes in the PCH breed . Finally, within the PPH breed, the c.-3669G>C GG genotype carriers  show a significantly (p < 0.05) elevated mean kappa-casein (0.79 g/L) and total milk protein content (1.77 g/100 mL), as compared with horses bearing other genotypes. Moreover, an increased CSN3 protein content was noted for the CC genotype of c.-2925C>G SNP (p < 0.05). No significant relationship with gene expression or milk composition traits was recorded for the CSN1S1 gene 5’-flanking variants. The detailed results of the association studies are presented as The association study of those polymorphisms that occurred with sufficient frequency within each horse breed  revealed several statistically significant relationships. In the case of the Although equids (horses and donkeys) are currently considered minor dairy species, research indicates that the consumption of equine milk is one of the oldest forms of horse utility  estimated for particular casein amounts in cows’ milk  in equine milk is variable, even within the same horse breed  and ~ 0.7 polymorphic site per 100 bp (CSN3). The vast majority of the genetic variants were present in a large number of horse breeds, which may suggest that these polymorphisms are phylogenetically old. Interestingly, many of these variants were present even in small indigenous horse breeds that originated from a very limited number of founders, such as the PPH and the HUC breeds . A similar high level of casein gene coding sequence variation was described recently for the Icelandic Horse breed, which is also considered to be an old, indigenous horse breed with a closed registry (no outside blood is accepted) . However, the observed profiles of gene expression measured at the relative mRNA stage were different from the results assessed at protein level. These results are in agreement with the position of many earlier papers, indicating there is no linear relationship between mRNA abundance given the protein synthesis rate. This mainly due to the diversity of translation efficiency and the numerous mechanisms that affect gene expression processes  are needed to fully characterize the functionality of equine casein gene 5’-flanking regions.JC: Experimental design, animal genotyping, results data analysis, manuscript preparationMM and LW: Molecular technique optimization, animal genotyping, manuscript revisionPP: Gene expression studies, manuscript revisionGC-R and JW: Basic milk composition analyses, manuscript revisionKP and BK: Milk casein fraction analyses, manuscript revisionAB: Statistical analysis, manuscript revisionESM 1(PDF 115 kb)ESM 2(PDF 430 kb)ESM 3(PDF 401 kb)


Background and study aims The standard radiofrequency ablation (RFA) protocol for Barrett’s esophagus (BE) encompasses an intermediary cleaning phase between two ablation sessions. A simplified protocol omitting the cleaning phase is less labor-intensive but equally effective in studies based on single ablation procedures. The aim of this study was to compare efficacy and safety of the standard and simplified RFA protocols for the whole treatment pathway for BE, including both circumferential and focal devices.Patients and methods We performed a retrospective analysis of prospectively collected data on patients receiving RFA between January 2007 and August 2017 at two institutions. Outcomes assessed were: 1) complete remission of dysplasia (CR-D) and intestinal metaplasia (CR-IM) at 18 months; and 2) rate of esophageal strictures.Results One hundred forty-five patients were included of whom 73 patients received the standard and 72 patients received the simplified protocol. CR-D was achieved in 94.5 % and 95.8 % of patients receiving the standard and simplified protocol, respectively (P = 0.71). CR-IM was achieved in 84.9 % and 77.8 % of patients treated with the standard and simplified protocol, respectively (P = 0.27). Strictures were significantly more common among patients who received the simplified protocol (12.5 %) compared to the standard protocol . The median number of esophageal dilations was one.Conclusion The simplified RFA protocol is as effective as the standard protocol in eradicating BE but carries a higher risk of strictures. This needs to be taken into account, particularly in patients with higher pretreatment risk of strictures, such as those with esophageal narrowing from previous endoscopic mucosal resection (EMR). Barrett’s esophagus (BE) is a premalignant condition characterized by the replacement of the native esophageal squamous epithelium by columnar epithelial with intestinal metaplasia (IM)2for circumferential RFA or two ablations at 15 J/cm2for focal RFA) is removed with cleaning devices, followed by a second round of ablation with an energy setting identical to the firstRFA protocol varies between institutions. The most commonly applied protocol incorporates a cleaning phase, whereby debris from the ablated mucosa (one ablation at 12 J/cm2-clean-1 × 12 J/cm2) versus simplified (2 × 12 J/cm2) protocol without cleaning were found to be equally effective based on subjective assessment of the percentage of BE regression at follow-up2-clean-2 × 15 J/cm2) or simplified (3 × 15 J/cm2-no clean) focal protocols and found no significant difference between the twoTo reduce procedural time and increase tolerability of RFA, van Vilsteren et al performed two randomized studies to evaluate the efficacy of a “simplified protocol” which omits the cleaning phase. In the first study, circumferential ablations with standard (1 × 12 J/cm2-no clean) achieved CR-D and CR-IM in 94 % and 87% of patients, respectively, but were complicated by strictures in 11 % of patients, who required a median of two dilation sessions2for the simplified protocol was thought to be too aggressive and following a consensus meeting in Amsterdam, a lower dose of 3 × 12 J/cm2was suggested. Therefore, most European centers have now adopted the lower 3 × 12 J/cm2dose for focal ablations. Currently there is still no universally defined regimen for RFA for dysplastic BE and data are lacking on efficacy and safety of the two protocols when they are assessed over the course of the entire RFA treatment pathway. The aim of this study was to investigate efficacy and safety of the standard and simplified protocols for treatment of BE including both circumferential and focal treatments. We hypothesize that the simplified protocol will be as effective as the standard protocol in achieving CR-D and CR-IM but may carry a higher risk of strictures.Following this evidence, many centers have adopted the simplified protocol as it is easier and better tolerated by patients, however, the randomized studies only referred to efficacy and safety of individual treatments and not the overall treatment pathway. A retrospective study of 83 patients showed that multiple treatments over time with the simplified protocol  or high-grade dysplasia (HGD) and residual BE post-endoscopic mucosal resection (EMR) for low-risk BE-related early cancer. BE was defined as presence of endoscopically visible columnar epithelial with biopsy confirming IM. For LGD, RFA was performed once LGD was confirmed by two gastrointestinal pathologists on two separate endoscopies. For flat HGD, RFA was performed after histological confirmation from a single endoscopy by two gastrointestinal pathologists. Low-risk intramucosal carcinoma (IMC) was defined as well-differentiated IMC  without lymphovascular invasion. Dysplasia was classified according to the Vienna ClassificationPatients were scoped with GIF-FQ260Z, GIF-H290Z or GIF-HQ290 endoscopes . BE was graded according to the Prague classificationRFA was performed using the BARRX ablation devices . RFA was repeated at roughly 3-month intervals. Circumferential ablation was performed with an appropriate-size balloon selected after estimation of esophageal diameter using a sizing balloon. Sizing and treatment balloons were introduced blindly over a guidewire. In patients with previous EMR, a smaller balloon was used as per manufacturer’s recommendation.2, followed by cleaning of mucosal slough with esophageal cap, patient extubation, cleaning of ablation device with wet gauze, and finally a second ablation at 12 J/cm2(1 × 12 J/cm2-clean-1 × 12 J/cm2). Focal ablation using the standard protocol was performed with two ablations at 15 J/cm2, until coverage of the whole segment, followed by cleaning of the mucosal slough with the focal device, patient extubation, cleaning of the RFA device with wet gauze and finally further treatment with two ablations at 15 J/cm2per BE area (2 × 15 J/cm2-clean-2 × 15 J/cm2).Circumferential ablation with the standard protocol was performed for each segment of the BE starting from the proximal end with one ablation at 12 J/cm2without cleaning (2 × 12 J/cm2-no clean) for each segment of the BE until coverage of the whole BE area. Focal ablations were performed with three consecutive ablations at 12 J/cm2without cleaning (3 × 12 J/cm2-no clean). Focal ablations were performed using the BARRX 60, BARRX 90, BARRX Ultra and BARRX Through-the-scope (TTS) devices.Circumferential ablation using the simplified protocol was performed with two consecutive ablations at 12 J/cmDuring follow-up, argon plasma coagulation was performed at the discretion of the endoscopist to ablate small BE-islands. Uptake of the simplified protocol varied between the institutions with Cambridge adopting the simplified protocol from November 2012, while Nottingham adopted the simplified protocol in January 2015. This change in practice led to some patients receiving a hybrid protocol with the standard protocol during early treatments and the simplified protocol during later visits. In this study, we only included patients who were treated exclusively with either the standard or simplified protocols. We also excluded patients who were treated with the more recent BARRX Express device, which does not require sizing of the esophageal lumen. These cases were excluded due to lack of agreement among users about the optimal protocol to be used, and because of the variation in practice even within the same endoscopy unit.The interval between follow-up post-RFA was dependent on baseline histology. Patients with LGD were seen at 6 months following complete pathological response and then at 12 months and every 2 years thereafter. Patients with HGD or IMC were seen at 3 months following complete pathological response, and then at 6 months, and annually thereafter. At follow-up, white-light and narrow-band imaging were used to inspect the gastroesophageal junction (GEJ) to identify BE islands. Quadrantic biopsies were taken at the GEJ to detect IM or dysplasia and every 2 cm within the neo-squamous epithelium to detect buried IM. If IM was found at the GEJ at the first post-RFA follow-up, circumferential ablation of the GEJ was performed with focal BARRX device. Focal IM in a single GEJ biopsy at the second or subsequent endoscopic follow-up was not considered BE recurrence and was not an indication for repeat RFA.Outcomes measure were: 1) complete remission of dysplasia (CR-D) and complete remission of IM (CR-IM) at 18 months; and 2) rate of esophageal strictures. CR-D was defined as eradication of dysplasia within 18 months, but with persistent columnar-lined epithelium with IM. CR-IM was defined as complete eradication of both dysplasia and IM. Complete remission of dysplasia or IM was defined as absence of dysplasia or IM on two consecutive biopsies from the last RFA session. We also assessed the rate of strictures post-RFA. Stricture was defined as symptomatic esophageal narrowing requiring dilation to improve symptoms or to allow endoscope passage. Asymptomatic narrowing passed by the endoscope was not considered strictures. Patients were only considered to have an RFA-related stricture if the stricture developed after commencing RFA. Patients who had previous stricture secondary to prior-EMR were not considered to have a stricture, unless the stricture recurred after RFA. Esophageal dilation was performed with a through-the-scope device with the aim of providing symptomatic relief rather than achieving a specific size of the esophagus. The size of the dilatation balloon was selected based on the estimated diameter of the esophagus. If required, open biopsy forceps were used to size the stricture. We also assessed whether age, gender, maximum length of BE, prior-EMR, extent of prior-EMR, baseline histology, type of RFA protocol, and different ablation devices were predictors of stricture formation. For patients who received prior-EMR, the number of EMR specimens performed during a single session was recorded as a continuous variable and acted as a surrogate for the extent of prior-EMR.t-test and Mann-Whitney test were used to compare means between groups. Rates of CR-D, CR-IM and strictures between groups were compared using Chi-squared test. Binary logistic regression was used to assess for predictors of stricture formation.P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS .Normality testing was performed using the Shapiro-Wilk test. For descriptive statistics, mean  was used for parametric variables and median  was used for non-parametric variables. Independent student’sIn total, 311 patients underwent RFA between July 2007 and August 2017. Of them, 166 patients were excluded due to ongoing RFA treatment (n = 102), combination protocol (n = 20), loss to follow-up (n = 18), RFA for non-Barrett’s disease (n = 11), BARRX Express device used (n = 8), treatment interrupted because of other co-morbidities (n = 6) and patient refusal of further treatment (n = 1) . A totaWhen stratified by protocol type (standard vs simplified), there was no significant difference between the groups in terms of age, gender ratio, BE maximum length, rate of rescue EMR and number of RFA sessions within 18 months. There was no significant difference between the groups in terms of grade of dysplasia, however, the number of LGD cases in the standard protocol group was smaller, in keeping with the fact that RFA for LGD was only recently approved by the British Society of GastroenterologyP = 0.71). Similarly, there was no significant difference in CR-IM between the standard (84.9 %) and simplified (77.8 %) protocol (P = 0.27). Twelve patients (8.3 %) developed visible nodular lesions during follow-up and required rescue EMR. Among them, only two patients had their BE upstaged. One patient had RFA performed for LGD but required rescue EMR 6 months later, which showed HGD. This patient achieved CR-IM at the end of the protocol. Another patient with baseline HGD required rescue EMR, which showed upstaging of disease to IMC. This patient did not achieve CR-D at the protocol endpoint. The remaining 10 patients who received rescue EMR did not have their disease upstaged  and simplified (95.8 %) protocol (pstaged .P = 0.008)  developed esophageal strictures. One patient in the standard protocol group developed a stricture  compared to nine patients  in the simplified protocol group ( 0.008) . A mediP = 0.029). Extent of prior EMR had no association with risk of developing strictures . With regard to the type of device used, we considered a total of 371 ablations performed. Type of ablation device  also was not associated with risk of stricture (P = 0.78)  .We observed serious adverse events (SAE) in two patients treated with the standard protocol. One patient developed symptomatic atrial fibrillation within 36 hours after two ablations  and required pharmacological cardioversion. Another patient was admitted within 48 hours after RFA with severe chest pain and dysphagia, which was not related to esophageal stricture and was managed conservatively with analgesia and intravenous fluids. We also observed one SAE in a patient treated with the simplified protocol who developed melena 48 hours after RFA. Gastroscopy showed no evidence of active bleeding and he was discharged after observation for 24 hours.2-cleaning-2 × 12 J/cm2) resulting in a higher rate of BE eradication2-cleaning-2 × 15 J/cm2) resulted in better efficacy without compromising the complication rate2-cleaning-2 × 12 J/cm2was still favored in US-based studies for focal ablationRFA for BE has been widely available for over a decade, however, there is geographical variation in the protocol used in clinical practice and there remains uncertainty about the optimal energy dosimetry. Early studies of focal RFA used a regimen with a single ablation, followed by cleaning of the ablation zone and subsequent application of a single ablationIt was then hypothesized that the intermediary cleaning phase using the esophageal cap could inherently lead to mucosal edema, decreasing efficacy of the second cycle of ablations. In two randomized trials based on single RFA procedures, Van Vilsteren et al showed that the simplified protocol without cleaning for both circumferential or focal RFA was equally effective as the standard protocol2) and standard (2 × 15 J/cm2-cleaning-2 × 15 J/cm2) regimen using focal RFA for dysplastic BE with a pre-specified non-inferiority margin of –15 % and showed that the simplified protocol was non-inferior to the standard protocol in achieving BE surface regression after two ablation sessionsP = 1)Kunzli et al then compared the efficacy of the simplified protocol using focal ablation device only (BARRX 90) for the whole treatment pathway. Once again, the simplified protocol was effective in BE eradication compared to historical literature, but induced esophageal strictures in 11 % patients. Although the median number of dilations required was two, three patients required at least eight endoscopic dilationsHowever, we found a higher number of strictures in patients treated with the simplified protocol. The rate of strictures was significantly higher in the simplified protocol (12.5 %) compared to the standard protocol (1.4 %). This could be secondary to the “stacking” of energy with consecutive ablations which penetrate deeper into the esophageal wall, leading to more extensive damage and stricture development. Although the randomized trial by Pouw et al showed no significant difference between the rate of esophageal strictures, that study was powered to detect a difference between BE surface regression, and not to detect a difference between the rate of strictures. Further, this study also did not include patients undergoing circumferential ablationThe higher stricture rate for the simplified protocol needs to be considered in cases that have a higher pretreatment probability of stricture, such as those with preexisting stricture from previous EMR who could have already been treated with dilatation. For these cases, the standard ablation protocol could be used. The ablation device used in our study was the older-generation BARRX 360 circumferential device with separate sizing balloon, which is no longer commercially available and has been replaced by the BARRX 360 Express catheter. The findings from our study have potential implications for the new BARRX 360 Express device, which is longer and more compliant to the esophagus than the BARRX 360 circumferential device and has been shown to correlate in early clinical experience to a high rate of stricturesIn our current study, we selected a more liberal RFA protocol duration of 18 months compared to 12 months for the UK registry, and in other prospective studies, as we felt that this more accurately reflects the high demands and prolonged waiting list for endoscopy seen in large UK tertiary hospitalsOur study does have some limitations. First, it was not powered to detect a difference in stricture rate between the groups. Second, we only assessed short-term outcomes of RFA. Although we applied a rigorous protocol with two consecutive biopsies to confirm CR-D or CR-IM, overall follow-up was relatively short, therefore, our data are insufficient to provide information on the durability of RFA comparing these two protocols. However, the long-term durability of RFA in one of the two centers has been reported elsewhereIn conclusion, we showed that the simplified RFA protocol for the whole treatment pathway, including both circumferential and focal ablations, is just as effective as the standard protocol. The added benefit of fewer introductions of the device, shorter procedural time, and likely less discomfort for patients makes it favorable. However, the higher rate of strictures needs to be considered when making decisions on an individual basis, especially in patients with higher pretreatment probability of stricture, such as those with esophageal stenosis from previous EMR.


Bone is a highly vascularized and resilient organ with innate healing abilities, however some bone injuries overwhelm these attributes and require intervention, such as bone tissue engineering strategies. Combining biomaterials and growth factors, such as bone morphogenetic protein 2 (BMP2), is one of the most commonly used tissue engineering strategies. However, use of BMP2 has been correlated with negative clinical outcomes including aberrant inflammatory response, poor quality bone, and ectopic bone.n-acetyl glucosamine  scaffold was utilized in addition to the commonly used acellular collagen sponge (ACS) BMP2 delivery system in a murine calvarial defect model to investigate whether the innate properties of Talymed can reduce the noted negative bone phenotypes associated with BMP2 treatment.In the present study, a novel poly-Comparison of murine calvarial defect healing between ACS with and without Talymed revealed that there was no measurable healing benefit for the combined treatment. Healing was most effective utilizing the traditional acellular collagen sponge with a reduced dose of BMP2.The results of this investigation lead to the conclusion that excessive dosing of BMP2 may be responsible for the negative clinical side effects observed with this bone tissue engineering strategy. Rather than augmenting the currently used ACS BMP2 bone wound healing strategy with an additional anti-inflammatory scaffold, reducing the dose of BMP2 used in the traditional delivery system results in optimal healing without the published negative side effects of BMP2 treatment. Bone is a highly vascularized and resilient organ with innate healing abilities, however some bone injuries overwhelm these attributes and thus require significant medical intervention . TherapeCurrently bone tissue engineering uses cell therapy, biomaterials/scaffolds, and/or manipulation of osteogenic signaling pathways to overcome failures in the treatment of bone wounds –9. CombiACS releases BMP2 at a rate that is unable to sustain quality bone growth , 20. ConIn the present study, the novel Talymed scaffold was utilized in addition to the approved ACS BMP2 delivery system to investigate whether the innate properties of Talymed can reduce negative outcomes associated with the current BMP2 delivery system. Due to the enhanced BMP2 binding and anti-inflammatory qualities of Talymed, we hypothesize that bone regenerated using combined ACS/Talymed as a BMP2 delivery system will yield better healing outcomes as compared to the current clinical standard scaffold (ACS) alone.2O), medium (325 ng), high (542 ng) or scaled clinical (5000 ng) doses of BMP2 (Table Adult (8 week old) male and female C57BL6 mice (n = 80)  were randomized into one of eight surgical groups implanted with ACS alone or ACS overlaid with Talymed  matrix soak loaded with control , in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility where all husbandry and related services were provided by the Division of Laboratory Animal Resources. The animals were housed 5 to a cage which contained enrichment material and were allowed access to food and water ad libitum. All procedures and the reporting thereof comply with the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines [For each specimen, a critical-sized calvarial defect was performed as previously described , 26–28. idelines .Collected skulls were removed of soft tissue and bisected from the occipital protuberance to nasale in the transaxial plane. Micro-computed tomography (µCT) images of the defect areas were obtained via ex vivo Skyscan 1176  with a 0.5 mm thick aluminum filter at a voltage of 50 kV and current of 500 µA. Data were acquired at an isotropic resolution of 18 µm, rotation step of 0.5°, and 180° rotation. Reconstruction and analyses were performed on the blinded samples using NRecon and CTAn SkyScan software. A global threshold of 100–255 was used. Standard 3D morphometric parameters  were detBlinded, representative samples (n = 4) from each group were bisected in the coronal plane through the center of the surgical defect and prepared for paraffin sectioning by fixing in 3.7% formaldehyde for 2 days. Subsequently, samples were decalcified in 0.25 M EDTA at pH 7.4 for 21 days and then washed, dehydrated in graded ethanol (70–100%), cleared in xylene, and embedded in paraffin. Histology was performed on 3, 7 µm sections in the coronal plane at least 40 μm apart per sample for analysis of the defect area. Standard procedures were employed for hematoxylin and eosin, Masson’s trichrome , and picro sirius red (non-polarized and polarized) staining , 9. StaiTwo-way ANOVA Scaffold x Treatment with post hoc Bonferroni analyses were conducted for all comparisons where appropriate. Transformations of the data were performed where needed to correct for violations and meet the assumptions of homogeneity of variance or normality. Non-parametric analyses were utilized where necessary for unavoidable violations of assumptions. Differences were considered significant if p ≤ 0.05. Data are presented as mean ± SEM. A power study for experimentation was calculated based on an α = 0.05, β = 0.8, η > 0.4, necessitating 10 animals per group. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.Analysis of healing within the calvarial defects using standard morphometric measures of µCT scans revealed no difference between the ACS alone and ACS combined with the novel Talymed scaffold  Fig. a, b. TheHistological staining of the defects allowed for a more specific assessment of the healing process. Observation of the morphology via hematoxylin and eosin staining of the defect areas for the ACS/Talymed group indicates areas devoid of cells, and areas that lack the organization associated with both scar and bone Fig. a. As newMasson’s trichrome staining was used to quantify osseous healing within the defects. Assessment of the area of osteoid indicates that more healing occurred within the ACS only defects as compared to the ACS/Talymed defects (p = 0.032) Fig. . The mosPicro sirius red staining was used to assess collagen within the healing defects. A greater area of total collagen was found within the ACS only defects as compared to the defects filled with combined ACS/Talymed (p < 0.001). BMP2 treatment also precipitated a greater area of total collagen as compared to control (p ≤ 0.003). An interaction between the scaffold and the BMP2 treatment can be observed with the reduction of total collagen in the ACS only scaffolds with increasing BMP2. At the highest dosage of BMP2, the defect areas are disorganized in both scaffolds as compared to the dense nature of the tissue within the healing defects treated with the lower doses of BMP2 Fig. a, b. PolOur hypothesis that adding an overlay of Talymed to the currently employed ACS BMP2 delivery system as a means of reducing negative outcomes was refuted by our µCT analysis. The combined scaffold did not produce better healing or more bone fill than the traditional delivery system. This outcome agrees with previous studies that found that the osteoinductive BMP2 along with an osteoconductive matrix such as ACS precipitates the greatest amount of bone growth , 31, 32.The importance of BMP2 dose was highlighted in our more specific histological assessment of healing. Despite the noted large acellular areas, there was greater cellular infiltration in the combined ACS/Talymed group. Though Talymed has been shown to augment healing in cutaneous wounds by enhancing cellular activity and possessing anti-inflammatory and non-immunogenic properties, these characteristics do not seem to translate into quality bone regeneration , 23, 24.Assessment of the collagen within the healing defects again indicated the superiority of the ACS scaffold without the addition of Talymed. Not only did the ACS scaffold present the most total collagen, it also included the most of all types/maturity levels of collagen fibers. The lower doses of BMP2 included fibers of all maturity levels indicating areas of active turnover and remodeling. The clinical dose of BMP2, on the other hand, presented with the least amount of all fiber types including mature fibers. This may indicate that healing and turnover have ceased in these areas resulting in maintenance of the acellular space, ectopic bone, and a reduction in overall collagen matrix.Though Talymed can retain BMP2 better than ACS, this slower release may not be as important as BMP2 dose for healing , 12, 33.


It was discovered that the 1D topological edge states are present in single-unit-cell FeSe film on SrTiO3, but are absent when more than 50% Se atoms are replaced by Te atoms. Stress induced displacive phase transformation exists in FeSe/SrTiO3 film when Te atoms substitute Se atoms in the bottom (top) layer in single-layer FeSe under 3% strain respectively. The 1D topological edge states are present under 3% (1.8%) strain in FeSe/SrTiO3 films with Te substitution Se in the bottom (top) layer in single-layer FeSe, even up to 5%, respectively. This indicates that the bonding angle of Se-Fe-Se (Te) and the distance of Te (or Se) atoms to the Fe plane are correlated with the topological edge states. Our findings provide an effective interface system that provides both superconducting and topological states, opening a new route for realizing 2D topological superconductors with proximity effect.Using first principles theory, we investigated the behavior of the one-dimensional (1D) topological edge states of high temperature superconductiviing FeSe/SrTiO The Fermi surface of single-layer FeSe/STO have been measured by angle-resolved photoemission spectroscopy (ARPES) experiments, showing electron pockets at the M point and no hole pockets at the Γ point. This experimental observation is inconsistency with theory in band structure. Despite intense experimental9 and theoretical efforts10 have been devoted, some of the essential fascinating new-style class of 2D superconductor is still elusive. Recently, much attention has been paid to the potential being of topological phases in FeSe13. Theoretical and experimental results have presented for an antiferromagnetic (AFM) quantum spin Hall (QSH) state in FeSe14. Previous low-temperature scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) study have demonstrated that the superconducting transition temperature (Tc) increases with the substitution of Se composition by Te which reaches 14.5 K at about 40% Se substitution16, which is higher than the maximum Tc ~9 K of bulk FeSe. 1UC FeTex1−Sex films (0 < x < 0.6) on STO(001) substrates were prepared by molecular beam epitaxy (MBE) and the superconducting properties were studied by in situ STS study and ex situ transport measurement17.Single-unit-cell (UC) FeSe films have been successfully grown on SrTiO14, it’s necessary to test the potential existence of topological phases in high-temperature superconductiving FeSe/STO films with Te substitution to Se. The properties of FeSe films with Te substitution could be affected by the key factors including interfacial strain, structural deformation, interfacial charge transfer and so on. However, the interfacial strain is the most dominant factor amongst these. The in-plane lattice constant of FeSe exactly matches the substrate with a perfect coherent interface. While Se atoms are substituted by Te, strain will be induced to the film, so that strain is suggested to play an important role in affecting the interaction of topological band states.Considering the fact of both theoretical and experimental results for an AFM QSH state in FeSeMotivated by these recent discoveries, we have carried out a systematic study of the topoelectronic properties of 1UC FeSe/STO films with Te substitution as a function of concentration change of Te substituting and strain, based on first-principles calculations. More than 50% Te substitution Se atoms in both top and bottom layers in single-layer FeSe will lead to the disappear of the 1D Dirac edge state. These calculation results allow us to conclude that tensile strain induces displacive phase transformation when Te atoms substitute Se atoms in the bottom (top) layer in single-layer FeSe under 3% strain respectively. 1D topological edge states of 1UC FeSe/STO films with Te substituting Se on bottom (top) layer is presented under tensile strain 3% (1.8%), respectively, even up to 5% strain. This study provides a good foundation for experimental study that leads to potential applications in spintronics.14. In our calculation, we assumed that the bottom Se atom in single-layer FeSe is directly above the top of O atom in the STO substrate. Its electronic band structures and topoelectronic properties were calculated with the framework of the PBE-type generalized gradient approximation using VASP package as described before21. Spin orbit coupling (SOC) was included in the self-consistent electronic structure calculations. All calculations were implemented with a plane-wave cutoff of 400 eV on a 9 × 9 × 1 Monkhorst-Pack k-point mesh, and a vacuum layer that is over 15 Å to avoid the interaction between neighboring slabs. For structural relaxation, all the atoms are allowed to relax until atomic forces are smaller than 0.01 eV/Å. The experimental lattice constant is a = 3.90 Å22. In order to test the influence of SrTiO3 antiferrodistortion on surface electronic structures, we chosen 2 × 2 × 1 supercell. Previous study indicated that the band structure of FeSe/STO with chequerboard AFM configuration coincides exactly with the ARPES spectra within the whole Brillouin zone, so that we chosen chequerboard AFM configuration in our calculations (neighbouring Fe atoms have opposite spin directions). Figure 14, so we set the z spin direction for Fe atoms in our calculation. We used the Wannier90 package23 to calculate the topoelectronic edge states of 1D zigzag nanoribbon as described previously21. The tight-binding Hamiltonian with maximally localized Wannier functions (MLWFs) was fitted to the first-principles band structures. The Coulomb repulsion is characterized by a spherically averaged Hubbard parameter U, which depends on the spatial extension of the wave functions. In our calculation the U was set to be 0.6 eV14. Considering that GGA is usually regarded to underestimate the binding in weakly bonded systems of inhomogeneous systems, we used van der Waals (vdw) correction in our calculations and applied the semiempirical DFT-D2 method proposed by Grimme24.The experimental interface configuration of FeSe/STO is complex. There are two possible atomic structures of FeSe/STO: the bottom Se atom in single-layer FeSe is directly above the top of either O or Ti atom in the STO substrate as can be seen in Fig. 2-terminated SrTiO3 (001) surface in six atomic layers, where the bottom Se atom in single-layer FeSe is directly above the top O atom in the STO substrate. The vertical distance between O and Se atoms is 3.188 Å. The bond length between top (bottom) Se and Fe atoms is 2.389 (2.387) Å. The Fe-Se-Fe angle for top (bottom) Se atoms is 109.338° (109.520°). The calculated magnetic moment is 2.37 μB on each Fe atom.We first modeled the interface structure of FeSe monolayer on TiOa and b of FeSe/Te on STO are same. Duo to the effect of substrate, the symmetry of this system would be lowered, the electronic structures maybe have a small difference along MX and MX΄ directions. The corresponding band structures with Hubbard-U and vdw corrections are shown in Fig. 4 and previous calculations22. The outspread flat bands below the Fermi level come from the dz2, dxz and dyz orbitals of the Fe atoms, and the pz orbital of the Se atoms. We demonstrate the band of free standing 1UC FeSe monolayer in Fig. Figure 27. Spin symmetry breaking along the FM edge leads to two asymmetric edge states for both the left and right edges. The edge states with bright lines are presented around Fermi level and Dirac point with the valence and conduction bands connecting is appeared in Fig. 3 substrate and FeSe monolayer induces charge transfer and an interfacial electric field, which slightly modify the relative shape in “bulk” band structures while keep Dirac edge state.1D ferromagnetic (FM) edge states of a FeSe/STO ribbon indicated in Fig. x1−Sex films (0 < x < 0.6) on STO (001) substrates were reported with superconducting properties examined by combined in-situ STS study and ex-situ transport measurements17. In experiments, atom doping is an out of order distribution. The simulation modeling of such disorder is very complicated and time consuming. For simplicity, we firstly assumed that the Te replaces either the bottom or top Se atoms in 1UC FeSe. In this substitution we could distinguish which one between Se and Te atoms is benefit to induce 1D Dirac edge state in this system. Firstly, we consider that Te atoms completely substitute bottom Se atom in single-layer FeSe  Se (Te) and the Fe atoms is 2.368 (2.542) Å. The Fe-Se (Te)-Fe angle for top Se and (bottom Te) atoms is 107.66° (97.92°). The calculated magnetic moment is 2.23 μB on each Fe atom. Te atom substituting changes bond length and bond angle with Fe atom, and decreases interaction in FeSeTe/STO interfacial system slightly.1UC FeTeTe/STO film are shown in Fig. 2 feature, whereas the bands along X- M line still show hole pockets, which are the dxz and dyz features. The Te substitution enhances the feature of electron pockets at Γ point slightly. In addition, this Te substitution induces the spatial inversion symmetry breaking. This is similar to the external electric field induced Rashba band splitting in the surface state of system. The 1D edge states of a FeSeTe/STO ribbon with FM edge shown in Fig. The bands for FeSeTe/STO, as indicated in Supplementary Fig. Te/STO by 0.111 Å. The bond length between top (bottom) Te (Se) atoms and the Fe atom is 2.53 (2.36) Å. The Fe-Te(Se)-Fe angle for top Te and (bottom Se) atoms is 97.92° (107.66°). The calculated magnetic moment is 2.24 μB on each Fe atom which is almost same as 2.23 μB in FeSeTe/STO interface system.Subsequently, we considered Te atom substituting Se atom on the top layer of FeSe  on the band details. In our calculation, we assumed that Te atoms substitute Se atoms (on both top and bottom layers) with 0.25%, 50%, 50% and 75% concentration in single-layer FeSe of 2 × 2 × 1 supercell as shown in Fig. 0.75Te0.25/STO, exhibiting 1D Dirac edge state inside the “bulk” band gap, while others do not show the Dirac edge states. Figure Next we focused on the effect of Te substitution concentration . When the strain increases to 3% (with lattice constant a = 4.018 Å), e.g. the bond length between Te (Se) atoms and the Fe atom is 2.57 (2.40) Å and the Fe-Te(Se)-Fe angle is 102.90° (113.41°). The interfacial distance decreases to 2.943 Å with an increased interface energy of 0.05 eV. The decreased interfacial distance and the increased interface energy enhance the interfacial coupling. In particular, stress-induced displacive phase transformation could be found while the thin film is under a strain of 3%, where the bottom Te atom in single-layer FeSe is directly above the top Ti atom in the STO substrate , two Dirac edge points could formed in 1D band structure of 1UC FeSeTe/STO film as shown in Fig. Te/STO film is also found under a strain of 3%  is calculated by the equation35fn is the Fermi distribution, ψnk is the eigenstate of eigenvalue Te/STO film under 3% strain with a = 4.018 Å. The Chern number (C) is expressed asCs is spin Chern number and it is expressed asThe time-reversal symmetry is preserved in the conventional QSH state while it is broken in the AFM system. However, AFM QSH state is presented if spin-up and spin-down channels are decoupled from each other. 3D AFM topological insulatorsBy integrating the Berry curvatures over the first Brillouin zone (BZ), we can gain the spin Chern number with −1 and then the spin Hall conductance can be obtained from the spin Chern number asCorrespondingly, an AFM QSH state is confirmed with quantized value of spin Hall conductance within the energy range of the SOC gap in Fig. 36. The quantum spin Hall phase could exist until the exchange field strength reaches a critical value. When the bulk band gap closes and then opens, the system enters the quantum anomalous Hall phase37 characterized by 1 (or −1). The non-trivial spin Chern number requires the existence of an edge state at the sample boundary without considering any symmetry36. Spin Chen Number is an effective tool to characterize the different topological phases of quantum spin Hall system without time inversion symmetry.It was found that the QSH state characterized by nonzero spin Chern numbers ±1 persists when the time reversal symmetry is broken with exchange field3 substrate, local tension exists at the interface of heterojunction materials, which leads to charge transfer. This leads to a Rashba effect that is resulted from the internal electrical field which is introduced by interfacial charge transfer when they are grew on the STO substrate. The differential charge density at the interface is defined as ρ = ρFeSe/STO − ρFeSe − ρSTO, where, ρFeSe/STO, ρFeSe, and ρSTO are the charge density of FeSe/STO respectively, and FeSe and STO are at the same surface lattice constant. The differential charge density at the interface of FeSe/STO film is presented in Fig. −3 e per cell from STO substrate to FeSe film. In practice, the actual charge transfer is larger because defects in substrate results in additional charge transfer. The differential charge density at the interface of two mentioned systems FeSeTe/STO and FeSeTe/STO are presented in Fig. Te monolayer has a different charge density due to more electrons transfer from Fe atoms to Se and Te under 1.8% strain. This is confirmed in our Bader analysis showing a charge transfer of 0.03 e from Fe atom to Se and Te atoms. This charge transfer generates an internal electric field at the interface region. The electric field leads to bands along M-X and M-X′ directions with an obvious band splitting around M point. The amplitude of this splitting is enhanced in FeSeTe/STO and FeSeTe /STO films than that in FeSe/STO. In order to further verify the charge transfer and dipole effect of SrTiO3 substrate, we also calculated electronic structure for a free-standing monolayer FeSeTe  and the distance of Te or Se atoms away from the Fe plane are correlated with the topological edge states.The strain-induced different Rashba states, that originate from their different response to strain, can be understood from deformation potential theory3 films without and with Te substitution by first-principles theory. Our calculations show that, although the bands are changed by interface electronic field, the 1D topological edge states appear within the gap without Te substitution. Interfacial Se atom layer is beneficial to form the1D topological edge states. Te substitution to Se atoms larger than 50% in both top and bottom layers in single-layer FeSe will lead to absence of original Dirac point. Stress-induced displacive phase transformation occurs in FeSe/SrTiO3 films with Te substitution under 3% strain. For the case of Te submitting the Se atom on the bottom layer of FeSe, the films on SrTiO3 exhibit no 1D topological edge states with a strain less than 3%. For the case of Te submitting the Se atom on the top layer of FeSe, the films on SrTiO3 exhibit 1D topological edge states under a strain of 1.8–5%. Since the 1D topological edge states of FeSeTe/SrTiO3 film are more sensitive to strain than that of FeSeTe /SrTiO3 film, we propose that Se atom layer should be used as a more practical interfacial atom layer on STO than Te atom layer. We also found that the bonding angle of Se-Fe-Se (Te) and the distance of Te or Se atoms away from the Fe plane are correlated with the topological edge states. These results provide an effective interface system that supports both superconducting and topological states, shedding light on realizing 2D topological superconductors on oxide substrate.In conclusion, we have studied the electronic properties of high-temperature superconductiving FeSe/SrTiOSupplementary Dataset 1


Marine microscopic particles profoundly impact global biogeochemical cycles, but our understanding of their dynamics is hindered by lack of observations. To fill this gap, optical backscattering measured by satellite sensors and in-situ autonomous platforms can be exploited. Unfortunately, these observations remain critically limited by an incomplete mechanistic understanding of what particles generate the backscattering signal. To achieve this understanding, optical models are employed. The simplest of these models—the homogeneous sphere—severely underestimates the measured backscattering and the missing signal has been attributed to submicron particles. This issue is known as the missing backscattering enigma. Here we show that a slightly more complex optical model—the coated sphere—can predict the measured backscattering and suggests that most of the signal comes from particles >1 µm. These findings were confirmed by independent size-fractionation experiments. Our results demonstrate that the structural complexity of particles is critical to understand open-ocean backscattering and contribute to solving the enigma. Particulate optical backscattering is key to studying the oceanic carbon pump though it remains unclear what particles are detected. Here the authors show that complex particles larger than 1 µm help reproduce all the measured backscattering across the Atlantic Ocean and explain the majority of the signal. The biological pump thus modulates atmospheric CO22 and supports deep water ecosystems and the fisheries upon which humans depend. Understanding this pump is of the utmost importance.Microscopic organic particles in the open ocean support a suite of processes, known as the biological carbon pump, which is ultimately responsible for transferring atmospheric CO9. Optical scattering is quantified by the volume scattering function (VSF) that measures the fraction of incident light deflected by particles in a given direction per unit distance10. By integrating the VSF in the forward and backward directions, one obtains the forward- and backscattering coefficients that are the most commonly measured optical proxies for particle concentration. Although the particulate backscattering coefficient (bbp) is only a small fraction (1–2%) of the total scattering (or of the particulate beam attenuation coefficient cp11), it has the great advantage of being directly linked to space-based measurements12. In addition, bbp can be measured in situ by miniaturised instruments installed on autonomous robotic platforms that sample at high vertical and temporal resolution from the surface to the mesopelagic zone (200–1000 m)13. Thus, optical backscattering measurements can be exploited to better understand the biological carbon pump.To achieve this understanding, measurements of the light scattered by marine particles are crucial16 and phytoplankton carbon18, from which algal physiological parameters can be derived19. The bbp spectrum provides a proxy of the particle size20 and can be used to analyse the phytoplankton community structure and derive group-specific carbon biomass21. Yet, the biogeochemical quantities derived from these algorithms suffer from large uncertainties. For example, the performance of POC and phytoplankton carbon empirical models can vary widely18.The particulate optical backscattering coefficient can be used to estimate, through empirical relationships, the concentration of particulate organic carbon (POC)bbp arise due to an incomplete mechanistic understanding of which particles are detected by bbp. The bbp coefficient has been mainly modelled using Mie theory25 assuming that marine particles can be represented as homogeneous spheres. Although this assumption is sufficient to model the forward scattering of particles27, this approach severely underestimates the phytoplankton backscattering30 and the particulate backscattering in the open ocean31. This mismatch between theory and observations is known as the missing backscattering enigma32. To explain this missing backscattering, submicron detrital particles have been suggested as the major source of oceanic particulate backscattering34. Hence, it is currently assumed by many that particulate forward- and backscattering coefficients detect different oceanic particles.Difficulties in interpreting 42 or as non-spherical particles46. Heterogeneous coated spheres represent the simplest way to model the bulk structural complexity of marine particle populations. Coated spheres can reasonably represent the external and internal cellular structures  of marine spherical and non-spherical phytoplankton, and account for differences in their chemical nature42. Coated spheres can also be used to simulate aggregates of living and detrital particles48 and algal colonies49. Coated-sphere models produce backscattering signals that can be an order of magnitude higher than those predicted by Mie theory for the same overall diameter and volume-averaged particle composition . Coated models, however, require parameters that quantify the thickness and chemical composition of each part of the particle and the choice of these parameters is critical32. Non-homogeneous spherical models thus have not been applied to study oceanic backscattering due to the high variability in the size and nature of natural particles. Prudence has been suggested for these more complex approaches32.Alternative approaches have been proposed to model marine particles as heterogeneous coated spheres25, as well as to directly compare our results with previous results based on the homogeneous-sphere assumption27. We then examined the contributions to modelled scattering of different size fractions and confirmed the findings by additional optical measurements collected through independent size-fractionation experiments. Our results show that particles larger than 1 µm contribute the majority of the bbp measured across the Atlantic Ocean and that backward and forward scattering therefore detect different characteristics of particles within similar size ranges. We anticipate our results will open the door to a new way of interpreting marine optical backscattering measurements.Here we show that the open-ocean missing backscattering can be found in the structural complexity of marine particles. We have applied a coated-sphere model to estimate the backscattering of particle populations across the Atlantic Ocean and through mid-ocean gyres  and backscattering (bbp) coefficients  were tightly correlated with measured values regardless of the refractive index (n) assumed  could not reproduce simultaneously the measured particulate beam attenuation  with assigned refractive index (n2)  too low for marine particles 51 or unrealistic (n1 < 1). Combinations with n2 equal to 1.22 were also disregarded because they were too high for algal cellular constituents50. The selected parameterisations thus included 12 combinations of 4% ≤ tk2 ≤ 15% and 1.12 ≤ n2 ≤ 1.20, with n2 decreasing as a function of tk2 according to a power law  Fig. . The ension Fig. . This selaw Fig. . Corresp042 Fig. . These 1cp and bbp coefficients for an independent dataset  > 1.56 × 10−3 m−1), bbp predictions were less accurate , to the ratio between bbp and total scattering , which measures the efficiency with which light is backscattered by particles25. The bbp-to-cp ratios for a coated sphere can be 40 times higher than for homogeneous spheres for particles with diameters between 1 and 12 µm, and 8 times for submicron particles . This modelled value was consistent with those derived from our , it is instructive to attempt to model the scattering of a complex particle by only using homogeneous spheres. Figure 46 that could modify the contribution to the optical backscattering by the different size fractions. In our analysis, we assumed all marine particles to be coated spheres with similar characteristics because we did not have data to distinguish coated from homogeneous particles and to characterise their morphology. However, we performed a sensitivity analysis to understand how many homogeneous and coated spherical particles within the same population could still accurately reproduce the in-situ backscattering measurements. We found that modelled backscattering coefficients reproduced measured values until homogeneous particles represented up to 25% of the total number of particles measured  were accurately predicted when the volume-averaged refractive index was set equal to 1.07 (Bias = −0.01).The coated-sphere model that was parameterised using surface data underestimated the backscattering in the mesopelagic region of the Atlantic Ocean. Figure bbp was higher than 1.56 × 10−3 m−1 , then new information of particle characteristics might be extracted from optical measurements. This new information could also help explain why predictions of POC are less noisy when using algorithms based on cp rather than on bbp16, and why performance of empirical algorithms for phytoplankton carbon varies widely18.A coated-sphere model might therefore disclose additional information on particle characteristics from optical data. This hypothesis is also supported by the higher uncertainties in the modelled backscattering coefficients that were found for the AMT22 samples when m−1 Fig. . These Am−1 Fig. . Algal pm−1 Fig. . Thus, tcp than bbp56. Particle and phytoplankton growth and loss rates can be derived from cp diel cycles57, and the correlation between cp and bbp observed in oceanic waters58 may suggest the same retrievals could also be possible by satellite observations of bbp. However, cp and bbp diel cycles are out of phase59 and diel phytoplankton carbon-specific backscattering and attenuation coefficients exhibit different behaviours60. Understanding the contribution of phytoplankton to in-situ measurements of cp and bbp might thus increase our capability to predict oceanic carbon biogeochemical rates. We therefore suggest that a potential new avenue of research could be to identify what changes in cell ultrastructures and composition govern diel variations of optical scattering properties.Understanding which particle characteristics determine optical scattering coefficients in natural waters may also shed light on why community production is better predicted from 50. The volume-averaged refractive index equal to 1.06 reveals that particle populations across the Atlantic Ocean are mainly of organic origin50. Although we may compare modelled coated-sphere thicknesses and compositions  and measured64 polarised components of the Mueller scattering matrix65, the range of selected coated-sphere parameterisations may be further constrained  and how they vary with depth and latitude. We also need to characterise better the detrital matter and particle aggregates. For example, it is possible that detritus constitutes a large fraction of oceanic particles32, future efforts should also be directed to test and constrain complex optical models with in-situ measurements of particle and cellular characteristics. Only when we will understand the sources of oceanic bbp, we will be able to exploit the full potential of optical backscattering observations for investigating the biological carbon pump. Our study opens a new direction towards achieving this ultimate goal.Our findings clearly show that the structural complexity of particles is key to explain mechanistically the sources of open-ocean backscattering. In addition to measuring the enigmatic open-ocean submicron particles−3 µm−1), and particulate beam attenuation and optical backscattering coefficients at the wavelength 532 nm (cp(532) and bbp(532), respectively). Here, we use the particulate beam attenuation coefficient as a measure of the total scattering because these two quantities are numerically close at 532 nm due to negligible particulate light absorption25.Measurements were collected during two Atlantic Meridional Transect (AMT) cruises carried out in October–November 2012 (AMT22) and in September–November 2016 (AMT26). Both cruises covered the Atlantic Ocean from the United Kingdom to South America, by encompassing a wide range of oceanic conditions, from sub-polar to tropical and from eutrophic systems to mid-ocean oligotrophic gyres  to optical scattering measurements. A total of 22 additional stations were sampled, at the same time as the ship’s CTD casts, in flow-through mode from the 5-m underway ship’s seawater supply  and at the DCM were used to test the homogeneous-sphere assumption for marine particles and parameterise the coated-sphere model. AMT22 and AMT26_1µm samples were used as independent datasets to validate model results. AMT26 samples collected in the mesopelagic region of the Atlantic Ocean were used to discuss coated-sphere model advantages and limitations.N = 134 from casts + 44 from underway) were directly collected in 500-ml acid-washed amber bottles and immediately prepared under a vertical flow hood for the analysis. Samples were kept in a cold and dark place during the analysis. Measurements were conducted with a Multisizer III Coulter counter (Beckman Coulter) fitted with 20-μm and 100-μm apertures, in multiple 50-µl and 1-ml replicates, respectively. The ESD size ranges were 0.588–12 μm and 2–60 μm for the 20-μm and 100-μm apertures, respectively, each distributed in 256 logarithmically spaced size bins. The number of replicates was selected, sample by sample, to achieve an overall error <15% in a given size range , which corresponded to a detection limit of 45 counted particles .During AMT22, a total of 134 PSDs were measured on board, in multiple 2-ml replicates (from 2 to 26), through a Multisizer III Coulter counter (Beckman Coulter) fitted with a 70-μm aperture. The measured size range of equivalent spherical diameters (ESDs) was 1.4–42 μm and distributed in 200 logarithmically spaced size bins. During AMT26, seawater samples  as sources of error. Specific to the AMT26, the PSDs obtained from the 20-µm and 100-µm apertures were combined in a single PSD by merging the measurements from the two apertures at around 2.14 µm, where they presented similar bin width and upper/lower limits. The resulting PSD spanned from 0.588 to 60 µm by 360 logarithmically spaced size bins with associated uncertainties.For both AMT22 and AMT26 samples, the PSD was calculated for each aperture by summation of all the replicates. The total analysed volume was 4–52 ml with the 70-µm aperture, 150–600 µl with the 20-µm aperture, and 3–15 ml for the 100-µm aperture. The accuracy of measurements within each size bin was calculated through the standard law of propagation of uncertaintyBefore each cruise, the Coulter counter apertures were calibrated using suspensions of recommended Beckman Coulter calibration spheres. During AMT26, the instrument performance was checked using suspensions of standardised spheres of 3.6 µm of diameter (Beckman Coulter) and provided counting accuracy within 10% for both 20-µm and 100-μm apertures. Blank references of 500-m seawater filtered through 0.1-μm Millex sterile polyvinylidene difluoride syringe filters (Millipore) were recorded daily for both 20-μm and 100-μm apertures. During AMT22, blanks were acquired through repeated (three times) filtration of surface seawater on 0.2-µm syringe filters.Examples of PSDs from AMT22, AMT26, and AMT26_1µm with combined uncertainties are shown in Supplementary Figure c(532)) were measured with an ac-s spectrophotometer  for underway samples (AMT22 and AMT26_1µm), or through 0–250 m casts of an ac-9 spectrophotometer  at the same time of CTD measurements and water sampling. The ac-s and ac-9 had the same optical configuration and beam acceptance angle67, and collected data along a 25-cm path length. Both underway and cast measurements were repeated after filtration through a 0.2-µm filter in order to determine the contribution of the coloured dissolved organic matter, as well as calibration drifts31. The beam attenuation coefficient of particles (cp(532)) was obtained by subtraction of the 0.2-µm filtered signal from c(532), following established protocols52.Bulk beam attenuation coefficients at 532 nm , with three ECO-BB3 backscattering sensors . During AMT26, the ECO-BB3 was installed on the rosette sampling system and provided 0–500 m β measurements. For AMT22 and AMT26_1µm underway samples, β coefficients were acquired in a flow-through chamber. Data were collected and processed following established protocols52. The relative uncertainty associated with the extrapolation of the optical backscattering coefficient from a single angle measurement is <10%69.The angular scattering coefficient at 532 nm was measured at a central angle of 124°, cp(532) and bbp(532) measurements were quality checked52. Underway data were binned into 1-min intervals and the median value was calculated for the timing corresponding to PSD water sampling. The cp(532) and bbp(532) coefficients acquired during upward casts were binned in 1-m intervals and the median was calculated for values within a 5-m window centred at the depth of the Niskin bottles fired for the PSD samples. Before 1 m binning, bbp(532) profiles were smoothed with a moving-median filter (five-point window). To assess the robustness of the optical measurements used in the analysis, we checked consistency through inter-comparisons among them and versus independent simultaneous measurements when available  with minima and maxima as defined from PSD measurements  × 10−6 m for AMT26 and (1.4–42) × 10−6 m for AMT22 samples). We did not extrapolate the PSDs for particles with diameters <Dmin that consequently were not modelled. For each individual particle, Qc and n is the volume-averaged refractive index38, which represents the characteristics of the coated sphere with refractive indices (relative to that of seawater54) of its core and coat, n1 and n2, respectively, and fractional thickness of the coat tk2; the term πD2/4 indicates the particle cross-sectional area (units of m2); and F(D) is the concentration of particles for a given diameter as obtained from the PSD measurements (units of particles m−4). The values of n2 were varied between 1.02 and 1.2250, whereas those of tk2 varied between 2 and 30% of the radius of the particle. The volume-averaged refractive index of the particle was forced to be equal to 1.06, which was the single value that best fitted the beam attenuation measurements through the homogeneous-sphere assumption (see Results). As a consequence, the refractive index of the core n1 varied as a function of n2 and tk2. A total of 609 combinations of tk2 and n2 were tested with the coated-sphere model. Homogeneous spheres were represented as a coated sphere with tk2 = 0 and n1 values between 1.02 and 1.2250. Particles counted within each size bin of the measured PSD were considered as monodispersed and non-absorbing populations at the selected wavelength25. We expect the results of this modelling analysis to be more sensitive to the structural complexity of the spheres than to the impact of light absorbing material on bbp39, and thus to be representative also of other portions of the visible spectrum.The beam attenuation and optical backscattering coefficients of particles were modelled at the wavelength 532 nm as follows:Qc for homogeneous and coated spheres were generated by using the scattnlay software70, for angles between 0 and 180° (0.1° increments) and logarithmically spaced ESDs between 0.4 and 70 µm. The Qc values both for homogeneous and coated spheres were corrected for the acceptance angle effect to be consistent with WETLabs ac-9 and ac-s in-situ measurements67.Look-Up-Tables of 70) and 70. First, for each set of particle parameters  we computed the following elements of the scattering matrix:70 and the starred quantities indicate complex conjugates.The values of Each We then computed the Mueller matrix components of particle populations composed of modelled coated spheres and the measured PSDs, To compare the shape of the VSFs with existing measurements, we normalised i and j are not simultaneously equal to 1.Finally, to compare the polarised components with existing datasets, we normalised each component of the scattering matrix by cp(532) and bbp(532) coefficients was calculated by propagation of the errors associated with PSD measurements66. Modelled cp(532) and bbp(532) coefficients were then compared with in-situ measurements through match-up analysis. The model performance was evaluated by calculating the systematic error, in logarithmic space, as follows:r) was also calculated between log-transformed quantities and the significance was verified by a two-tailed Student’s t-test .The combined uncertainty of modelled cp(532) and bbp(532) of the size fraction from Dmin to D were defined, respectively, as:27The cumulative percent contributions to Supplementary InformationPeer Review File