Gadus morhua) in oceanic waters and compared it with results obtained from a standardized demersal trawl survey. Detection of eDNA originating from Atlantic cod was highly concordant (80%) with trawl catches. We observed significantly positive correlations between the regional integrals of Atlantic cod biomass (kg) and eDNA quantities (copies)  and between sampling effort-normalised Catch Per Unit Effort (kg hr−1) and eDNA concentrations (copies L−1) . These findings extend the potential application of environmental DNA to regional biomass assessments of commercially important fish stocks in the ocean.Environmental DNA (eDNA) has emerged as a powerful approach for studying marine fisheries and has the potential to negate some of the drawbacks of trawl surveys. However, successful applications in oceanic waters have to date been largely focused on qualitative descriptions of species inventories. Here we conducted a quantitative eDNA survey of Atlantic cod  and demersal trawl surveys for detecting Atlantic cod in oceanic waters. Their results suggest that this approach could contribute to regional assessments of commercially important fish species. Gadus morhua) is an iconic example that demonstrates how poorly constrained data and uninformed decision making can result in catastrophic stock decline and ensuing economic and social problems1. Traditional stock assessments of demersal fish species have relied primarily on trawl surveys, which have provided a valuable stream of information to decision makers2. However, there are some notable drawbacks of demersal trawl surveys including cost3, gear selectivity/catchability4, habitat destruction5 and restricted coverage .The successful management of commercial fisheries relies on standardised surveys to estimate the quantity and distribution of fish stocks. Atlantic cod  has emerged as a potentially powerful alternative for studying ecosystem dynamics. The constant loss and shedding of genetic material from macrogranisms imparts a molecular footprint in environmental samples that can be analysed to determine either the presence of specific target species16. However, known variations between eDNA production18 and degradation22 rates is anticipated to complicate these relationships in natural systems. Furthermore, in oceanic systems, large habitat volumes and strong currents are likely to result in physical dispersal of DNA fragments away from target organisms23. These confounding factors have been previously considered to restrict the application of quantitative eDNA monitoring in oceanic settings24.Positive relationships between eDNA quantities and fish biomass and abundance have been demonstrated in experimental systems25, visual surveys26, echo-sounding27 and trawl surveys28. However, studies that quantify target eDNA concentrations of commercial fish species with standardised trawl surveys in marine environments are much scarcer28. In this context, direct comparisons of eDNA concentrations with biomass and stock assessment metrics, such as Catch Per Unit Effort (CPUE), are necessary to understand the applicability of eDNA monitoring to contribute to fisheries management efforts.Despite these potential constraints, numerous studies in marine environments have found positive relationships between eDNA quantities and complimentary survey efforts including radio-tagging2, particularly in the Faroe Bank region. Fisheries management closed the Faroe Bank to all fishing gears in 2009, except for minor jigging during summertime. Understanding the recovery and spatial distribution of Atlantic cod is extremely relevant for the Faroese fishing economy. Here we used qPCR detection to measure the concentrations of Atlantic cod eDNA in bottom water samples around the Faroe Islands and compared this with biomass estimates obtained from a parallel standardised demersal survey. We apply these data to test the hypothesis that there is a positive correlation between the regional biomass of Atlantic cod and eDNA concentrations in oceanic waters.The present study focuses on the application of quantitative eDNA monitoring targeting Atlantic cod because it is a species of commercial importance that has been subjected to strong fishing pressure. Atlantic cod is a valuable commodity for the Faroese fishing economy, accounting for an export value of ~0.9 billion Danish Krona in 2018 . Sustained demersal surveys between 1994 and 2018 have shown a notable decline in Atlantic cod biomass in Faroese watersG. adus morhua (Atlantic cod) we performed cross-amplification tests on related non-target species collected from the survey area (Table Melanogrammus aeglefinus) and saithe (Pollachius virens) were present at biomass levels one order of magnitude lower than Atlantic cod. Norway pout (Trisopterus esmarkii), whiting (Merlangius merlangus) and blue whiting (Micromesistius poutassou) were present at two orders of magnitude lower and poor cod (Trisopterus minutus) and silvery pout (Gadiculus argenteus thori) at three orders of magnitude lower. DNA extracted from tissue specimens amplified for Atlantic cod at a Cq value of 18.8 ± 0.56, no amplification was observed for non-target species. Due to the low abundance of poor cod it was not possible to collect a specimen for cross-amplification tests. However, considering it’s negligible biomass and regional distribution, compared to that of Atlantic cod  primers for ea Table . AnalysiG. morhua qPCR primer  water samples collected at the Faroese national aquarium (Føroya Sjósavn). DNA extracted from water samples of three separate tank systems, each containing Atlantic cod, successfully amplified with the .5L water−1, respectively. Regions of intermediate biomass are the East coast (<150 m), South and East shelf (<150–200 m), with CPUEs of 91.4 (626), 27.5 (92.3) and 11.0 (55.9) kg h−1, respectively. Low biomass regions were identified on the edge of the Faroe Bank  and further to the East, between 200 and 500 m . Despite temporal fluctuations in total catches, this regional distribution of Atlantic cod has been quite consistent over the trawl-survey period  show a strong regional distribution of Atlantic cod around the Faroe Islands  and North (max = 1531 kg h−1). Negligible CPUE’s were recorded in the East deep region (<3 kg h−1), the East Shelf region (<10 kg h−1) and Bank Edge (<8 kg h−1)  Fig. .−1; excluding these negligible trawl catches establishes concordance between detection methods at 80%. Less than 6% (2/35) of sampling locations exhibited positive eDNA detection discordant with positive trawl detection. To assess association between trawl and eDNA detection methods we calculated the mean square contingency coefficient (Φ). Considering all data, Φ was calculated at 0.48, increasing to 0.62 if negligible trawl catches (<10 kg h−1) are classified as negative trawl detections.Considering the paired trawl-eDNA stations, Atlantic cod was caught at 27 trawling locations (77%). The spatial distribution of detection rates is shown for the trawl Fig.  and eDNA−1), followed by the North region . None of the samples from the East deep and Bank edge region displayed positive amplification . The highest biomass of 14.4 × 103 kg per region was found in the West together with the highest concentration of Atlantic cod-derived eDNA (151.2 × 103 copies per region). No eDNA was detected in the East deep and Bank edge regions, corresponding to negligible trawl biomass of <10 kg per region. Assuming a median size of 3.24 kg individual−1  and (copies per litre) was significantly positive with a smaller regression coefficient  on a station-by-station basis.To test our central hypothesis we evaluated the relationship between eDNA concentrations and trawl-biomass of Atlantic cod in the standardised survey region Fig. . We dete2 standardised survey area in Faroese waters. On the basis of historical (25 years) CPUE data obtained from standardised trawl surveys we were able to identify eight distinct regions that were targeted for a quantitative eDNA survey  characterising an ~100,000 km4 copies L−1 of Atlantic cod eDNA in Faroese waters, comparable to the 0–104 copies L−1 recently reported in the Baltic Sea28. There have been few studies that have attempted to apply qPCR-based quantification of target-species eDNA concentrations in open sea systems29. Whale shark and mackrel tuna eDNA concentrations have been shown to vary from 0 to 106 and 102 to 108 copies L−1, respectively, in the Arabian Gulf29. Studies that have aimed to compare eDNA concentrations with alternative survey methods, including echo-sounding27 and visual observations29, have found a good degree of spatial concordance between the methods.Concentrations of Atlantic cod eDNA in bottom waters in the survey area exhibited significant regional variability. We observed a range of 0–10−1 and copies L−1, Fig. 28, we observed statistically weak correlations between CPUE and eDNA concentrations when assessed on a station-by-station basis . It is worth noting that even this weak regression coefficient characterising the station-by-station comparison could be potentially biased due to a skewed distribution of eDNA copy numbers that result from numerous non-zero values for eDNA copies. Removing non-zero eDNA copy numbers results in an insignificant correlation on a station-by-station basis. Thus it appears that at a fine scale, eDNA signals are diluted and degraded to a level that confounds quantitative relationships whilst still remaining amplifiable, and thus amenable to species detection by qPCR and next generation sequencing approaches.In the present study we observed significantly positive correlations between DNA concentrations and biomass of Atlantic cod, both in terms of regional integrals  was sufficient to establish statistically robust regional relationships between eDNA and CPUE for Atlantic cod.These findings support our initial presumption of a regional approach and that quantitative relationships between eDNA and trawl-surveys are weak at fine-scale resolution in the ocean. For management purposes, stock estimates often rely on integrating multiple trawl catches over a larger spatial areaThe Faroe bank region has exhibited significant temporal variability in stocks of Atlantic cod over the last 25 years  on the Faroe Bank. Quantitative eDNA monitoring may thus have the sensitivity to track regional stock fluctuations in the ocean. Our findings extend the potential application of eDNA to regional biomass assessments of commercial fish stocks in the ocean.eDNA has emerged as a valuable monitoring tool for fisheries science and managementDemersal trawling was carried out on board Magnus Heinason  as part of the standardised spring demersal survey conducted by the Faroese Marine Research Institute. During a five-week period from February–March 2018, 128 one-hour bottom-trawls were carried out on the Faroe Plateau and Faroe Bank. Trawl doors were of the Thyborøn type, and the bridle length was either 60 m (<140 m) or 120 m (>140 m). The length from the codend to the headrope was 40 m, and the distance between doors was either 70 m (60-m bridles) or 130 m (120-m bridles). Trawling gear was a 116-ft box trawl with a mesh-size of 135 mm. The headrope was located 4.5–5 m above the bottom. A net of 40-mm mesh and 8 m long was placed inside the codend and tows were conducted by day. The survey catch was sorted by species. The total weight of the survey catch and of the sorted species was recorded. Individuals of larger specimens were collected for weight and length measurements.Seawater samples of 1.5 L were collected at 35 of the trawling positions Fig.  from Nis−1). Workbench area on-board was covered with aluminium foil and rinsed with a 20% commercial bleach solution, followed by ultrapure water. The foil was replaced after each sampling event.Careful measures were taken on-board to eliminate contamination. Upon recovery the CTD rosette and Niskin sampling bottles were thoroughly rinsed with fresh water on the deck. The Niskin bottles were removed from the sampling frame and transported to a CTD control lab isolated from the deck area where they were mounted on wall brackets for further processing. Prior to sub-sampling the exterior of the Niskin bottles and sampling nozzle were rinsed with a sodium hypochlorite solution  followed by ultrapure water (18.2 MΩ cmSub-sampling bottles were 2 L LDPE bottles that had been thoroughly cleaned beforehand with a 10% HCl solution (24 h soaking), 20% commercial bleach solution (24 h soaking) and subsequently rinsed three times with ultrapure water. Individual sub-sampling bottles were stored in double plastic bags in locked plastic crates that had been washed beforehand with a 20% commercial bleach solution and rinsed with distilled water. The neck diameter of the LDPE sub-sampling bottles was selected such that it formed a perfect seal with the base of the sampling nozzle of the Niskin bottle. The interior of the sub-sampling bottle was therefore not exposed to the atmosphere during sampling aside from the few seconds it took to remove the cap and dock with the Niskin Nozzle. Each sub-sample bottle was rinsed three times with sample water and then filled to a 1.5 L graduation mark. The 2 L LDPE sample bottles were immediately capped and placed back in double plastic bags and frozen on board at −20 °C to eliminate contamination risks associated with filtering on the research vessel. Sample blanks were taken periodically alongside field samples by filling clean polypropylene bottles with ultrapure water and freezing. All subsequent sample processing took place in a sterile environment at a molecular biology institute ashore.Upon return to a sterile laboratory samples were stored at −20 °C in a separate wing physically isolated from the molecular laboratory workspace. Samples were subsequently processed in a wet-lab of the same wing. Samples were defrosted at room temperature. Prior to removing the cap, the exterior of the sample bottles were thoroughly washed with tap water (3 min each), followed by a sodium hypochlorite solution and ultrapure water. Immediately prior to filtering, the water samples were homogenised by vigorous shaking for a period of 2 min. Samples were filtered using a peristaltic pump (40 rpm) and particulate material harvested on a 0.2 μm Sterivex filter . Sterivex filters were chosen because they are encapsulated in a plastic cartridge eliminating contact of the filter and sample material with the atmosphere. Residual sample volume in the Sterivex cartridge was evacuated by expelling from the cartridge with a single-use sterile 50 mL luer-lock syringe . The female luer-lock inlet of the Sterivex filter was sealed with a male Luer integral lock ring plug . The exterior of the cartridge was wiped with a 10% sodium hypochlorite solution, followed by ultrapure and isolated in a sterile 50 mL Falcon tube. The 50 mL Falcon tubes were stored at -80 °C. Operational field blanks were processed in an identical manner. In between samples, peristaltic pump tubing was rinsed with 1 L of sodium hypochlorite solution  and 2 L of ultrapure water.DNA was extracted from the Sterivex cartidges  using a modified protocol of the Qiagen DNeasy Blood and Tissue Kit . All bench space, pipettes and instruments associated with the extraction were cleaned before use with 70% ethanol, followed by RNase away (Qiagen #19101).Sterivex cartridges were removed from the freezer and allowed to defrost at room temperature for 20 min. A final check on residual volume was performed by expunging air through the Sterivex cartridge with a 50 mL sterile syringe. The male nipple of the Sterivex cartridge was flame sealed and extraction reagents were added directly inside of the cartridge using sterile filter pipette tips. Extraction proceeded according to the manufactuer’s instructions with the following modifications. A volume of 720 µL of buffer ATL and 80 µL of proteinase K was added directly to the interior of the Sterivex cartridge. The female inlet was capped with a male luer-lock cap. The Sterivex cartridges were then placed in a rotary spinner and incubated at 56 °C for 2 h. The cartridges were rotated 90° around their central axis every 30 min to ensure even coverage of the filter roll with extraction solution. The lysis soluition was removed from the Sterivex cartridge using a sterile 3 mL luer-lock syringe and transferred to sterile DNase-free 2 mL Eppendorf tubes. The Eppendorfs were pulse vortexed (10 s) and spun down in a mini-centrifuge. Subsequently, 600 µL of extraction solution was transferred to a clean 2 mL Eppendorf tube, followed by 600 µL of buffer AL solution. These Eppendorf tubes were pulse vortexed (10 s) to mix and centrifuged. Taking each Eppendorf in turn, 600 µL of ethanol was added, followed by pulse-vortexing and centrifugation. This extraction solution was added to the Qiagen DNEasy Blood and Tissue kit spin columns in 3 × 600 µL aliquots. The spin column procedure followed the manufacturers instructions.−1 .DNA was eluted from the spin column in 120 µL of nuclease-free water following an incubation period of 5 min at 37 °C. The flow-through from the first elution step was pipetted back onto the column for a second elution under identical conditions. DNA extracts were frozen at −20 °C until further processing. For each batch of extractions an extraction control was included to test for laboratory and reagent contamination. All extraction reagents were added to a sterile Sterivex cartridge and extraction carried out in an identical manner alongside field samples. DNA concentrations in the extracts were measured with a Qubit Fluorometer and Qubit dsDNA HS assay (Q32854), following the manufacturers instructions. DNA concentrations in 1.5 L bottom water samples ranged from 0.16 to 2.3 ng µLGadus morhua speciation assay . An Applied Biosystems StepOnePLUS real-time PCR platform was used for amplification. Each PCR reaction mixture of 20 µL contained 5 µL of DNA template, 10 µL 2 × qPCR Mastermix , 1 µL Gadus morhua primer/probe (TaqMan hydrolysis probe), 1 µL Internal Extraction Control primer/probe (TaqMan hydrolysis probe) and 3 µL of nuclease-free water. Primer and probe sequences are deemed proprietary (Techne) but were designed to target the mitochondrial D-loop region of Atlantic cod. PCR reactions were performed under thermocycler conditions of 2 min at 95 °C and 50 cycles of 10 s at 95 °C and 60 s at 60 °C. Fluorogenic data was collected through the FAM and VIC channels.Quantitative real-time PCR of Atlantic cod eDNA was carried out using a commercially available 37. Inhibition was not detected in any of the ocean bottom water field samples.DNA extracts were not diluted for inhibition checks due to the potentially low yield of target DNA in field samples. Inhibition was tested for in all sample and blank PCR reactions using multiplex PCR and an internal control template (sequence: proprietary-Techne) and primer-probe (sequence: proprietary-Techne) supplied with the Techne Atlantic cod speciation kit (TKIT06035). A volume of 5 µL control template was added to DNA extracts. A comparison of Cq values between samples with internal control template and nuclease-free water with internal control template was used to diagnose inhibition, whereby a Cq shift ≥ 3 cycles was considered as inhibitoryG. morhua, and NTC-IEC was used to confirm amplification of the internal control used for comparison with field samples to test for inhibition. None of the NTC samples amplified for G. morhua template. Positive amplification controls were included from the kit-supplied positive control template. Extraction blanks were included with each batch of DNA extraction to control reagent contamination. Extractions were carried out in an identical manner and simultaneously with sample extractions, substituting a sample filter for an unused sterivex filter cartridge. Field blanks were comprised of 1.5 L of ultrapure water collected alongside ocean samples on board the research vessel. Neither extraction blanks nor field sample blanks exhibited amplification with the G. morhua qPCR primers.The qPCR assays were set up to include six no template controls (NTC), three contained nuclease-free water in place of template DNA (NTC), and three contained nuclease-free water + internal control template (NTC + IEC). All six NTC wells were used as negative controls for the amplification of −1. A 10-point concentration series, with ten technical replicates at each concentration was used to estimate an R2 value of 0.99 and PCR efficiency of 81%  identical in sequence to the mitochondrial target region. Standard curves were generated by serial dilution of the positive control template to create a concentration series of 2, 20, 200, 2000, 20000, 200000 copies µL−1 = Copies per reaction × [(Evol/Rvol)/Svol], where Evol and Rvol are the extraction volume and PCR reaction volume (µL) and Svol is the filtered sample volume (L). According to this equation and the methodological set-up LOD and LOQ were 48 and 320 copies L−1.LOD and LOQ were determined from an 10-point concentration series, with ten technical replicates at each concentration. LOD was defined as the lowest concentration at which 95% of the technical replicates exhibited positive amplification. LOQ was determined at the lowest concentration at which the relative standard deviation of back-calculated concentrations was <35% (Supplementary Methods). LOD and LOQ were 3 and 20 copies per reaction. Copies per litre were calculated according to the equation: Copies LFor each field sample, operational sampling blank and laboratory extraction blank, four technical replicates were amplified. Positive amplifications were classified as those samples in which at least ¾ technical replicates amplified above the LOQ. Averages were calculated from these technical replicates and analytical error expressed as ± 1 s.d. (Supplementary Table G. morhua) is based on in-silico analysis of the mitochondrial D-loop region. Additionally we confirmed specificity from the analysis of tissue samples of other Gadidae species collected from the survey area . Aquarium tank water was collected from three separate display tanks, each containing Atlantic cod. Sample filtration, DNA extraction and qPCR amplification were carried out as described above. A sample blank of ultrapure water was taken simultaneously. Positive amplification of Atlantic cod eDNA from tank water was observed from all three tanks and the sample blank was negative  is that spatial autocorrelation does not exist. Z-scores from Moran’s I were calculated according to Eq. 42.All statistical analyses were conducted in the R environment−1) and average Atlantic cod DNA copies per region were evaluated using Type I (OLS) and Type II (RMA) regressions. Type II regressions are preferred when the x variable cannot be assumed to be free of variance43. Atlantic cod biomass data was log10-transformed to satisfy normality assumptions required for Type I and Type II linear regression. Normality was tested using a Shapiro-Wilk normality test at a significance level 95% (α = 0.05). An ordinary least squares (OLS) method was used for Type I regression. In the Type II regression a Ranged Major Axis (RMA) method was used. Bivariate normality was checked using a Shapiro-Wilk normality test (α = 0.05) to satisfy the assumptions of RMA Type II regression. Statistical significance of regression coefficients were assessed from parameteric p- values (two tailed) at a significance level of (α = 0.05) Regression coefficients A permutation test (99 permutations) was used to determine the significance (α = 0.05) of the slopes and correlation coefficient of Type I-OLS and Type II-RMA regressions. Type I and type II regressions were performed using the R-package lmodel244.The correlation between total Atlantic cod biomass per region vs total Atlantic cod DNA copies per region and Catch Per Unit Effort . The two detection methods were compared in a 2 × 2 contingency table, where a = presence–presence, b = presence–absence, c = absence–presence, d = absence–absence. The phi coefficient is analytically equivalent to the Pearson’s product moment correlation (Eq. Association of presence–absence data was calculated from mean square contingency coefficientFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryPeer Review File


Cucurbit[8]uril (CB[8]) mediated assembly of extended aryl viologens (EVs) into optically tunable dimers is reported for the first time. 2·(EV[X]R)2) in the ground and excited states was characterized by NMR, isothermal titration calorimetry, and steady-state spectroscopic measurements.Cucurbit[8]uril (CB[8]) mediated assembly of extended aryl viologens (EVs) into optically tunable dimers is reported for the first time. We show that the modular design and synthesis of a new class of π-conjugated viologen derivatives with rigid aromatic or heteroaromatic bridging units as well as electron donating molecular recognition motifs enable their self-assembly into 2 : 2 complexes with CB[8]. The quantitative dimerization process involving these two molecular components in an aqueous solution enables excimer-like interactions between closely packed charged guests giving rise to distinct spectroscopic behavior. The nature of these dimers (CB[8] Achieving precise control over the spatial arrangement of organic photoemissive molecules is of interest in the area of photovoltaic device construction, light-emitting diodes,n]urils (CB[n]) are a family of macrocyclic host molecules often used in synthetic and materials sciences on account of their variety of sizes and capability to complex different types of guest molecules in an aqueous environment.Cucurbit[i.e. phenyl (EV[Ph]), naphthyl (EV[Np]) and thiophene (EV[Th]) were synthesized in a three-step procedure, as depicted in 22In order to take advantage of strict 2 : 2 complex formation through the CB[8] binding motif we explored the introduction of an extended π-bridging unit between the two pyridyl groups as shown in 2·(EV[X]R)2 would not only be influenced by the host-guest interactions in their ground state but also in their excited state as an excimer-like complex. Previous observations have been made for molecular packing of naphthalene, anthracene and phenanthrene under pressure or in the solid state with a red shift of the energies of their electronic transitions (lower energy), and with broadened and featureless spectral bands.et al. and many other reports are focused mostly on covalent dimers where the dimeric units are not free to rotate or rearrange.We report here the extent of inter-chromophoric interactions in the ground and excited states based on their steady-state electronic transitions and fluorescence lifetimes as a function of their binding with CB[8]. We hypothesized that the emissive excited state properties of the dimeric complexes CB[8]1H NMR and isothermal titration calorimetry (ITC). Typical NMR experiments are carried out at 100 uM at room temperature where the 2 : 2 complexes form instantly. Guest complexation with the hydrophobic cavity of CB[8] typically results in an upfield shift of the encapsulated protons and a downfield shift of protons in close proximity to the carbonyl portals as shown in Ha was observed , typically around –70 kJ mol–1.–1 to –70 kJ mol–1 suggesting 2 : 2 binding with CB[8] (see the ESI1H NMR (Ka) for the EV and CB[8] complexes were found to be within 5.5 × 106 M–1 to 3.8 × 107 M–1, indicating strong host–guest binding. Importantly, when EV[Np]R bearing electron withdrawing groups (R = CF3 and Br) was mixed with a stoichiometric amount of CB[8], the complexes formed were not found to bind in a 2 : 2 manner, but rather yielded 1 : 1 or n:n complexes as confirmed by NMR and ITC measurements  and fluorescence lifetime (τ).Complexation inside the CB[8] cavity affects the local environment of extended viologens, which in turn alters their optical properties. It has been reported that encapsulation in CB[8] causes a red shift in the absorption and emission bands, as well as an increase in the fluorescence quantum yield  and a broad, featureless emission band with a maximum at λem = 456 nm (ΦPL = 0.65). Upon mixing with 1 eq. of CB[8], bathochromic shifts of over 10 nm in the absorption and emission spectra are observed, as well as strong enhancement in the fluorescence quantum yield (ΦPL = 0.92). Similarly, for EV[Np]Me  and an emission band at λem = 487 nm (ΦPL = 0.68). Upon mixing with 1 eq. of CB[8], a 25 nm red-shift in the absorption spectra and a 31 nm red-shift in the emission spectra are observed, as well as a small decrease in the fluorescence quantum yield (ΦPL = 0.58). The UV/vis absorption spectra of EV[Th]Me  and a maximum in the emission band at λem = 453 nm (ΦPL = 0.62). Upon mixing with 1 eq. of CB[8], a 10 nm red-shift in the absorption spectra and a 91 nm red-shift in the emission spectra are observed.The ground-state UV/vis absorption spectra of EV[Ph]Me . The emission spectrum observed for EV[Th]Me complexed with CB[8] has characteristics of typical excimer-like species; here, where 2 : 2 complex formation is evident, two thiophene bridging moieties are electronically coupled in the excited state. Correspondingly, monomeric EV[Th]Me and de-aggregated 2 : 1 complexes with CB[7] have high quantum yields of 0.62 and 0.67, respectively (see the ESI–1) for the CB[8]2·(EV[Np]Me)2 complex and 137 nm (6187 cm–1) for CB[8]2·(EV[Th]Me)2 complex indicates that the dipole moments of the photoexcited states have been changed significantly, suggesting the formation of intramolecular charge transfer states. Broad, unstructured red-shifted emission is characteristic of charge–transfer transitions as highlighted by Wasielewski and co-workers.Unlike the cases of EV[Ph]Me and EV[Np]Me, a huge decrease in the fluorescence quantum yield is recorded for EV[Th]Me of the EVs alone and in the presence of 1 eq. of CB[8] was carried out to gain insight into the possible EV–EV interactions and their dynamics in the excited state. knrad on account of the mechanical protection from quenchers present in solution.2·(EV[X]Me)2) complexes for X = Ph and Np give rise to a notable decrease in both krad and knrad. In the case of the (CB[8]2·(EV[Th]Me)2) complex we observed lower radiative but higher non-radiative decay rates in comparison to free guests  simulations were performed, followed by quantum chemical calculations. Molecular interactions in these MD simulations include both coulombic repulsion and π–π attraction between the pairs of EVs, leading to the observed dynamic behavior of the complexes (see the ESIdc) and shifts (ds1 and ds2) between the rings compared with the geometry optimized structures  and green (EV[Np]Me) emission, respectively. For these two EVs, the fluorescence spectra exhibited red-shifted, broad, structureless bands with fluorescence quantum yields as high as 0.92 and long fluorescence lifetimes of up to 17 ns. On the other hand, EV[Th]Me with a heteroatom in the bridging moiety, resulted in an emission spectrum typical of excimer-like structures with weak orange luminescence. Here in the benzene-naphthalene–thiophene series, stronger π–π interactions through an increase of aromatic surfaces and, consequently, the restricted rotation or rearrangement in the cavity of CB[8] molecules affect the formation and optical efficiency of excited dimers. Our results suggest that CB[8]-extended aryl viologen supramolecular complexes are a new way to form distinct dimeric entities through a straightforward, synthetically accessible modular approach. These systems represent a platform for studying excited dimers in the excited state such as excimers or exciplexes. Furthermore, systematic modification of a bridging unit of extended aryl viologen derivatives will lead to a range of systems with long-lived excited states. Finally, on account of such interesting optical properties, these distinct dimers can find a myriad of applications in imaging.6We have described the synthesis of a new family of extended viologen compounds with increasing π-surfaces between the two pyridinium units. The binding properties of this family of molecules with CB[8] are assessed through There are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.


The purpose of this n = 3), according to polishing protocol: Laboratory-machine polishing with different siliconcarbide paper grits (G1: 320) → (G2: 800) → (G3: 1200) → (G4: 2000) → (G5: 4000). Chairside-hand polishing using a series of Sof-Lex spiral (G6) and abrasive polishing points (G7). Glossmeter was used to determine the SG at 60° incidence angle. SG was measured before and after polishing. Three-dimensional (3 D) noncontact optical profilometer and scanning electron microscopy (SEM) analysis were performed. Data were analyzed using ANOVA (p = .05). A total of 147 block-shaped specimens (40 mm length × 10 mm width × 2 mm thick) were made from conventional RCs , bulk-fill RC (Filtek Bulk Fill), fluoride-releasing RCs  and discontinuous microfiber-reinforced RCs (Alert and everX Flow). Each group was subdivided into seven subgroups (p < .05). Specimens polished with 4000 grit paper showed the highest SG (93 GU) values among all the groups tested. Significant differences in SG (ranged 3–93 GU) were found according to the type of polishing protocol and RC  and unpolished surfaces. A glossy and perfectly smooth surface is a requirement for a desirable esthetic appearance . It alsoGloss is an important property and is used primarily as a measure of surface shine . The gloin vitro study was to determine the effects of different polishing laboratory-machine and clinical protocols on the SG of a seven different commercial RCs . The null hypotheses were that no difference in SG would be found among the polished RCs or among the different polishing protocols when used on the same RCs.Nevertheless, very few studies have investigated the influence of different surface polishing protocols on the SG of a new commercial RCs used for direct restorations. Thus, the aim of this 2. The specimens from each RC were then divided into seven groups (n = 3) according to the polishing protocols. Laboratory-machine polishing was performed using different silicon paper grit sizes (G1: 320) → (G2: 800) → (G3: 1200) → (G4: 2000) → (G5: 4000) at 300 rpm while water-cooled using an automatic polishing machine  for 1 min. Chairside-hand polishing was performed using a series of Sof-Lex spiral   and abrasive polishing points   with a low-speed handpiece  underwater cooling and with constant moving repetitive stroking action. One side of the specimen surface facing the mold was polished and this side was named as the polished side. The other side of the specimens facing the glass slide and mylar strip remained unpolished and was named the unpolished side. After polishing, specimens were rinsed with water and stored dry at room temperature before testing.A total of 147 block-shaped specimens (40 mm length × 10 mm width × 2 mm thick) of seven different commercial RCs , were man = 3) per each material involving five different laboratory-machine polishing paper grits and two chairside-hand polishing protocols. The SG was assessed for polished and unpolished sides.In total, there were seven groups  with a square measurement area of 6 mm × 40 mm area. The average of five measurements was recorded per surface.n = 3) from each of the polishing protocols.Three-dimensional (3D) noncontact optical profilometer  using Vision64 software was used to observe and capture images of the polished surfaces (n = 2) were stored in desiccator for 1 d. Then, they were coated with a gold layer using a sputter coater in vacuum evaporator  before the SEM examination. SEM observations were carried out at an operating voltage of 5 kV and working distance of 3–6 mm.Scanning electron microscopy  provided the characterization of the microstructure of the investigated RCs. Polished specimens (4000 grit) from each material  using analysis of variance (ANOVA) at the p < .05), however, some interaction existed between the groups. The polishing protocol was always a significant and stronger factor than the type of material. Specimens polished with 4000 grit paper showed significantly the highest SG (93 GU) values among all the groups tested. As seen in p < .05). SG after polishing with Sof-Lex spiral was significantly higher than surfaces polished by abrasive polishing points (p < .05), regardless of the material used. In four of the seven polishing protocols, Filtek Bulk Fill composite presented a higher SG values than did the other tested RCs. The 3D images after each polishing protocol are shown in The SG mean values of the tested RCs after various polishing protocols are shown in SEM analysis showed typical microstructure of each tested material with different particulate fillers size and shape in polymer matrix . This suIn this study, seven restorative RCs were selected based on different filler concepts in each material. The SG investigated after being polished using different polishing protocols. Both the type of material and the polishing protocols significantly affected the SG values and hence the null hypotheses were rejected. High gloss for a RC gives a natural, esthetic appearance to a restoration. According to ISO semigloss surfaces like RCs should be measured with 60° angle of illumination, which was applied in this. This was found closer to the angle from which the average person will observe the surface . The higGloss reduction of RCs following brushing test was observed in previous studies ,20. The Many researchers showed the correlation between SG and surface roughness ,23–25. TAs recommended by Roeder et al., in our study we measured the SG of materials before and after polishing to homogenize the specimens . It was By comparing SG values obtained with different polishing protocols, it can be clearly observed that laboratory-machine polishing with 2000 and 4000 silicon paper grits obtained glossier surfaces than chairside-hand polishing protocols . In addiAccording to the American Dental Association (ADA) professional product review, 40–60 GU was identified as a typically desired gloss based on observations from an expert panelist . Cook anThe limitation of this study is that specimen preparation was done by two investigators and this might have had an effect on the pressures exerted during the polishing procedures although the polishing time was controlled. A negative control group of RC whose roughness was provided using a diamond finishing bur on the surface is missing and this will be evaluated in the near future.According to the research methodology used, the SG of evaluated RCs is influenced by the polishing protocol used. The smoothest and most glossy surfaces were obtained with laboratory-machine polishing protocol (4000 grit).


We report a theoretical and experimental study of the aggregation kinetics of oppositely charged nanoparticles. Kinetic Monte Carlo simulations are performed for symmetric, charge-asymmetric and size-asymmetric systems of oppositely charged nanoparticles. Simulation results show that both the weight and number average aggregate size kinetics exhibit power law scaling with different exponents for small and intermediate time of evolution. The qualitative behavior of the symmetric and the size asymmetric system are the same, but the charge asymmetric system shows anomalous behavior for intermediate to high particle concentrations. We also observe a strong dependence of power law exponents on the particle concentration. Radius of gyration of the cluster that indicates how nanoparticles inside a cluster are distributed around the center of mass of the cluster shows a non-monotonic time evolution with pronounced peak at higher particle concentration. The dependence of particle concentration on aggregation kinetics as observed by predictive numerical simulation is further verified experimentally by monitoring the time evolution of aggregate size of nanoparticles assemblies of Poly (methacrylic acid) (PMMA) nanoparticles functionalized with oppositely charged ligands. These size and charge tunable asymmetric polymeric nanoparticles were synthesized by modified miniemulsion technique. The integrated approach for studying nanoparticles aggregation as described here renders new insights into super structure formation and morphology optimization which can be potentially useful in the design of new materials, such as organic photovoltaics. These self-assembled structures can be effectively used in various industrial applications such as waste water treatment2, sensing3, drug delivery4, NP based solar cells5, etc. Tuning of NP shape, size, interaction etc. gives a control over the super structure formed by them9. Aggregation of NPs is strongly affected by the competition of inter-particle interactions such as van der Waals, electrostatic, and magnetic interactions, which in turn depends on the material property of NPs, dielectric constant of solvent, temperature, pH, and presence of external fields. Out of various inter-particle interactions, electrostatic interactions are somewhat unique, because they are long-ranged and, can be either attractive or repulsive. Also, their interaction strength and interaction range can be easily tuned by varying the charge on the NPs and ionic strength, respectively13. Such tunability of electrostatic interaction has many implications on the aggregation kinetics and self-assembly of oppositely charged NPs14.Bottom-up self-assembly to build functional complex structures using nanoparticles (NP) as building blocks has received tremendous research attention in last few years15; homo-aggregation is the aggregation of the NPs with identical characteristics, whereas in hetero-aggregation, NPs with different physical and chemical properties aggregates to form clusters. Naturally, hetero-aggregation is more complex and is therefore not yet completely understood18. Recent experiments on the synthesis of NPs of different size, charge, and chemical composition have provided new insight into the mechanisms of aggregation processes21. Experimentally aggregation kinetics of NPs is studied using Dynamic Light Scattering(DLS)23, time-resolved DLS24 and Small-angle Neutron Scattering(SANS)26. Charged NPs hetero-aggregation has received much more attention due to ease of controlling the charge on NPs and its application in ionic colloidal crystals, organic optoelectronics and flocculation techniques32. However, synthesis of monodisperse and precisely charged NPs at large scale with high reproducibility is always challenging and needs in-depth theoretical understanding and predictive modeling. Several theoretical models33 to explain aggregation of sub-micrometer spherical particles have been developed and experimentally validated, the most common being DLVO theory. Computer simulation plays a crucial role to understand the internal dynamics of the system and provide a complimentary tool for the rational design of chemical reactors for high-throughput synthesis of nanoparticles.Aggregation of nanoparticles can be broadly classified into homo-aggregation and hetero-aggregation12. Using importance sampling of configuration space, MC simulation efficiently reaches the final equilibrium state by skipping several metastable states. But during the evolution to reach equilibrium, system passes through various non-equilibrium complex structures which are very different from equilibrium structures34. For instance, various studies have shown that system of oppositely charged nanoparticles are often trapped in non-equilibrium “gel”-like (percolated) structures at high packing fractions, and do not evolve to minimum energy crystalline structures due to slow system dynamics37. In one of our previous study, we have found that these kinetically trapped percolated structures formed by self-assembly of electron conducting and hole conducting polymer NPs are the bulk heterojunction morphologies for efficient organic solar cells37.Monte Carlo(MC) simulation is very useful tool to study the final equilibrium structure of super lattice formed by aggregation of NPs and the effect of various interactions on these structures39 and is argued to be more efficient than Brownian dynamics simulation because it employs a longer time step without compromising the numerical stability. The predictive modeling is validated experimentally by synthesizing size and charge tunable functionalized polymeric NPs with modified mini-emulsion method. Polymeric NPs functionalized with oppositely charged surfactants are chosen as model system to study aggregation behavior as they can be easily synthesized with different sizes and charges by tuning polymer’s molecular weight, polymer to surfactant ratio, sonication time, solvent evaporation rate etc. The time evolution of the aggregate size of these PMMA NPs is studied by DLS.In this paper, using kinetic Monte Carlo (kMC) simulations, we study the effect of charge and size asymmetry on aggregation kinetics of oppositely charged NPs. kMC scheme used in this simulation has been used in various other studiesσA and σB and valence zA and zB, respectively. The system containing total NT NPs satisfy the electroneutrality condition nAzA + nBzB = 0 and nA + nB = NT, where nA and nB are number of NPs of type A and type B respectively. NPs interact via electrostatic interactions, which are modeled using a pairwise screened Coulomb potential between ith and jth NP at distance r given by40Implicit-solvent simulations are performed on the solution containing two types, type A (+) and type B (−), of oppositely charged spherical NPs of diameter i, j = A, B and σij = (σi + σj)/2 is the average diameter of ith and jth NPs. We define dimensionless Bjerrum length as σ0 = (σA + σB)/2. ε, ε0, kB, T and cs are the dielectric constant of solvent, permittivity in free space, Boltzmann constant, absolute temperature and salt concentration respectively. In simulation we have used truncated and shifted form of both electrostatic and soft-core potential to improve computational efficiency, that is,rij,cut = 2.5σij and BT.The soft-core repulsion between NP is modeled using Lennard-Jones (LJ) potential given by38:η is the packing fraction, defined as the fraction of volume occupied by NPs.Random distribution of NPs inside cubic box of length a around the center of NP. We choose a random point ; θ and φ are the polar angle and azimuthal angle, respectively.Trial displacement of the center of a randomly picked NP to the surface of a sphere of radius E is the energy change for the trial displacement.Move is accepted or rejected using the Glauber transition probability Simulation algorithm comprises of following stepsNT trial moves. Time step Δt of a sweep is related to the step size a as σ0, characteristic diffusion time scale is t is calculated by assuming the energy change in each sweep is small. In previous kMC study39, a = 0.02σ0 is observed as the proper step size and we have used this value of a in our simulation. Note that the current approach do not explicitly account for changes in diffusion coefficient on aggregation, as elaborated in previous studies39. However, this limitation is not expected to be of much consequence in the current work, since we focus on early stage kinetics of NP aggregation when the aggregate size is relatively small except for the high particle concentration case where percolation occurs.One kMC sweep consist of μl of each positive and negative NPs. Similarly in set 2, 50 μl of each positive and negative NPs were mixed in distilled water to get 200 ml solution. Average aggregate size(MN) and zeta potential(ζ) of NPs was recorded by Malvern Nanosizer DLS instrument.Chloroform, SDS, CTAB, PMAA and acetone were purchased from Sigma–Aldrich. All the materials were used as obtained. Millipore water was used in all parts of experiment including NP synthesis and DLS study. We synthesized both positively and negatively charged PMAA NPs of different concentrations with mini-emulsion technique using anionic surfactant, SDS (sodium dodecyl sulfate) and cationic surfactant, CTAB (cetyltrimethyl ammonium bromide), respectively. In a typical fabrication process, SDS/CTAB and PMAA are separately dissolved in distilled water and chloroform, respectively. Polymer solution was heated to 50 °C to ensure complete solubility. The NPs were then formed by adding the polymer solution to the aqueous SDS/CTAB solution under probe sonication with constant heat environment at 50 °C and stirring at 1000 rpm by hot plate. After complete cycle of probe sonication for 6 minute, dispersion was kept on heating at 65 °C for 45 minute to remove chloroform. To perform aggregation kinetics experiment, we prepared two sets by mixing both positively and negatively charged NPs together. In set 1, 200 ml was prepared by mixing distilled water with 25 Symmetric NPs: Both type A and type B have same size (σA = σB) and equal and opposite valence (zA = −zB).Charge Asymmetric NPs: Both type A and type B have same size (σA = σB) but unequal valence (zA = −2.0zB).Size Asymmetric NPs: type A and type B have unequal size (σA = 1.5σB) but equal and opposite valence (zA = −zB).We numerically studied the aggregation kinetics for the following three sets of oppositely charged NPs:η = 0.005,0.025,0.06). Aggregate size of a cluster is calculated by computing center to center distance(r) between two NPs and two NPs are considered to be part of the same cluster if r < 1.5σ0. We computed mean average aggregate size  was not captured due to initial fast clustering and only β2 values are given in Fig. η = 0.06 in size asymmetric case where aggregation is slow due to large size of the NPs. As η increases, both β1 and β2 increases for all the three cases. But, there is no substantial difference in β2 values for η = 0.025 and η = 0.005 in charge asymmetric case. Furthermore, for all the three sets, we observed β2 > β1 for all η, but again in charge asymmetric case this difference is not very predominant as η increases. The behavior of exponents β1 and β2 in charge asymmetric case for intermediate to high η hints towards anomalous behavior of charge asymmetric system. We further plotted Mw vs t/τ0 and observed similar time dependence behavior as MN vs t/τ0, therefore we write β1 and β2 respectively, except for charge asymmetric case where η = 0.025 is smaller than η = 0.005. Also, MN exponents is distinctly visible from Mw exponents for the charge asymmetric case.Figure mentclass2pt{minimmentclass2pt{minimsee Fig.  for charβ1 and η increases is due to decrease in the average separation between NPs. This results in an increase in the Columbic force between NPs and hence faster aggregation at initial time for large η values is observed. Strong Columbic attraction due to high charge on NPs leads to much faster initial aggregation in charge asymmetric case as compared to symmetric and size asymmetric case , one NP of high valence (charge asymmetric case) is surrounded by many NPs of opposite charge which leads to more screening and excluded volume effect42 at intermediate time. This leads to large reduction in the strength of electrostatic interaction between the clusters and hence slower agglomeration of clusters. The increase in the size of agglomerated cluster due to the merging of smaller clusters is dominated by slower agglomeration of clusters in charge asymmetric case despite the fact that the asymmetric case have bigger clusters to agglomerate as compared to the symmteric case. Thus, we observe a crossing of aggregate size between symmteric and charge asymmetric case  for large η values which represent the presence of a single large percolated cluster. Also, at initial time PDI value is higher for larger η which indicates that aggregation is faster for high η. This further substantiates our point that initial time aggregation behavior is not captured for higher η values. Another important point to notice is that the time corresponding to the maxima of PDI . This also leads to large decrease in charge/size ratio of newly formed aggregates in charge asymmetric system as compared to symmetric system and hence at intermediate times, aggregation become slower in asymmetric system. This happens because of the interplay between the rate of agglomeration of clusters and the size of agglomerating clusters. Further, NPs within a cluster rearrange themselves to minimize free energy and due to interplay between the time scale of aggregation and time scale of rearrangement leads to a non-monotonic behavior in


Tear film regulatory properties were analyzed with the spinning drop method and the regulation of SFTA3 transcripts was studied in HCE and HCjE after incubation with proinflammatory cytokines as well as typical ocular pathogens by real-time RT-PCR and ELISA. The results reveal that human ocular tissue as well as tears of healthy volunteers express SFTA3 whereas tears from patients with DED showed significantly increased SFTA3 levels. In vitro wounding of HCE cell cultures that had been treated with recombinant SFTA3 demonstrated a significantly increased wound closure rate and rSFTA3 reduced the surface tension of tear fluid. The results indicate that SFTA3 at the ocular surface seemed to be involved in wound healing and the reduction of surface tension.The study aimed to characterize the expression and function of SFTA3 at the ocular surface and in tears. Ocular tissues, conjunctival (HCjE) and human corneal (HCE) epithelial cell lines as well as tearfilm of patients suffering from different forms of dry eye disease (DED) were analyzed by means of RT-PCR, western blot, immunohistochemistry, and ELISA. A possible role of recombinant SFTA3 in corneal wound healing was investigated performing  In almost 86% DED is the result of a diminished outer lipid layer at the surface of the tear film2, a condition called evaporative DED. A quantitative deficit of tears, on the other hand, is considered to be less common. The lipid component of the tear film is mainly secreted by the meibomian glands of the eyelids. Under physiological conditions, the lipid component prevents evaporation and early break-up of the tear film and thus, protects the eye surface3. Together with the lacrimal glands and accessory lacrimal glands of the eyelids which produce the aqueous component of the tear film, meibomian secretions (meibum) generate a barrier against pathogenic microorganisms4.In Central Europe, about 15% to 20% of the human population are suffering from dry eye disease . Exogenous influences  and endogenous factors  are the main causes of this disease. DED is based upon a discontinuity of the tear film7. In the lung surfactant proteins regulate surface activity and have immunological properties with regard to both adaptive and innate immunity10.A further component of the tear film comprises proteins of the surfactant family  that are well known from the lung and have previously also been described at the eye surface, in tear film and in the lacrimal gland11 and SFTA3 (SP-H)12 were identified and characterized. SP-G has physicochemical properties similar to the already described proteins SP-B and SP-C. Moreover, the ability to interact with lipid systems combined with a potential surface-regulatory function of SP-G has been discussed. Surfactant-associated protein 3 (SFTA 3) was first identified by bioinformatic analyses and named surfactant protein H (SP-H) or SFTA313. This protein shows no significant sequential or structural similarities with other surfactant proteins or other known proteins in general. The proposed physicochemical properties of SFTA3 show similarities with the well-characterized surfactant proteins B and C. Molecular dynamic (MD) simulations and protein modeling analyses suggest that SFTA3 may constitute a surface-active substance. Despite the absence of known structurally conserved immune regulatory domains SFTA3 might still play a possible role in the immune defense and during inflammation processes12. Stimulation experiments with a lung epithelial cell line (A549) revealed that SFTA3 gene expression is significantly influenced by the presence of immunomodulating cytokines (IL-1β and IL-23) as well as bacterial lipopolysaccharide (LPS)12. In a dose-effect analysis with IL-1β and IL-23 a reduced expression of SFTA3 was shown, while LPS induced SFTA3 expression. It was hypothesized that LPS simultaneously triggers the production of inflammatory cytokines like IL-1β and IL-23 production in resident alveolar macrophages and SFTA3 induction via TLR in alveolar epithelial cells. Thus, gram-negative bacterial infection apparently promotes the SFTA3 pathway in alveolar epithelial cells, whereas inflammatory cytokines like IL-1β and IL-23 secreted by alveolar macrophages apparently limit this process12. A recent study on the impact of recombinant SFTA3 protein on alveolar macrophages showed an increased phagocytosis rate, suggesting a possible immunological function of the protein inside the lung14.Recently, two novel surfactant proteins SFTA2 (SP-G)In the present study we investigated whether SFTA3 is also part of the tissues of the human ocular surface, the lacrimal system as well as tears. Furthermore, we examined the expression and secretion of SFTA3 (SP-H) at the human ocular surface and in tear fluid samples of patients with dry eye disease (DED).in vitro scratch assay and physicochemical tear film regulatory properties was analyzed with the spinning drop method.The objective of this study was to determine the expression and production of SFTA3 at the healthy human ocular surface and to investigate potential differences in the presence of dry eye disease (DED). SFTA3 expression was analyzed by western blot analysis, ELISA as well as by immunohistochemistry and immunofluorescence in different tissues of the ocular surface and in human tear fluid from patients suffering from DED in comparison to healthy controls. The activation and regulation of SFTA3 transcription was studied in cell culture experiments after incubation with ocular pathogens and cytokines by real-time RT-PCR and ELISA. A possible contribution of SFTA3 in corneal wound healing was investigated by an et al.15. All tissue samples were obtained from body donors  donated to the Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Germany. All body donors signed a testamentary permission in time. The study included proper consent and approval, complied with the tenets of the Declaration of Helsinki, and was approved by institutional review board regulations. The samples were obtained from the body donors within a time-frame of 5–20 h post mortem. Prior to dissection, the history of each cadaver was studied. The donors were free of recent trauma, eye and nasal infections or diseases involving or affecting lacrimal function. Moreover, with regard to the whole body, donors that had been affected by acute infection, tumors, recent trauma or surgical operations were not used in this study. After the dissection of cornea, conjunctiva, lacrimal gland, eyelid and efferent tear ducts , one half of each specimen was fixed in 4% paraformaldehyde for later paraffin embedding. The other half was used for molecular-biological investigations and was thus immediately frozen at −80 °C. Bronchial mucosa and lung samples served as positive control tissues.The preparation of the samples were performed as previously described by Schicht et al.15. Tear fluid samples were collected with Schirmer strips at the Department of Ophthalmology, Friedrich Alexander University Erlangen-Nürnberg, Germany. These specimens were obtained in compliance with good clinical practice and with informed consent (54-2532.1-35/13). Ethical approval was obtained from the Ethics Committee of the University of Erlangen-Nürnberg. Written consent was received from all patients and subjects after explanation of the procedures and study requirements. Tears from 21 control subjects  were included in the study. They showed no symptoms for dry eye disease or ocular discomfort, did not use any artificial tears or lubricant eye drops and did not suffer from any autoimmune disorders or other eye diseases including ocular allergies and had no history of eye surgery or contact lens wearing. 39 eyes of 39 patients  with moderate to severe dry eye (DEWS dry eye severity level 2)16 were also enrolled in the study. Inclusion criteria were as follows: (i) Ocular Surface Disease Index questionnaire Score (OSDI Score) >40, (ii) tear break-up time (TBUT) ≤10 s, (iii) Schirmer test with anesthesia ≤10 mm, (iv) lid-parallel conjunctival folds (LIPCOF) >2. A more detailed description of these points is given below. For the ELISA, patients were divided into two subgroups: evaporative dry eye  and aqueous-deficient dry eye . EDE patients had a tear break-up time of ≤5 s and a Schirmer test ≤10 mm. ADDE patients showed a tear break-up time of ≤10 s and a Schirmer test ≤5 mm. Exclusion criteria consisted of a medical history of trauma or infection, ocular allergies, pregnancy, lactation, history of refractive surgery/ocular surgery/any other surgery within the previous 6 months, immunosuppressive medications, or the use of contact lenses within 14 days prior to ophthalmological examination. Moreover, patients wearing punctum plugs, patients with history or evidence of epithelial keratitis derived from herpes simplex infection, recent varicella infection, corneal or conjunctival viral disease, acute corneal, conjunctival, or palpebral bacterial infection; or ocular fungal infection were excluded from this study.Tear fluid extraction were performed as previously described by Schicht et al.15. SV40-transformed human corneal epithelial cells 17 as well as a human spontaneously immortalized epithelial cell line from normal human conjunctiva 18 were cultured as monolayer and used for further stimulation experiments. HCE and HCjE cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM)/HAM’s F12 (1:1)  supplemented with 10% fetal calf serum  in a humidified incubator containing 5% CO2 at 37 °C. For stimulation experiments, cells (1 × 106) were seeded in Petri dishes and cultured until confluence was reached. Cells were washed with phosphate buffered saline (PBS) and changed to serum-free medium for 3 h. Afterwards, cells were either treated with recombinant proinflammatory cytokine interleukin (IL)-1β  or tumor necrosis factor (TNF) α  for 6 h, 12 h, 24 h, or 48 h each, or with different dilutions of bacterial supernatants of Staphylococcus aureus (SA) or Pseudomonas aeruginosa (PA) for 6 h, 24 h or 48 h. Tryptone Soy Broth (TSB) was used to incubate the bacteria. Therefore, incubation of cells with TSB that had not been exposed to bacteria served as a medium control in the bacterial supernatant group. In the cytokine group incubation with the cytokine solvent alone yielded the controls. All experimental procedures were performed under normoxic conditions. On completion of each experiment, cells and culture supernatants were collected and stored at −80 °C until they were processed for RNA extraction (cells) or analysis of SFTA3 secretion (culture supernatants). For in vitro simulation of wound healing, a scratch experiment was conducted using confluent HCE cells. The confluent monolayer was scratched three times with a sterile 100 μl pipette tip, washed three times with PBS and incubated with serum-free cell culture media for different time spans . All experimental procedures were performed under normoxic conditions. On completion of each experiment, cells were harvested by using the peqGOLD TriFast System and stored at −80 °C until they were processed for RNA extraction.Cell culture experiments were performed as previously described by Schicht et al.15. Laboratory strains of S. aureus and P. aeruginosa were grown overnight at 37 °C by shaking in Tryptone Soy Broth . Bacteria were centrifuged (Scanspeed 1730R) twice at 6,000 rpm/3393 rcf for 30 min. Supernatants were filtered twice using filters impermeable to bacteria . Aliquots of the supernatants were proven to be sterile by overnight incubation on agar. TSB growth medium (diluted 1:100) served as medium control.Extraction of bacterial supernatants was performed as previously described by Schicht et al.19 and Schicht et al.15.Patient examination after OSDI Score, LOPCOF and TBUT was performed as previously described by Jacobi et al.20. The OSDI was already successfully used to measure the outcome in a randomized controlled trial21.The OSDI is a subjective symptom questionnaire that measures the severity of dry eye disease. It includes 12 items regarding visual function, ocular symptoms, and environmental triggers queried for the past week. The questionnaire is standardized and well described by Schiffmann et al. was used22. Here, degree 0 means that no permanent conjunctival fold exist. Degree 1 describes the permanent presence of one single fold which does not exceed the height of the normal tear meniscus. With degree 2, the LIPCOF disintegrates into 2 or several small parallel folds that remain lower than the normal tear meniscus. If there are several parallel conjunctival folds exceeding the height of the normal tear meniscus, degree 3 is present.LIPCOF were evaluated by slit-lamp examination. The classification of LIPCOF according to Höh 23. A stopwatch was used to record the time between the last complete blink and the first indication of tear film break-up. Thereafter, the patient was instructed to blink normally again. Three measurements were taken as recommended by the DEWS II report24, and the average was calculated.For diagnosing tear film stability, a standardized measurement of the tear film break-up time was taken. 5 μl of non-preserved 2% sodium fluorescein were instilled onto the bulbar conjunctiva (the secretion of reflex tears was prevented by using a micropipette). Then the patient was instructed to blink normally without squeezing several times to distribute the fluorescein and then refrain from blinking until told otherwise. The slit lamp magnification was set at 10× and a Wratten 12 yellow filter was used to enhance the observation of the tear film over the entire corneaet al.15.Schirmer’s test with anesthesia  was performed as previously described by Schicht Conjuncain-EDO®), a Schirmer test strip  was placed in the lower outer fornix and then the patient was instructed to close his/her eyes. After 5 minutes, the strip was removed from the eye and the length of the wetted Area was measured21. In addition, the tear fluid-soaked Schirmer strip was transferred to a 1.5 ml reaction tube and stored at −80 °C for further experiments. For extraction of the tear fluid, the Schirmer strips were transferred to a 0.5 ml tube punctured at the bottom with a cannula. The tube was placed in a larger (1.5 ml) tube and centrifuged at maximum rpm (17900 rcf) for 5 minutes. The centrifugal force pulled the tear fluid out of the Schirmer strip, through the central “pore” in the bottom of the smaller tube and into the outer 1.5 ml tube25.After topical anesthesia with one drop of oxybuprocaine-HCL . Total RNA was extracted from the samples by use of the RNeasy Mini Kit .18 primer (Fermentas) were used for each reaction. The ubiquitously expressed β-actin and HPRT which proved amplifiable in each case with the specific primer pair, served as the internal control for the integrity of the translated cDNA.In addition, total RNA was extracted from cultivated HCE and HCjE . Crude RNA was purified with isopropanol and repeated ethanol precipitation, and contaminated DNA was destroyed by digestion with RNase-free DNase I . The DNase was heat-inactivated for 10 min at 65 °C. Reverse transcription of all RNA samples to first-strand cDNA  was performed according to the manufacturer’s protocol. Two micrograms total RNA and 10 pmol Oligo (dT)Conventional PCR was performed according to the following protocol: 1. 95 °C for 5 min, 2. 95 °C for 30 sec, 3. 62 °C for 30 sec, 4. 72 °C for 30 sec, 5. 72 °C for 5 min. Steps 2. −4. were repeated 30 times. Primers used for conventional PCR: SFTA3 sense 5′-ATG AGA GCC GGG TTT TCT GAC-3′, antisense 5′-TGC AGT ATG AAT AAT TAA CAT C-3′ (282 bp), as well as for quantitative  PCR were: SFTA3 real sense 5′-TGG TGT TCC AAA TAT TGC AGA-3′, antisense 5′-GTT CAT CCG AGG CCA AGA-3′ (92 bp) and 18S human sense 5′-GGT GCA TGG CCG TTC TTA-3′, antisense 5′-TGC CAG AGT CTC GTT CGT TA-3′. PCR products were confirmed by Sanger-sequencing, using the BigDye Terminator v3.1 Cycle Sequencing Kit . To estimate the amount of amplified PCR product, we performed a β-actin PCR with specific primers: sense 5′-CAA GAG ATG GCC ACG GCT GCT-3′, antisense 5′-TCC TTC TGC ATC CTG TCG GCA-3′ (275 bp) for each analyzed tissue.® system (Roche). The PCR reaction mixture contained 10 μL LightCycler480® 5x probe mastermix, 0.25 μL of each primer and 2 μL of each cDNA, 0,4 µl Universal ProbeLibrary  probe #13 for SFTA3 or #22 for 18S (10 µM) and 7,1 µl nuclease free water. On each 96-well plate qPCR was performed with a cycle of 5 min at 95 °C, 55 cycles of 15 s at 95 °C, 30 s at 60 °C and 1 s at 72 °C, to confirm amplification of specific transcripts. SFTA3 and 18S primers as well as the corresponding UPL probes (see above) were generated by using the ProbeFinderTM software . A standard curve was generated by serial dilutions of cDNA from non-stimulated cells. To standardize mRNA concentration the transcript levels of the housekeeping gene small ribosomal subunit (18S rRNA) were determined in parallel for each sample, and relative transcript levels were corrected by normalization based on the 18SrRNA transcript levels. All qPCRs were performed in triplicate, and the changes in gene expression were calculated applying the ΔΔCt method.Gene expression was analyzed with quantitative Real-Time RT-PCR (qPCR) using a LightCyler480et al.12.Western blot analysis was performed as previously described by Schicht 26. The protein concentration was measured with a protein assay based on the Bradford dye-binding procedure . The total protein (30 µg) was then analyzed by western blot. Proteins were resolved by reducing 15% SDS-polyacrylamide gel electrophoresis and horizontally transferred at room temperature for 2 h at 0.8 mA/cm2 onto a nitrocellulose membrane with a pore size of 0,1 µm. Bands were detected with a polyclonal primary antibody to SFTA3 (1:200) and a secondary antibody  using chemiluminescence . To verify the specificity of the self made antibody employed, two additional commercial SFTA3 antibodies were used: goat anti SFTPH – D14  and Goat anti SFTPH – C20 . Protein extracted from human lung and bronchoalveolar lavage (BAL) served as positive control. The molecular weights of the detected protein bands were estimated using standard proteins  ranging from 10 to 170 kDa.For Western blot analysis tissue samples (standardized ratio: 100 mg wet weight/400 µm buffer containing 1% SDS and 4% 2-mercaptoethanol) were extracted as previously describedet al.12.Immunohistochemistry analysis was performed as previously described by Schicht For immunohistochemistry, specimens taken from healthy body donors tissues were embedded in paraffin, sectioned (5 µm) and dewaxed. Immunohistochemical staining was carried out with a self made polyclonal primary antibody against SFTA3. Antigen retrieval was performed by microwave pretreatment for 10 min and non-specific binding was inhibited by incubation with porcine normal serum (Dako) 1:5 in Tris-buffered saline (TBS). Each primary antibody (1:50–1:100) was applied overnight at 4 °C. The secondary antibodies (1:200) were incubated at room temperature for at least 2 h. Visualization was achieved with aminoethylcarbazole (AEC) for at least 5 min. Red staining within the tissue indicates a positive antibody reaction. After counterstaining with hematoxylin the sections were coverslipped in Aquatex . Two negative control sections were used in each case: one was incubated with the secondary antibody only, and the other one with the primary antibody only.Tear fluid of healthy donors and patients suffering from DED as well as ocular tissue samples were analyzed by quantitative sandwich ELISA. Analysis was performed by using the Human Putative Protein SFTA3 (SFTA3) ELISA kit and the regarding protocols from Cusabio Biotech Co., Ltd. . Quantification was accomplished by comparison with the 2-fold standard dilution series and the determined values for antigen concentration ranging from 15 ng/ml to 0 ng/ml. Subsequently, each sample was approximated to ng/mg.www.kruss.de. It is an optical tensiometric method that measures the geometry of a rotating drop in the heavy phase. When a heavy phase and a light phase are situated in a horizontal, rotating capillary, the drop radius perpendicular to the axis of rotation depends on the interfacial tension γ between the phases, the angular frequency ω of the rotation and the density difference Δρ. In the classic approach according to Vonnegut’s equation, the interfacial tension can be calculated from the measured drop diameter d (=2r) with a given speed of rotation and with known densities of the two phases (www.kruss-scientific.com). Adding surface active substances changes the geometry and stability of the drop, thus enabling determination of the density-dependent influence of added proteins on the surface tension. A spinning drop interfacial tensiometer  was used to measure the surface and interfacial tensions of the tear film containing various concentrations of SFTA3 at room temperature.The method itself is described by et al.12.Wound healing assay was performed as previously described by Schicht HCE cells were grown in the above-mentioned medium until confluence was reached. Using pipette tips, the cell layer was scratched several times, creating “wounds” of similar width. The cells were washed twice with PBS to remove debris, and fresh medium was applied. Images of wounded areas were taken and areas were marked for later observation. Cells were subsequently stimulated with 300 µg/ml rhu-SFTA3 (n = 4). The negative control (n = 4) contained no rhu-SFTA3 while BSA (300 µg/ml) (n = 3) served as a protein control. The previously imaged areas were photographed again after 24 hours of stimulation. The wounded area was assessed at 0 hours as well as 24 hours using Adobe Photoshop. Stimulated samples were compared to the controls.U-test (nonparametric data) or two-sided Welch’s t-test (parametric data). Significance was defined at *P < 0.05, **P < 0.01 or ***P < 0.001. All data are presented as mean ± standard error of the mean (SEM).The statistical analysis (significance) was evaluated by using an unpaired, a two-sided Mann-Whitney www.ncbi.com).SFTA3 mRNA was detected in tissues of the ocular surface and lacrimal duct system by RT-PCR. Tissues of lung (control), eyelids, lacrimal gland, conjunctiva and cornea were positive for SFTA3 mRNA Fig. . The loaHuman tear film samples were tested for presence of the SFTA3 protein using Western blot analysis Fig. . The antSFTA3 showed a distinct distribution pattern in tissue of the ocular system. In all investigated tissue samples antibody reactivity against SFTA3 was discovered Fig . 3–5 μm Cornea: SFTA3 reactivity was present in the cytoplasm of basal epithelial cells Fig. . Also enComparative quantification of the SFTA3 content in tear fluid of the two DED forms yielded an average value of 1.157 ng/mg (n = 14) in samples of patients with aqueous deficient dry eye  and of 0.669 ng/mg (n = 14) in patients suffering from evaporative dry eye (EDE). Healthy donors exhibited an average SFTA3 protein concentration of 0.196 ng/mg (n = 7) Fig. . Hence, −1 as determined by Spinning Drop Method (n = 3). BSA as loading protein control shows no effects  from 50 to 46 mN/mcts Fig. .in vitro. The corneal epithelial cell line (HCE) expresses SFTA3 and scratching of HCE cells cultured as a monolayer leads to a significant increase in SFTA3 expression. Real Time RT-PCR analysis showed a significant increase in SFTA3 mRNA expression within the first hour after scratching  revealed an increased gap closure by HCE cells in the presence of 5, 10, 100 and 500 μg/ml SFTA3 compared to 0 µg/ml  and the immortalized alveolar type II cell line A54912. Further data obtained from our ELISA experiments disclosed that cells of the eyelid and the lacrimal gland show a lower concentration of SFTA3 compared to cornea and conjunctiva.Western blot analysis of tear fluid revealed three distinct protein bands at 10, 13 and 26 kDa, respectively. This SFTA3 expresion pattern is in accordance with findings in lung tissue, bronchoalveolar lavage (BAL) and the immortalized alveolar type II cell line A549“in vitro” ocular surface model for further SFTA3 studies. SFTA3 was detected in acinar cells of the lacrimal gland, as well as in conjunctival epithelial cells and efferent tear duct epithelial cells. Goblet cells were always SFTA3-negative. In addition, SFTA3 was shown to be secreted into the tear film. This corresponds to previous reports on SFTA3 being detected in bronchial epithelium and alveolar parenchyma. As far as the cornea is concerned, it is important to point out that SFTA3 was detected in the basal epithelial cells and in the endothelial cells, only. This indicates that the SFTA3 produced by the basal epithelial cells is not secreted into the tear film, but rather serves a different function. Which consequences this may have for organisms, must be clarified by further research. We were able to show that SFTA3 is synthesized by meibocytes and the epithelial cells of the excretory ducts of the meibomian glands. It can therefore be assumed that SFTA3 is a secretory product of the meibomian glands and interacts with the lipids to facilitate the secretion process. This is in line with previous reports on meibocytes producing different proteins that interact with the lipids of meibum27.The results clearly show that the two established cell lines HCE and HCjE produce and secrete SFTA3 and can be used for further stimulation experiments. The cell lines are useful as an 12.Interestingly, we found that the SFTA3-protein concentration is increased in tears from patients suffering from different forms of DED. We suppose that there are several possible explanations for this. For example, SFTA3 expression may be increased in order to counter balance the increased evaporation in the case of EDE in which the lipid layer is diminished. As shown by immunofluorescence, SFTA3 is localized in the plasma membrane of HCE and HCjE cells, indicating its ability to interact with hydrophobic groups and therefore may possess surface active properties. We believe that SFTA3 can be considered an additional regulatory factor to influence surface tension. This assumption is supported by previous molecular dynamic (MD) simulations, which showed a theoretical interaction potential for SFTA3 with lipid systems28. However, to date, it remains unclear whether this increase is the cause or an effect of DED. The observed increased expression of surfactant proteins could, for example, result in a destabilization of the tear film. On the other hand, it possibly gives rise to several other proinflammatory mechanisms 30 that further exacerbate the severity of DED symptoms. It is as yet unclear, if the observed higher SFTA3 concentration in the tear film of DED patients is beneficial for the stabilization of the tear film.Beside its function to regulate the relative loss of tear fluid, another explanation for the DED-related increase of SFTA3 in the tear fluid may be a potential role in humoral immunity. As it is known that DED is accompanied by inflammation and sometimes bacterial infection, these pathologic states may cause an increased expression of SFTA3. A general increase in the expression of all surfactant proteins  in patients suffering from DED has been described earlierThe spinning drop analysis showed that additional recombinant SFTA3 reduces the surface tension in tears. This indicates the high potential of SFTA3 to interact with a membrane system and supports our hypothesis that SFTA3 is able to interact with lipid systems. Therefore, it is tempting to speculate that high concentrations of SFTA3, in the tear film of DED patients reduce the surface tension and destabilize the tear film.in vitro data suggest a potential role of SFTA3 in the context of post-injury/healing processes in the human cornea. Accordingly, we found a short term regulation of SFTA3 after epithelial damage as demonstrated by quantitative Real Time PCR after in vitro scratch assay. SFTA3 expression reached its maximum one hour after induction of epithelial damage. Then, it declined and gradually returned to its basal level 6 hours after the scratch. Incubation with rhu-SFTA3 resulted in a significant reduction of the wound area after 96 h. Another peptide that was recently reported to be involved in epithelial healing in the context of dry eye disease is trefoil factor family peptide 3 (TFF3). Compared with TFF3, which remains on its basal expression level within the first hour after the scratch and reaches its most obvious change in expression six hours after setting of the lesion31, SFTA3 displays a quite early and temporally limited reaction to epithelial damage. Our results confirmed the assumption that SFTA3 might be involved in the initiation of repair mechanisms within the corneal epithelium, maybe contributing to the release of other wound healing factors like EGF, FGF or TFF3. Because of the importance of this finding additional experiments using a mouse model for corneal damage/wound healing are crucial in order to determine the exact relevance and function of SFTA3.Beside its yet unknown involvement in the context of DED, our 6. HCE- and HCjE cells were incubated with bacterial supernatants of SA and PA as both bacteria are common causes of bacterial keratitis, an inflammatory process that can lead to loss of vision32. Although our results revealed no considerable effect on SFTA3 expression after stimulation with bacterial supernatant of SA and PA, it cannot be excluded that the expression of SFTA3 is dependent on the immunological activation state and/or the presence of distinct bacterial strains. Therefore, the use of bacterial strains directly isolated from the ocular surface may represent a more suitable model for bacterial infections of the eye in cell culture experiments.To reveal the influence of bacterial infection and proinflammatory cytokines on SFTA3 expression in ocular tissue we performed experiments using HCE and HCjE cell lines as previously described. These cells are well established and known to produce surfactant proteins in general33. Interestingly, stimulation of HCE- and HCjE cells with IL-1β or TNFα resulted in a down-regulation of SFTA3 mRNA in vitro. But as a significant effect on protein level could only be demonstrated for the conjunctival epithelial cell line HCjE 48 h after stimulation with TNFα, it is possible that these two cytokines are no direct modulators SFTA3 expression.Recent studies have shown that the pro-inflammatory cytokines IL-1β and TNFα are elevated in DED patientsThe reduction of SFTA3 expression by inflammatory components suggests that SFTA3 does not play an important role in the context of ocular immune responses.Up to now, considerations concerning the function of SFTA3 are still highly speculative. We are convinced, however, that SFTA3 is a regulator of surface tension and has rheological potential. Combined with the wound healing effects SFTA3 seems to constitute an important factor for the homeostasis of the ocular surface.


In addition, inner structures of extracted inclusions were detected by applying of RTO technique. The large size calcium-silicate based inclusions are confirmed high possibilities originating from mold flux and/or tundish flux entrapment, which are less affected by the liquid steel composition; while the smaller ones are generally endogenous inclusion precipitating during the refining or solidification process that strongly depending on the liquid steel composition and temperature.Metallographic, electrolytic method and RTO(room temperature organic) technique were applied in the present study to more accurately determine non-metallic inclusion in a ultra-low carbon interstitial free(IF) steel and further to confirm their origination in a Compact Strip Production Process (CSP Process) continuous casting (CC) slab. Results show that inclusions detected by metallographic method usually appear relative smaller size and are mainly Al The surface quality of rolled strips is influenced by each operation of the foregoing process, including refining, continuous casting, reheating, hot rolling, pickling and cold rolling,. Many researchers8 attempted to promote the strip surface quality by modifying inclusion composition and morphology based on thermodynamic calculation. Two of the authors’ previous works8 have tried to modify the Al2O3 based inclusion into liquid ones to improve their deformability during CSP rolling process. But it seems still not enough to solve the surface quality problem completely. For example, a number of SiO2-CaO based inclusions were frequently detected in some strip cracks, as shown in Fig. The TSCR(Thin-slab Casting and Rolling) process is with high casting speed thus the fluctuation of meniscus in the mold is much more volatile and steel cleanness is more difficult to control. Therefore, there are more negative effects of inclusion on surface quality for the rolled strip. In fact, the relative poor trip surface quality is one of the biggest challenge to produce the ultra low carbon Interstitial free(IF) steel by applying TSCR process. Many researchers2 based ones.In this manuscript, two technology process, with Ca-treatment and without Ca-treatment respectively, to produce ultra-low carbon interstitial free(IF) steel were carried out in CSP Process. Moreover, inclusions in a casting slab were detected by applying metallographic,electrolytic method and RTO technique in the present study in order to obtain more information of inclusions in a slab and to further confirm the origin of the non-metallic inclusions, in particular the CaO-SiOThe present trials were carried out in a 120 ton ladle of a steel plant in China. Two kinds of routes listed in Table 2. Generally, there are enough inclusion particles in the solution after the electrolyisis for 4 hours. Thereafter, the inclusions in the remaining solutions are separated and collected with the help of a centrifuge. Then the collected inclusions are dispersed by ultrasonic and this process repeated three times to remove the electrolyte completely. Finally, the collected inclusions are transferred on a conductive taps for SEM/EDS detection. In the present work, approximating 50 extracted inclusions per specimen were detected by EDS to determine their composition.Two methods, i.e., traditional metallographic and electrolytic method, were applied to detect the non-metallic inclusion in a casting slab to obtain more information. For the first method, the steel samples were cut into 15 mm × 15 mm × 15 mm cubic specimens and then were ground and polished for SEM observation. Inclusions on the cross-section plane of each steel sample were detected and analyzed by SEM-EDS to obtain information of inclusions such as morphology, size and chemical compositions, etc. 20–30 inclusions per specimen were detected under 1000–5000 times magnification by this method. For the second method, the steel samples were cut into cylindrical shape with 10 mm in diameter and 80 mm in length. The apparatus for non-aqueous solution electrolysis is schematically shown in Fig. 9In order to determine the inner structures of extracted inclusions, a RTO technique was also developed to wrap and cut the collected inclusions. First, the collected inclusions extracted by electrolysis were laid on a clean copper plate in a monolayer, which was then used as a cathode (another pure copper plate was used as an anode). The cell reactions on the anode and cathode for RTO technique are as reactions 3) and (4), respectively. The main steps of the RTO technique are schematically illustrated in Fig.  and (4),2O3-MgO based inclusion or Al2O3 based inclusion determined by EDS; and the other type is with a cubic morphology which is usually TiN type inclusion. The inclusion diameters are several micro-meters. Additionally, some complex duplex inclusions, i.e., TiN precipitating around oxide inclusion as a core, are also detected.Figures 2O3 based inclusion, generally with irregular morphology which are very different from those in route A as shown in Fig. Figure 2O3-MgO ternary phase diagram at 1873K, respectively, as almost all inclusions are mainly composed of CaO, Al2O3 and MgO. It can be seen that majority of inclusions in route A are CaO-Al2O3 based inclusion locating in the liquid region in the phase diagram. Also, there are only a small amount of Al2O3 or MgAl2O4 spinel type inclusions as shown in Fig. 2O3 or MgAl2O4 spinel with high melting temperature as shown in Fig. 2 based inclusions were detected while no SiO2 content was projected in the phase diagrams.Figure 2O3-MgO based inclusion owing to Ca-treatment. However, oxides in route B are almost not modified at all due to no Ca-treatment and higher oxidizing refining slag. It should be pointed out that there are a large number of TiN precipitating in the slab of both routes since they have very similar Ti and N content.In brief, the oxide inclusions in a slab of route A and route B show large differences from each other. In route A, majority of oxide inclusion have been modified into CaO-AlTable 2O3 based inclusion determined by EDS, in which Fig. 2O3, Fig. 2O3 spinel, and Fig. 2O3 based cluster. It should be pointed out that the above types of inclusion are detected both in route A and route B. Figure 2O3 based inclusion observed in route A and it was not detected in route B. Figure 2 based inclusions those are seldom detected by traditional metallographic method comparing to those inclusion morphology and composition presented in Figs 2 based inclusion usually has a irregular morphology and relative large size, majority of their size is more than 20 μm. Figure 2 based inclusion, in which Ca and Si distribute almost homogenously and a small amount of Na is also detected within the inclusion. In addition, many small crystals are precipitated on the surface of the inclusion.Figure 2O3-based inclusions of route A and route B, respectively, which are very dense and no core observed by SEM within them. Figure 2O3-based inclusions from route A and route B respectively. They are composed of many small particles with similar inner structure though they are extracted from two different routes. Figure 2O3 based one from route B. Figure 2O3 based inclusion while the later one is Al2O3 based determined by EDS. Figure 2-based inclusion extracted from route A and from route B, respectively, in which no inner secondary dendrites precipitated within the inclusion, remaining as a glassy phase, while there are some small crystals precipitating on the edge or central part in the Fig. 2 appear very large different in different part due to different crystallization.Figure 2-CaO-Al2O3 ternary phase diagram since majority of inclusions are mainly composed of CaO, Al2O3 and/or SiO2. The CaO-SiO2 based inclusions are much more compared with that presented in Fig. 2 based inclusions present a huge composition variety scope in the phase diagram. The reason will be explained in the following paragraphFigure 2O3 based, CaO-Al2O3 based, TiN or TiOx based. In contrast, a large number of CaO-SiO2 based inclusions up to approximating 40% were detected by applying of electrolysis method while it is nearly not observed by former method.Figure Figure The reasons for the difference are that, one can usually detect the small size endogenous inclusions using a metallographic method since they have a larger number density. While it is easier to find out the large size inclusions by electrolysis method, since all the collected inclusions, including large size extraneous inclusion and small size endogenous inclusion, mixed together. In addition, some small size inclusions are perhaps missed during the collection process.2O3 based or TiN containing inclusion in ultra-low carbon IF steel, many researchers have reported thermodynamics for their precipitation8. Here we would not discuss them any longer since it has been well understood. The SiO2-CaO based inclusion, however, was not very well understood as this kind of inclusion is not likely to be an endogenous inclusion due to a very low Si content in liquid IF steel. It is very necessary to clarify the origin of SiO2-CaO based inclusion.Regarding Al2 based inclusions containing some amount of alkaline oxides such as Na2O and K2O, shown as in Fig. 2 based inclusions are detected free of alkaline oxide those are possible from tundish flux entrapment since they are very similar in composition. Also, this kind of CaO-SiO2 based inclusions have a possibility originating from mold flux entrapment since some alkaline oxides have relative low boiling temperature thus they are likely to be removed during the entrappment process due to the high temperature. Further, CaO-SiO2 based inclusion compositions are largely scattered on the diagram, as shown in. Figure 2 of CaO-SiO2 based inclusions fluctuate in a very huge region. The causes for this phenomena are perhaps as follows:(1) the CaO-SiO2 based inclusions might come from different slag or flux entrapment, such as mold flux and tundish flux, which have different CaO/SiO2 in composition; (2) the different degrees of chemical reactions between the liquid steel and entrapped slag/flux drops, such 4[Al] + 3(SiO2) = 2(Al2O3) + 3[Si], and/or [Ca] + (SiO2) = 2(CaO) + [Si], may cause the CaO/SiO2 of inclusion change; and 3) the entrapped extraneous inclusions would crystallize during the solidification process to precipitate crystal phases with different CaO/SiO2, such as 3CaO·SiO2, 2CaO·SiO2, 3CaO·2SiO2·CaF2, 2CaO·Al2O3·SiO2 and so on with higher CaO/SiO2, and glassy phase and some SiO2 based phase with relative low CaO/SiO2.Compared to the composition of tundish flux and mold flux listed in the Table et al.’s numerical simulating results10, the level fluctuation of CSP mold is much more than that in traditional plate caster mold due to its rapid casting speed, which suggests the possibilities for mold flux entrapment increase a lot. In addition, the tundish flux entrapment should be also paid attention since a large number of CaO-SiO2 based inclusions are detected very similar composition to those of tundish flux, which means they are most likely to originate from tundish flux entrapment.As reported in Song 2O3 based or TiN based in composition, whereas those extracted by electrolytic method usually have larger size and are much more calcium-silicate based. The three dimensional morphology and inner structures of non-metallic inclusion can be determined by combined with electrolysis method and RTO technique. It is very helpful for us to fully understand the formation and origin of inclusion in a continuous casting slab by combing these methods.Metallographic, electrolytic method and RTO technique are applied to analyze the inclusion characteristics in an IF steel continuous casting slab. The morphology and composition of the inclusions obtained by different manners are significant different: the inclusions detected by metallographic method usually appear relative smaller size and are mainly Al2 based inclusions are verified the main cause of the surface defect in the rolled strip.The large size calcium-silicate based inclusions are confirmed high possibilities originating from mold flux and tundish flux entrapment, which are less affected by the liquid steel composition; while the smaller ones are generally endogenous inclusion during the refining or solidification process which strongly depend on the liquid steel composition and temperature. The large size CaO-SiO