Introduction: Catheter ablation (CA) is a common treatment for atrial fibrillation (AF), but the knowledge of optimal ablation sites, and hence clinical outcomes, are suboptimal. Increasing evidence suggest that ablation strategies based on patient-specific substrates information, such as distributions of fibrosis and atrial wall thickness (AWT), may be used to improve therapy. We hypothesized that competing influences of large AWT gradients and fibrotic patches on conductive properties of atrial tissue can determine locations of re-entrant drivers (RDs) sustaining AF.Methods: Two sets of models were used: (1) a simple model of 3D atrial tissue slab with a step change in AWT and a synthetic fibrosis patch, and (2) 3D models based on patient-specific right atrial (RA) and left atrial (LA) geometries. The latter were obtained from four healthy volunteers and two AF patients, respectively, using magnetic resonance imaging (MRI). A synthetic fibrotic patch was added in the RA and fibrosis distributions in the LA were obtained from gadolinium-enhanced MRI of the same patients. In all models, 3D geometry was combined with the Fenton-Karma atrial cell model to simulate RDs.Results: In the slab, RDs drifted toward, and then along the AWT step. However, with additional fibrosis, the RDs were localized in regions between the step and fibrosis. In the RA, RDs drifted toward and anchored to a large AWT gradient between the crista terminalis (CT) region and the surrounding atrial wall. Without such a gradient, RDs drifted toward the superior vena cava (SVC) or the tricuspid valve (TSV). With additional fibrosis, RDs initiated away from the CT anchored to the fibrotic patch, whereas RDs initiated close to the CT region remained localized between the two structures. In the LA, AWT was more uniform and RDs drifted toward the pulmonary veins (PVs). However, with additional fibrotic patches, RDs either anchored to them or multiplied.Conclusion: In the RA, RD locations are determined by both fibrosis and AWT gradients at the CT region. In the LA, they are determined by fibrosis due to the absence of large AWT gradients. These results elucidate mechanisms behind the stabilization of RDs sustaining AF and can help guide ablation therapy. Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is characterized by rapid and irregular activations of the upper chambers of the heart . AF is iCatheter ablation (CA) is a well-established strategy for the restoration of sinus rhythm in AF patients who do not respond well to anti-arrhythmic drugs, with success rates of up to 70% in patients with episodes of AF lasting less than 1 week . It typically relies on the insertion of a catheter into the atria, where it delivers high amounts of localized energy for the destruction and isolation of arrhythmogenic substrates without affecting the surrounding areas . The widRecent advances in catheter and electro-anatomic mapping technologies have enabled the development of more patient-specific ablation strategies that complement standard approaches, such as PV isolation (PVI), in persistent AF. These can directly target: (i) AF sources (ectopic triggers or RDs) identified invasively using basket catheters  or non-iAtrial fibrosis is the most studied example of an arrhythmogenic substrate in AF patients and it has been reported to correlate with both AF incidence and post-ablation recurrence . MoreoveIn order to predict optimal target locations for ablation, a better understanding of the mechanistic influence of fibrosis on AF should be considered in combination with other structural factors, which have been shown to also influence the dynamics of RDs. Theoretical studies have highlighted the role of fiber orientation , surfaceThese findings show a potential of AWT gradients as a marker for identifying RD locations in the atria. However, neither the influence of AWT gradients on the RD dynamics in realistic atrial geometries nor comparative effects of AWT gradients and fibrotic patches on RDs have been investigated. Reconstruction of the atrial wall from imaging  providesThis study aims to investigate the mechanistic influence of two structural factors: (i) AWT and (ii) fibrosis on the dynamics of RDs sustaining AF. To this end, computational simulations of atrial electrophysiology will be performed on (1) an idealized 3D atrial slab with a sharp change in thickness and (2) realistic RA and LA geometries obtained from MRI of six patients. Our working hypothesis is that competing influences of AWT gradients and fibrotic patches on conductive properties of atrial tissue determine anchoring locations of RDs in 3D atrial models.Table 1. In Study 1, we perform simulations in an idealized 3D atrial slab with a varying thickness step to evaluate the mechanistic influence of AWT on the RDs dynamics (Study 1a). A single cylindrical fibrotic patch was subsequently included in the slab to investigate the competing effects of fibrosis and AWT (Study 1b). In Studies 2 and 3, we extend these simulations to realistic models of atrial geometries derived from MRI data, which were created using the general workflow shown in Figure 1. We first investigate the role of AWT and morphology of the RA (Study 2) and LA (Study 3) on the RD dynamics. The effect of a single cylindrical fibrotic patch on the RD dynamics in the RA is also investigated (Study 2b). Finally, we incorporate atrial fibrosis from patient-specific LGE MRI into our LA models to evaluate its influence on RDs relative to the effects of AWT and atrial geometry (Study 3b).The study consists of three parts summarized in All simulations were performed by solving the standard monodomain equation:V (mV) is the membrane potential and t is time (ms). D is the tensor with diffusion coefficients (mm2 ms-1) that characterizes the spread of voltage within the tissue, Cm (pF) is the membrane capacitance, and Iion is the total membrane ionic current (pA). Our model was isotropic and the diffusion tensor was replaced by a scalar diffusion coefficient D = 0.1 mm2 ms-1, carefully chosen to match an atrial conduction velocity (CV) of 0.60 m s-1 typical of AF . In Study 1a, we investigated the mechanistic influence of thickness gradients on the RD dynamics . RDs were initiated using a cross-field protocol, where S2 pulse was applied at multiple distances from the AWT step, starting from 6 mm on the thick side to 13.5 mm on the thin side, and moving with a step of 1.5 mm . The trajectory of the RDs tip was tracked for 10 s in each simulation. Study 1a was also repeated using the CRN model to check the independence of results on the model choice .A thickness step was introduced at the middle of the slab, with thickness of the right-hand side fixed at 7.5 mm and that of the left-hand side was varied between 5.7 and 3 mm . This was done for slab H2 with the step height of 2.7 mm. The rationale of placing the fibrotic region on the thinner side of the step was to mimic conditions when fibrotic tissue could be present in regions surrounding thick structures such as the crista terminalis (CT) in the RA. The cylindrical patch was divided into five concentric regions of gradually decreasing diameter representing increasingly dense fibrosis. RDs were initiated at multiple sites using the cross-field protocol, with varying relative distance from the step and the fibrotic patch: (i) 4.5 mm away from the step on the thick side , (ii) on the step , (iii) 7.5 mm , and (iv) 15 mm  away from the step on the thin side. The RD tip trajectories were analyzed to explore their dynamics in each case. The choice of 4.5 mm as the extreme limit of the RD initiation distance to the left of the patch was to ensure that the RDs was sensitive only to the AWT gradient and not the fibrotic patch. Likewise, the choice of 15 mm as the extreme limit on the right side was to ensure the sensitivity of RDs only to the fibrotic patch.In Study 1b, the comparative effect of AWT and fibrosis on the RD dynamics were also investigated by incorporating a cylindrical fibrotic patch of 9-mm diameter positioned 10.5 mm away from the AWT step on the thinner side .For Study 3b, the segmentation of fibrotic LA tissue was performed by analyzing the image intensity ratio (IIR), computed by dividing individual voxel intensities by the mean blood pool intensity. IIR values >1.08 were classified as interstitial fibrosis and >1.32 as dense fibrosis. The IIR threshold of 1.08 was obtained as an average of the previously proposed values of 1.2  and 0.97In the RA, due to lack of patient-specific fibrosis data, we added a synthetic 3D spherical fibrotic patch of diameter 9 mm, with similar labels 1-5 as done before for the 3D slab, in a region near the thick RA muscle bundle of the CT. This enabled a direct comparison of the effect of AWT and fibrosis on the RD dynamics in the RA (Study 2b). The choice of 9 mm diameter was based on calculating an average fibrotic patch size in the LA (Study 3).A single RD was initiated using a cross-field protocol at different locations within the atrial geometries. In the RA, RDs were initiated in the vicinity of the CT region at six to nine different locations, where a large AWT gradient was typically observed. In the LA, four initiation locations were selected across the atrial wall. Simulations were performed in both geometries with and without fibrosis for a duration of at least 3 s. For each simulation, movement of the RD was tracked by recording location of its tip (organizing center around which a RD rotates) for each time step over duration of the entire simulation . The resFigure 2A). However, with the introduction of a step change in AWT , in addition to meandering around the core, the RD also drifted in a direction perpendicular to the direction of AWT change. The direction of RD drift also depended on its initiation location in the slab. The RD tip trajectories for multiple initiation locations are shown in the Supplementary Figure S1 (H1-4), and the drift directions are summarized in Figures 2C,D.The RDs initiated in the 3D atrial tissue slab meandered to form a characteristic hypotrochoidal (“flower-like”) pattern, with the RD tip moving along the “petals” in the outward clockwise direction around a central core. In a slab with uniform thickness, the flower pattern was stable, its radius was 10.4 mm, and the RD tip motion to complete the whole pattern took 500 ms  were not influenced by the AWT gradient and remained stationary. For the default aFK model settings, RDs anchored to the AWT gradient in the range from 1.8 to 4.5 mm (H1-4), when they were initiated less than 4.5 mm from the step on the thick side, or less than 12 mm (H1-3)/15 mm (H4) on the thin side. Moreover, after an RD anchored to the step, its core moved vertically along the step at a specific distance from it. The core always drifted on the thinner side, and its distance from the step decreased with increasing the step height .The core of RDs initiated on the thicker side (L1) first drift toward the AWT step, eventually crossing it and drifting along it on the thinner side. The core of RDs initiated on the thinner side (L2-3), otherwise, drifted toward the step and along it, but did not cross over to the thicker side. Besides, RDs that were initiated far away from the step . The RD tip in the CRN-based model also meandered to form a flower-like pattern with radius of 12 mm and one complete rotation around the pattern taking about 700 ms. Similar to the aFK-based model, anchoring of the RDs to the AWT step was observed, indicating that the anchoring phenomena was model independent.Simulations of the 3D slab with the CRN atrial cell model demonstrated similar behavior of the RDs , and hence the direction of drift along the step (downward here) is determined by the cross-field initiation protocol. However, the anchoring of the RD to the thickness step does not depend on the wave’s chirality.The RD drift along the AWT step became faster as the height of the step (H1-4) was increased. The drift velocity (DV) was computed by measuring the vertical displacement of the RD core over time, which was done for each step height (H1-4) as shown in Figure 4 for 12 different locations of the RD initiation, with the AWT step height  and location of the synthetic fibrotic patch kept constant. In control cases of the AWT step only and the fibrotic patch only , the RDs either drifted toward and anchored to the step (9 out of 12 locations) or anchored to the fibrotic patch and drifted in a clockwise direction around it (4 out of 12 locations). However, in the presence of both , the majority of the RDs stabilized and anchored between the patch and the AWT step (8 out of 12 locations). Analyzing the RD trajectories in the latter case , we found that RDs initiated on the thicker side, which were insensitive to the fibrotic patch alone, now drifted toward the patch located in the thinner region due to the influence of the AWT gradient. Once the RDs reached the region between the ATW step and the fibrotic patch, they could either drift downward along the step or drift rightward and rotate clockwise around the patch. Opposing influences from the AWT step and the fibrotic patch in this case can cancel each other out, with the RD becoming stationary and anchoring between the two structures.Results of the simulations comparing the effects of AWT and fibrosis in the 3D slab are shown in Figure 4D), increasing the step height from H2 to H4 caused the RDs to anchor to the step . However, increasing the distance between the step and the RD initiation site (from 7.5 to 15 mm), while preserving the distance between the initiation site and the patch, resulted in the RD anchoring to the patch .For location R2 on the thinner side  and 2  showed a higher gradient in AWT compared to Persons 3  and 4 . Hence, Persons 1 and 2 had a more prominent CT region compared to Persons 3 and 4.The average AWT values computed in the CT region and the surrounding RA wall of Persons 1  or crossing over it to the posterior side of the RA . As an example, the 3D atrial voltage map in Person 1 is shown in Supplementary Figure S3. However, in the RA of Persons 3 and 4 , the CT regions had thickness comparable to the remaining RA wall. Hence, the RDs neither drifted toward the CT, nor anchored at any location . Instead, they either drifted toward the superior vena cava (SVC) or the tricuspid valve (TSV) or terminated.In these patients, 55% of the RDs initiated at different locations drifted toward the CT region, either anchoring to it , RDs initiated closer to the fibrotic patch either stabilized between the CT region and the patch , or anchored to the latter , depending on the distance from their initiation site to either structure. The critical proximity to the CT region and the patch is challenging to determine in realistic RA geometry because, unlike AWT in a rectangular slab, the CT region is not a simple straight line. However, in an example provided for Person 1 , RDs initiated at the middle between edges of the CT region and the fibrotic patch  , stabilized and anchored between them. Increasing the horizontal distance between the RD and the CT to 13 mm  led to the RD anchoring to the fibrotic patch. However, in the RAs with no distinguishable thickness gradient at the CT region , all RDs anchored to the fibrotic patch .When the RA simulations were repeated in the presence of a synthetic fibrotic patch, the RD trajectories were affected in all patients, as shown in Figure 8A) showed higher AWT in the LA roof (4.77 ± 0.60 mm) compared to the rest of the atrial wall (2.96 ± 0.31 mm). Therefore, in the respective simulations RDs drifted toward and along the LA roof  when initiated in its proximity, but drifted toward the PVs  if initiated further away. The LA of Person 6  was characterized by roughly uniform AWT (2.53 ± 1.20 mm), and RDs initiated in this geometry drifted either toward the PVs  or the mitral valve .The AWT maps computed for LA geometry of Person 5  compared to the other . In Person 5, LA simulations repeated with the presence of fibrosis showed either stable RD anchoring to the border zones of fibrotic tissue  or transient anchoring where RDs stabilized at a fibrotic patch in the first 1 s of simulation and then drifted to a new location in another fibrotic region and stabilized there . 3D atrial voltage maps for the cases shown in Figure 8Ci,ii are illustrated in Figures 9A,B, respectively. In one particular case, we also observed the breakdown of the primary RD into multiple wavelets meandering between fibrotic patches . However, in Person 6, simulations repeated with the presence of fibrosis in the LA showed either stable RDs forming macroscopic re-entry around the PVs  or RDs anchored to a fibrotic region  near the PV.In the LA, LGE MRI intensity-based segmentations yielded different fibrosis distributions in the two AF patients, as shown in The computational study performed on the 3D atrial tissue slabs and MRI-based realistic 3D atrial geometries enabled us to successfully evaluate the mechanistic influence of AWT and fibrosis distribution on the drift and anchoring of the RDs sustaining AF. In the 3D slabs, the AWT gradients acted as anchoring points for RDs in the absence of fibrosis, while additional fibrotic patches provided competing attractors for the RDs. The results obtained from the RA and LA model simulations confirmed that drift direction and anchoring points of RDs are determined by the interplay of competing influences of the patient-specific AWT gradients and fibrosis.Supplementary Figure S2). This suggests that the anchoring of RDs to regions with large AWT gradients is model independent.Simulations with both 3D atrial slabs and realistic 3D atrial geometries showed large AWT gradients provide anchoring locations for RDs. These results are in agreement with previous experimental observations in animal models of AF . MoreoveNote that, in addition to the influence of AWT gradients, anchoring of RDs to junction between thin and thick tissue observed by Yamazaki et al. could also be explained by the presence of pectinate muscles and activity of stretch-activated channels. Recent evidence also suggest that AF substrate can be provided by 3D structure of the atrial wall, which may cause epicardial and the endocardial discordance . The latIn this study, image-based RA and LA geometries from four healthy volunteers and two paroxysmal AF patients were used to assess the link between RD activity and regions of large AWT gradients. The subject-specific AWT was computed using previously published protocol from Our 3D model simulations using both atrial slabs and realistic atrial geometries revealed the interplay between the effects AWT and fibrosis on the dynamics of RDs. Previous imaging-based computational studies have reported anchoring of RDs at fibrotic patches in the LA . HoweverFigure 3B), the RDs are more likely to drift along a large gradient, anchoring to it. Therefore, atrial tissue regions between a fibrotic patch and a large AWT gradient experience both competing influences and are most likely to anchor RDs. In addition, our simulations in both 3D slab and RA showed that RDs anchored to the fibrotic patch only when they were initiated further away from the structural features  with large AWT gradients. Hence, anchoring of the RDs to either the AWT gradients or fibrotic patches may depend on (1) their comparative ability to provide suitable substrate and (2) their relative distance from the RD initiation site.Simulations performed in both the 3D slab and realistic RA geometries produced similar results, with RDs anchoring between a region with a large AWT gradient (such as the CT region) and local fibrosis patches. Hence, locations of the RDs stabilization can be strongly influenced by both the presence of fibrosis, as predicted by previous studies , and theOur results could explain why the correlation between LGE-derived fibrosis and RDs locations reported by Figure 8), who had high fibrosis burden, RDs were unstable and formed multiple wavelets meandering between fibrotic patches.In LA model simulations in the absence of patient-specific fibrosis distribution, RDs were mostly localized in regions surround the PVs and MV openings. Such a lack of influence of AWT on the RD stabilization in the LA could be explained by the absence of structural features with large AWT gradient in most patients. The only exception was Person 5, where the RD anchored to a large AWT gradient at the LA roof. However, when the LA simulations were repeated with LGE MRI-derived fibrotic patches, a clear stabilization of RDs near the patches was observed in all patients. In the exceptional case of Person 5  and 1.32 IIR (level 5) was obtained from previous studies validated by electro-anatomical mapping data , but thecal data . TherefoOur study did not consider the influence of other patient-specific factors such as atrial anisotropy and electrophysiological heterogeneity, which may contribute to drift of the RDs observed in the realistic LA and RA geometries . Atrial Note also that aFK model developed by Goodman et al. was based on data from tachypaced sheep atrial cells, due to a lack of equivalent human data. However, comparison of APD between the aFK model and in the CRN model for a remodeled human atrial myocyte showed a good agreement between the two models: less than 5% difference in APD in the considered range of frequencies between 6 and 10 Hz. Moreover, the main result of the study – that RDs anchor to regions with large AWT gradients – was qualitatively similar between the aFK and CRN models.Finally, LA wall dilation and heterogeneous wall thinning can occur with the disease progression from PAF to permanent AF . TherefoThis study elucidated the role of AWT as a substrate for RDs and marker for the identification of RD locations in the patient-specific atria, and compared the AWT effects with the respective effects of fibrosis. In the RA, RDs stabilized around structural features with large AWT gradients, while the addition of fibrotic patches provided an alternative attractor for the RDs. In the LA that had more uniform AWT distributions, RD locations were determined by the distribution of fibrotic patches or by anatomical features . These findings corroborate our hypothesis that anchoring locations of RDs are dependent on the relative influence of gradients in AWT and fibrosis, and suggests the non-invasive assessment of AWT and fibrosis using MRI may inform clinical interventions in AF patients.All authors have made substantial contributions to this study. AR and OA conceived and designed the study, and drafted the manuscript. AR substantially contributed to data analysis and computer simulations, and OA substantially contributed to the interpretation of the results. AR, MV, and OA contributed to data analysis, computer simulations, and manuscripts editing. All authors have also approved the final version to be published while agreeing to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PB and handling Editor declared their shared affiliation.


Scientific Reports 10.1038/s41598-018-31971-9, published online 10 September 2018Retraction of: Following publication, the journal received criticisms regarding the rationale of this study and the plausibility of its central conclusions. Expert advice was obtained, and the following issues were determined to undermine confidence in the reliability of the study.in vitro model does not support the main conclusion of the paper that Rhus Tox reduces pain. The qualitative and quantitative composition of the Rhus Tox extract is unknown. Figures 1G and 1H are duplicates; and figures 1I and 1J are duplicates. The majority of experimental points reported in figure 3 panel A are duplicated in figure 3 panel B. The collection, description, analysis and presentation of the behavioural data in Figure 3 is inadequate and cannot be relied upon.The As a result the editors are retracting the Article. The authors do not agree with the retraction.


When the functionality of UHPC is considered, at case study level, two design options of a bridge are tested, which use either totally CC (CC design) or CC enhanced with UHPC (UHPC design). The results show that the UHPC design could provide a reduction of 14%, 27%, and 43% of carbon, material, and water footprint, respectively.There is a common understanding that the environmental impacts of construction materials should be significantly reduced. This article provides a comprehensive environmental assessment within Life Cycle Assessment (LCA) boundaries for Ultra-High-Performance Concrete (UHPC) in comparison with Conventional Concrete (CC), in terms of carbon, material, and water footprint. Environmental impacts are determined for the cradle-to-grave life cycle of the UHPC, considering precast and ready-mix concrete. The LCA shows that UHPC has higher environmental impacts per m The construction industry is recognized as an issue of the global environmental impact . Cement The innovation of Ultra-High-Performance Concrete (UHPC) is increasingly used ,12 for iHowever, UHPC provides a significant reduction in the ratio of mass to load-bearing capacity . It is pEnvironmental performance of construction materials could be comprehensively described by using LCA  based onThis article systematically investigates carbon, material, and water footprint of UHPC in comparison with CC, aiming to facilitate the decision-making process, undertaken by the construction planners. The footprints are assessed within cradle-to-grave life cycle stages according to EN 15804 .Specifications of concrete are regulated in DIN EN 206 . Two mixUHPC is known for its high compressive strength of more than 150 MPa with low water/cement ratio . SchmidtFor the CC mixtures, the average mixture of concrete in Germany is used as it has been described for the environmental product declarations (EPD) of the concrete by the German Federal Association of Concrete . For theThe Life Cycle Inventory (LCI) of UHPC in comparison with CC is described based on the Life Cycle Stages according to EN 15804 . LCI InpA1 life cycle stage of the concrete represents cradle-to-grave inventory (A1–A3) of its ingredient, for instance, A1–A3 production process of cement. Cement production includes raw material extraction (A1), transport of the raw materials (A2), and production process (A3), e.g., manufacturing of clinker, and cement grinding. Production process of quartz sand and quartz powder is defined within GaBi XIV construction materials . This prA2 life cycle stage represents the transport of concrete ingredients to the concrete plant. Transporting types and distances of cement, aggregate, fly ash, plasticizers are taken from an LCA study conducted by the German ready-mixed concrete association , while iA3 life cycle stage comprises the concrete mixing process. Concerning CC, life cycle inventories of electricity, diesel and fuel oil demand are analyzed according to data from a technical report of energetic optimization of concrete production of the ready-mix concrete plant done at the University of Stuttgart . The ele3. The modeling of the heat treatment is based on the expert’s interviews. Therefore, an amount of 3 L/m3 of fuel oil is required. 3 concrete.In addition, A3 life cycle stage of precast concrete includes compacting and heating treatment of the concrete. The research project of the Technical University of Cottbus  describeLCI of the A4 life cycle stage of CC and UHPC are the same. The ready-mix concrete is transported by a truck mixer from the concrete plant to the construction site. The GaBi database  does not3/h, with a utilization rate of 50%. This is equivalent to an operating period of 0.012 h/m3. Precast concrete is either installed by using a revolving tower crane or by using a mobile crane [The transport of the precast concrete is modeled by using a truck with a total weight of 34–40 t and a capacity of 27 t with fuel consumption of 0.46 L/km . Environ3/h according to the recommendations from experts. In terms of UHPC, there is no documented practical experience concerning the demolishing process [3/h.C1 life cycle stage of UHPC and CC refers to the deconstruction measures. They differ from each other in the type, the capacity, and the fuel consumption of excavator. CC could be demolished using an excavator with a total weight of 30 t and fuel consumption of 18 L/h . The cap process . A more Transport to the recycling plant represents C2 life cycle stage. In Germany, the construction waste is usually transported by trucks with a total weight of 40 t . The disLife cycle stage of C3 includes the treatment of the construction waste. The waste of CC is going to be pre-crushed with an excavator and transported to the processing plant by a wheel loader, related technical specifications are described by Heyn and Mettke . In case100) for defining the carbon footprint [2 equivalents. CFs are documented in the fifth assessment report (AR5) by the Intergovernmental Panel on Climate Change (IPCC) [The Joint Research Center (JRC) of the European Commission recommends using characterization factors (CFs) of a 100-year time horizon global warming potential (GWPootprint . The care (IPCC) . The elereport AR by the IMaterial footprint is defined in terms of Raw Material Input (RMI) and Total Material Requirement (TMR). RMI measures cumulative raw materials used within the whole process chain of a product ,56. UsedThe TMR thus measures the total amount of abiotic and biotic primary material required over the complete life cycle ,58. RegaCFs of RMI are developed by the Sustainable Resource Futures (SURF) group in the Center for Environmental Systems Research (CESR) at the University of Kassel ,59,60, wWater footprint is assessed according to the AWARE method , which i3/m2 * month of any basin is related to the world average to calculate regionalized CFs, which are available online http://www.wulca-waterlca.org/aware.html.AWARE aims at identifying the risk of freshwater depletion for any other water user in the same area. The resulting indicator Availability-Minus-Demand with the unit m3 world eq/m3. For instance, the German CF of 1.36 [The CFs represent “a relative value of the impact score of water consumption” ,  are tested ,64. The However, the design of the bridge is wholly described by Brühwiler et al. . An overThe system boundaries include the whole life cycle from the raw material extraction to the end-of-life phase of the bridge within the two design options. LCA calculations are done within the GaBi database. Regarding materials, which are not available in the GaBi database, i.e., steel fibers, they are modeled within the ecoinvent database . For theIn terms of the module A4, for the transportation modeling, van, truck, and road train are used for the transportation modeling. The choices of the transport type depend on the amount of transported material. Transportation distances are defined according to Lünser . An addiA5 represents the installation of precast and ready-mix concrete at the construction site. Diesel demand of the construction equipment and the amount of liquefied petroleum gas to install the bituminous asphalt sheeting are defined according to the expert’s interviews.The lifespan of the bridge, use phase B4, is assumed to be 90 years . Mainten3 in terms of CC [3 according to the experts’ interviews. A milling machine with 2.54 L/m3 diesel demand could be used for the deconstruction of the road surface, and the deconstruction of the sealing could be done using an excavator with 0.20 L/m2 diesel demand and 0.13 L/m2 liquefied petroleum gas demand [3 diesel demand, excavator with 0.30 L/m3 electricity demand of 18.26 L/m3 for other processes within a waste plant [3 and simultaneously 21.21 MJ/t of electricity is needed for other processes within the waste plant.For the deconstruction life cycle stage, C1, crowbar, excavator loader, hydraulic hammer, wheel loader, and cutters are assumed to be used; They require a diesel consumption of 17.6 L/mms of CC . To conss demand . The cons demand . Life cyte plant . Regardi3 of UHPC in comparison with CC will be discussed in ISO 14040  defined 3 of UHPC in comparison with 1 m3 of CC are shown in LCA results of the carbon footprint of 1 mLCA results of the material footprint (measured in RMI and TMR) are shown in The evaluation of the considered footprints shows that the UHPC (M2Q) has the highest environmental impact. The analysis results of UHPC ready-mix concrete (M3Q) show slightly lower environmental impacts but they are still higher than CC results. About CC, the concrete of compressive strength class C50/60 has more environmental impact. Furthermore, as a commonality of the three footprints can be seen that the highest environmental impact occurs in the production stage A1–A3. These contributed to more than 95% to the material footprint and 90% to the carbon and water footprint. Dominant processes of the carbon and water footprint are the production of cement and steel fibers, as well as the aggregates of the material footprint. The micro steel fibers have been also identified for the largest unused per used extractions.Type of cement.Production process of micro steel fibers.The sensitivity analysis will examine how the footprints are sensitive to changing the two dominants variants as follows:The CEM I 52.5 R-HS/NA is used as the type of cement for the UHPC . This isThe main part of steel production for instance in Germany takes place in the blast furnace route . Pig iroSensitivity of the footprints to the changing type of cement for all mixtures of UHPC and CC and type of production process of steel fibers for UHPC mixture will be shown in this section. Regarding material footprint, the sensitivity analysis indicates that a potential saving of 62% could be done within the UHPC mixtures as shown in 2 eq. when the UHPC is considered to enhance the design of the bridge (UHPC Design) as shown in The carbon footprint of the bridge with CC (CC-Design) could be decreased by about 40 t COWater footprint could be reduced by more than 30% when the UHPC design is considered, as shown in 3 of concrete mixtures . In contrast to the results for the environmental footprint of 1 mmixtures , the anaThis article provides a comprehensive LCA of carbon, material, and water footprint of UHPC in comparison with CC using precast and ready-mix concrete. The GaBi construction materials database is used altogether with openLCA software, which enhances significantly quantifying environmental footprints of constructions. Results are analyzed at the construction material level to enhance sustainability aspects within design of constructions. Higher environmental impact of UHPC is shown when only the quantitative aspects are considered for the formulation of the functional unit. At the case study level, results are analyzed to test the footprints of UHPC in relation to the functionality provided. Better environmental performance of UHPC could be indicated within the assessment of a practical application such as a bridge. Cumulative raw material demand in terms of RMI and total primary material extraction in terms of TMR, in addition to water use of UHPC, are provided for the first time by this article. UHPC shows relative lower footprint values when the use phase is considered. However, higher environmental impacts are shown within the end-of-life phase according to the high compressive strength. Policies and standards related to UHPC use need to be developed according to the current increase in the usage of new concrete technologies. Because of the relative high cost of UHPC, an economic analysis, e.g., life cycle costing of UHPC in comparison with CC, could be a crucial issue to be considered for future studies.


Ultraviolet light at a wavelength of 254 nanometers was used to irradiate the reactor. The results showed that azithromycin removal was significantly lower in the presence of ultraviolet radiation alone 58% with the removal efficiency than the case that ultraviolet radiation was used with sodium persulfate 98%. The best azithromycin removal conditions were obtained at the removal efficiency with the initial concentration of antibiotic 5 mgL-1, the concentration of persulfate 1mmol, and the contact time 30 min. and pH = 7. The rate of decrease in the concentration of residual azithromycin is increasing with increasing sodium persulfate concentration and decreasing the initial azithromycin concentration. This research can help to apply the integrated use of advanced oxidation processes to idealize decomposition-resistant compounds removal processes and to better understand the parameters affecting the removal.Azithromycin is among the broad-spectrum antibiotics that is widely available in various environmental systems and could have destructive effects on the ecosystem and human health due to its bacterial resistance. In this study, removal of azithromycin from wastewater using an advanced oxidation process of ultraviolet light with and without persulfate was investigated and effective parameters for the management of each of the processes were evaluated. The effect of different parameters including the concentration of Azithromycin antibiotic at levels 5, 15, 45 mgL Population growth and social and economic development have caused serious damage to the environment in the past 50 years . Most meThe expired or unused drug compounds may enter the landfill site. If the right technology is not used in the designing, developing or equipping these sites, their leakage to the surface and groundwater and contaminating the drinking water sources will be inevitable. Biological solids from the sediments in pounds of wastewater treatment plants contain high concentration of these compounds , 8. More Azithromycin is the result of a change in the erythromycin structure. This compound has different chemical and antimicrobial properties compared to erythromycin, for example, it is more stable in the acidic environments and it is better diffused in tissues. A large amount of this drug is not metabolized in the body and is largely eliminated by the bile , 17. VarThe aim of this study was the comparative removal of azithromycin from sewage using two ultraviolet and ultraviolet/sodium peroxide processes based on the formation of hydroxyl and sulfate radicals. Parameters such as initial concentration of antibiotic, initial persulfate concentration, reaction medium, and reaction time were used as effective factors on system conductance and after obtaining the optimal conditions for the best removal efficiency experiments were evaluated on real wastewater as well. By obtaining the COD, the removal efficiency of other organic compounds in the sewage was also measured by the combined process of ultraviolet and persulfate radiation. Also, nitrate, phosphate, and bicarbonate anions removal efficiency was also calculated from this process.Chemicals38H72N2O12) 98%, Analytical grade, CAS Number: 117772-70-0 was purchased from the Farma Pharmaceuticals Company Tehran, Iran. The molecular structure of azithromycin is shown in 2S2O8, 99%), CAS Number: 7775-27-1, of the Sigma Aldrich Company HPLC grade methanol , CAS Number: 67-56-1, acetonitrile , CAS Number: 75-05-8, sodium hydroxide , CAS Number: 1310-73-2, and sulfuric acid , CAS Number: 7664-93-9, were purchased from Merck-Germany. Standard Stocks Solution of 1000 mgml-1 of antibiotic was prepared by solving 100 mg of azithromycin antibiotic in 100 mL of methanol. Standard Stocks Solution of 50 mmol was prepared by solving 11.9g Sodium persulfate in one liter of deionized water with a resistivity less than 5 Ω. , and also solid phase extraction (SPE) tubes were purchased from INOPAK, Korea. Low pressure mercury lamp  was purchased with a protective quartz coating .Azithromycin . The low pressure mercury lamp  with a quartz protective tube was submerged in the middle of the reactor’s central cylinder. The effect of ambient light was prevented by wrapping this set in an aluminum foil. The continuous mixing of the specimen was performed by the magnetic stirrer. After preparation of the reactor, an appropriate volume of antibiotic stocks and sodium persulfate solutions were added. Using the sulfuric acid and sodium hydroxide 0.1 N, the pH of the sample was adjusted within the desired range. By turning the mercury lamp on and during the contact time 30, 60, 90 minutes; the entire contents of the reactor were passed through the cartridge. In order to ensure the decomposability, all tests were repeated twice and the standard deviation was considered to be less than 5%.-1 was reported. To compare the two methods of ultraviolet and ultraviolet/persulfate, all conditions were considered the same.In this study also for investigating the effectiveness of advanced oxidation (ultraviolet/persulfate and ultraviolet radiation) on the actual wastewater of hospitals a wastewater specimen was collected from hospital in Shahr-e-Kord and analyzed after the transfer to the laboratory. The specimen was first filtered through 0.45 µm filter in the laboratory. The steps of SPE tube preparation, extraction and analysis of specimens were carried out according to the analytical method described in the next section. The concentration of antibiotic studied in the hospital wastewater 5.04 mgLAnalytical methodsHigh Performance Liquid Chromatography equipped with fluorescence detector  and column C18  was used in analytical experiments. A mixture of methanol and 0.1% formic acid in the volumetric ratio of (90:10) was used as the mobile phase in a uniform flow of 0.3 ml/min . The injThe extracted specimen was collected in a Microtube 2 mL for HPLC injection. 20 μL of the extracted specimens were injected into an HPLC device through a 20 μL loop. Using the equation and calibration diagram, the amount of residual azithromycin was calculated after the advanced oxidation process and using the following equation 1, the amount of azithromycin removal efficiency was measured. The other analysis such as COD, bicarbonate, nitrate, and phosphate were performed based on the protocol in standard methods for the examination of water and wastewater; .Efficiency (%) = The obtained data were entered into the SPSS software and according to the type of data distribution ANOVA and Bonferroni tests were used. The significance level was considered as pv < 0.05. Comparison of Azithromycin removal by direct photolysis UV alone and UV/persulfate-1 obtained under the same conditions in terms of pH = 7, PS = 1 mmol, time = 30 min. 58% and 98% for UV alone and UV/sodium persulfate, respectively. Decrease the amount of Azithromycin followed first order kinetics. In general, the reaction rate constant obtained from the equation 2, was increased when ultraviolet radiation was used alone 0.03 min-1 compared to the case that the ultraviolet light was used with sodium persulfate 0.13 min-1. Increasing the efficiency in UV/sodium persulfate process could be due to the production of sulfate radicals due to the simultaneous use of sodium persulfate and ultraviolet radiation along with the presence of hydroxyl radicals. As a result, the degradation and removal of azithromycin in optimal conditions is increased from 58%  to 99% (UV/persulfate). Azithromycin removal in any of the processes  is shown in et al. on sulfamethazin showed that among the ultraviolet, persulfate, ultraviolet and persulfate and ultraviolet and hydrogen peroxide removal methods the highest removal efficiency was associated with 96.5% the ultraviolet and persulfate process (30). Similar results were observed by Yang Deng et al. on fluorophenicol showed that compared to the UV and UV/persulfate methods, the UV/persulfate method removed 98.4% fluorophenic over an hour  from 0.13 min-1 at a concentration of 5 mgL-1 to 0.061 min-1 at a concentration of 45 mgL-1 of the azithromycin antibiotic. Azithromycin removal efficiency was compared at three levels of antibiotic concentration 5, 15, 45 mgL-1 by one-way ANOVA. According to the -1 of azithromycin; the p-value was 0.00 (first type error). With the increase in antibiotic levels, the decrease in antibiotic elimination efficiency is observed. The possible justification of these changes can be because the available sulfate radicals are insufficient to degrade the high concentrations of azithromycin antibiotic (K=) -0.0016  + 0.133 for 0.135 min-1 at a concentration of 1 mmol to 0.14 min-1 at a concentration of 4 mmol of the sodium persulfate. These changes are presented in the The reactions constant was increase according to the equation K = 0.0017 [PSet al. in their study on the removal of sulfamethazine and fluorophenicol from water by persulfate and ultraviolet concluded that the concentration of 1 mmol of sodium persulfate was the optimal concentration  which is one of the advantages of the UV/persulfate method. As it can be observed in the et al. in a study conducted on levoflacxin by ferrous and persulfate, they also concluded that they have a better efficiency in near-neutral pH state , which indicates oxidant demand of the organic compounds and decreasing the removal efficiency of azithromycin in the raw wastewater . The competition between the mentioned above compounds with azithromycin content can be shown as follows.The nitrate in the wastewater is converted to the nitrate radicals by UV/persulfate process, and decreases with the efficiency 44%. Due to the creation of an internal filter, it reduces the amount of sulfate radicals and, as a result, reduces azithromycin removal efficiency compared to the time when the synthetic sewage without nitrates was analyzed . The con3-> COD> PO43-> NO3-HCO-1, sodium peroxide concentration of 1 mmol, pH = 7 and the contact time of 30 min were obtained as optimal values. The ultraviolet radiation process removes 60.21% and UV/ sodium persulfate process removes 98.30% of azithromycin antibiotic in synthetic wastewater under the optimal conditions. This study proved that the idealization of the removal process can affect the efficiency of the system so that the removal efficiency in optimal conditions of the real wastewater was obtained as 70% and with the increase of the contact time to 3 h it approached to 90%. The study demonstrated the use of advanced UV/persulfate oxidation is a good way to remove azithromycin and similar antibiotics in the hospital and pharmaceutical plant’s wastewater. We propose more studies to apply this oxidation method and to determine the operation parameters for similar antibiotics in pilot and full scale in the future. The use of UV/persulfate process on a laboratory scale was a safe method to remove azithromycin. In this study, the effect of azithromycin concentration, persulfate concentration, pH and contact time on azithromycin removal efficiency was investigated. In the conducted experiments the azithromycin antibiotic concentration of 5 mgL


The aim of the study was to estimate the influence of the different levels of Cu, Zn, and Mn nanoparticles on the activity of aminopeptidases in turkey. An experiment was carried out on 144 turkey hen Hybrid Converter. The birds were divided into groups given standard- and nanoparticle-supplementation of different level of copper , zinc , and manganese , covering respectively 100%, 50%, and 10% of the physiological demands for those minerals in the diet. The activity of aminopeptidases  after supplementation of minerals was determined in the breast and thigh turkey muscle. The strongest effect of interaction among minerals supplementation form and dose on the activity levels of the aminopeptidases in thigh muscle was observed for nano-Cu already at the lowest dose of 2 mg/kg. In this dose (covering 10% of the birds’ demand) nano form of supplementation significantly increased the activity of Ala-, Leu-, and ArgAP , compared to standard form of supplementation . In turn, in breast muscle, after supplementation covering 10% of the demand with the nano-Cu, nano-Zn, and nano-Mn compared to the standard form, we did not observe any significant difference in the activity levels of any of the investigated aminopeptidases, except for AlaAP under Zn supplementation. Supplementation with the 20 mg/kg of Nano-Cu (100% of demand) and with 10 mg/kg of Nano-Cu (50% of demand) inhibited the activity of all of the three aminopeptidases in thigh muscle. Supplementation of the minerals in nano form into the diet, especially of Cu and Zn in the dose covering 10% of the demand is relevant to maintain homeostasis in turkey muscles, as indicated by the activity of the aminopeptidases. Poultry meat belongs to a group of high-quality food products because of its valuable dietetic and nutritional properties, providing the necessary proteins and trace minerals into the human diet ,2,3. FeeThe maintenance of cellular homeostasis involves lysosomal enzymes, including aminopeptidases, responsible not only for the accumulation and circulation of proteins and microelements in the animal body. Changes in the aminopeptidase activity affect the bioavailability of the nutrients from the feed, composition of the muscle, and—in case of production animals—the final product quality . Trace eZinc, copper, and manganese are significant supplements in commercial poultry diets, and are provided traditionally in feed using inorganic salts such as oxides and sulfates. The availability of minerals from feed materials of plant origin, as well as from traditional inorganic sources—i.e., oxides, sulphates, or carbonates—is relatively low, while the requirements of modern poultry production for microelements are very high . One of It is also crucial to avoid the oversupplementation of the Cu, Mn, and Zn in the poultry diets. This is important aspect since the oversupplementation of those minerals negatively affects the animals’ performance ,14. ThusThe aim of this study was to evaluate the influence of supplementation of the diet with different levels of nanoparticles and traditional form—i.e., Cu, Zn, and Mn—on the activity of the selected, most indicative aminopeptidases: alanyl: AlaAP, leucyl: LeuAP, and arginyl: ArgAP in turkey meat.n = 8 birds).The experiment was conducted on a turkey farm located in the north-eastern part of Poland, using 144 turkey hen hybrid converter (2 with a wood chip bedding. The stocking density was at the level of 4.8 birds/m2 for the first 6 weeks and 3.2 birds/m2 from seven weeks onward until the end of production cycle (98 days). The experimental building was equipped with automatically controlled lighting, heating, and ventilation system. Light programs and temperature were in accordance with the recommendations of hybrid turkeys . Birds had ad libitum access to drinking water and feed mixtures. All birds were fed according to the diet shown in the The birds were kept in pens, each with an area of 3.7 mThe vitamin premix was added to the feed as a second step of the diet preparation. It covered the total (100%) of the demand for the two minerals not being a treatment, all provided in a standard form, while the treatment mineral was present in the premix in the form and dose respective to the specific treatment described below.2O3 were obtained as a powder from Sky Spring Nanomaterials Inc. . They were applied either in the form of a powder or of a colloid prepared based on the miliQ water. The treatments receiving standard form of the minerals included copper in the form of CuSO4, zinc in the form of ZnO and manganese, as MnO.In order to test the effect of mineral supplementation form in turkey feeding, the birds were divided into two groups. Control groups received a standard form of the studied minerals, while the test groups received the mineral in the nanoparticles form. The birds in both groups were supplemented with different levels of copper , manganese , and zinc  respectively covering 100%, 50%, and 10% of the physiological demands for those minerals in the diet. The nanoparticles of Cu, ZnO, and MnM. pectoralis) and thigh  muscle meat samples were collected immediately after slaughter (max 40 min) and frozen in liquid nitrogen (−80 °C). Then, the samples were homogenized in 0.1 M phosphate buffer (pH 7.0) with 0.1% Triton X100 according to the modified method of [The turkey breast , leucylaminopeptidase, and arginyl-amino-peptidase (ArgAP-EC 3.4.11.6) were determined by the method of  in obtaip ≤ 0.05) were computed using the LSMEANS option.A generalized linear mixed model analysis was performed on all measured parameters: AlaAP, LeuAp, and ArgApP including supplementation form, dose, and their interaction as fixed factors. A separate model was run for each of the minerals: Cu, Mn, and Zn in each of two types of muscle—breast and thigh—the validity of the models was tested by using Akaike’s information criterion. PROC GLIMMIX of SAS v 9.3  includinThe activities of alanyl-aminopeptidase (AlaAP-EC 3.4.11.2), leucylaminopeptidase (LeuAP 3.4.11.1) and arginyl-amino-peptidase (ArgAP-EC 3.4.11.6), as subjected to the different levels of supplementation in nano- and standard form were analyzed in turkey breast and thigh muscle. Obtained results have been presented respectively in The strongest effect of interaction between Cu supplementation form and dose on the activity levels of all aminopeptidases was observed in thigh muscle already at the lowest dose 2 mg/kg. The lowest dose of Cu (10% of the demand) in nanoform increased the activity of Ala-, Leu-, and ArgAP significantly higher than standard form, as well as of all other applied treatments . SupplemIn turn, in breast muscle, after supplementation covering 10% of the demand with the nano-Cu compared to the standard form, we did not observe any significant difference in the activity levels of any of the investigated aminopeptidases. In case of higher doses of Cu supplementation, at the level of 50% provision of the nano-Cu in this muscle increased the LeuAP activity compared to standard form, while at the level of 100% nano-Cu decreased the activity of AlaAP compared to standard form .As presented in the In the breast muscle, provision of the Nano-Zn at the level of 2 mg/kg caused the AlaAP activity decrease when compared to provision of this mineral in the same dose in the standard form , as wellNo effect of interaction between Mn supplementation form and dose on the activity levels of any aminopeptidase neither in thigh nor in breast muscles was observed . A mediuObtained results in our study demonstrated that supplementation of minerals, especially Cu in the nano form into turkey diet significantly influenced the aminopeptidases activity. It should be noted that changes in the activity of the aminopeptidases are conditioned by different factors covering the dosage and form of supplementation of minerals like Cu, Zn, or Mn which are essential bioactive compounds ,15. For Among three used minerals  the strongest effect of interaction of supplementation form and dose on the activity levels of the aminopeptidases was observed in case of Cu—already at the lowest dose 2 mg/kg. The activity of Ala-, Leu-, and Arg-AP in nanoform increased significantly more than in standard form in case of thigh muscle. In turn, in breast muscle, after Cu, Zn, and Mn supplementation we did not observe any significant difference in the activity levels of any of the investigated aminopeptidases, except for AlaAP under Zn suppelentation. Supplementation with the 20 mg/kg of Nano-Cu (100% of demand) and with 10 mg/kg of Nano-Cu (50% of demand) clearly inhibited the activity of all of the three aminopeptidases, which can even lead to accumulation of the investigated minerals in the body and can affect negatively the performance in turkey production.We concluded that supplementation of the minerals in nano form into the turkey diet, especially of Cu and Zn in the dose covering 10% of the demand is relevant to maintain homeostasis in turkey muscles, as indicated by the activity of the aminopeptidases.


Additionally, UV absorption, fluorescence, and circular dichroism (CD) spectroscopy were applied to investigate the mechanism of the interactions and to study the conformational change of mucin. We found that the TMD NSs could adsorb on the mucin layer and affect its viscoelasticity. The results indicated that the SL WS2 NSs exhibited the highest initial absorption rate and the maximum absorption amount, while the SL MoS2 NSs exhibited the highest initial desorption rate. During the adsorption, the viscoelasticity variations of the mucin layer caused by the WS2 nanosheets were weaker than those caused by the MoS2 nanosheets. Furthermore, the conformational changes of mucin caused by the SL MoS2, SL WS2, and SFL MoS2 NSs were higher than those resulting from other TMD NSs. These findings provide important information on the interactions between TMD NSs and mucin and provide useful insights into the interfacial behavior of TMD NSs before they enter tissues.Ultrathin 2-dimensional transition metal dichalcogenides (TMDs) have become a class of high-potential materials in biomedicine due to their intriguing properties. They have been applied to solve biomedical challenges, such as biosensing, bioimaging, drug delivery, and cancer therapy. However, studies of the interactions between these materials and biomolecules are insufficient. Mucous tissue serves as a barrier to foreign hazardous substances and a gel layer for substance exchange. The main organic matter of mucous tissue is mucin, so it was selected as a model biomolecule to study its interactions with six different TMD nanosheets (NSs), including single-layered (SL), few-layered (FL), and small few-layered (SFL) MoS Mucus is secreted by specialized goblet cells in the columnar epithelium and serves many functions including lubrication, maintenance of a hydrated layer over the epithelium, a barrier to hazardous substances, and a permeable gel layer for the exchange of gases and nutrients with the underlying cells and tissues  are fundamentally and technologically intriguing  should be conducted before the biomedical applications of such novel inorganic nanomaterials. However, there are only a few studies on the interaction between TMD NSs and proteins via molecular simulation .Mucin from bovine submaxillary gland was purchased from Shanghai Yuanye Biotech Co., Ltd, China. TMD NS aqueous dispersions (0.1 mg/mL) were purchased from XFNANO Materials Tech Co., Ltd, and the layered NSs were prepared via the lithium intercalating method. The product information of these TMD NS aqueous dispersions was provided by the supplier and listed in The TMD NS aqueous dispersions were diluted to 0.01 mg/mL with ultrapure water. Thereafter, the hydrodynamic diameter and the electrophoretic mobility (EPM) of the TMD NSs was measured using a zetasizer .F) and dissipation (D) became stable, samples were added following the stages below. (I) The formation of mucin: 0.2 mg/mL mucin aqueous solution flowed over for ~20 min and coated the bare gold chip, forming a layer of mucin on its surface. (II) The rinsing of the mucin layer: ultrapure water was flowed over the mucin for ~10 min to remove the unstably combined mucin. (III) The interaction of TMD NS and the mucin layer: a 0.1 mg/mL TMD NS dispersion was flowed through the channel for ~45 min, giving a sufficient interaction with the mucin layer. (IV) Desorption: ultrapure water was flowed into the channel again. The signal of F and D at the seventh overtone of each experiment was recorded for the study.QCM  was applied to monitor the real-time mass and viscoelasticity change of the mucin layer during its interaction with the TMD NSs. Prior to the experiments, the bare gold chips were cleaned following the protocol in the QCM with a dissipation monitor (QCM-D) manual. Briefly, the gold chips were treated in a UV-ozone cleaner  for 30 min to destroy the organic matters and then soaked in 75% ethanol and cleaned in an ultrasonic cleaner for 30 min, followed by thorough rinsing with ultrapure water and drying with nitrogen. During the QCM experiments, the flow rate was set as 60 μL/min maintaining a laminar flow in the sensing module. Ultrapure water flowed through the channel at the beginning of each experiment, and after the signal of frequency  was used to real-time monitor the refractive index of the materials during the interactions between the TMD NSs and mucin layer. The bare gold SPR chips were cleaned using the method mentioned in the “2.3. QCM study” session. At the beginning of each experiment, the twelve-way valve was set to “Load,” and the 100 μL sample loops were filled with a 0.2 mg/mL mucin aqueous solution. Ultrapure water flowed as a running buffer at the rate of 5 μL/min to maintain a laminar flow. After the stable signal of the SPR angle was achieved, the 12-way valve was set to “Inject,” and the mucin solution flowed over the bare gold chip and formed a mucin layer on its surface. The twelve-way valve was set to “Load” again after the mucin solution was exhausted, and the sample loops were filled with 0.1 mg/mL TMD NS dispersions. Then, the flow rate was set to 2 μL/min, and the twelve-way valve was set to “Inject,” resulting in a slow flow of the TMD NS dispersions over the mucin and a sufficient interaction with it. The experiment was terminated after the TMD NS dispersions in the sample loops were exhausted.UV absorption spectra, fluorescence spectra and CD spectra were applied to investigate the interactions between the TMD NSs and mucin in the aqueous phase. TMD NS dispersions and mucin aqueous solutions were fully mixed and interacted for 2 min at room temperature, and then the spectroscopy spectra of the mixtures were measured. In the UV and fluorescence spectroscopy experiments, the final concentration of mucin was 0.1 mg/mL, while in the CD measurement, it was 0.5 mg/mL. The UV absorption measurements were performed using a TU-1810 spectrophotometer , and the fluorescence spectra were evaluated using a Cary Eclipse fluorescence spectrometer . A J-810 CD spectrometer  was used for CD spectrum determinations. The TMD dispersions with the same concentration as in the mixtures were used as a reference sample in all of the spectroscopy measurements.2 nanosheets were shown in 2 and SL WS2 had the biggest diameters and the smallest thicknesses, while SFL MoS2 and SFL WS2 were smallest in diameter and biggest in thickness. FL MoS2 and FL WS2 were in the middle. Herein, “flakiness” is noted to describe “the ratio of diameter and thickness,” so the single-layered TMD NSs were the highest in flakiness, while the SFL TMD NSs were the lowest.The interactions between proteins and nanomaterials are related to the physicochemical properties of the nanomaterials, including size and surface charge  in odd overtones are measured to directly reflect the mass change of the materials on the surface of its sensing module via Sauerbrey equation . The attenuation of oscillation amplitudes at a certain overtone is described as the dissipation (D), which gives information on the surface viscoelasticity , the trend of F and D variation is the same. In stage I, when the mucin aqueous solution flowed over the gold chip surface, the F value descended while the D value ascended owing to the increase of the mass and viscoelasticity of the materials on the surface, which indicated the adsorption of mucin on the surface of the QCM sensing module and the formation of a mucin layer. In stage II, ultrapure water was introduced to remove the unstably combined mucin. The F value ascended as the D value descended because the slight desorption of mucin from the gold surface. The average weight of the mucin layer formed on the sensing module was 197.7 ng/cm2. In stage III, the F value decreased and the D value increased when TMD NS dispersions were flowed over the mucin layer, indicating an increased mass and viscoelasticity. In stage IV, an increase of the F value and a decrease of the D value were observed because of the desorption of the mucin and TMD NSs, which demonstrated a decrease of the mass and viscoelasticity. The main difference existed in stage III. When the MoS2 NS dispersions flowed over the mucin layer , and the D values continued to increase. During “stage a,” the adsorption and desorption of the MoS2 NSs, as well as the desorption of the mucin, occurred simultaneously, but the desorption rate of the MoS2 NSs and mucin was higher than the adsorption rate of the MoS2 NSs. The duration of the “stage a” change trend was SL MoS2 > FL MoS2 > SFL MoS2, indicating that the MoS2 NSs with higher flakiness had a smaller initial adsorption rate, and they were more prone to cause the desorption of the mucin. On the other hand, a “stage b” was divided at the beginning inside the stage III. When FL WS2 and SFL WS2 interacted with mucin . Additionally, the SL MoS2 exhibited the highest initial desorption rate.DA as the total change in dissipation in the adsorption stage, i.e., the maximum D in the adsorption stage minus the D value at its beginning. This index reveals the influence of the TMD NSs on the structure of the mucin in the whole adsorption stage. SFL MoS2 was highest in this index, while SFL WS2 was the lowest. In dDA/dt is the initial rate of dissipation change in the adsorption stage, which was defined as a variation of D value in the first 150 s of the adsorption stage. This index reflects the rate of viscoelasticity change and further reveals the intensity of the influence of the TMD NSs on the mucin structure at the initial stage of adsorption. The dDA/dt value of SL WS2 was much higher than that of the other TMD NSs, indicating that it could rapidly affect the structure of mucin. In DA/ΔFA is the variation of the dissipation value accompanying the change in unit frequency in the adsorption stage, which gives information about the viscoelasticity change caused by per unit mass change. The −ΔDA/ΔFA value of FL MoS2 had the highest of all the TMD NSs, while the SFL WS2 had the lowest −ΔDA/ΔFA value. In general, the −ΔDA/ΔFA value of the WS2 NSs was smaller than that of the MoS2 NSs, indicating that when the TMD NSs in the same mass adsorbed onto the mucin, the WS2 NSs caused less influence on its viscoelasticity than the MoS2 NSs. Based on the above experimental results, suitable TMD NSs can be selected for specific biomedical applications as demanded.The viscoelasticity changes of the protein hydrogel layer can be caused by the changes in the structure of protein , and the Hill coefficient n. These parameters can be obtained through the Hill equations as follows:The relative strength and cooperativity of the interaction of the TMD NSs and mucin were further studied based on the quantification of several key parameters, including the binding dissociation constant Qmax is the saturation value of Q, which can be derived through non-linear fit of fluorescence quenching  have a stronger association with mucin. The Hill coefficient n describes the binding cooperativity between the TMD NSs and mucin (2-mucin complex exhibited cooperative binding with positive cooperativity (n > 1), implying that an increased amount of mucin adsorbed onto the surface of SL WS2 will enhance the binding strength between the proteins and the surface. In contrast, the FL MoS2-mucin, FL WS2-mucin, and SFL MoS2-mucin complexes displayed anti-cooperative binding with negative cooperativity (n < 1). This indicates that the more mucin that is absorbed on the surface of these TMD NSs, the weaker binding strength will be.Where uenching . KD is tnd mucin . The SL 2, FL WS2, and SFL WS2 did not exhibit much variation, while SL MoS2 and SL WS2 caused the CD spectra of mucin to redshift, and SFL MoS2 slightly enlarged the peak of CD spectra. These experiment results were in accordance with the trends of UV spectra variation as mentioned.CD spectroscopy is an effective tool for rapid determination of secondary structure of proteins (Greenfield, 2 increased the percentage of α-helix, and decreased the percentage of β-sheet. In contrast, the interaction of mucin with other TMD NSs made its percentage of α-helix higher, and β-sheet lower, and SL MoS2 and SL WS2 caused larger-scale variation. These results gave the information of conformational change of mucin during its interactions with the TMD NSs.The analysis of secondary structure variation of mucin was conducted via CD Pro software. As shown in 2 can quickly adsorb on the mucin layer, and the viscoelasticity variations of the mucin layer caused by the MoS2 nanosheets were more intense than those caused by the WS2 nanosheets. It was observed in the spectroscopic probing that SL MoS2, SL WS2, and SFL MoS2 caused higher the conformational changes of mucin than the other three TMD NSs. TMD NSs interact with mucin molecules by hydrophobic effect. The TMD NS-mucin complex probably fell off from the gold surfaces after the thorough washing, thus resulting in the increased F value and the decreased D value (2 exhibited the highest initial desorption rate.According to the results mentioned above, here a possible mechanism of the interactions between the TMD NSs and mucin was proposed, as shown in  D value . In thisN-acetylgalactosamine, N-acetylglucosamine, fucose, galactose, and sialic acid, and the glycosyls are arranged in a “bottle brush” configuration around the protein core (Bansil and Turner, F value and a decreased D value (Mucin consists of a protein core and glycosyls in its common structure. The protein core is arranged into distinct regions. The central glycosylated region is comprised of a large number of tandem repeats that are rich in serine, threonine and proline, while the regions with an amino acid composition more representative of globular proteins are located at the terminals. The central glycosylated region is highly glycosylated consisting of ~80% carbohydrates primarily  D value .2 exhibited the highest initial absorption rate and the maximum absorption amount, while SL MoS2 exhibited the highest initial desorption rate. During the adsorption, the viscoelasticity variations of the mucin layer caused by the WS2 nanosheets was weaker than that caused by the MoS2 nanosheets. Furthermore, the conformational changes of mucin caused by SL MoS2, SL WS2, and SFL MoS2 were higher than those from other TMD NSs. This work provides insights on the interactions between TMD NSs and mucin. Suitable TMD NSs can be selected for specific biomedical applications based on the findings in this study.In summary, we demonstrated the interactions between mucin and TMD NSs using QCM-D, SPR, and spectroscopic methods, including UV absorption spectroscopy, fluorescence quenching spectroscopy, and CD spectroscopy. The results indicated that the TMD NSs adsorbed on the mucin layer and affected its viscoelasticity and refractive index. SL WSBL, RH, and RS designed research. BL and TY performed research. All the authors analyzed data and wrote the paper and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Electron microscopy observation confirmed that the amorphous phase was present in the fusion zone and amorphous–crystalline phase was present in the heat-affected zone. Higher nanohardness and reduced Young’s modulus values were demonstrated for laser welds using higher impulse energy (2.78 J) and impulse peak power (1000 W).Laser-beam fusion welding processes enable the increase of the size of metallic glass parts, and therefore facilitate the application of this new material in different products. To get exceptional properties of the material after the welding process, characteristics of the weld structure in the heat affected zone and in the fusion zone should be predicted. The main goal of this work is to study the structure and specific characteristics of the Zr Recent years have seen the intense development of bulk metallic glasses (BMGs). Presently, maximum dimensions of manufactured parts reach diameters of tens of millimeters, yet the thickness of homogenous metallic glass is still overly thin for BMGs to find industrial applications. Sufficient dimensions and the appropriately high thermal stability of specimens are required for BMGs to be used in selected engineering processes. The development of methods enabling the joining of BMGs is the precondition of their industrial application ,2,3.Generally, in some specific cases the properties of BMGs are diversified and better than the properties of their equivalents having the crystalline structure. Due to a chemical uniformity of their structure BMGs are characterized by a high strength and corrosion resistance. The values of their Young’s modulus are similar to those of their crystalline equivalents, yet they do not show the anisotropy of elastic properties and cannot accommodate a plastic strain. Amorphous materials have a lower electrical conductance than the crystalline alloys with the same chemical composition. For this reason, BMGs can be applied as functional materials and materials having favorable (desired) characteristic structures ,6,7,8,9.The making of small-sized specimens is connected, among other things, with the limited glass forming ability (GFA) of test alloys. For this reason, numerous research works are concerned with various methods enabling not only the production, but also the joining of BMGs.40Ni40P20, Pd40Cu30Ni10P20, Zr55Al10Ni5Cu30, and Zr41Ti14Cu12Ni10Be23) using welding processes in the supercooled liquid phase and using welding methods with the liquid phase content [Until today, it has been possible to join BMGs having various chemical compositions  and the heat affected zone (HAZ) during laser welding.The analysis of related reference publications revealed that tests involving the joining of BMGs performed using the laser beam were undertaken as early as in 2003. Li et al.  successfm et al.  used an o et al.  successfThe reduction or even the entire elimination of crystallization during laser welding was presented by Wang et al. . The resCrystallization occurring in the HAZ is the solid-state reaction differing from the crystalline phase formation taking place in the FZ ,21. CrysThe technology enabling the joining of BMGs was selected through the analysis of reference publications. Because of the availability of necessary equipment and the possibility of the adjustment of related welding parameters, the selected method was that of laser welding performed using a TruLaser Station 5004 laser station.l) to the glass transition temperature (Tg) has a decisive influence on the fusion zone structure. The HAZ  is more susceptible to crystallization than the weld metal, as the heating time in the solid state is longer than the cooling time of the molten material [x) may trigger changes of mechanical properties.The BMG joint made using the laser welding method consists of the FZ (weld), the HAZ, and the PM  ,9. Aftermaterial . In addiWhen adjusting laser welding parameters, the primary emphasis is given to the obtainment of desired weld morphology and penetration depth. Usually, the microstructure and the mechanical properties of the FZ and the HAZ are tested using X-ray-based methods, thermal analysis, calorimetric tests, electron microscopy, and hardness measurements ,24,25,262Cu phase precipitation. The priority of the Zr2Cu crystallization could be attributed to the fact that Cu and Zr were the primary compound constituents and that the Zr-Cu atomic pair was characterized by negative heat of mixing, the difference of atomic radiuses and electronegativity. In addition, it was observed that the Zr2Cu phase precipitated easier on the boundary between the amorphous-structure matrix and the reinforced particles.The HAZ of Zr-based laser-welded BMGs tend to undergo crystallization ,27. The 55Cu30Ni5Al10 alloy. The procedure for obtaining BMGs in the form of plates involved: the production of an initial alloy in the form of an ingot, then casting the molten alloy into a copper mold. The tested ingot was prepared on the basis of pure Zr, Al, Cu, and Ni elements with a total weight of 30 g, using an induction generator in a ceramic crucible. The alloy composition represents nominal atomic percentages , then raising the crucible plug, and introducing the molten alloy into the copper mold by the protective gas pressure. The investigated material was cast in form of plate with the dimension 20 × 10 × 2 mm.Welding tests were performed with TruLaser Station 5004 device . The parameters of the laser beam were selected on the basis of multiple tests. The TruLaser Station is a pulsed laser welding station. It enables positioning the working head by means of numerically controlled line guides. A stereoscopic microscope enables precise positioning of the head in the melting area and proper laser beam positioning on the welded material.The device is integrated with a TruPulse 103 laser resonator of the average impulse power reaching 95 W and the maximum impulse power of 6 kW, as well as with a radiator enabling the exchange of resonator-generated heat with the environment. A laser beam generated in the resonator is transported to the working head via an optical fiber of a core diameter reaching 300 µm.The parameters of the TruLaser Station used in this work are presented in the The laser beam parameters used in the process are presented in the The different weld zones are presented in The PANalytical XPERT PRO MPD X-Ray diffractometer  equipped with a copper anode X-ray tube was used to identify the phase composition of the HAZ and the FZ. The operating parameters of the lamp were 30 mA and 40 kV. For the purpose of the phase composition analysis, diffraction patterns were performed in the angular range 2Θ from 10° to 120°. Glassy structures were examined by X-ray diffraction (XRD) with the use of a Seifert—FPM XRD 7 diffractometer with Co Kα radiation at 35 kV. The data of diffraction lines were recorded by means of the stepwise method within the angular range of 30° to 90°. The diffraction pattern was obtained with step 0.04° 2Θ and 3 s per step. The X-ray wavelengths were applied at 1.79 Å  and 1.54Constituent elements of zirconium-based alloys are characterized by high affinity for oxygen, leading to greater amounts of impurities in alloys, easy formation of nuclei, and an increase in crystalline phase, ultimately resulting in the impairment of glass forming ability. The production of Zr-based BMGs is a complex process. Oxidation and crystallization may occur as early as when preparing a preliminary alloy, during casting, or even when preparing test specimens.The analysis of X-ray spectra enabled the preliminary determination of specimen structures and the repeatability of results. The related test results revealed the good GFA of Zr-Cu-Ni-Al type alloys.55Cu30Ni5Al10 BMG  for oxygen, leading to the reduction of GFA, as well as to an excessively long time at which the HAZ was exposed to high temperature.The X-ray diffraction tests of the ZrTransmission Electron Microscope (TEM) was used to verify the BMG structure and to analyze the nanocrystalline precipitates in the amorphous materials. The TEM-based test results did not provide complete information on the material structure. The tests involved the sampling of representative local fragments of specimens. It should be noted that the preparation of the specimens could be accompanied by crystallization or oxidation, affecting the structure of the materials subjected to microscopic observation.55Cu30Ni5Al10 in the form of 2-mm-thick plate in the as-cast state are presented in The identification and the analysis of the nanocrystalline features in the zirconium-based BMG and in the laser welds were performed using High Resolution Transmission Electron Microscope (HRTEM). Exemplary images of the amorphous-nanocrystalline structure, electron diffraction and the HRTEM image of the structure of selected areas of alloy Zr55Cu30Ni5Al10 as an example. The observation in the bright field . Other surface imperfections revealed in AFM-based images included nuclei of metallic phases or defects generated during mechanical surface preparation.The literature date revealed that the correlation between properties and the structure was described using similar dependences regardless of materials, chemical compositions or technologies used when making specimens. The scientists , based o55Cu30Ni5Al10 BMG are presented in Measurement results concerning the reduced Young’s modulus and the nanohardness in the tested zones of the welds made of the ZrCurves determined in relation to the individual zones of the weld made of the Zr-based BMG, using present laser welding parameters no. 2 are presented in v in the FZ and HAZ differed slightly, whereas Er in the FZ and HAZ differed by 16.49 GPa in relation to the specimen made using laser beam parameters no. 1 and by 3.24 GPa in relation to the specimen made using laser beam parameters no. 2. Similar results related to the Hv measurement could imply the homogenous structure in the FZ and HAZ.Higher nanohardness and reduced Young’s modulus values were determined in relation to the laser weld made using an impulse energy of 2.78 J and an impulse peak power of 1000 W. H55Cu30Ni5Al10 alloy in the form of 2-mm-thick plates and rods with 2 mm diameter were obtained.The application of effective pressure die-casting into copper molds enabled the creation of bulk metallic glasses on zirconium matrix. The Zr55Cu30Ni5Al10 BMGs after laser welding performed using a laser beam having a power of 700 W and 1000 W revealed that the structure was amorphous-nanocrystalline. The nucleation of crystalline phase Zr2Cu were the most prevalent in the HAZ.X-ray diffraction tests of ZrThe microscopic test results of as-cast plates revealed the presence of single regular crystals in the amorphous phase. The size of the crystals was restricted within the range of 5 to 70 nm. The structural observations of selected HAZ areas of the weld revealed the presence of single approximately 200-nm-sized crystallites in the amorphous matrix, whereas HRTEM-based observations confirmed the amorphous structure of the FZ.r in the FZ and HAZ differed by 16.49 GPa in relation to the specimen made using laser beam parameters no. 1 and by 3.24 GPa in relation to the specimen made using laser beam parameters no. 2.The weld area of HAZ revealed slightly lower nanohardness than the nanohardness of smooth FZ and PM surface areas. EOn the basis of research results analysis, the following conclusions have been voiced:


Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions. It haultivate . In the ultivate . As a reultivate ,7. For dHowever, it is difficult to obtain a high-quality contig from a short-reads output by Illumina Hi-Seq and others, and the overlapping regions among different bacterial species make it even more difficult to assemble for metagenome analysis . In addiThe combinations of protein genes in evolutionally close species are expected to show higher similarities ,13. TherThroughout this paper, bacteria are expressed not with their scientific names, but with the RefSeq accession numbers used by the National Center for Biotechnology Information (NCBI), as shown in If all  of the protein IDs are not picked up through the previous step, the sum of the lengths of the amino acid sequences would be smaller than in reality; therefore, the value would be much smaller than 100%.The results of the analysis were output in a CSV  file containing 11 columns (15 if options were included) as follows: (1) The protein’s accession ID; (2) the amino acid sequence in the MD5 format; (3) the length of the amino acid sequence; (4) the database used for the analysis; (5) the protein-domain ID in the database; (6) the name of the protein domain; (7) the starting position of the protein domain on the amino acid sequence; (8) the end position of the protein domain on the amino acid sequence; (9) the expected e-value of the protein domain; (10) whether the protein domain is matched (matched = T: true); (11) the analysis data. This research used the protein domain ID and the name of the protein domain. We divided the file into columns and provided the respective species, the types, and the numbers of the domain from Refseqs. This file had 8596 lines, 325 columns, and 8596 protein domains, see We made a cluster dendrogram for the pvclust package of the R data-analysis software ,20,21. TFor the 325 genomes acquired from the NCBI RefSeq, 100 bp fragments were randomly cut from the whole genome sequences so that the total length of the fragments would be ten times the length. The sequence of the fragments was translated into amino acid sequences of 6-frame. From these amino acid sequences, we searched for the protein domains using the InterProScan. Next, we created a matrix that showed the sum of the numbers of each domain that was found in the proteins of each species. Then, two files were created. The first had the values 1 or 0 and, where the number of a domain was 1 or more, the value was 1. The other file showed a logarithmic value of “the original number of domain plus 1.” A phylogenetic tree was similarly created to calculate the correlation coefficients for combinations of protein domains using those files.Bacterial 16S rRNA sequences were using NCBI RefSeq for 325 species. We confirmed the existence of 16S rRNA sequences by searching the corresponding genome. A phylogenetic tree was created based on the DNA sequences of these 16S rRNA sequences for 325 bacteria species using the maximum likelihood method for using Molecular Evolutionary Genetics Analysis 7 (MEGA 7) ,23. The 296C2 = 43,660 patterns) [We also obtained the gene sequences of 296 species within the 325 species. atterns) .An amino acid sequences phylogenetic tree was also created for DNA gyrase subunit B. RefSeqs divided each of them into files using one protein ID. Among the 296 species, three sequences  were very short. We also found that six sequences  were exactly the same, and therefore, these six species were excluded from further analyses. A phylogenetic tree was created for the amino acid sequences of DNA gyrase subunit B of those 287 species using MEGA 7, see To compare the cluster dendrograms and the phylogenetic trees, the correlation coefficients for the domain combinations and the pairwise distances for index sequences (DNA sequences for 16S rRNA and DNA and amino acid sequences for DNA gyrase subunit B) were calculated. Those results were dot-plotted using software R for every combination of two among the 322 species.The 143 environmental metagenome data were downloaded from the DNA Data Bank of Japan (DDBJ) sequence read archive (DDBJ SRA: DRA005425). These data were obtained by periodically collecting seawater from March 2012 to May 2014 in the northeastern coastal area of Japan. After the seawater was filtered using 0.2, 0.8, and 5 μm pore filters, the DNA samples were extracted from the bacteria that was attached to the filter. The 100 bp paired end sequencing was performed with the illumina Hiseq 2000 sequencer . Information on the collection date, filter size, sampling point and sampling depth is shown in The protein domains were obtained from 325 species. A total of 322 species were used for comparison (except for three that formed clusters that were not close to other species in the phylogenetic tree generated for the DNA sequences of 16S rRNA). The correlation coefficients of the domain combinations and the pairwise distances of the DNA sequences of 16S rRNA were dot-plotted using R for all of the combinations of any two species among the 322 species. The results are presented in The correlation coefficients were obtained for each of the two data sets—one in which all of the values were converted to 0 (absence) or 1 (presence), see Similarly, we made comparisons based on the information for 16S rRNA. For 296 species from which DNA sequences of DNA gyrase subunit B were obtained, the correlation coefficients for the domain combinations and pairwise distances of DNA sequences of DNA gyrase subunit B were similarly dot-plotted using R. Furthermore, for 284 species for which the amino acid sequences of the DNA gyrase subunit B were obtained (the three species that formed a cluster apart from the other 284 species in the created tree were excluded), the correlation coefficients for the domain combinations and pairwise distances of the amino acid sequences of the DNA gyrase subunit B were similarly dot-plotted using R.The results are shown in In each of the 500,000 reads from the metagenome data from the northeastern coastal area of Japan, the average number of reads for which the protein domains were identified by Pfam were 50,089 (10.02%), 19,566 (3.91%) and 8093 (1.62%) for the 0.2, 0.8 and 5 μm filters, respectively, and this suggests that more motif’s data were identified from samples using a smaller filter size. A heat map analysis that calculates the frequency of occurrence of each domain indicated that most of the samples were clustered by the sizes of the filters, see Subsequently, a cluster analysis was carried out in detail using the pvclust package. As a result, the samples were divided into four clusters, which were corresponded to the filters’ sizes, one to 0.2 μm, another to 5 μm and the other two clusters to two 0.8 μm, see Finally, a principal component analysis (PCA) was performed in order to determine the factor that caused the 0.8 μm samples to split into two clusters. Sea depth (surface (1 m) vs. surface chlorophyll maxima; SCM (10–20 m)), see The correlation analysis revealed that the cluster dendrogram that was generated based on the combination of the protein domains is somewhat similar to the phylogenetic tree for the DNA or amino acid sequence of 16S rRNA and DNA gyrase subunit B, see In order to verify the effectiveness of the protein motif-based method, we used actual environmental data from the Tohoku coastal area of Japan. The filter samples with larger pore sizes had fewer protein domains, as found by the Pfam database search. Other studies explain that this result is due to the large number of DNA sequences that are derived from the phytoplankton in the filter with a large pore size, and therefore, the sequence of the metagenome might contain a large amount of non-coding regions intron and intergenic sequences ,28.A PCA analysis was performed to determine a factor of 0.8 μm pore filter samples, which were divided into two clusters by cluster analysis. When the samples were classified by seasons, the samples were plotted more accurately, see