The results from our analyses indicate that at least for the given systems, force field and simulation protocol, the two methods diverge in their predictions. The possibility of a force field-dependent deficiency is examined as a possible source of the observed discrepancy.Both molecular mechanical and quantum mechanical calculations play an important role in describing the behavior and structure of molecules. In this work, we compare for the same peptide systems the results obtained from folding molecular dynamics simulations with previously reported results from quantum mechanical calculations. More specifically, three molecular dynamics simulations of 5 μs each in explicit water solvent were carried out for three Asn-Gly-containing heptapeptides, in order to study their folding and dynamics. Previous data, based on quantum mechanical calculations within the DFT framework have shown that these peptides adopt  Type I and II β-turns are the most common types of β-turns found in proteins, with the ideal torsion angles  in each of these categories being  and , respectively, whereas, the corresponding values for their “mirror-image” type I’ and II’ β-turns are  and  [esidues) –5. They s i to i+ with the[β-turns , the almβ-turns can be considered structurally as no well-behaved model systems, as they occasionally adopt random conformations, it is both the nature and position of specific residues that favors the propensity towards β-turn formation usually by stabilizing the formation of β-hairpin motifs [β-turns [β-turns which are common among observed protein structures [φ,ψ angles of this turn type are typically constrained in a small region of the Ramachandran plot for L-amino acids [i+1 and i+2 of type I’ β-turns indicate that, among others, Asn is generally favored in position i+1 and Gly in position i+2 [β-turn and β-hairpin conformations, though not as effectively as the D-Pro-Xaa loop-forming segment [β-hairpin promoter sequence D-Pro-Gly is balanced to some extent by an enthalpic advantage of the Asn-Gly sequence, as the rigid nature of the D-Pro-Gly segment may prevent energetically favored contacts between side-chains [β-turns, no extensive computational investigation has been made on the folding of short peptides containing this segment. A recent study by Kang and Yoo [β-turn structures using ab initio quantum mechanical (QM) calculations within the DFT framework, indicated that Asn-Gly type I’ β-turn was indeed the preferred structural motif for all the three heptapeptides. The optimized torsion angles for every β-turn type in each heptapeptide as obtained from their work [ψGly value that despite residing within the ±30° deviation limit, has a slightly increased flexibility compared to the rest of the angles.Although short peptides that appear in n motifs –12. Anal[β-turns , 10–12. ructures , 7. The no acids . Studiestion i+2 . Analysi segment –23. Intee-chains . However and Yoo , who exaeir work  at the Sβ-hairpin-like [β-turn motif [β-turn conformations in water, and examined to what extent the AMBER99SB*-ILDN force field, combined with the TIP3P [Folding molecular dynamics (MD) simulations using empirical force fields have matured to the point of becoming a powerful and useful analytical tool for studying the structure and dynamics of a wide variety of peptide systems. Our perspective on the current literature is that among all extensively tested and validated force fields , the AMBpin-like , 37–39. pin-like . So far,rn motif —possiblyrn motif , 42. Thehe TIP3P  water moβ-turn structures we conducted three MD simulations for the same three capped heptapeptides that Kang and Yoo used in their work: Ac-Ala-Ala-Asn-Gly-Ala-Ala-NHMe (hpNG-1), Ac-Leu-Val-Asn-Gly-Gln-Tyr-NHMe  [Asn-Gly-Leu-Phe-NHMe  [In order to study the propensities of the Asn-Gly segment to form ry 1EST)  and Ac-P β-turn) . The sys β-turn) –38 and i β-turn) , 47 for  β-turn)  and the  β-turn) , 29, whi β-turn)  was applΤ step of 20 K) over a period of 32 ps. Subsequently, the system was equilibrated for 10 ps under NpT conditions without any restraints, until the volume equilibrated. This was followed by the production NpT runs with the temperature and pressure controlled using the Nosé-Hoover Langevin dynamics and Langevin piston barostat control method as implemented by the NAMD program, with adaptive tempering applied through the Langevin thermostat, while the pressure was maintained at 1 atm. The application of the adaptive tempering in combination with the choice of the NpT ensemble guarantees the absence of significant variations of the system’s mean density . The Langevin damping coefficient was set to 1 ps-1 and the piston oscillation period set to 200 fs with a decay time of 100 fs. The production run was performed with the impulse Verlet-I multiple time-step integration algorithm [−6. A cutoff for the van der Waals interactions was applied through a switching function, acting between 7 and 9 Å. The SHAKE algorithm [−8 was used to restrain all bonds involving hydrogen atoms. Trajectories were obtained by saving the atomic coordinates of the whole system every 1 ps.The simulation protocol for all the peptides was the following: the system was first energy minimized for 1000 conjugate gradient steps followed by a heating-up phase to a final temperature of 320 K  between the DFT-derived φ,ψ torsion angles (from ref 24) and their corresponding torsion angles from each frame of the trajectory (or from each frame of a dPCA-derived cluster). In the second step, the distributions of these angle deviations were calculated and the resulting histograms plotted. In the final step, we treated these distributions as a sum of independent Gaussian distributions, and we obtained numerical values for the means (μ) and standard deviations (σ) of those peaks residing closest to the reference DFT angle values  parameters was performed with the nlsLM function of the R package) [A significant part of this work focused on analyzing the extent to which the ulations  and the ulations  agree wipackage) . These vnot been observed in a given trajectory. These probabilistic estimates are presented in the form of graphs that depict the probability of observing a new/different structure, as a function of the minimal RMSD—of this (thus far unobserved) structure—from all those structures already observed. The results from the application of Good-Turing statistics are shown in The heptapeptides—as will be discussed in the next section—demonstrate a highly dynamic behavior throughout the molecular dynamics simulations. Their structural flexibility/disorder leads to a rugged folding landscape and, consequently, to the absence of a well-defined gradient towards a would-be ‘native’ structure. The implication of this finding is that the whole configurational space available to the peptides is accessible during the simulations. This immediately raises the issue of convergence and statistical significance of our results. To tackle this problem, we apply a recently proposed probabilistic method  for estiβ-turn is rather dramatic: the curves fall quite fast to negligible punobserved values for RMSDs of the order of 1 Å. The apparent difference between these two cases may be attributed to the fact that the four-residue central parts are structurally more stable, possibly promoting the formation of secondary-structure-like patterns, whereas the peptides' termini are highly mobile and disordered.To clarify these graphs, the high probability of unobserved species for low RMSD values indicates that if we were to continue the simulation  it would be highly probable to observe structures, which although similar to some of the already observed ones, they would still be slightly different from them. As the RMSD from the already observed structures increases, the corresponding probability decreases. It is therefore the exact form of this graph and the inclination of each curve towards low probability values that inform us to what extent the trajectories have been sampled or, in other words, how significant is the structural variability that has been missed due to limited sampling. Focusing on the application of Good-Turing statistics in our trajectories, the results have been obtained from multiple calculations and are organised in two groups. To conclude, the results drawn from the analysis of our 5 μs trajectories indicate that the length of the simulations is probably sufficient and guarantees a reasonable sampling of the peptides’ configurational space for the given force field. A similar type of analysis has also been made but using only structures whose corresponding adaptive tempering temperature was ≤ 360 K, thus corresponding to more stable (from the simulation’s point of view) conformers. As shown in β-turn motifs for each heptapeptide can be seen in To place our observations on a structurally firm ground, we calculated the free energy landscapes of the heptapeptides using the dihedral angle Principal Component Analysis (dPCA) method to identify the prominent conformations. Various studies have already indicated that dPCA is a high resolution, powerful and more appropriate method for studying such highly flexible systems –56. We rhpNG-1, 34.7% of the total frames were assigned by the program CARMA to clusters, while for hpNG-2 and hpNG-3 the corresponding percentages were 36.7% and 31.8%, respectively. The most highly populated cluster recorded for each heptapeptide occupied only 11.9%, 20.1% and 20.7% of the clustered frames , clearly indicating that the peptides demonstrate a very dynamic behavior. β-turn motifs in each cluster. There is a structural variability concerning the preference among turn types and the positional preference of each amino acid residue in positions i+1 and i+2. Asn-Gly turns, not only failed to be the dominant structural motif, but the analysis also revealed the presence of a plethora of β-turn motifs as well. Examination of clusters’ representative structures reveals that structures can vary among different β-turn and unfolded conformations. β-turn, clearly demonstrating the large diversity of the turn types that have been sampled. Many representative structures corresponding to significantly populated clusters adopt a β-turn conformation, but the presence of unfolded random coil representatives is nonetheless considerable. Asn-Gly is not favored to be the turn segment among the representatives, as there is a variety in the positional preference of each residue for this site.As it can be seen in β-turn motifs, β-turns sampled in each trajectory taking into account both the different combinations of i+1 and i+2 positional preferences and the different types of β-turns. Of the turns located, βIV turns seem to be the most prominent turn motif in each trajectory, but this can be attributed to the fact that this miscellaneous type does not follow any of the stringent criteria being used for turns’ classification scheme [βVIII turns’ sampling on the other hand is limited, resulting only in a negligible amount of observations. As for the most common classical turn motifs βI, βI’, βII, βII’, we must also focus on the various analyses of turns derived from experimental data, apart from the DFT calculations, so that we examine the possibility that our simulations have accurately described the peptides’ folding behavior. X-1-Asn and Gly-X+1βI turns appear to be the most frequently sampled turn motifs in each trajectory, in agreement with the overall positional potentials which show a distinctive preference for Asn in positions i and i+2 [i forming a hydrogen bond with the main-chain nitrogen of residue i+2, stabilizing consequently type I β-turns [i to i+3 form another turn-like structure known as the “Asx-turn”, which is commonly observed in type I β-turns [φ = -90°, ψ = 0°) for position i+2 in βI turns [αL conformation in position i+3 of type I turns [-1-Asn and Gly-X+1βI turns in our trajectories. Regarding type II’ turns, although poorly sampled, especially in the case where Asn-Gly occupies the turn position, Gly-X+1 has a relatively increased preference as a turn segment for this turn type, in agreement with the crystallographic data where Gly shows a major preference for position i+1 in βII’ turns due to φ,ψ restrictions around +60°, -120° respectively [βII’ Gly-X turns in model peptides containing the Asn-Gly segment [βII’ turns (followed by βI turns) [+1 populations. Finally, the Asn-Gly segment is slightly to moderately favored as a turn segment depending on the heptapeptide, showing a rather increased preference towards βII and βI’ turns . Although Gly residue very often occupies the i+2 position in both of the aforementioned turn types (as it most readily adopts the αL and γL conformations) [i+1 of βII turns in protein structures [βI’ turns contrarily, the Asn residue occurs very frequently in this position, as several studies have already indicated [φ,ψ constraints around the γL region of the Ramachandran plot [βI’ turns is apparent in our results, contrary to the consistent intrinsic preference of this dipeptide for the turn segment in βI’ turns as shown by numerous above-mentioned studies.To have a more comprehensive understanding of the preferences among the different n scheme . βVIII t and i+2 , 7, 10.  β-turns . AdditioI turns) , justifyructures . In βI’ ndicated –20, 23, βI’ turn motif . In contrast, the present MD analysis shows that Asn-Gly turns are overall , these residues do not occupy proper pair-facing positions that strongly influence stabilization interactions [βI’ turns is not unequivocally established, as previous NMR and MD studies implied either the concomitant presence of other turn motifs [Although the general pre-existing conformational preferences of the amino acid residues are important for the turn formation, side-chain to side-chain or side-chain to backbone interactions also play a key role in the folding and stabilization of turns and by extent hairpin motifs, especially in the context of a three-dimensional protein structure , 19–23. ractions , 24. Thermations , 64. Thi studies . Though  studies , 64, 65,ractions . Finallyn motifs  or the in motifs .ab initio level might overshadow the structural distribution of these systems.Having said the above and seeing our results in a positive light, given that MD simulations are superior in sampling a large conformational phase space, they might adequately interpret at first sight the behavior of such short and flexible peptides isolated in water solvent. Despite arguing in favor of our MD results though, our aim at this point is not to dispute the validity of the QM calculations, but to note that generating single structures on the d = 0° indicate similarity with the DFT model , while values far from d = 0° indicate large deviations. To further quantify these observations, we treated the distributions of these deviations (green curves in d = 0°) and obtained numerical values for their means (μ) and standard deviations (σ) through a nonlinear regression fitting performed with the nlsLM function of the R package . The outliers are again ψAsn in βII’ hpNG-1, ψAsn in βII’ hpNG-2 and ψAsn in βII’ hpNG-3, which show a very significant deviation from the ideal β-turn values, far exceeding the ±30° limit set by Thornton and coworkers.To complete this set of comparisons, we also calculated the deviations (in degrees) between the mean Asn/Gly torsion angles from molecular dynamics and the corresponding ornton’s  definitiψAsn βII’ angles disputes the validity of our simulations. The fact that molecular dynamics utterly fail to sample energetically allowed—as indicated by QM calculations—angle values, along with the absence of a strong propensity towards the Asn-Gly βI’ conformation, might underlie a force field discrepancy. Indeed, previous studies on the Asn-Gly β-hairpin Trpzip-2 model peptide using the 99SB*-ILDN force field, showed the inability of this force field variant to sufficiently fold and stabilize the β-hairpin conformation [Although, as mentioned above, the results from our MD simulations, under the scope of the application regime they have been designed for, manifest a substantial convergence with data concerning the general amino acid frequencies in turns, such conspicuous disparity with the experimental/DFT results regarding ormation , 42. Thiφ,ψ angles in each cluster’s frame from the corresponding DFT-calculated value. β-turns and a representative structure for each cluster. A closer look at the histograms reveals a significant divergence of the Asn/Gly φ,ψ peaks from the d = 0° value for several clusters, showing that Asn-Gly turns have not been adequately sampled in the major dPCA clusters. The cases where this torsion angle analysis indicates convergence with the DFT data can also be verified through a comparison with the results shown in hpNG-1, for example, have significant populations of Asn-Gly type II and I β-turns respectively, as the sharp Asn/Gly φ,ψ peaks lying around d = 0° indicate, but the Asn-Gly βI’ motif has not been sampled at all  of Asn-Gly turns sampled in the clusters. The results are in good agreement with the previously observed populations of βII’ turns have been identified. As for the Asn-Gly βΙ’ turn, although it is present, it is definitely not a predominant structural motif of the dPCA clusters. A question that arises at this point is this: Can the clusters be characterized by a single structural motif? In other words, can a highly populated motif be representative of a cluster’s structural ensemble? If we look in β-turn type, they cannot be unequivocally identified by a single motif. As can be seen in To compare our previous observations with the results obtained from the dPCA we calculated, as described above, the deviations of the d at all . Clusterectively , whereasectively . S8 Fig β-turns in each cluster gives rise mainly to coil or non-classical βIV representative structures, suggesting that clusters are barely characterized by native-like conformations. While the tendency towards β-turn formation is in general present, the structural diversity observed in our trajectories (and clusters) precludes us from identifying adequately large populations of specific turn motifs that would be indicative of a propensity towards specific β-turn structures, such as the type I’ Asn-Gly turns. That being said, along with the incapability of the force field to sample certain torsion angles—and therefore conformations—identified as energetically favoured by the DFT calculations [To summarize, turn populations in clusters are indicative of what is mentioned above: the small fraction of stable, folded ulations , cast doβII’ turns that—although meagerly reported in previous statistical analyses of database [βI’ turns, as previously has been confirmed by several experimental and statistical analyses [β-hairpin peptide, the Trpzip-2, giving no dominant cluster but a small fraction of folded structures under equilibrium conditions, possibly due to backbone torsion parameterization and problems in describing local conformational preferences [β-hairpin formation could not be stabilized, as well as that there is an increase in a-helix propensity due to an imbalance of the local conformational preferences [α-helical conformations, and therefore of an inadequate balance between various secondary structure elements, thus making the stabilization of the β-hairpin unachievable.As previously mentioned, the inability to sample Asn-Gly database –5, 7—havanalyses –20, 23, ferences , 42. In ferences , 42. Theferences , 42. ThihpNG-1 peptide, but this time using the 99SB-ILDN force field instead of the 99SB*-ILDN variant. Results from the aforementioned studies on the Trpzip-2 showed that although 99SB-ILDN underestimates ΔΗF, it achieves a relatively better folding of the peptide [β-turns was introduced in the force field during its optimization to better sample α-helical structures. Analysis of this 99SB-ILDN trajectory with the PROMOTIF program and comparison with the results shown in hpNG-1 entries in I’: 0.41→0.47%, 2.34→2.54%, 0.38→0.31%, II’: 0.17→0.08%, 0.04→0.03%, 7.35→6.75%, implying that there is no profound variation in the performance between the star-corrected force field and its uncorrected counterpart. This is not necessarily an unexpected outcome. Despite the success of the 99SB-ILDN variant  in folding Trpzip-2 in previous studies, the increased flexibility of our systems leads correspondingly to an increased difficulty of tracking these systems computationally. Note, however, that this remark should not be taken trivially. Constantly increasing computing power has concomitantly facilitated the application of high quality, longer simulations . At least for such short peptides, such long simulation times eliminate obscurities associated with insufficient sampling and allow these studies to be seen as an acid test for the ability of force fields to successfully interpret physical reality. In this spirit, the above reported discrepancies add one more piece of evidence to the existing literature by showing that two force fields give a totally different view contrary to QM and X-ray determined results. This, by extent, points towards the necessity of numerous independent case studies for such systematic inaccuracies to be detected, despite the long-term validation a force field might have undergone.Given the long and successful history of applications of the AMBER99SB family of force fields to peptide folding , 69, we  peptide . The aimζ basis sets, such as the 6-31G(d) used in ref 24, frequently suffer from inaccuracies due to basis-set superposition (BSSE) and basis-set incompleteness (BSIE) errors. These problems affect not only energies but also properties derived from them such as geometries, as previous studies on peptides and protein fragments have already indicated [βII’ turn in our case, may account for inconsistencies in the DFT calculations that were performed. That being the case, we could argue that our MD analysis might after all adequately provide a physically correct description of the structures and dynamics of the Asn-Gly heptapeptides.Having noted, though, the above conclusions and aiming to provide the full scenery of the complications accompanying this work, should we solely attribute such discrepancies to the parametrization of the force field? The comparison between DFT and MD rises and falls with the quality of the DFT calculations as well. The differences between these two methods, especially in terms of the potential energy surface calculation, are widely known to the scientific community, and disagreement is not unexpected. Despite the approximations used in MD simulations, the reliability of QM calculations is also not unquestionable. Concerning our case, although the hybrid meta-GGA M06-2X functional  coupled ndicated –78. Despβ-turn is the most preferred motif in aqueous solution for the three heptapeptides containing the Asn-Gly segment. The picture painted by our molecular dynamics analysis, however, is fundamentally different: according to the simulations, the peptides are highly flexible with multiple relatively shallow free energy minima that allow the peptides to quickly interconvert between structurally diverse conformations. The dPCA-based free energy landscapes in β-turn formation. As β-turns containing Asn/Gly as one of their central residues occupy the largest part of the respective MD trajectories with cumulative frequencies reaching ≈85% for two of the peptides (becoming ≈45% if the type IV turns are excluded). The residue-specific preferences for positions i+1 and i+2 on the β-turns vary significantly between the four central residues, with the Asn-Gly segment preferring the aforementioned positions almost equally with segments formed by neighboring residues (X-1-Asn and Gly-X+1). Type I β-turns appear to be the most prominent MD-derived motif in each trajectory, while Asn-Gly βI’ turns show relatively significant populations only for the hpNG-2 and hpNG-3 peptides. In agreement with the dPCA results, the torsion angle analysis also demonstrated the structural malleability of the peptides and their pronounced tendency to form various types of β-turns. But it was also this more detailed analysis that allowed us to convincingly demonstrate the inability of the AMBER99SB*-ILDN force field to sample the Asn-Gly βII’ motif. This apparent force field discrepancy was also confirmed through a comparison between MD and the ideal β-turn values obtained by the Thornton group. This comparison , and can thus be argued that we could be completely wrong in assuming that these specific peptides should actually sample, for example, the  studies , 23, 63  studies –5, 7 thaS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigResults from the application of Good-Turing statistics to the three trajectories, for both the full-length peptides and their four-residue central part, obtained using only structures associated with temperatures ≤ 360 K.(TIF)Click here for additional data file.S2 Figφ,ψ torsion angle values of residues i to i+3 for (A) βΙ, (Β) βI’, (C) βII and (D) βII’ DFT-obtained hpNG-1 turn structures. Areas in red, yellow, beige and white represent the core, the allowed, the generous and the disallowed regions respectively. Non-glycine residues are depicted here with black square signs and glycine residues with black triangles. Figures were generated using PROCHECK.Ramachandran plots showing the (TIF)Click here for additional data file.S3 Figφ,ψ torsion angle values of residues i to i+3 for (A) βΙ, (Β) βI’, (C) βII and (D) βII’ DFT-obtained hpNG-2 turn structures. Areas in red, yellow, beige and white represent the core, the allowed, the generous and the disallowed regions respectively. Non-glycine residues are depicted here with black square signs and glycine residues with black triangles. Figures were generated using PROCHECK.Ramachandran plots showing the (TIF)Click here for additional data file.S4 Figφ,ψ torsion angle values of residues i to i+3 for (A) βΙ, (Β) βI’, (C) βII and (D) βII’ DFT-obtained hpNG-3 turn structures. Areas in red, yellow, beige and white represent the core, the allowed, the generous and the disallowed regions respectively. Non-glycine residues are depicted here with black square signs and glycine residues with black triangles. Figures were generated using PROCHECK.Ramachandran plots showing the (TIF)Click here for additional data file.S5 FigNG-1 for every turn type. Shown from left to right is the number of the cluster along with its population, the occupancy of β-turn motifs in each cluster, the histograms showing the distribution of deviations (in degrees) between the reference DFT φ,ψ values of the 4-residue central part and the respective φ,ψ values obtained from the simulation, and the corresponding backbone representative structures of each cluster. Representatives with random coil conformation are depicted here only with their Ala-Asn-Gly-Ala part.Results from the torsion angles analysis of the five most prominent dPCA clusters of hp(TIFF)Click here for additional data file.S6 FigNG-2 for every turn type. Shown from left to right is the number of the cluster along with its population, the occupancy of β-turn motifs in each cluster, the histograms showing the distribution of deviations (in degrees) between the reference DFT φ,ψ values of the 4-residue central part and the respective φ,ψ values obtained from the simulation, and the corresponding backbone representative structures of each cluster. Representatives with random coil conformation are depicted here only with their Val-Asn-Gly-Gln part.Results from the torsion angles analysis of the five most prominent dPCA clusters of hp(TIFF)Click here for additional data file.S7 FigNG-3 for every turn type. Shown from left to right is the number of the cluster along with its population, the occupancy of β-turn motifs in each cluster, the histograms showing the distribution of deviations (in degrees) between the reference DFT φ,ψ values of the 4-residue central part and the respective φ,ψ values obtained from the simulation, and the corresponding backbone representative structures of each cluster. Representatives with random coil conformation are depicted here only with their Val-Asn-Gly-Leu part.Results from the torsion angles analysis of the five most prominent dPCA clusters of hp(TIFF)Click here for additional data file.S8 Figβ-turns among a set of 500 randomly selected structures from every dPCA cluster of the three heptapeptides. Clusters in which β-turns were not identified among the set of structures are omitted. Shown at the horizontal axis are clusters’ numbers and the different turn types. The vertical axis shows the % occupancy of the different turn types in each cluster. The structural assignment was performed using the PROMOTIF program.The above barcharts show the occupancy of Asn-Gly (TIFF)Click here for additional data file.


Once internalized, receptors reach the sorting endosome and are either targeted for degradation or recycled to the plasma membrane, a process mediated at least in part by tubular recycling endosomes (TREs). TREs may be efficient for sorting owing to the ratio of large surface membrane area to luminal volume; following receptor segregation, TRE fission likely releases receptor-laden tubules and vesicles for recycling. Despite the importance of TRE networks for recycling, these unique structures remain poorly understood, and unresolved questions relate to their lipid and protein composition and biogenesis. Our previous studies have depicted the endocytic protein MICAL-L1 as an essential TRE constituent, and newer studies show a similar localization for the GTP-binding protein Rab10. We demonstrate that TREs are enriched in both phosphatidic acid (PA) and phosphatidylinositol-4,5-bisphosphate P2), supporting the idea of MICAL-L1 recruitment by PA and Rab10 recruitment via PIP2. Using siRNA knock-down, we demonstrate that Rab10-marked TREs remain prominent in cells upon MICAL-L1 or Syndapin2 depletion. However, depletion of Rab10 or its interaction partner, EHBP1, led to loss of MICAL-L1-marked TREs. We next used phospholipase D inhibitors to decrease PA synthesis, acutely disrupt TREs, and enable monitoring of TRE regeneration after inhibitor washout. Rab10 depletion prevented TRE regeneration, whereas MICAL-L1 knock-down did not. It is surprising that EHBP1 depletion did not affect TRE regeneration under these conditions. Overall, our study supports a primary role for Rab10 and the requirement for PA and PIP2 in TRE biogenesis and regeneration, with Rab10 likely linking the sorting endosome to motor proteins and the microtubule network. The internalization of receptors and lipids from the plasma membrane is an essential process in all mammalian cells . Once inOne of the mechanisms proposed for efficient endosomal sorting is the use of a large surface membrane area to segregate select molecules and target them to distinct pathways . Indeed,TREs containing MICAL-L1 have been implicated in the trafficking of receptors both from the SE toward the perinuclear endocytic recycling compartment (ERC) and from the ERC toward the plasma membrane . HoweverRecently, Rab10 has also been identified as a component of tubular endosomes . Indeed,A–F. By performing 3D surface volume analysis, we were able to avoid interference from non–membrane bound protein and focus almost exclusively on the surface volume of the endosomal proteins. In addition, the serial sections ensured that we measured true overlap and not merely proteins that overlapped in the X-Y axis (see G). The Rab10 overlap with MICAL-L1 was considerably lower, likely resulting from the more intense overall Rab10 staining pattern that includes nontubular endosomes in addition to the TREs. Even the total volume overlap, which measures the total surface volume of each protein and the percentage of overlap, was about 20%, further highlighting the degree of overlap on TREs. Moreover, overlap between MICAL-L1 and Rab10 was further validated using super-resolution Structured Illumination Microscopy . The PA marker also localized primarily to long, tubular structures, but unlike PLCδ was largely devoid of localization to the plasma membrane and to perinuclear spherical vesicles (B). The localization of endogenous Rab10 closely mirrored that of the PA marker (C) and similarly was absent on the plasma membrane and perinuclear spherical vesicles. Significantly, endogenous Rab10 was found on TREs that were marked by both PIP2 and PA , indicating that these phospholipids are both enriched on TRE.Given the requirement for PA in recruiting MICAL-L1 to TREs , and thein vitro . As demoin vitro A. The PAvesicles B. The loA marker C and simA–C). When MICAL-L1 was depleted by siRNA , the apparent volume of Rab10 TREs was not decreased . Indeed, the apparent volume of Rab10 TREs displayed a small but significant increase in the absence of MICAL-L1 . Since Syndapin2 interacts with MICAL-L1 and colocalizes with it on TREs, we similarly knocked down Syndapin2 and measured the apparent Rab10 and MICAL-L1 TRE volume. As demonstrated, Syndapin2 depletion led to reduced overall expression of MICAL-L1 . As a result, significantly fewer MICAL-L1 TREs were measured , likely owing to the instability of MICAL-L1 on membranes and its degradation  upon MICAL-L1 or Syndapin2 knock-down. As anticipated, in Mock-treated cells, MICAL-L1 and Rab10 displayed a partial overlap on the same TREs , A–C. Whradation . Howeverradation .Figure 3J; quantified in Fig. 4K). Compared with Mock-treated cells (A–C), Rab10 knock-down cells (D–F) displayed a significantly decreased apparent volume of MICAL-L1-containing TRE . Since Rab10 functions with EHBP1, a protein that interacts with PIP2 and reportedly affiliates with TREs . When the localizations of Rab10 and MICAL-L1 were analyzed, significantly reduced Rab10- and MICAL-L1-TRE were observed, with Rab10 displaying an apparently more cytoplasmic localization . Indeed, membrane fractionation experiments demonstrated that significantly more Rab10 could be detected in the cytosolic fraction and less Rab10 was detected in the membrane fraction when EHBP1 expression was impaired by EHBP1 siRNA knock-down . These data suggest that both Rab10 and EHBP1 are required for TRE generation and/or maintenance.Given the surprisingly superfluous requirement for MICAL-L1 and Syndapin2 expression to maintain Rab10-containing TRE, we next asked whether Rab10 is required for the generation of MICAL-L1-containing TREs . To thisFig. 4ith TREs , 31, we A; quantified in Fig. 5I), significantly fewer TREs were observed in Rab10 KO MEF cells . Indeed, the total apparent tubular volume of MICAL-L1-marked TRE was typically two- to threefold higher in WT MEF cells than in their MEF Rab10 KO counterparts . Significantly, when the Rab10 KO MEF cells were “rescued” by transfection of WT Rab10 (+red fluorescent protein-Rab10) into these cells as opposed to a green fluorescent protein control, the apparent volume of MICAL-L1-marked TRE increased by ∼ three-fold . Overall, these data support the notion that Rab10 is a major protein involved in the generation of TRE.To further address the role of Rab10 in TRE biogenesis, we examined MICAL-L1-containing TRE in mouse embryonic fibroblast (MEF) cells derived from Rab10 knockout (KO) embryos . CompareFig. 5A–F). Similarly, TREs marked by Rab10 were significantly impaired upon PLD treatment but recovered rapidly upon PLD washout (G–L). These data further support the idea that MICAL-L1- and Rab10-TREs mostly mark the same tubular endosomes.We have previously demonstrated that phospholipase D (PLD) inhibitors may be used to acutely disrupt the TRE network within 30 min of treatment, likely by affecting levels of PA and potentially also PIP2 . It is i washout , G–L. ThM; quantified in Fig. 6N). We then calculated and compared the apparent MICAL-L1-TRE volume between Mock and Rab10 knock-down cells under control conditions (dimethyl sulfoxide [DMSO] treatment), PLD inhibitor treatment, and following inhibitor washout . As expected, the apparent MICAL-L1-TRE volume was reduced by over 50% in DMSO-treated cells upon Rab10 knock-down compared with Mock-treated cells. PLD inhibitor treatment further reduced the apparent TRE volume levels in both Mock-treated and Rab10 knock-down cells. Most significantly, whereas the Mock-treated cells displayed a massive recovery in TRE regeneration upon washout, the Rab10 knock-down cells did not, and the TRE levels remained as low as the DMSO treatment baseline . These data are consistent with the requirement of Rab10 for TRE biogenesis. We also tested the role of EHBP1 in TRE regeneration, since like Rab10, it appeared to be necessary for the biogenesis and/or maintenance of these endosomes. Accordingly, we used DMSO, PLD inhibitors, and washout of the inhibitors to compare apparent TRE volumes  in Mock-treated and EHBP1 knock-down cells. As expected, EHBP1 knock-down significantly reduced the apparent volumes of TREs marked by either MICAL-L1  or Rab10 . Also as anticipated, PLD inhibitor treatment further reduced TRE levels of both Mock-treated and EHBP1 knock-down cells. However, somewhat surprisingly, upon PLD inhibitor washout the EHBP1 knock-down cells (O-P) displayed TRE regeneration almost identical to that seen in Mock-treated cells (R–S). These data suggest that Rab10 is a key protein involved in the biogenesis, maintenance, and regeneration of TREs. However, although EHBP1 appears to be required for TRE biogenesis and/or maintenance, unlike Rab10, EHBP1 appears to be expendable for TRE regeneration.Using this system, we now addressed the role of Rab10 in TRE regeneration by knocking it down with siRNA. As demonstrated by immunoblot, Rab10 expression was significantly reduced with siRNA , only the presence of Rab10 is necessary for acute regeneration of TREs following PA depletion. EHBP1 has been implicated in fission of tubular endosomes , 41, a fMycoplasma infection.The HeLa cervical cancer cell line was obtained from the american type culture collection and grown in Dulbecco's modified Eagle's medium (high-glucose) containing 10% FBS, 1× penicillin–streptomycin (Invitrogen), and 2 mM glutamine. The MEFs derived from Rab10 knockout (KO) embryos were previously described  and growThe following antibodies were used: anti-MICAL-L1 , anti-Rab10 , anti-EHBP1 , anti-Syndapin2/Pacsin2 , anti-EEA1 , anti-pPKCα , anti-GAPDH-horseradish peroxidase (HRP) , mouse anti-rabbit IgG light chain–HRP , Alexa Fluor 568–conjugated goat anti-rabbit , Alexa Fluor 568–conjugated goat anti-mouse , and Alexa Fluor 488–conjugated donkey anti-mouse . The PLD inhibitors CAY 10593  and CAY 10594 were purchased from Cayman Chemical Co and were typically used at 50 μM for 30 min at 37 °C.Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and were then scraped off plates with a rubber policeman into ice-cold lysis buffer . Protein levels of postnuclear lysates were quantified using the Bradford assay (Bio-Rad) for equal protein level loading. For immunoblotting, 20 to 30 μg of protein per lysate was separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and blocked for 30 min at room temperature in PBS with 0.3% Tween (PBST) plus 5% nonfat dry milk. The membranes were then incubated overnight at 4 °C or for 1 h at room temperature with primary antibodies diluted in PBST. Membranes were then washed three times with PBST and incubated at room temperature with appropriate secondary antibodies in PBST for 30 min. The membranes were then washed again three times with PBST, before being subjected to enhanced chemiluminescence.g. Supernatants (cytosolic fractions) and pellets (membrane fractions) were boiled in 4× Laemmli sample buffer, and proteins samples were separated and visualized by SDS-PAGE and immunoblotting.HeLa cells plated on 10-cm plates were treated with EHBP1 siRNA or mock transfected (48 h) using Lipofectamine RNAiMAX according to the manufacturer's instructions. Cells were then resuspended with Cellstripper (Corning #25-056-CI) and centrifuged. Cell pellets were resuspended with Homogenization Buffer  containing protease inhibitors  and homogenized on ice. Homogenates were centrifuged for 1 h at 4 °C at 100,000https://imagej.nih.gov/ij/docs/menus/analyze.html#gels. Statistical significance was calculated using a Student's t-test with the Vassarstats program (http://www.vassarstats.net).The adjusted relative density of the immunoblots was measured in Fiji ImageJ based on the method in the following protocol: HeLa cells were grown either on coverslips or 35-mm culture dishes for 24 h in Dulbecco's modified Eagle's medium containing 10% FBS with 2 mM L-glutamine and 1× units/ml penicillin/streptomycin. The cells were then subjected to siRNA treatment with human MICAL-L1 , Syndapin-2 , Rab10 , or EHBP1  oligonucleotides from Dharmacon for 48 h at 37 °C using Dharmafect , following the manufacturer's protocol.MEFs were cultured on coverslips and transfected with either green fluorescent protein or red fluorescent protein-Rab10 using GeneExpresso Max transfection reagent (Excellgen) for 48 h.Red fluorescent protein-Rab10 was a gift from Dr Barth Grant. The PLCδ1 PH domain P2) and MICAL-L1 CC (marker for PA) have been described previously , 51.HeLa or MEF cells were treated as indicated in the text and then fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then rinsed three times in PBS and incubated with primary antibody in PBS containing 0.5% bovine serum albumin and 0.2% saponin for 1 h at room temperature. Cells were then washed three times in PBS and incubated with the appropriate fluorochrome-conjugated secondary antibodies diluted in PBS containing 0.5% bovine serum albumin and 0.2% saponin for 30 min. Cells were then washed three times in PBS and mounted in Fluoromount. Z-Stack confocal imaging was performed using a Zeiss LSM 800 confocal microscope with a 63×/1.4 numerical aperture oil objective, and more than 10 cells from three independent experiments were processed using the IMARIS software.t-test with the Vassarstats program (http://www.vassarstats.net). Snapshots and/or videos were obtained from the IMARIS program and used as representative images.Z-Sections of images (8–12 slices) acquired from the confocal microscope were imported into IMARIS x64 9.1.2 software  coupled with custom MAT-LAB (2009 and 2014) programming for 3D surface rendering and quantitative analysis, as indicated. Briefly, the image display was adjusted for both of the channels , and the rendering quality was set to 100%. Surfaces were created by selecting the source channel and smooth surface detail set at 0.198 m. Background subtraction was set to 0.743 m, and the threshold was reduced for surfaces to fully cover all voxels. The surface area and volume of the surfaces generated were quantified by IMARIS for both of the channels, and the values were exported into Excel for graphical and statistical analysis. To quantify apparent surface overlap volume between two surfaces (MICAL-L1 and Rab10), the IMARIS XT bundle Kiss and Run was first integrated with MATLAB and launched in IMARIS. The 3D surface-reconstructed images were then processed for Kiss and Run analysis using the surface–surface overlap module, which uses a surface mask for the target and tracks the surface and determines the overlap for each surface independently. This particular Xtension program analyzes contact events between surfaces that are defined by having at least one overlapping voxel. The volume of overlap for each surface was then quantified and exported to Excel for further analysis. The mean and the SE of the mean were calculated from data obtained from three independent experiments with at least 10 images taken per treatment. Statistical significance was calculated using a Student's Structured illumination microscopy (SIM) images were collected with a Zeiss Elyra PS.1 illumination system (Carl Zeiss) using a 63× oil objective with numerical aperture of 1.4. Two laser lines were used in acquisition of images: 488 and 568 nm. Three orientation angles of the excitation grid were acquired for each Z-plane, with Z spacing of 110 nm between planes. SIM processing was performed with the SIM module of the Zen Black software (Carl Zeiss). The processed SIM images were then exported in TIF format.All data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


The aim of this study was to optimize a Question Prompt List which is designed to improve communication about the heart failure trajectory among patients, family members, and health care professionals.Data were collected in a two-round Delphi survey and a cross-sectional survey, including patients with heart failure, their family members, and health care professionals working in heart failure care in Sweden and the Netherlands. Acceptability for and demand of the Question Prompt List were assessed.A total of 96 patients, 63 family members and 26 health care professionals participated in the study. Regarding acceptability, most of the original questions were found to be relevant by the participants for inclusion in the Question Prompt List but some cultural differences exist, which resulted in two versions of the list: a Swedish version including 33 questions and a Dutch version including 38 questions. Concerning demand, participants reported that they were interested in discussing the questions in the revised Question Prompt List with a physician or a nurse. Few patients and family members reported that they were worried by the questions in the Question Prompt List and hence did not want to discuss the questions.This Question Prompt List has successfully been adapted into a Swedish version and a Dutch version and includes questions about the HF trajectory which patients, their families, and health care professionals perceived to be relevant for discussion in clinical practice. Overall, patients and family members were not worried about the content in the Question Prompt List and if used in accordance with patients’ and family members’ preferences, the Question Prompt List can help to improve communication about the heart failure trajectory. Discussing the Heart Failure (HF) trajectory with patients and their families is recommended in clinical guidelines worldwide –3. HealtOne way to improve communication about sensitive topics in clinical practice is by using a communication tool . A tool heartfailurematters.org, an educational tool provided by the Heart Failure Association of the European Society of Cardiology that has a specific section which intends to help patients and families to discuss living with HF and plan for the end-of-life [Previous research on QPLs are mostly from cancer care where such a tool has helped cancer patients and their families to give cues that they wished to discuss prognosis and end-of-life care with health care professionals . Special-of-life . In othe-of-life , 18 howeRecently, our research group including researchers from Sweden and the Netherlands, developed a QPL with the aim of improving communication about the HF trajectory in HF care. The first version of the QPL was developed in a co-design process  in SwedeTo ensure the QPL is connected to clinical practice and has the potential to be used in the “real world”, further testing among patients, family members and health care professionals is needed. Accordingly, we know from previous studies in cancer care that QPLs developed in one country might need to be adapted for other countries depending on differences in how cultures approach sensitive topics . Also, aThe aim of this study was to optimize a Question Prompt List which is designed to improve communication about the heart failure trajectory among patients, family members, and health care professionals.How do the participants perceive the title, the pictures, and the introductory text in the QPL?Are the questions in the QPL relevant, are they clearly formulated, or do they need to be re-formulated?Are there questions missing in the QPL?Are the participants interested in discussing the questions in the QPL with a physician or a nurse?What are the participants’ reasons for discussing/not discussing the questions in the QPL?This study was conducted in two steps Fig.  with thePurposeful sampling  was usedTo report on the participants’ reactions to and perceived appropriateness of the QPL (acceptability), patients with HF, their family members and health care professionals in HF care in Sweden and the Netherlands were invited to take part in a two-round Delphi survey.In the first round, participants were asked to comment on their reactions to the title, the pictures, and the introductory text in the QPL. They were asked to report on the relevance of each of the 45 questions in the QPL, using a Likert scale from 0 to 4 . They were also asked to report on the clarity (yes/no) and need for re-formulation of each question. Furthermore, they could suggest additional questions to be included in the QPL (Table A revised QPL was developed for the second round with 1) questions which the majority of the participants found relevant and thought should be kept on the list, 2) questions that were suggested for deletion because they were perceived as not relevant, and 3) questions that were added by participants. In the second round, participants were asked to state for each question whether the question should be kept, deleted or added. After this second round, a revised version of the QPL was made by the research group.To test the demand of the QPL, a cross-sectional survey was performed. A questionnaire including the revised questions in the QPL was sent out to a new sample of participants to assess the estimated use of the QPL . The parDescriptive statistics were used to analyse the data in the study. The Delphi technique was used as a tool to receive feed-back on possible areas that could be optimised in the QPL. Round 1 questions that were perceived as not relevant by 25% or more of the participants were specifically discussed in the research group concerning formulations and content. Some of these questions were re-worded and added to round two for participants to comment on; some were suggested for deletion. Questions that were perceived as relevant by 50% or more of the participants in rounds 1- and 2 were kept in the QPL. The opinions of the participants had the biggest impact on the results, but the researchers also took on an active role in the analysis, contributing their knowledge to the optimizing of the QPL. The researchers’ role in the analysis of data concerned i. e. bringing in knowledge from relevant literature when designing the QPL, deciding on the ordering of the questions in the list as well as their final wording, using suggestions from all participants.Ethical approval was granted for this study by the Swedish Ethical Review Authority in Linköping . The Medical Ethical Committee of the University Medical Centre Groningen (UMCG) concluded that no additional approval of the committee was needed (METC 2018/169). Written informed consent was obtained from all patients before commencement of the study.Box 1 An example of one of the questions and the different response alternatives in the cross-sectional surveyA total of 71 participants were approached and 57 responded in the first round . Patients had a mean age of 70 years (± 9), 78% were male and 89% were married or living with a partner. Most of the patients (56%) were in NYHA-class III and 72% of them had an ICD or CRT device. Family members had a mean age of 67 years (± 11), 71% were female and most of them (93%) were living together with the patient. Only one family member was the child of a patient. The professionals  had a mean age of 43 years, 54% were female, and the majority of them (85%) worked at a hospital. In total, 4 patients, 6 family members and 5 health care professionals did not respond in the first round.In the second round, 68 participants who responded in the first round were approached, and 51 participants responded . In total, 5 patients, 6 family members and 6 health care professionals did not respond.Overall, most of the participants in both samples endorsed the title, the two pictures and the introductory text of the QPL. Comments were made such as: the title could be shorter, and the picture on the front page  was found to be “too positive” with a tone that did not correspond with the serious content of the QPL. There were Dutch patients/family members who did not recognise themselves in the couple shown in the picture. There were also smaller changes suggested for the introductory text.Forty-five of the questions in Sweden and thirty-nine in the Netherlands were perceived as relevant and clear by the participants, but for some of the questions, re-formulations were suggested to make them clearer Fig. . Most ofAfter Delphi round 1, the research groups in both countries discussed the results and discussed which questions should be kept, merged and/or re-formulated as they had similar content, which questions could be deleted from the QPL and also which new questions could be added that were found to be missing in the QPL.In the second Delphi round, the Swedish QPL included 49 questions of which the researchers suggested that 33 should be kept, 12 questions deleted, and 4 new questions added. In the second Delphi round in the Netherlands, the QPL included 59 questions of which the researchers suggested that 33 should be kept, 12 questions deleted, and 12 new questions added. The participants were asked to state if they agreed on the suggestions.In round 2, the participants agreed that 38 proposed questions in Sweden and 45 questions in the Netherlands should be kept in the list. In the research group discussions, some questions with similar content were merged or deleted which resulted in a final QPL for Sweden including 33 questions and 38 questions for the Dutch QPL. Based on the participants’ answers questions that that were deleted were for example “is it possible to predict how long someone has left to live?”, “what happens in the body when you die from HF?”, and “how do I reply to the question “Am I going to die now?”.A total of 115 patients .If participants did not want to discuss a question, they most often gave the reason that they already had enough information. If participants reported they did not want to discuss a question because they would be worried or they did not want to think about the future, it was often the same participants reporting this answer on more than one question.In this section Fig. , each quIn this section Fig. , each quIn this section Fig. , each quIn this section Fig. , only thIn this section Fig. , only thPatients and family members in both countries provided additional comments to describe their reasons for why they did or did not want to discuss certain questions in the QPL at this moment. For some it seemed too early to discuss several questions at this point in the HF trajectory but later, it could be important to get more information. Some patients expressed how they wanted to live ‘in the here and now’, not worrying about possible deterioration in the future. On the other hand, participants also commented that it could be too late to have this kind of conversation, if the patient, for example, was very old.This study describes the optimizing of a QPL to improve communication about the HF trajectory in HF care. The QPL was previously developed in a co-design process  togetherThe acceptability testing  focused Our findings show that patients and families in our study were not upset by the idea of discussing the more sensitive questions concerning the HF trajectory. Health care professionals, such as physicians, nurses and other relevant professionals, need to discuss the HF trajectory with patients in order to improve their understanding of their illness and address possible care needs of patients and their families , 2, 29. A QPL needs to be used with care as patients have different approaches to cope with HF and also want different amounts of information depending on their current situation . Some paWe found in our acceptability testing of the QPL that most of the original 45 questions were perceived as appropriate by the participants in both Sweden and the Netherlands and there were no huge differences in the participants’ opinions. Nevertheless, some cultural discrepancies exist in the content and in the amount of questions, resulting in two versions of the QPL: a Swedish version including 33 questions and a Dutch version including 38 questions. The Dutch QPL included more questions in the sections “Heart Failure in daily life”, “Support when deteriorating”, and “End-of-life”. The Swedish QPL included more questions in the section covering “Questions for family members”.We suggest that the questions in the Swedish and the Dutch QPLs can function as a blue-print for others to use. These QPLs can provide possible questions to be included in future QPLs, but we recommend that cultural adaptation of the questions should be done to fit the intended culture optimally and also provide the opportunity for each country to add additional questions that might be missing in the original QPL . AdditioThe participants’ interest in using the QPL in clinical practice was assessed, and participants in both Sweden and the Netherlands reported that they were interested to discuss questions from all the five sections in the QPL. Some participants, particularly those who felt they lacked information, commented that the QPL could help them to select questions and initiate a discussion. This is in line with previous research concluding that providing a QPL in clinical practice can increase the number of questions a patient asks if the QPL is tailored to the patient’s needs , 23, 38.Importantly, few patients and family members reported that they were worried by the questions in the QPL and hence did not want to discuss the questions. This needs to be considered in clinical practice since one commonly reported barrier from health care professionals why they avoid conversations about the HF trajectory is the belief that patients might become worried if they initiate discussions about prognosis or end-of-life issues , 32.For participants who did not want to discuss the questions in the QPL, the most reported reason was because they already had all the information that they wanted about the HF trajectory. Interestingly, all patients included in this study were visiting a HF clinic in Sweden or the Netherlands, where they were provided with dedicated time with professionals specialised in HF care. Nevertheless, around 30% of the participants declared a need for more information about the HF trajectory, indicating that we can do better in providing this kind of information to patients and their families.We found in the open-ended answers from patients and family members that the timepoint at which they wanted to discuss certain questions in the certain sections in the QPL differed. Some participants wanted to discuss, for example end-of-life issues later in the HF trajectory, and perceived that it was too early at the time the questionnaire was administered. Hence, we suggest that there must be repeated opportunities for discussion when the patient and family members want more information. These repeated opportunities for discussion are also important as we know that many HF patients are not aware of their poor prognosis or the severity of HF , 40. ManFamily members reported that they would like to discuss questions in all the sections of the QPL; sometimes they reported even higher needs for information than the patients did. Information needs may change between patients and their family members during the HF trajectory, whereas patients tend to want less information the closer they are to the end-of-life , but theStudy participants were recruited from regions which are seen as average regions for each country. They consisted of a diverse group of patients, their families and health care professionals from Sweden and the Netherlands. Although our study population was selected from HF clinics, we still realise that participants were approached to be in a research study. Maybe only patients, families, and health care professionals with an open approach to discussing the HF trajectory were represented in the samples, but as we included four different samples from two different countries, we believe this risk is minimized.The research team, involving 6 nurses, 1 physician and 1 behavioral scientist, provided their clinical experience and research knowledge of HF- and palliative care to inform the planning and performance of the study.In the cross-sectional survey, the participants were asked if they would like to discuss the questions in the QPL with a physician or a nurse, as these are health care professionals that they in general meet the most. Additionally, we argue that the QPL is suitable for use by several other health care professionals as well, such as social workers, and psychologists. Accordingly, in the cross-sectional survey the participants were asked if they wanted to discuss the questions in the QPL “right now”. This may have affected the results and the final questions in the QPL.There were some differences between the countries in sample sizes and NYHA class in the cross-sectional study. The Dutch samples included more patients and family members compared to the Swedish samples and a bigger proportion of the Dutch patients had more symptomatic patients in NYHA class III.One possible limitation in data analyses in the cross-sectional survey was the use of predetermined answering options in the questionnaire. If only open answers would have been allowed it could have produced a more rich data, possibly providing more important insights into the reasons why or why not patients and their family members wanted to discuss the different questions in the QPL.The QPL, designed for communication about the HF trajectory, has successfully been adapted into one Swedish version and one Dutch version. It includes questions that patients, their families, and health care professionals perceived as relevant to discuss with health care professionals about the HF trajectory. Overall, patients and family members were not worried about the content in the QPL and if used in accordance with patients’ and family members’ preferences, the QPL can help to improve communication about the heart failure trajectory. The QPL is ready to be used in clinical HF care in Sweden and the Netherlands. Further research is needed to examine the optimal way of delivery of the QPL into clinical practice and to examine how patients/family/professionals experience using the QPL in daily practice.Additional file 1. The final questions in the two versions of the QPL. SW, Sweden, NL, The Netherlands.Additional file 2. The questions that were suggested to be deleted or added in the Swedish and Dutch Delphi rounds. SW, Sweden, NL, The Netherlands.


Skin manifestations of sarcoidosis occur in up to 30% of cases, and may be the sentinel sign of the disease, with the skin being sometimes exclusively affected. While this may facilitate an early dermatologic diagnosis, heterogeneity in the cutaneous morphologies of sarcoidosis complicates recognition and affirms its reputation as a “great imitator”. Here, we present a case of a verrucous version of sarcoidosis that may be misdiagnosed because it can mimic other inflammatory and neoplastic skin disorders. Although it is a rare variant, its presence should alert clinicians to the likelihood of systemic involvement of cutaneous sarcoidosis. Skin manifestations of sarcoidosis occur in up to 30% of cases, and may be the sentinel sign of the disease . CutaneoA 63-year-old man presented with a one-year history of progressive and itchy perineal lesions. On physical examination, the patient had extensive verrucous plaques and nodules of the gluteal region, perineum extending to the inner thighs . Skin biSarcoidosis-specific cutaneous lesions are characterized by variable clinical presentations. This fact makes it difficult for dermatologists to appropriately classify this condition on macroscopic evaluation . VerrucoHistologically, these cases showed marked verrucous epidermal hyperplasia with dermal involvement by both typical noncaseating sarcoid granulomas . Only twOur case highlights that verrucous versions of sarcoidosis may be misdiagnosed because it can mimic other inflammatory and neoplastic skin disorders. Although it is a rare variant, its presence should alert clinicians to the likelihood of systemic involvement of cutaneous sarcoidosis.


The presence of traces of heavy metals in wastewater causes adverse health effects on humans and the ecosystem. Adsorption is a low cost and eco-friendly method for the removal of low concentrations of heavy metals from wastewater streams. Over the past several years, graphene-based materials have been researched as exceptional adsorbents. In this review, the applications of graphene oxide (GO), reduce graphene oxide (rGO), and graphene-based nanocomposites (GNCs) for the removal of various metals are analyzed. Firstly, the common synthesis routes for GO, rGO, and GNCs are discussed. Secondly, the available literature on the adsorption of heavy metals including arsenic, lead, cadmium, nickel, mercury, chromium and copper using graphene-based materials are reviewed and analyzed. The adsorption isotherms, kinetics, capacity, and removal efficiency for each metal on different graphene materials, as well as the effects of the synthesis method and the adsorption process conditions on the recyclability of the graphene materials, are discussed. Finally, future perspectives and trends in the field are also highlighted. Both natural and anthropogenic sources linked to industrial activities such as electroplating, metal smelting, fertilizer industries, mining operations, pesticides, and paper manufacturing result in wastewater streams contaminated with various heavy metals. Discharging these streams into the environment leads to the absorption and accumulation of heavy metals into living organisms resulting in severe health complications. Therefore, the current stringent regulations, requires the removal of heavy metal from wastewater streams to extremely low levels. The WHO guideline  for the Several technologies are being used or proposed for removing heavy metals from wastewater streams including chemical precipitation , ion excAlthough graphene can be prepared by bottom up approaches such as CVD, epitaxial growth, templating, and organic synthesis, graphene materials used in adsorption applications are prepared by top-down methods via oxidation of graphite to produced graphene oxide (GO) that can be thermally or chemically reduced to obtain reduced graphene oxide (rGO). Further modification of GO and rGO via the development of nanocomposites with metal oxides and organic molecules enhances their sorption characteristics.This review focuses on the removal of heavy metals from wastewater using various graphene materials. Although there are already few articles that review the adsorption of various metals on graphene materials, this article is more comprehensive and it also accounts for the large number of very recent articles appeared in 2018 and 2019, which represent about 60% of the total number of publications. Moreover, this review includes a concise section on the synthesis of various graphene materials and composites in order to classify the various graphene materials and establish the correlation between the synthesis method, structure, and the adsorption characteristics of the graphene materials. Moreover, the key adsorption parameters associated with each metal on various graphene-based materials are summarized in tables and the performance of the key graphene materials are highlighted and discussed. It is very evident that application of graphene materials as adsorbents for metal removal is a rapidly growing research field as can be seen by the very large increase in number of publications in 2018 and 2019, This review begins with a concise discussion on the synthesis of GO, rGO and their nanocomposites with organic, inorganic, and polymers, which have been used for metal removal. Then an overview of the typical methodology to study adsorption and the models used for analysis of the process thermodynamics and kinetics are provided and a comprehensive analysis of the performance of various graphene-based materials for the adsorption of various metals is carried. Finally, an outline of the major challenges for the commercialization of graphene-based adsorbents for heavy metals and highlights the future research directions are presented. GO and rGO and their foam/aerogel structures andHybrid of GO or rGO with metallic, organic, inorganic, and polymeric materialsThe graphene materials used for the removal of heavy metals are classified into two broad categories that include:While there are few studies on the use GO and rGO in powder, aerogel, and 3D foam structure, most of the reports are on using hybrids of GO and rGO with organic, inorganic, and/or polymers. In this section, we summarize the synthesis methods for GO, rGO and hybrid graphene materials.2 in graphite to a mixture of sp2 and sp3 in GO. The oxygen content of graphite oxide is defined by the C/O ratio which ranges between 1:1 in highly oxidized GO to 4:1 in less oxidized GO depending on the oxidation method and conditions. The presence of these oxygen groups makes graphite oxide very hydrophilic such that it readily exfoliates to GO in aqueous solution. rGO is prepared by thermal, hydrothermal, or chemical reduction of graphite oxide or GO. The reduction of GO to rGO reduced the oxygen content to about C/O ratio of 10. The structure of GO and rGO as well as the schematic for their preparation is shown in Although graphene can be obtained directly from graphite using mechanical cleavage or solution assisted exfoliation, the dominant type of graphene used in adsorption application is prepared via the graphite oxide route not only because its potential for large scale production, but also because it produces a functional form of graphene that is attractive for adsorption applications. In this route, GO is produced by exfoliation of graphite powder, which is produced through solution oxidation/intercalation of graphite using acids and oxidants. The first report on the synthesis of graphite dates back to 1958 by Brodie using nitric acid and potassium chlorate . CurrentAlazmi et al.  investigIn addition to microscopic characterization of GO and rGO, characterization using spectroscopic techniques such as XPS and FTIR and diffraction techniques such as XRD and SAD are commonly used to determine the structure of GO and rGO and the presence of oxygen-containing functional compounds. Other characterization techniques have been performed. The synthesis of the adsorbents was been carried out with a variety of methods and with great rigor and control. Heat treatment at different temperature ranges, vacuum promoted exfoliation, physical incorporation of additives, and surface modifications using oxidative agents are a few techniques used in the synthesis processes . For mor3O4 are very commonly applied not only to enhance the adsorption characteristics, but also to allow the retrieval of the adsorbent for regeneration at the end of the batch adsorption using magnetic recovery. Nevertheless, many studies have further modified the magnetic GO or rGO with other metallic, organic or inorganic materials. A concise review of the synthesis of these different hybrid graphene materials applied for the adsorption of heavy metals is provided.To improve the adsorption characteristics of GO and rGO and to enable their effective application in selective adsorption of heavy metals, a few strategies are applied. These strategies include the formation of hybrid structures of GO or rGO with oxides of transition metals such as iron, Zn, Cu, Ni, and Co, organic materials such as cellulose and chitosan, inorganic materials including metal organic framework (MOF), zeolites, and silica nanostructure, polymers and mixtures of two or more of these groups. Among these strategies, the development of magnetic nanocomposites of GO and rGO with Fe3O4 or Fe3S4. mGO is synthesized via impregnation or co-precipitation methods [3O4, FeSO4, Fe(NO)3, or iron acetate on the surface of dispersed GO under sonication, see One of the significant challenges faced when using nanosorbents is their separation from the treated solution. Centrifugation, sedimentation, filtration, and other traditional separation methods deem to be ineffective and time consuming. On the other hand, magnetic GO (mGO) is synthesized via impregnation or co-precipitation methods ,25,26,27 methods ,25,26,27In the impregnation method, a GO suspension is mixed with pre-synthesized magnetic nanoparticles and sonicated at room temperature until a homogenous suspension is achieved. The mGO is then collected by centrifugation followed by freeze-drying, 4·7H2O to create GO/Brianyoungite GO/BY) composite with hollow spherical and flake-like morphologies. Wen et al. [3O4/GO/Cu-ZEA via solid state dispersion of Cu-ZEA and Fe3O4 into GO suspension followed by sonication. Lingamdinne et al. [Zhu et al. used ZnSOn et al.  incubaten et al.  fabricate et al.  mixed Hy2+, Cu2+, and As3+. Shahzad et al. synthesized EDTA-MCS/GO via 2-step process in which the first step yielded (MCS/GO), which was dried and mixed with Na2EDTA to produce the composite [Graphene-based hybrids have been widely used in the removal of heavy metals. Some of the materials used to make these composites include chitosan and cellulose. Cellulose is combined with GO to form Ethylenediaminetetraacetic acid (EDTA)-functionalized magnetic chitosan (CS) graphene oxide (GO) nanocomposites (EDTA-MCS/GO) through a reduction precipitation method . This coomposite . Yang etomposite  cross-liomposite . 3O4 and adding it to graphene to develop polypyrrole–graphene (PPy–GO) nanocomposites [3O4-encapsulated poly(C3N3S3) polymer/rGO nanocomposite following the method shown in Zhu et al. fabricated a magnetic composite by polymerizing Femposites . Yang etmposites . Tadjaromposites . Sulfurimposites . Najafabmposites . Li et amposites . Zare-Domposites . Nano simposites  on GO tooC, mg/L), equilibrium concentration , maximum adsorption capacity , adsorption percentage (%), the kinetics are studies by measuring the adsorption capacity at a given time t , solution volume , weight of GO , and the final concentration of metal ions . The equations are based on the mass balance of the reaction, as follows:Water solution containing various concentrations of heavy metal ions are used to carry out the adsorption studies typically at pH 5.5 and room temperature. After equilibrium is achieved, the following parameters are measured or calculated: initial concentration  > Cu (II) >> Cd (II) > Zn (II). The adsorption affinity is not dependent on the ionic radius of the heavy metals but more on their relative positions in the electro chemical series. However, the capacity can increase or decrease with temperature depending on heat of adsorption, i.e., exothermicity or endothermicity of the process . tC) and adsorption capacity (tQ) versus adsorption time (t) and various kinetic models are used to regress these data to find the kinetic model that best describes the collected data. For the adsorption of heavy metal ions on GO-based substances, two kinetic models are commonly applied, pseudo-1st-order kinetic model and pseudo-2nd-order kinetic model.Studying the kinetics of the adsorption process is crucial not only to identify the adsorption mechanism but also to ensure the proper design of the adsorption process in terms of the required residence time. In general, experiments are designed to collect data on the change of the adsorbate concentration , more functional groups are available and electrostatic interactions between the heavy metal ions and GO are stronger, but the optimum pH for maximum ion uptake may vary depending on the type of the heavy metal ion. GO modified with metal oxides and organic molecules are among the highest capacities, while inorganic composites (Mg−Al layered double hydroxides GO nanocomposite) when dispersed in water, it swells and forms viscous gel as shown in 0~1 mg/L. The kinetic of the arsenate adsorption on the various graphene materials provided in An important and key aspect for the commercial application of the adsorption processes that are based on costly adsorbent, which is often neglected in research studies, is the regeneration and re-usability of the adsorbent. Few studies have investigated the regeneration and reuse of the graphene adsorbents using either a strong alkali (NaOH) or strong acid (HCl) in the desorption step. For As (III), M-nOG, EDTA-MCS/GO, and GO-f were found to be reusable. M-nOG was observed to retain 94.5% of the first cycle capacity after 5 cycles . Moreove3O4 [Because of the toxicity and abundance of lead in wastewater streams, there are large number of studies focused on the adsorption of Pb (II) on GO with several of studies investigating the regeneration of the graphene adsorbents. The properties of the graphene adsorbents and their lead (II) adsorption properties are summarized in 3O4 ,59,60,613O4 . The trends for the best synthesis method to yield a high capacity adsorbent were very hard to distinguish. The best capacities were achieved by modifying graphene with polymers and inorganics ,64. The The most economic synthesis methods are those using fewer chemicals and production steps. One-pot hydrothermal, dispersion and co-precipitation with few components would serve as a better synthesis option especially when coupled with good performance. For instance, 3D G/chitosan/nickel ferrite NC was synthesized hydrothermally in a few steps utilizing sustainable components like chitosan and is one of the best performing graphene-based adsorbents in the removal of lead from aqueous solutions with a maximum capacity of 957.3 mg/g . 2O4-G, NiFe2O4-G, Fe3O4-GS, EDTA-mGO, EDTA-MCS/GO, DTPA/MGO, GOMCS-IL, and IT-PRGO, Mg−Al LDH / pRGO [3O4-GS and EDTA-mGO maintained more than 80% of their original adsorption capacity [2O4-G and CoFe2O4-G had 98% and 100% desorbing capacity and the adsorption efficiency was almost retained during 3 repeated cycles [The potential for the regeneration of the lead loaded adsorbents has also attracted much attention and several studies have investigated the reusability of the sorbents after regeneration. These include: CoFeH / pRGO ,73,74,75capacity ,77. The capacity . EDTA-MCcapacity . The adscapacity . Similarcapacity . IT-PRGOcapacity . Mg−Al Lcapacity . NiFe2O4d cycles ,66. Basid cycles . It wort4Cl was 17,900 mg/g which is extremely high, although the conditions and results of the study may not be realistic [Despite the large number of adsorption studies for the removal of lead (II) using graphene oxides and derivatives of graphene oxide, almost all the equilibrium adsorption results are best-modeled using the Langmuir isotherm. Most of the kinetic results follows pseudo-second order kinetics with exception of Mg-Al LDH/pRGO and pRGO, which followed Elovich kinetics model , HMO@GO ealistic . The adsorption capacity, removal efficiency, and the isotherm models for the adsorption of Cadmium (II) on different graphene materials are summarized in The most common routes used for synthesis of graphene composites used for Cadmium removal were the solvothermal, hydrothermal, dispersion, and co-precipitation methods. However, nanoscale zero-valent iron-reduced graphene oxide composite was prepared via plasma-reduction method and graphene oxide embedded calcium alginate was prepared via drop-wise formation method to create bead like structures . The bes2O4, G-CoFe2O4, GO-DPA, rGO-NZVI and GO-f are found to be reusable with a slight decrease in capacity with number of cycles. For example, the Cadmium adsorption capacities of DTPA/MGO-based composites synthesized following the route shown in 2O4 and G-CoFe2O4 retained 98% and 100% of their capacity during 3 repeated cycles [Regeneration and reusability of the graphene materials are examined by several groups and mGO- DTPA, G-NiFed cycles . GO-DPA d cycles . rGO-NZVd cycles . The recd cycles . 2O3, GO and DTPA forming a compound with a higher metal binding and electrostatic forces thus improving adsorption capacity [Magnetized DTPA-mGO has been synthesized by combining Fecapacity .4Cl and Zinc [Cadmium is another highly toxic heavy metal, where the kinetics of its adsorption with various conventional adsorbents including Amberlite IR-120 synthetic resin—101 mg/g , modifieand Zinc . The entxR@TiO2 [The adsorption properties of Ni (II) on graphene-based materials are presented in xR@TiO2 . The adsxR@TiO2 .The synthesis process of 2, 20-dipyridylamine (DPA) was a simple precipitation in the presence of nitrogen gas. Dry dichloromethane and phosphorous pentachloride were added to GO and mixed to yield a highly sensitive and stable composite for the adsorption of nickel . The reported capacities of 76, 104, 181 mg Ni (II)/g GO are linked to the high initial adsorbate concentrations of 51, 60, 100 mg/L, respectively . These vThe results of studies on the adsorption characteristics of Hg (II) ions on GO (s) are presented in All studies were on batch experiments. The most common synthesis methods were hydrothermal, solvothermal, and diffusion methods. Some adsorbents were fabricated through a mechanical route whether ultra-sonication , electroHigh adsorption capacities of mercury in the range of 168–700 mg/g have been obtained with different graphene composites ,133,134.The Hg (II) capacities on conventional adsorbents are frequently much lower with adsorption capacities ranging from single digit values to over 200 mg/g. Other reported capacities on conventional adsorbents include walnut shell derived activated carbon—151 mg/g .3O4 to facilitate the decontamination process by simplifying the separation process described in Utilization of the magnetic properties of FeComposite adsorbents have higher adsorption capacities than pure graphene oxide. A novel adsorbent was designed by chemically modifying graphene oxide (GO) with 2-pyridinecarboxaldehyde thiosemicarbazone (2-PTSC) as ligand for removing Hg (II) from aqueous solutions with capacity reaching 555 mg/g. During this experiment the effect of pH, mercury concentrations and time on adsorption capacity were investigated. Moreover, EDTA-mGO, Ag/graphene, GO/2-PTSC, GO/2-PTSC with US, GO-f were identified as reusable adsorbents. Almost half of the studies investigated the regeneration of the adsorbents. Some studies reported excellent regeneration after 6 cycles but had lower maximum capacities in comparison to the rest of the adsorbents ,137. Reu3O4 was a component additive and the resulting adsorbent material had more than one active surface adsorption site. On the other hand, the kinetic data are well represented by the pseudo-2nd order model with two exceptions where the double-exponential and intraparticle diffusion models were followed [The adsorption capacities of Cr (III) and Cr (VI) on various GO-based adsorbents are presented in followed .Ak and Bk, for each step respectively. This model is represented by Equation (4):tq—amount of chromium adsorbed at time t, eq—amount of chromium adsorbed at equilibrium e. Ad and Bd—amounts of rapidly and slowly adsorbed fraction of chromium (mg/L), respectively, m—mass of adsorbent.The double exponential kinetic model assumes the adsorption reaction occurs in two steps comprising a fast stage step 1 and a slow stage step 2, with first order rate constants, 2SO4 which introduces more active sites on the surface of the adsorbents [G-PDAP is a sulfuric acid doped diaminopyridine polymer prepared in-situ on the surface of GO through mutual oxidation-reduction process as detailed in sorbents .Ultrasonication was a popular synthesis route with organic graphene composites with competitive results ,80,141. 3O4/SiO2-GO-PEI was recycled several times without a significant decrease in its adsorption capacity [All these high capacities have the initial adsorbate concentration greater than 200 mg/L. The ability to regenerate and reuse the GO-based adsorbents has been reported. In a few cases, Fecapacity . GONF wacapacity  and rGO-capacity . The moscapacity ,149.th cycle of Cr (VI) removal [GEC’s adsorption capacity decreased slightly from 92.5% to 87.5% after 7 cycles, indicating it is suitable adsorbent for Cr (VI) removal . For chi removal . Moreove removal . DAP–RGO removal . The reg removal . Upon an removal . nd order model. Only one study followed the Double Exponential kinetic model [Removal of copper ions or copper complexes from water are most widely studied among all other metal ions due to its extensive occurrence especially in the microelectronics industry effluents. ic model .The highest copper uptake capacities shown in 3O4, Fe3O4/TiO2, CS-IIP and Fe3O4/HAP/GQDs were found to be reusable. EDTA-mGO retained a 97.6% removal efficiency after five cycles [In addition to high adsorption capacity, it is important for the expensive GO derived adsorbents to have the ability to be regenerated and reused many times, whilst retaining a high capacity. In this regard EDTA-mGO, FA-mGO, EDTA-MCS/GO, DTPA/MGO, GO-DPA, chitosan/GO, GO-CTPy, Fee cycles  and FA-me cycles . i), simple emulsion and Co-precipitation (mGOp) [Magnetic graphene oxide is prepared through the Impregnation Method (mGOn (mGOp) ,71,134. Various methods were used to fabricate or synthesize the graphene composites used for the removal of copper. The three most common were solvothermal, hydrothermal and dispersion methods. In some cases, a combination of the methods was used to synthesize different components of the adsorbents ,70. The th cycle for metal ions [3O4 and FTG were 9% and 7.4% after 5 cycles [EDTA-MCS/GO showed high magnetic sensitivity in the presence of an external magnetic field after 4 cycles, which indicated good reusability status . DTPA/MGtal ions . The adstal ions . It also5 cycles . CS-IIP’5 cycles . The adsorption capacities for copper using the more conventional adsorbents are generally much lower than the high values reported in 5 remained about 95% with both Au(III) and Pd(II) after 3 cycles, showing that it is possible to successfully reuse CSGO5 for the adsorption of both metals [h metals .GO provides an economically viable and efficient method to treat wastewater containing harmful heavy metals. Various methods are available for synthesizing composite GO materials providing materials that are tailored for removal of specific species. The carboxyl, hydroxyl, and epoxy functional oxygen groups on GO facilitate the adsorption of ions by providing active binding sites. This review analyzes the metal adsorption results from over one hundred and eighty recent studies using GO and GO-derived materials for the removal of more than 10 metal ions. The adsorption capacities of different metals are analyzed and compared with the capacity of conventional adsorbents. In almost every case, the metal adsorption capacity using GO materials are considerably higher. Although the production of GOs is expensive at present, these high capacities may lead to lower cost for treatment of wastewater streams polluted with metal ions as not only is the required adsorbent amount lower, but also the size of adsorption equipment will be less and hence the plant capital cost will be less. There are no studies that have considered how to recover the valuable metals for re-use which is an important topic that needs further research.New research directions started in the last few years for GO hybridizations with different organics to enhance the metal uptake and speed the adsorption kinetics. More detailed mechanistic studies have been utilizing UV and ultrasound to facilitate the adsorption process. More research is required on new functional groups and composites that increase the maximum adsorption capacity of GO and improve its reusability. More fixed bed column studies are required and both testing of the graphene materials for selective removal of metals is still absent from the current literature as the current studies focus on testing single metals. Moreover, the effect of water salinity on the adsorption of heavy metals is important for treatment of oil- and gas-produced water, where high concentrations of monovalent salts exist. Another key issue is the ability to regenerate and retain the adsorption capacity of the GO-based adsorbent in an economic manner and recover the desorbed metal  for reuse. There are almost no studies which have utilized the use of fixed bed columns experiments as almost all studies have been on batch experiments. Further studies in this area and scale-up are also required with estimation of the cost of the material and its impact on the economic viability of large-scale adsorption processes.