Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease for which age is the most important risk factor. Different mechanisms associated with aging, including stem cell dysfunction, have been described to participate in the pathophysiology of IPF. We observed an extrapulmonary effect associated with IPF: increase in cell senescence of bone marrow-derived mesenchymal stem cells (B-MSCs).INK4A, p21, and β-galactosidase activity. Mitochondrial function and DNA damage were measured. Paracrine induction of senescence and profibrotic responses were analyzed in vitro using human lung fibroblasts. The reparative capacity of B-MSCs was examined in vivo using the bleomycin-induced lung fibrosis model.B-MSCs were obtained from vertebral bodies procured from IPF patients and age-matched normal controls. Cell senescence was determined by cell proliferation and expression of markers of cell senescence p16In our study, we demonstrate for the first time that B-MSCs from IPF patients are senescent with significant differences in mitochondrial function, with accumulation of DNA damage resulting in defects in critical cell functions when compared with age-matched controls. Senescent IPF B-MSCs have the capability of paracrine senescence by inducing senescence in normal-aged fibroblasts, suggesting a possible link between senescent B-MSCs and the late onset of the disease. IPF B-MSCs also showed a diminished capacity to migrate and were less effective in preventing fibrotic changes observed in mice after bleomycin-induced injury, increasing illness severity and proinflammatory responses.We describe extrapulmonary alterations in B-MSCs from IPF patients. The consequences of having senescent B-MSCs are not completely understood, but the decrease in their ability to respond to normal activation and the risk of having a negative impact on the local niche by inducing inflammation and senescence in the neighboring cells suggests a new link between B-MSC and the onset of the disease.The online version of this article (10.1186/s13287-018-0970-6) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease characterized by a progressive and irreversible loss of lung function though accumulation of scar tissue –3. Its aAging is considered the main risk factor for IPF –11. Alonn = 7), old donors , and IPF patients   of the University of Pittsburgh. As previously described, B-MSCs were isolated from bone marrow fragments from cadaveric vertebral bones . B-MSCs Female 11-week-old C57BL/6 mice  were treated with 2 U/kg bleomycin hydrochloride solution  dissolved to 1 U/ml in sterile saline and delivered by direct injection into the trachea using a 0.9-mm needle. While under anesthesia, a cell suspension of 500,000 human B-MSCs in 100 μL culture medium was injected intravenously. Control group mice received the same volume of sterile medium solution  and STATA version 13 (Stata Corporation). Comparisons between control and IPF B-MSCs were made using a Mann-Whitney test. Kruskal-Wallis and Dunn tests were used for between-group comparisons. For time-dependent observations, we used mixed-effect models with a robust variance estimator to calculate the difference between groups in experimental outcomes.B-MSCs isolated from IPF patients showed morphological changes characterized by increased cell size accompanied by replicative senescence in comparison with B-MSCs from age-matched controls. Quantification of B-MSC proliferation by measurement of DNA staining showed decreased cell proliferation Fig. , which wINK4A and p53 were moderately increased when compared with age-matched controls [To confirm the senescence phenotype of B-MSCs from IPF patients, we investigated the presence of other markers of senescence. Measurements of senescence-associated βgalactosidase  activity showed significantly higher positivity in B-MSCs derived from IPF patients compared with the control group Fig. . Furthercontrols , 20 , and maximal respiratory capacity on the injection of the mitochondrial inner membrane uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Finally, the complex I inhibitor rotenone was administered to determine the nonmitochondrial oxygen consumption rate (OCR). IPF B-MSCs showed lower OCR at basal and maximal respiration conditions and after injection of mitochondrial complex inhibitors in comparison with control cells Fig. .Fig. 6Mi. As a consequence of lower oxygen consumption and glycolytic rates, total adenosine triphosphate (ATP) content in IPF patients decreased compared with the control group  readings at baseline and after FCCP treatment were lower in IPF B-MSCs compared with controls, suggesting there was no glycolytic reprograming in these cells Fig. . In factoup Fig. .d-glucose (2-DG)  Fig. , whereasDG) Fig. . These rDG) Fig.  and IPF DG) Fig. .To assess changes in the capabilities of human B-MSCs to alter the severity of the lung injury, we evaluated the ability of age-matched control and IPF B-MSCs to prevent the development of bleomycin-induced lung fibrosis and then compared this with the response mediated by B-MSCs isolated from young donors. Two regimens of cell infusion were examined: a preventive regimen with infusion of cells 2 h after bleomycin injection, and a therapeutic regimen with cell infusion at day 7 post-bleomycin.Weight loss was used as a measurement of illness severity. As previously reported, mice in the preventive and therapeutic regimens that received young B-MSCs were protected against weight loss compared with bleomycin-treated mice without B-MSC infusion Fig. . Mice reINK4A and p53  from IPF B-MSCs or age-matched controls. After 48 h of treatment, human lung fibroblasts showed increased expression of markers of senescence, including β-galactosidase activity Fig. , and uprp53 Fig. . In parap53 Fig. .Fig. 8StIPF is an age-related systemic disease with a predominant lung phenotype. There is compelling evidence that, for unknown reasons, the lung can be the main target of systemic alterations such as telomere mutations, alterations in proteostasis, and mitochondrial dysfunction. It has been proposed that the reparative capacity of B-MSCs may be decreased with age . In our Aging is a process that affects all cells, including mesenchymal stem cells. It has been suggested that, in aged mesenchymal stem cells and aged lungs, several pathways are altered that could increase the risk of IPF . We haveA decrease in cell proliferation, mitochondrial dysfunction, telomere attrition, and cellular senescence are identified as hallmarks of aging . Our resIn the present study, we have demonstrated that B-MSCs from IPF patients have important differences in mitochondrial function, increases in DNA damage that result in cell senescence, and defects in critical cell functions when compared with age-matched controls. IPF B-MSCs show signs of accelerated senescence that suggests a link between aging and the late onset of the disease. Given that MSCs exhibit decreased function with age and disease, this confirms the possible risk of the use of autologous stem cells in patients with IPF.MSCs, like other cells in IPF patients, have multiple defects that can result in an increase in the severity of the disease. We have identified extrapulmonary changes in the bone marrow-derived mesenchymal stem cells (B-MSCs). Although there is evidence in animal models, no human studies have assessed the function of IPF B-MSCs compared with age-matched old control donors. In our study, we demonstrate for the first time that B-MSCs from IPF patients are senescent with significant differences in mitochondrial function and accumulation of DNA damage resulting in defects in critical cell functions when compared with age-matched controls. Senescent IPF B-MSCs have the capability to stimulate paracrine senescence by inducing senescence in normal aged fibroblasts, suggesting a possible link between senescent B-MSCs and the late onset of the disease. Despite IPF being a disease with a respiratory phenotype and a major representation in the lung, our results show systemic consequences of the disease.Additional file 1:Supplementary figures. (ZIP 623 kb)


We identify graphene layer on a disordered substrate as a system where localization of phonons can be observed. Generally, observation of localization for scattering waves is not simple, because the Rayleigh scattering is inversely proportional to a high power of wavelength. The situation is radically different for the out of plane vibrations, so-called flexural phonons, scattered by pinning centers induced by a substrate. In this case, the scattering time for vanishing wave vector tends to a finite limit. One may, therefore, expect that physics of the flexural phonons exhibits features characteristic for electron localization in two dimensions, albeit without complications caused by the electron-electron interactions. We confirm this idea by calculating statistical properties of the Anderson localization of flexural phonons for a model of elastic sheet in the presence of the pinning centers. Finally, we discuss possible manifestations of the flexural phonons, including the localized ones, in the electronic thermal conductance. Usually, FPs are considered in the context of the suspended graphene. Here we argue that graphene layer placed on the top of the supporting SiO2 substrate gives an opportunity to observe Anderson localization2 for the FPs.Most of the research in graphene emphasizes the relativistic character of its electron spectrum. However, graphene is also interesting due to its out-of-plane  vibrational phonon modes. Flexural phonons (FPs) are a unique addition that Van der Waals heterostructures have brought into microscopic physics3. If the velocity component normal to the surface of the cylinder vanishes, i.e., σR is proportional to a(ka)3, where a is the radius of the cylinder and k is the wave vector. This is the conventional Rayleigh scattering result4 for two dimensional (2d) geometry. However, when pressure is constant, the boundary condition reads Φ(a) = 0, and this influences forcefully the scattering. Unlike the Rayleigh scattering, the zero angular harmonic is involved, and as a result the cross section diverges at small k as ∝To get an idea, let us recall the known facts about a long-wave acoustic wave scattering on a cylinder. The output depends drastically on the boundary conditions for the velocity potential Φ on the surface of the cylinder2 substrate, it has been concluded that in this system the layer is suspended between hills of the substrate8. We have checked that scattering of the FPs from areas attached to the substrate is similar to the scattering from a rigid obstacle9. The zero harmonic is also involved, and the scattering cross-section diverges as σfl = 4/k.In graphene, the substrate cannot scatter effectively the usual acoustic waves, longitudinal and transverse, because graphene itself is one of the most rigid substances. The other thing are the out of plane phonons. From the analysis of intrinsic and extrinsic corrugation of monolayer graphene deposited on SiOni. Taking into consideration that the spectrum of the FPs ω(k) = αk2 is quadratic, i.e., velocity is linear in k, one obtains a scattering rate τ−1 = vσflni, that is finite in the low-energy limit. Thus, for the FPs one may expect localization of the low-energy modes with ω(k) < τ−1. This is in striking contrast with localization of acoustic modes, which is known to happen only at high enough frequency14.In this work we study statistical properties of the out of plane excitations for a pinned-suspended flexible sheet. Whether pinning centers are located in the vicinity of the maximal heights of the substrate where the interaction with the layer is the strongest, or there are charges on the substrate which interact strongly with their images, will be not important for our purposes. First of all, we are interested in the scattering rate of the FPs in the presence of the randomly located pinning centers with concentration 15 indicate, however, towards a possibility of the detaching a graphene sheet from a substrate to relieve its strain by slipping. (This is manifest by straightening of the cantilever). In the case of the SiO2 substrate, both experiment and theory agree that for typical magnitude for corrugations, the graphene layer is partially detached from the substrate. Moreover, the theoretical considerations17 justify the use of a contact force that is finite when graphene is conforming to the substrate and zero otherwise. The basic experimental facts7 which lead to the conclusion that graphene layer deposited on SiO2 is partly freely suspended are as follows: The long-range corrugation of the substrate with the correlation length of about 25 nm is also visible on the graphene sheet, but with a smaller amplitude than on the substrate. Mesoscopic corrugations with smaller length of about 15 nm not induced by the substrate were also identified. These short range corrugations are similar in height and wavelength to the ones observed on suspended graphene19. In addition, the picture of partially suspended graphene, and the presence of the FPs in the graphene on the SiO2 substrate, has been confirmed by the transport20 and thermal measurements22.Let us comment upon the graphene layer deposited on the top of the corrugated substrate. Naively, the membrane-like layer either follows the substrate or hovers over the surface at some distance. Measurements with the use of cantileversHere, we demonstrate that the FPs in a pinned-suspended flexible sheet are more similar to disordered electrons rather than to acoustical phonons. The main point here is that the pinning centers are effectively rigid obstacles for the FPs. As we have already explained, this leads to the non-vanishing scattering rate at small energies. Let us touch upon this point in more detail.h is displacement in the out of plane direction; the term describing the barrier is on the right-hand side of the above equation. As a result, a flexural phonon with an energy smaller than ω0 cannot enter the area of pinning. We have checked that such an inclusion is equivalent to a rigid obstacle. For not small ka, the cross-section σfl(k) = 4f(ka)/k. Interestingly enough, for f(ka) ≈ ka, for a discussion see Section I in METHODS. Therefore, the limiting cross-section is ≈4a, i.e., twice larger than the width of the obstacle3. (This form of σfl(k) is, of course, valid only when the energy of the phonon is much less than the pinning potential, i.e., 23 we assume that the pinning potential ω0 is about few meV).Usually by a rigid obstacle one understands an inclusion with the Young’s modulus much higher than that in surrounding area. For graphene, which itself is very rigid, this is not an issue. However, the pinning potential introduces an energy barrier of a finite height for the flexural modes:T2. The rate of energy exchange between electrons and FPs, as discussed below in section “Thermal transport”, is proportional to T3. Therefore, the openness of the FPs, cannot effectively destroy their localization at low temperatures.Let us now touch upon a subtle question of the openness of the ensemble of the FPs which can be relevant for their localization. In the discussed model, the low-energy FPs cannot penetrate into the areas of strong contact with the substrate. Therefore, the most relevant channel connecting the FPs with the other degrees of freedom is the interaction with conducting electrons. The effect of this interaction can be estimated by comparing the amount of heat stored in the FPs with the rate of cooling of electrons. At low temperatures, most of the heat in the graphene layer is stored by the FPs, as they are the softest modes. The amount of this heat is ∝W. Instead, there is concentration of the pinned sites. Furthermore, the FPs are described by the square of the Laplacian, rather than by the Laplacian in the case of electrons. To understand the general properties of the FPs in the presence of random pinning scatterers, we solve the equation of motion for the out-of-plane displacements using finite difference method on a 2d square lattice. We study a model in which the graphene sheet is completely attached at the pinning centers. For that, we use discretized LHS of Eq. h = 0 at randomly chosen pinned sites, so that one pinned site represents an attached area of the size ≈2a 2 which approximately equals 0.08 K for the parameters mentioned above. We found out here that typical energy scale for strong localization, 2 substrate We estimate the size of an attached area to be mentclass2pt{minimd is that the critical point is at e2/(2πħ). At a finite lloc, which is analogue of a correlation length at a quantum phase transition. A sample of size 24. A consequence of strong fluctuations of wave function amplitude in the critical region is the multifractality .As is well known, localization is a quantum critical phenomenon. The peculiarity of 2i.e., the “metal-insulator” transition with the mobility edge), or there is a crossover from strong to weak localization (WL)?Turning back to disordered FPs, the first question that needs to be answered is: Is there a transition to delocalized states at a certain energy . From our simulations, it is clear that low-energy modes are localized: the IPR scales with the sample size to a finite value. For higher energies, the behavior of the wave functions changes, see Fig. lloc starts to exceed the sample size. We are particularly interested in studying the FPs in this region when d Anderson model does not constitute a truly critical system, thanks to exponentially large (but still finite) localization length at d electrons at large 26. As we shall see, similar physics holds also for our system of the FPs.To figure this out, we first studied the dependence of the Inverse Participation Ratio (IPR) on the sample size We proceed as follows: From the size dependence of the IPR at a given energy as shown on the Fig. 28 equal to L is due to the WL corrections. Thus, for each concentration of the pinned sites we can prescribe for different energies ω the corresponding value of the phonon “conductance” gph, using the expression for the fractal dimension D2 for electrons. We defer the discussion of the dependence of D2 on the sample size, D2(L), to the end of section “Weak multifractality of eigenfunctions”.d, the well developed theory connects the behavior of various physical quantities with the value of the conductance25. We have calculated numerically the same quantities for the FPs, using the values of gph extracted from IPR, and found a very good agreement with the theoretical predictions existing for the disordered electrons in the case of the GOE. Below we present some results of such calculations.For disordered electrons in 2P(s) for localized states has almost Poissonian statistics, while for metallic states it is of the Wigner-Dyson form. Next, in 2d it becomes especially interesting to study the variance N in a strip of width Ω around the energy ω: d = 1 and 3, in two dimensions this quantity is directly related to the WL corrections29. Figure d = 2; for details the reader is referred to section “Energy level statistics”.First, we have checked  are of comparable strength for the discussed scales.Let us estimate energy of the FPs at which a crossover from strong to WL occurs. Strong localization, mentclass2pt{minimf ≈ ka > 1, the energy of the strongly localized FPs can be a few times larger. As we have already mentioned, a reasonable estimate for 2 is ≈0.3 K. Furthermore, effects of the weak localization noticeably expand localization of the FPs. One may easily show that, as compared to the strong localization, the weak localization increases momenta of the FPs which undergo localization by a factor ln(L/l). . Correspondingly, weak localization boosts the energy of the localized FPs by a factor ln2(L/l). For a standard micron size sample, So far, we have discussed point-like pinning centers only. In reality, the size of the attached areas can be comparable with the distances between them. Then, owing to the factor 36. For graphene on a random substrate, FPs not only scatter from the points of contact with the substrate, but also excite acoustic phonons. One may check, following the calculations of ref.37 for disorder-assisted scattering, that at low temperatures the effect is vanishingly small.In writing Eq.  we have Y0), while a bending rigidity κ0 is relatively modest. The anharmonicity transfers the strong in-plane rigidity into the bending one. The effect is controlled by the temperature, and is of the infrared origin. For small wave vectors κR(q) ~ κ0(q/qth)η− with the scaling exponent η ≈ 0.8–0.8539. The transition scale is given by qth ≈ 1.6 nm−1. As a result, effective bending rigidity of an atomically thin graphene ribbon that are 10–100 micrometers in size at room temperature can be thousands times larger than at T = 043. .Next, as it is well known anharmonicity yields a strong effect on the bending rigidity in graphene. The point is that the in-plane rigidity of graphene is extremely high  ≈ κ0. In graphene, the energy of a FP with the wave vector qth is equal to 2 substrate. Furthermore, in our analysis, we were mostly interested in FPs with the frequency exceeding Strong effects caused by the renormalizations, which have been mentioned above, correspond to the vanishing wave vector 2 substrate. We argue that traces of localization of the FPs may have been observed in the experiments at low temperatures.We shall discuss now the implications of the FPs on the thermal transport of a graphene layer placed on a corrugated SiO2 at low temperatures, refs49, has not been fully understood, yet. Theoretically, the heat flux from electrons to the FPs is known37 to be ∝δ = 3 observed in refs46 at low temperatures and far away from the neutral point. At concentrations of the electric carriers, electrons or holes, n ~ 1012 cm−2 the alternative explanation of δ = 3 with the use of the result obtained for the case of the unscreened deformation potential49 is not realistic; a discussion of this point is given below in section “Electron-phonon interaction at low temperatures”.The temperature behavior of overheating in graphene on the top of SiO46 for cooling rate of graphene at the lowest temperatures 52 that neither the Fermi liquid nor renormalization-group corrections in disordered 2d electron systems can modify the Lorenz number imitating the electron heat diffusion contribution, Moreover, we believe that the existence of localized FPs may explain the experimental result of ref.four powers of the FP-momenta. However, the localized FPs are not goldstone modes anymore. For localized FPs, the momenta that enter into the matrix elements of the electron-FP interaction should be substituted by the inverse of the localization length. As a result, two powers of frequencies in the expression for the heat flux P saturate at ω−2. The diverging integration should be cut-off at energies typical for the localized FPs. As a result one gets a contribution to the cooling rate of the order Let us comment upon 59. However, a layer placed on the top of a corrugated substrate, which has been discussed in the present work, is very different from the disordered membranes considered so far. First, disorder here is external rather than internally quenched. Next, disorder pins rigidly the height of randomly chosen points of the layer, rather than acting on the metric and curvature tensors describing the deformation of the membrane. Questions of localization of the FPs to the best of our knowledge have not been addressed previosly.Studying effects of disorder on the properties of elastic membranes has a long history2 substrates are expected to play an important role in applications related to thermal transport and for ultrasensitive bolometry. While the graphene sheet is pinned at random points, the FPs may exist in between due to the corrugation typical for this substrate surface. We have argued that in this system Anderson localization of low-energy FPs develop. We showed that the ensemble of flexural phonons in a pinned-suspended flexible sheet is statistically identical to an ensemble of disordered electrons, despite the very different underlying mathematical descriptions. Localization of flexural phonons should be important for thermal transport in such hybrid systems even at not very low temperatures. Traces of localization of the FPs may have been already observed in the experiments. Let us note that we have already shown that FPs give a significant contribution to dephasing rate of electrons in graphene60. Here, we have argued that localization of the FPs opens interesting perspectives for thermal transport.Graphene layers on top of SiO9, the scattering cross-section of the FP by a rigid obstacle of the radius a equals f(z) is given by the following expression:f(z) with asymptotes f(0) = 1 and f is important for the effectiveness of the weak localization in samples of large size.According toThe spatial distribution of wave functions is conveniently characterized by inverse participation ratios:Pq〉 shows the scaling behavior with the system size L:After sample average, 〈Dq = 0, while in a metal Dq = d. At a critical point, Dq is a fractional which leads to anomalous scaling behavior in 〈Pq〉. This is a manifestation of the wave function multifractality, which is a consequence of the spatial correlations of the wave function.Obviously, in the insulating state d, the inverse participation ratios scale asIn 2Dq from dimension 2 is determined by a small parameter 1/πg. The above result 6 was first obtained by Wegner27 via the renormalization group calculations for a system of disordered (non-interacting) electrons; see also28. The dimensionless parameter g is a conductance of a sample measured in quantum units. In analogous to weak localization, the phenomena is coined “weak multifractality”.Here, the deviation of q = 2. The corresponding inverse participation ratio, Pq = 2, was denoted as IPR. This quantity allowed us to extract the phononic “conductance”, which we used for the statistical analysis of our system. Note that, because of the absence of the genuine critical point in 2d, g becomes size-dependent, i.e., g(L) = g0 − (1/π) ln L/l where l is the mean-free path of FPs at a given energy, and g0 = g(l). This implies that for each scale L one can use the standard formula given above, but with slowly varying g(L) in the exponent. This is possible because corrections to g are not large in a finite size sample, and owing to the slow dependence of g on spatial scale L. With this procedure, we have obtained an excellent agreement between the theory of the logarithmic corrections to the conductivity and our numerical results as it is shown in Fig. We study a model of random pinning centers, in which the graphene sheet is completely attached at the pinning centers; see Fig. ωn, such as the distribution function of level spacing P(s), and the level number variance Several quantities are introduced to measure the fluctuations of energy levels P(s) = exp(−s). In our system of the FPs the crossover from the localized to delocalized behavior is illustrated on Fig. Random Matrix Theory (RMT) could be used to describe these quantities in ergodic systems . Here, we will focus on the case of Gaussian Orthogonal Ensemble (GOE). Then the distribution function of level spacing is well described by the Wigner surmise: N〉 = Ω/Δ, and cβ is a known constant. This, however, is far from the true behavior  and, hence, D → D + δD, where Kravtsov and Lerner recognized in29), we use Eq. Q and s, this yields11 for fitting the numerical data presented in Fig. Now, instead of splitting this expression into two parts as it was done iny:y〉 = 1.Porter and Thomas were first who studied the distribution of eigenfunction amplitudes within the RMT framework. Their result shows simply Gaussian distribution, which leads to the following distribution of the intensities d-dimensional disordered system. The eigenfunction intensity r0 is distributed as:The supersymmetric field theory was applied to the study of the eigenfunction statistics in a d = 2, and not too large y, one can calculate perturbatively the deviations from the RMT distribution P(y). The corrected distribution function was found by Fyodorov and Mirlin in (31):y can be seen in Fig. For 46 the T3 law has been observed down to 0.5 K for sample D3. Such a behaivior corresponds to the dirty regime with negligible screening of the e-ph deformation potential according to the Table 1 presented in49. We believe, however, that applicability of the screenless approximation for the e-ph interaction at such low temperatures and concentration of carriers n ~ 1012 cm−2 is highly questionable.Some recent experiments interpreted the cooling rate in terms of the weakly screened electron-phonon (e-ph) interaction down to very low temperatures. In particular, in49, temperature above which screening becomes irrelevant in the dirty regime 49, one finds that crossover from the T5 to the observed T3 behavior should happen at κ for an effective dielectric constant . Here we expressed the result in terms of TBG using the fact that in graphene the inverse of the screening radius is of the order of the Fermi momentum. The unscreened T3 behavior may occur at temperatures higher than Tdis. (For samples D1 and D3 in (46) one has Tdis ≈ 40 K).To illustrate our point, let us estimate, followingTBG ≈ 80 K. Thus, one may expect for the crossover temperature κ. We, therefore, believe that this mechanism may be discarded for explanation of the T3 scaling of the electron-phonon heat flux observed at low temperatures.For the aforementioned sample D3,


The yield of bilayer and few-layer graphene obtained was quantified by Raman spectroscopic analysis, and the adsorption of the pyrene derivative on the graphene surface was studied by thermogravimetric analysis and X-ray diffraction. The whole procedure was rationalized with the help of molecular modeling.The search for graphene or few-layer graphene production methods that are simple, allow mass production, and yield good quality material continues to provoke intense investigation. The present work contributes to this investigation through the study of the aqueous exfoliation of four types of graphene sources, which are namely graphite and graphite nanoflakes with different morphologies and geographical origins. The exfoliation was achieved in an aqueous solution of a soluble pyrene derivative that was synthesized to achieve maximum interaction with the graphene surface at low concentration (5 × 10 The hydrophobic nature of graphene leads to its re-stacking and agglomeration in aqueous dispersion. In fact, solvents with surface tensions of near 40 mJ m−2 [Graphene has emerged as an exciting material that has been intensively studied throughout the last decade, revealing potential applications in various fields ,2,3. How0 mJ m−2 .−) and pyrene sulfonic acid salt, as well as a pyrene-terminated poly (PDMAEA) and poly(acrylic acid) (PAA) have been investigated for use in solar cells, as well as electrochemical and composites applications [The performance of water for the LPE of graphite can be greatly improved with the aid of amphiphilic molecules that strongly interact with water and with graphene simultaneously. These molecules help the dispersion of graphite flakes and graphene, preventing their agglomeration. Polycyclic aromatic hydrocarbons such as pyrene derivatives have been studied for the exfoliation and stabilization of graphene and FLG in water ,7. The aications ,9,10. Inb-poly((polyethylene glycol) acrylate) (polyPA-b-polyPEG-A) and a pyrene-terminated polyethylene glycol (pyrene-PEG) were used by Liu et al. [Other approaches, based on the direct exfoliation of graphite in water, have been reported. A pyrene-functionalized amphiphilic block copolymer, poly(pyrenemethyl acrylate)-u et al.  and Zhenu et al. , respectBiocompatible polymers have been functionalized with pyrene moieties to prepare aqueous graphene suspensions for biomedical applications ,14,15. PIn this paper, the synthesis of a pyrene derivative through a simple and low-cost functionalization methodology is presented. This synthesis presents a high yield of the modified pyrene that may be scaled up for large-scale production. The synthetized pyrene derivative is soluble in water, and it was tested for the exfoliation and stabilization of three different types of graphite nanoplatelets (GnP) , which w2/g, while GnPH5 has a nominal equivalent diameter of 5 µm, thickness of 15 nm, and an average surface area of 60–80 m2/g. GnP Micrograf, provided by Nacional de Grafite , is a micronized graphite with an equivalent diameter of about 10 µm, and Graphexel is a natural crystalline graphite with a large equivalent diameter of approximately 180 µm that was provided by Graphexel Ltd. . Copper (II) nitrate trihydrate (Cu(NO3)2·3H2O), anhydrous copper (II) sulphate (CuSO4), and potassium hydroxide pellets (KOH) were purchased from Sigma Aldrich . Maleic anhydride 99% pellets and pyrene 98% were purchased from Acros Organics . 4-nitrobenzaldehyde was obtained from Merck . Acetic anhydride (Ac2O) and sodium borohydride (NaBH4) were purchased from VWR chemicals . Ethyl acetate (EtOAc), petroleum ether 40–60 °C, ethanol absolute (EtOH) and acetonitrile (ACN) were purchased from Fisher Scientific . Anhydrous magnesium sulphate (MgSO4), dicholoromethane (CH2Cl2), and diethyl ether were obtained from Panreac . Deuterated dimethyl sulfoxide  was purchased from Euriso-top .Two grades of GnP were purchased from XG Sciences , grade C (GnPC), and grade H5 (GnPH5). GnPC has, according to the manufacturer, a size distribution ranging from very small (100 nm) to relatively large flakes (1–2 µm), an average thickness of approximately 2 nm, and a typical average surface area of 750 m1H and 13C nuclear magnetic resonance (NMR) ). The detailed information about the synthesis of the pyrene derivatives and its spectral characterization . Graphite suspensions were prepared by mixing approximately 5.0 mg of graphite in 10 mL of the pyrene derivative solution using an Ultrasonic processor UP100H from Hielscher  equipped with a sonotrode MS7D. Ultrasound energy was applied to the suspensions for 1 h at maximum power. The suspensions were centrifuged  to remove larger aggregates, and the supernatant was collected. These stable suspensions were analyzed by UV-visible spectroscopy on a Shimadzu UV-2401PC, using quartz cells with a 10-mm pathlength. The concentration was determined using the Beer–Lambert Law, and the extinction coefficient for these materials was calculated using a procedure reported elsewhere [The suspensions were sprayed on a glass slide using a XL2000 Airbrush  with a 0.6-mm nozzle and analyzed by Raman spectroscopy. Raman spectra were obtained on a Horiba LabRAM HR Evolution confocal microscope  using a laser excitation of 532 nm (2.33 eV). A 100× objective lens was used to focus the laser onto the sample. For each sample, 60 spectra were collected randomly over a sample area of 50 × 20 mm, and analyzed using the LabSpec 6 software . Lorentzian functions were used to fit the characteristic peaks of the spectra.2.Scanning transmission electron microscopy (STEM) samples were prepared by the deposition of liquid suspensions of exfoliated graphite samples on 400 mesh carbon coated copper grids  and dried using a hot plate. The samples were analyzed using a NanoSEM FEI Nova 200 microscope . The dried samples, which were obtained by the solvent evaporation of the liquid suspensions of exfoliated graphite, as well as the graphite starting material, were analyzed by scanning electron microscopy (SEM) using the same equipment. The thermogravimetric analysis (TGA) of these powder samples was performed on a Modulated TGA Q500 from TA Instruments , heating the samples at 10 °C/min under a constant flow of NX-rays diffraction (XRD) measurements were performed on a PANalytical X´Pert PRO XRD System  using Cu–Kα radiation with the X-ray tube operated at 45 kV and 40 mA. The XRD spectra were obtained by continuously scanning 2theta/omega axes in steps of 0.0098 2theta°, using a scanning line detector with Soller slits and a Ni filter. The X-ray beam was incident over the sample in line focus type using a 1° divergence slit and also Soller slits. The XRD samples were obtained from solvent drying from the bulk solution, powder samples, and by spraying the solution over a heated silica wafer.In the present work, an amphiphilic pyrene derivative was synthesized for the purpose of graphite exfoliation, combining the pyrene moiety for an effective π-π interaction with the graphene layers and a hydrophilic carboxylic group for affinity with water , which is typical for this isomer [13C NMR spectrum at δ 164.46 ppm (for the amide) and δ 166.84 ppm (for the carboxylic acid). The FT-IR spectra are also presented as −1 and 1331 cm−1, corresponding to the –NO2 asymmetric and symmetric stretching vibrations. After reduction to 2, the characteristic symmetric and asymmetric stretching vibrations of the primary amino group can be seen at 3445 cm−1 and 3379 cm−1. The reaction of product 2 with maleic anhydride leads to the final product 3 showing the characteristic intense bands for the stretching vibration of the carbonyl groups at 1711 cm−1.The structure of compound 3 was confirmed by the presence of the amide and the carboxylic acid protons at δ10.84 and 13.26 ppm, respectively, in the s isomer . The two2O). The pyrene derivative 3 (PY) was used to exfoliate the graphite nanoplatelets GnPH5, GnPC, Micrograf and the natural graphite Graphexel in water, and stabilize the FLG formed. The same exfoliation/centrifugation procedure was applied to the different graphite grades using distilled water without PY , and the concentration of FLG was determined by UV-visible spectroscopy. The results are presented in The TGA curves of the graphite starting materials Graphexel, Micrograf, GnPH5, and GnPC show thermally stable materials with a weight loss at 600 °C of 0.6 wt%, 0.1 wt%, 2.4 wt% and 5.4 wt%, respectively. GnPC presents a larger weight loss at 600 °C, which may be due to its smaller flake size and decomposition of the oxygen-containing groups. The weight loss of the corresponding FLGs, also at 600 °C, are 39 ± 2 wt%, 35 ± 2 wt%, 34 ± 1 wt%, and 12 ± 2 wt% respectively, and these large values are due to the decomposition of the PY molecules adsorbed on the FLG surface.−1, is present in all of the materials with conjugated C–C double bonds and reflects the in-plane bond stretching motion of pairs of C sp2 atoms [laser), but is sensitive to temperature, strain, and doping of the graphene sample. The D mode, which is observed near 1350 cm−1, is a breathing mode of A1g symmetry that is forbidden in perfect graphene, becoming active in the presence of disorder [2 carbon network disturbed by chemical bonding such as that observed adjacent to a graphene edge or a defect. The D band is highly dispersive as a function of the Elaser. The two-dimensional (2D) band is a second-order mode that is sometimes referred as an overtone of the D band, although it is not related to graphene defects, and is always strong in graphene, even when the D band is absent. The shape and position of the 2D band varies with the number of graphene layers [−1, while bilayer graphene requires four characteristic Lorentzian functions, each with a FWHM of ~24 cm−1. As the number of layers increases, the 2D band shifts to a higher wave number and becomes broader and more asymmetrical in shape. Its deconvolution becomes more complex, and for few-layer graphene (less than 10 layers), it may be fitted with three Lorentzian functions with a FWHM higher than 24 cm−1 each. For more than 10 layers, the 2D band becomes similar to that of graphite, and may be fitted with two Lorentzian functions [Raman spectroscopy is an important tool for the characterization of graphene and carbon-based materials. The Raman spectrum of single-layer graphene presents three main characteristic bands, associated with the G, D, and 2D (or G′) modes. The G mode, which is observed near 1580 cmp2 atoms . Due to disorder . Its pree layers . For sinunctions ,38. Figu−1 is almost absent compared to the G band (at 1581 cm−1), which indicates the good structural quality of the graphite. The 2D band position of Graphexel and GnPH5 occurs at approximately 2722 cm−1, and its shape is asymmetric, which is typical of the graphite 2D band [−1), which is typical of a more exfoliated material. The 2D band of all of these starting materials can be fitted with two Lorentzian functions, as shown in −1, which was deconvoluted with four Lorentzian functions , which may be related to the smaller flake size. Thus, it also has a higher edge-to-basal plane ratio, as well as a higher oxidation level, which is in agreement with the producer specifications and the TGA analysis. The 2D band position occurs near 2692 cm−1, showing high symmetry, although with lower intensity compared to the G band. The 2D band of the GnPC starting material can be fitted with two Lorentzian functions, as shown in The Raman spectra of the GnPC starting material that was exfoliated with PY are presented in A statistical analysis of the flakes formed in suspension by exfoliation of graphite in water using PY was performed by Raman spectroscopy. The analysis was based on the Raman spectra collected for 60 different flakes of each exfoliated material , and the spectra of bilayer and FLG were identified. The FLG and bilayer graphene spectra were identified based on the position of the 2D band and its deconvolution, and are illustrated in It should be pointed out that the GnPC starting material is itself a highly exfoliated material, as its 2D spectrum is quite different from that of graphite d. Jang eThe morphology of the graphite materials and corresponding PY exfoliated materials is illustrated in The Raman spectroscopy studies reported above demonstrated that the application of energy to graphite in a PY aqueous solution produces a GnP stable suspension with more that 70% of the suspended GnP flakes exfoliated into FLG. The question arises now concerning the nature of the interactions between PY and the FLG surface. In particular, how PY is adsorbed at the FLG surface, how many PY molecules can be accommodated on a given area, and if they form a monolayer or stacks of several adlayers? Also, does PY tend to stack and form organized structures itself? In order to answer these questions, we used X-ray diffraction (XRD) and molecular modeling studies. X-ray diffraction studies were performed on a set of materials produced from FLG/PY suspensions by water evaporation; molecular modeling studies were performed that helped understanding the nature of the graphene/PY interactions.A GnPH5 PY suspension was prepared as described in the experimental section, and the GnPH5 PY powders were obtained by water evaporation from the bulk suspension in an oven at 90 °C. A PY solution with the same concentration used for exfoliation was prepared, and the PY powder was collected by solvent evaporation. The XRD profiles presented in The PY in powder form, which was obtained after dissolution in water and drying, does not display any peaks in the XRD difractogram, showing that this pyrene derivative does not form organized structures upon solvent evaporation. Thus, the results obtained indicate that when FLG and PY molecules are present in solution, an ordered structure is formed additionally to that observed for the original GnPH5. If these structures originate on PY organization/stacking, then they must be formed from several PY adlayers in order to produce a 3D crystal, and grow from the FLG surface.The same GnPH5 PY suspension was used to prepare a sample by spray drying that was also analyzed by XRD. The deposition/solvent drying method may strongly influence the molecular organization in the powder formed. Here, the additional XRD peaks observed on GnPH5 PY from slow drying, which are absent on the GnPH5 PY obtained by fast drying, indicate that PY stacking occurred from solution only under slow-drying conditions.A graphene hexagonal flake possessing a closed-shell electronic ground state, 546 carbon atoms, and 5-nm radii was chosen as the substrate. The use of flakes, as opposed to periodic systems, allows the explicit study of edge effects, the possibility of side switching, and the possibility of solvated systems. The adsorption of a variable number of the functionalized pyrene molecules, namely 1, 2, 4, 6, 7, 8, 9, 10, 11, and 12, was computed. For this, we performed molecular dynamics (MD) simulations using the molecular mechanics MMFF94 force field as implemented in the software Tinker. For simplicity and computational efficiency, the graphene substrate was held fixed during the classical simulations. A simulated annealing technique with initial and final temperatures of 500 K and 300 K respectively was chosen in order to improve the sampling of the potential energy surface during the simulations. Simulations were run for 20 ns with a RESPA integrator that allowed the use of a time step of 2 fs. Snapshots of the trajectory were selected at 2 ps intervals, yielding 10,000 different adsorption geometries that were optimized with the MMFF94 force field. Two representative geometries for each system were selected; geometries with the lowest total energy and geometries with the lowest intermolecular energy and optimized at the PM6-D3H4 level with the program MOPAC16 in vacuum, giving comparable results . The relative position of the adsorbate with respect to the graphene flake was monitored through the MD simulations by following the Cartesian coordinates of the nitrogen atom of PY, as nitrogen atoms with positions up to 5 Å from the flake plane were found to correspond to PY molecules directly adsorbed onto the surface. The typical number of adsorbed neutral pyrene molecules during the MD simulations was found to be eight. The maximum number of molecules that this flake can accommodate in one adlayer was found to be 10, but this was seldom found during the MD simulations. The geometries for the lowest energy minima found at the MMFF94 level were optimized at the PM6-D3H4 level (not shown) and at the GFN-xTB level methods for PY in its neutral (carboxylic) and anionic deprotonated (carboxylate) states; these are presented in For neutral PY in vacuum, the binding energy changes considerably, from −38 kcal/mol for 1 adsorbed PY to ~50 kcal/mol for 4 PY and 8 PY adsorbed molecules, respectively. This is due to the attractive interaction between PY molecules, which peaks for 4–8 PY molecules. When the carboxylate form of PY is considered, the binding energy for one molecule, −39 kcal/mol, is similar to the binding energy for neutral PY. However, in this case, it does not change with the number of adsorbed molecules, as in negatively charged PY adsorbates and the electrostatic repulsion counterbalances the hydrogen bonding and dispersion attractions. The differences between the neutral and the anionic form of PY are clear when the graphene flake is saturated with 10 PY molecules. In the neutral form, the lowest energy structure accommodates 10 PY molecules in more than one adlayer, while in the anionic form, the opposite is observed, i.e., one adlayer is more stable, as electrostatic repulsion inhibits the deposition of more PY adlayers.The optimized geometries at the GFN-xTB level for four, eight, and 10 deprotonated PY molecules in water are presented in The experimental TGA weight loss (WL) results have been compared with the estimate calculated considering the mass of PY in solution and the exfoliation yield of carbon in suspension (from UV-visible spectroscopy); these are presented in The calculations showed that the experimental WL is close to the estimated WL for all of the materials, considering that all of the pyrene derivative remains in solution. During solvent evaporation, the PY molecules may deposit randomly or in a regular manner, with the latter leading to the formation of stacked PY adlayers at the surface of the exfoliated material ,25. ThesA pyrene derivative was synthetized through a simple and low-cost functionalization methodology that leads to a high yield of the final compound, and can potentially be scaled up for large-scale production. The solubility of this pyrene derivative in water allowed its use for the exfoliation and stabilization of different types of graphite in aqueous media. The statistical Raman spectroscopic analysis showed the formation of bilayer and few-layer graphene after the exfoliation process of all of the types of graphite, and the STEM images showed the formation of thin flakes, in agreement with Raman spectroscopy. The XRD results showed the d-spacing of the larger FLG materials, similar to the graphite, and the molecular modeling demonstrated the maximum number of PY molecules that can be adsorbed to the surface of graphene. The estimated weight loss in the TGA analysis is similar to the experimental values, suggesting an excess of PY molecules adsorbed on the exfoliated material in more than one adlayer. The very low concentration of pyrene derivatives used in this work  to exfoliate graphite in water opens up the possibility of an efficient and cost-effective process to produce FLG. The application in biomedical areas requires further studies on the toxicity and biocompatibility of this new pyrene derivative. The FLG may be deposited directly from the aqueous suspension using techniques such as spray or spin-coating, or in the form of composites with water-soluble polymers or stable aqueous suspensions of polymers. The FLG may be collected from the aqueous suspensions by solvent evaporation and used in powder form in the production of polymer composites by melt-mixing methods.


Herein, hybrid k-carrageenan bio-nanocomposite films were fabricated by using two types of nanofillers, organically modified montmorillonite (OMMT), and cellulose nanocrystals (CNCs). Hybrid bio-nanocomposite films were made by casting techniques employing 4 wt% of CNCs, OMMT, and hybridized CNCs/OMMT in a 1:1 ratio. The rheological and morphological properties and thermal stability of all composites were investigated using rotational rheometry, thermogravimetry analysis, differential scanning calorimetry, field emission scanning electron microscopy, and transmission electron microscopy (TEM). The results showed that the hybrid CNC/OMMT bio-nanocomposite exhibited significantly improved properties as compared to those for the bio-nanocomposites with single fillers due to the nanosize and homogenous nanofiller dispersion in the matrix. Rheological analysis of the hybrid bio-nanocomposite showed higher dynamic shear storage modulus and complex viscosity values when compared to those for the bio-nanocomposite with individual fillers. The TEM analysis of the hybridized CNC/OMMT bio-nanocomposite revealed that more particles were packed together in the CNC network, which restricted the matrix mobility. The heat resistance and thermal stability bio-nanocomposite k-carrageenan film enhanced rapidly with the addition of hybridized CNCs/OMMT to 275 °C. The hybridized CNCs/OMMT exhibited synergistic effects due to the good affinity through interfacial interactions, resulting in the improvement of the material properties. Eucheuma cottonii) and consisting of repeating 4-linked 3,6-anhydro-D galactose and 4-linked β-D-galactose-4-sulphate units with anionic sulfate groups (OSO3−) disaccharide units [Biopolymers formulated with reinforcement materials such as natural fibers, nanoparticles, and synthetic fibers are currently being developed because of their unique properties to reduce the usage of synthetic plastics, particularly for short-term packaging and disposable materials ,2. K-carde units ,6. The ide units ,8,9. BioNanocellulose that can be extracted from abundant natural fiber exhibits properties such as relatively large surface areas, low density, high specific stiffness, high strength, and easy processability ,11,12. CNanocomposites based on polysaccharide and nanoclay reinforcements are widely used across various research fields as clay minerals are cheaper, and biodegradable nano additives used to enhance the properties of polymers materials are known as polymer–clay nanocomposites ,17,18,19In prior studies, k-carrageenan bio-nanocomposite materials were studied with a focus on the effects of the CNC and OMMT on the mechanical properties to determine the optimum nanoparticle loadings. Morphological studies show the well distribution of CNC and OMMT nanoparticles in the matrix. Hybridized CNC/OMMT nanofillers exhibit synergistic effects resulting in excellent mechanical properties, good water resistance, with decreasing absorption of water of the bio-nanocomposite in contrast to the bio-nanocomposites with single fillers . PreviouThe ultimate aims of this current work are to show how the hybridization of different organic and inorganic nanofillers affect the rheological behavior and thermal stability properties, and to extensively investigate the entanglement of the nanoparticles inside the matrix for applications depending on the location distribution of the nanofillers. Rheological properties of the materials are important during polymerization as well as downstream processing of the product . The measurement of the viscosity of the bio-nanocomposite can provide direct evidence for changes in the product quality due to the destabilization in a polymer .®; Hoffman Estates, IL, USA). Sulfuric acid (98%), Glycerol (99.5%), glacial acetic acid (99.5%), sodium chlorite (80%), and sodium hydroxide (99%), were obtained from SYSTERM-chemAR  and Sigma-Aldrich . Membrane tubes  were used for dialysis. Kenaf fiber was purchased from Kenaf Fiber Malaysia Sdn. Bhd.  and k-carrageenan was provided by TACARA Sdn. Bhd. . Methyl dihydroxyethyl hydrogenated tallow ammonium (OMMT 25–30 wt%) was purchased from AMCOL International Corp. , k-carrageenan–organically modified montmorillonite (k-carr–OMMT), and hybrid k-carrageenan–cellulose nanocrystal/organically modified montmorillonite (k-carr–CNCs/OMMT) bio-composite films was accomplished by the solution casting technique. K-carrageenan was first mixed with the 4% (w/w) nanofillers in distilled water. The suspension was heated in the range of 70–90 °C. The suspension was continuously stirred for another 30 min after the heating was removed. The thickness of the bio-nanocomposite film was controlled using a casting knife before being dried at room temperature to obtain the final film thickness in the range of 30–50 µm.The morphological observation of the fibers after each treatment was examined via field-emission scanning electron microscopy (FESEM) at a magnification of 1.00 K× and 15.00 K× . The morphology of the CNCs particles and bio-nanocomposite film in the water medium was analyzed by using transmission electron microscope , as described by Zakuwan and Ahmad .The rheological behavior of the polymers was carried out using a dynamic rotational rheometer  as described by Noranizan et al. . The sto−1. The thermal stability and degradation studies of the bio-nanocomposite films were performed using a thermogravimetric analyzer  under a nitrogen atmosphere. Each sample was heated from 25 °C to 600 °C at 10 °C·min−1. Thermal stability characteristics of the k-carrageenan bio-nanocomposite were determined using a differential scanning calorimeter . The heating processed up to 200 °C at a rate of 10 °C·minThe scanning electron microscopy (SEM) micrograph of the kenaf raw fiber after different chemical treatments are shown in The morphologies and sizes of the obtained CNCs were determined using TEM. TEM images of the CNCs produced after the acid hydrolysis in water are shown in The morphology of the hybrid CNCs/OMMT bio-nanocomposite films were analyzed using TEM. 3−) on k-carrageenan enable its interaction with the inorganic cations, with polymer chain intercalation [A rheological study of the bio-nanocomposites showed high complex viscosities and dynamic shear storage moduli for the CNCs, OMMT, and hybridized CNCs/OMMT. This study is useful for understanding the relationship between the nanofillers. calation . A decrecalation . Figure The variation in the complex viscosity values for the neat matrix and bio-nanocomposite films is shown in The thermal stability of the k-carrageenan matrix and bio-nanocomposite with the addition of CNCs, OMMT, and hybridization was investigated by using TGA. 42−) [Further degradation of the k-carrageenan matrix film occurred at approximately 228–236 °C. The addition of CNCs to the k-carrageenan matrix increased the decomposition temperature to approximately 264–278 °C at 4-wt% filler loading. The maximum degradation occurred at 270 °C. Furthermore, the addition of CNCs to k-carrageenan significantly increased the mass loss because the CNCs were less thermally stable compared to OMMT. Higher thermal decomposition with the incorporation of CNC due to high crystallinity, and had a higher aspect ratio of the CNC that was well dispersed within the matrix. This result shows that CNCs interacted with the matrix k-carrageenan and enhanced the thermal degradation of the corresponding bio-nanocomposites to a greater extent. Meanwhile, a drastically degradation rate due to the presence of negative sulfate groups (SO42−) . The add42−)  showed so, and the peak temperature, Tp, is important to determine whether a material is close enough to lose its structure during the heating process [p of 94 °C. A factor that influences the sealing strength is the hydroxyl groups in the plasticizers. Endothermic peaks for k-carr/CNCs were observed at 54–120 °C with a Tp of 94 °C. This proves that k-carr/CNCs show a higher transition temperature when compared to the neat matrix, with the lowest onset temperature and afforded better seal strength. The K-carr/OMMT bio-nanocomposite exhibited a transition temperature in the range of 54–115 °C with a Tp of 93 °C, which was lower than that of the neat matrix. Based on the TGA results, k-carr/OMMT only affords an increase in thermal stability  data  for the  process . All filtability . The DSCCellulose from kenaf and CNCs with average diameters and lengths of 12–15 nm and 101–260 nm, respectively, were obtained using alkaline treatment, bleaching, and acid hydrolysis. K-carrageenan bio-nanocomposite films reinforced with 4-wt% CNCs and OMMT were prepared via a casting technique. The hybrid bio-nanocomposite was prepared with the addition of 4-wt% CNCs/OMMT (1:1). Different types of nanofillers enhanced the thermal stabilities and rheological properties of the bio-nanocomposites. Hybrid bio-nanocomposite films showed a significant enhancement of various properties when compared with those for the k-carrageenan with CNC and k-carrageenan with OMMT bio-nanocomposite. Bio-nanocomposite films with CNCs/OMMT showed better rheological characteristics among the three formulations due to the entanglement of CNCs/OMMT inside the matrix phases, and the development of a network filler structure leading to the enhanced filler–matrix interactions. TEM data revealed that the OMMT particles were packed together in the CNC network, which restricted the matrix mobility. The hybrid bio-nanocomposite films exhibited synergistic effects with higher thermal degradation temperature when compared to the bio-nanocomposite films with single fillers. This improvement was associated with the homogeneous and well dispersed CNCs and OMMT, high aspect ratio for different dimensions, and the interfacial and ionic interactions between CNCs and k-carrageenan functional groups. It can be concluded from this study that a uniform mixing of the hybridization of CNC/OMMT with different polarities like CNCs and OMMT can increase the rheological behavior and that the thermal stability properties have the ability to make a fully biodegradable nanocomposite, and hence present a wide range of potential in packaging and coating applications.


Background: Applications of two-dimensional (2D) materials in electronic devices require the development of appropriate measuring methods for determining their typical semiconductor parameters, i.e., mobility and carrier lifetime. Among these methods, contactless techniques and mobility extraction methods based on field-effect measurements are of great importance.Results: Here we show a contactless method for determining these parameters in 2D semiconductors that is based on the photomagnetoelectric (PME) effect . We present calculated dependences of the PME magnetic moment, evoked in 2D Corbino configuration, on the magnetic field as well as on the intensity and spatial distribution of illumination. The theoretical predictions agree with the results of the contactless investigations performed on non-suspended single-layer graphene. We use the contactless PME method for determining the dependence of carrier mobility on the concentration of electrons and holes induced by a back-gate voltage.Conclusion: The presented contactless PME method, used in Corbino geometry, is complementary to the mobility extraction methods based on field-effect measurements. It can be used for determining the mobility and diffusion length of carriers in different 2D materials. The application of two-dimensional (2D) materials in electronic devices –6 requirB, perpendicular to the photogenerated carrier concentration gradient, the diffusing electrons and holes are deflected in opposite directions. Their current flows in a third, mutually perpendicular direction. The PME response decreases with the increase of recombination rate. Of course, the PME signal is stronger for higher μ. Therefore, investigations of the PME effect are used to determine carrier recombination and transport [There are a few phenomena which are or may be called PME effects. For example, in semiconductors the simultaneous action of light and magnetic field evokes specific electromotive forces . In the ransport –14 as weransport –17. The ransport –20 whichMette  proposeds) of the illuminated (front) and unilluminated (back) surfaces were previously developed in [s [s was possible only after calibrating the apparatus with standard samples of known semiconductor parameter values [The formula describing the PME magnetic moment, evoked in a bulk semiconductor wafer illuminated by weakly absorbed radiation as a function of τ, sample thickness, and surface recombination velocity  is taken into consideration. To test the theoretical predictions, we apply the induction technique in PME investigations of single-layer graphene on polyethylene terephthalate (PET) foil. The main aim of our work is to use the PME method for determining the dependence of μ on the concentration of electrons and holes induced by a back-gate voltage in graphene.00 light with photon energy greater than its optical energy gap. The transport of electrons and holes through 2D samples in the presence of a steady magnetic field under photogeneration and recombination is described by the solution of Maxwell's equations, the Poisson equation, the continuity equations and the transport equations  is the sum of azimuthal components of electron and hole current densities,  is the position vector, and r is the distance from the center of the light spot.where  as a function of parameters describing the experiment: magnetic field (B), beam radius (Rb) and maximum intensity (IV0) of the radiation incident upon a sample. These calculations were done for different values of τ and μ.Assuming an absorption coefficient of light appropriate for graphene (α = 1/137 ), the thMb = ) on the illumination intensity for different values of the beam radius. The Mb is a nonlinear function of Rb  and for μ smaller than about 1 m2V−1s−1, the  is proportional to the square of the carrier mobility  cm2V−1s−1 and sheet carrier concentration ne = 4.65(6)·1016 m−2 in the graphene were determined using the Van der Pauw method. For these measurements of resistivity and the Hall coefficient, the samples were equipped with 150 nm thick Au electrodes deposited using a Q150R ES rotary-pumped sputter coater with a film thickness monitor. For the PME-Corbino investigations of graphene with electrostatically tunable carrier density, a 150 nm thick Au film was also deposited on the back surface of the PET foil.The investigated graphene samples were supplied by Graphene Supermarket. The single-layer graphene films were grown by CVD processing on a cooper foil and transferred onto a 170 μm thick polyethylene terephthalate (PET) foil. The polycrystalline graphene films covered about 90% of the foil, with occasional holes and cracks. The presence of single-layer graphene was confirmed by Raman spectroscopy using an NTEGRA Spectra (NT-NDT) device with a wavelength of 532 nm. The carrier mobility μP = 100 mW). The laser diodes were biased with an ITC510 (Thorlabs) laser diode controller. In our experiments, the laser diode bias was sinusoidally modulated around a DC bias chosen to ensure that the waveform of the light output signal is sinusoidal with negligible distortion. The radiation intensity was changed using neutral density filters . The intensity was monitored using a Hamamatsu S3399 photodiode whose short-circuit current was measured using an EG&G 5110 lock-in amplifier. The time dependence of the illumination was determined using a Rigol Technologies DS1202CA oscilloscope. The spatial distribution of illumination  in a weak magnetic field [e = 1256(25) cm2V−1s−1 determined in the Van der Pauw measurements.nduction  and to tnduction . Such deic field . These ric field –28 and wVPME on the frequency (f) of sinusoidal modulation of the illumination presented in 2f2τ2 << 1 (see Equation S19 in The linear dependence of VPME voltage (induced in a measuring coil) and the investigated PME magnetic moment. However, the relative changes of a material parameter due to an external factor can be determined. Therefore, in this work, the PME-Corbino investigations of the dependence of carrier mobility on concentration of electrostatically tunable carrier density (N) were performed on a back-gated sample of graphene (e = 1256(25) cm2V−1s−1 and ne = 4.65(6)·1016 m−2 determined using Van der Pauw method).Unfortunately, the observed linear dependences of the PME response on experimental parameters –d have ngraphene  with knoVg, induces a sheet carrier concentration approximated by [0ε/we)Vg, where ε0 and ε are the permittivity of free space and the used dielectric, respectively; e is the electron charge; and w is the thickness of the dielectric. The authors of [It should be underlined that one of the most important properties of graphene –35 and omated by  (ε0ε/we). Therefore, the left axis in In our case, the energy position of the Fermi level of graphene was modulated by the field effect through the 170 μm thick PET foil. The relative permittivity of PET is ε = 3.5 at 71 kHz . Figure It was experimentally shown that the carrier lifetime only decreases slightly with carrier density in graphene . Taking The electron–hole asymmetry is concluded from the observed PME response in non-suspended graphene. It agrees with theoretical predictions  based onThe presented PME method is restricted to photogenerated carriers and does not account for several groups of carriers with distinct mobilities, and in particular, hot electrons. A brief review  presenteThe investigation of complex phenomena in 2D materials requires the combination of multiple experimental techniques. The contactless PME method, used in Corbino geometry, is complementary to the mobility extraction methods based on field-effect measurements. In the case of small carrier mobility and weak magnetic field, the PME magnetic flux is proportional to the fourth power of the diffusion length of carriers. The contactless PME method can be used to obtain scan maps of local values of 2D semiconductor parameters, i.e., diffusion length, mobility or lifetime of carriers, in future investigations.Possible difficulties in applying the proposed method may result from disturbances through the Corbino–Nernst effect from radial temperature gradients that are caused by heating of the center spot in the sample with the incident light beam. However, the distorting photovoltage and thermovoltage evoked due to illumination of the near-contact regions are eliminated in the proposed PME method. The existence of photogenerated hot carriers which can interact with the equilibrium free carriers must be taken into serious considerations in future experiments.File 1Theoretical description of the photomagnetoelectric effect in 2D materials in Corbino configuration.


Mutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA ) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues.We employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns.https://midb.pnb.uconn.edu.Here we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at The online version of this article (10.1186/s12864-019-6046-x) contains supplementary material, which is available to authorized users. RNU4ATAC, which encodes for U4atac snRNA, has been linked to microcephalic osteodysplastic primordial dwarfism type I (MOPD1), Roifman syndrome and Lowry-Wood syndrome [Splicing of introns is required for the expression of most eukaryotic genes. A subset of mammalian genes contains both major and minor introns, therefore requiring both the major  and the minor  spliceosome, respectively [syndrome . One wayE2f4, which is part of the E2F family that already had E2f1, E2f2, E2f3, E2f5 and E2f6 listed as MIGs in the U12DB  . HoweverNext, we investigated the terminal dinucleotides of all the identified minor introns, and found that ~ 72% of the minor introns were of the GT-AG type, whereas only ~ 24% and ~ 22% was of the AT-AC type in mouse and human, respectively Fig. d-e. The Finally, we wanted to compare the identity of MIGs between mouse and human. To this end, we curated the orthologs listed in the Ensembl and NCBI database, and found that 586 genes contained a minor intron in both mouse and human genome  in one tissue compared to all other samples to generate the UpSignature  that often serves a physiological function . HoweverCoa3. This suggests that splicing and indirectly expression of these MIGs solely relies on the minor spliceosome, yet retention of this single minor intron was tissue-specific  in the cortex and the kidney  in bone marrow, kidney and thymus by RNAseq  was often coincident with a cryptic 3′SS CAT7)  was ofteTo test whether these isoforms could also result in the production of tissue-specific proteins, we performed bioinformatics analysis to predict the fate of alternatively spliced transcripts. RNA transcripts are under tight quality control, such that aberrant splicing can result in activation of several degradation pathways. For instance, intron retention can result in nuclear retention of the transcript and subsequent degradation by the exosome . AlternaFinally, we also wanted to determine the prevalence of AS around minor introns in humans. Again, we found that cryptic splicing of minor introns (CAT6 and CAT7) was used at the highest levels in all tissues  . The inhrow Fig. d. In allSNRPE, E2F2, INSIG1), minor introns were retained at a higher level than the flanking major introns [We favor such a model, which would mean that splicing and/or retention of minor introns can behave like the splicing of major introns. This model is in contrast to the previous model of stochastic or inefficient minor intron splicing resulting in prevalent retention of minor introns. The latter ideas were based on comparison of minor intron retention levels to that of the flanking major introns. Briefly, Patel et al., employed this comparison to test the hypothesis that minor introns are excised more slowly than the upstream and/or the downstream major introns . They sh introns . Afterwa introns . However introns . While wCBP20 became progressively more efficient by increasing the length of the intron [P120 has served as a golden standard for studying the efficiency of minor intron splicing. Thus, the confluence of a naturally occurring preference for retention of shorter minor introns and the choice of minor introns that were studied for technical reasons might have led to the minor intron retention as a model for regulating all MIGs.The apparent differences in our findings and the subsequent models could be explained by the choice of MIGs that were interrogated. We found that retention of shorter minor introns was detected at a higher level than the longer introns Fig. a. This fe intron . Togethee intron . The elee intron . Since tRnpc3 resulted in leukopenia and a severely reduced thymus lacking medullary or cortical regions [Given our observation that splicing of minor introns is highly regulated, it stands to reason that it is also tissue-specific. Indeed, we found dynamic levels of minor intron retention across different mouse tissues  for the 5′SS and 3′SS of minor introns (AT-AC and GT-AG type) and major introns (GT-AG and GC-AG type) were downloaded from SpliceRack . MoreoveThe curated splice sites of all introns were then scored against the PWMs for minor introns and major introns by adding the scores of each individual element  would be considered the canonical translation. If these were not present, then any transcript with the longest CDS would be considered the canonical isoform. If there was no CDS, then the longest non-protein coding transcript was assigned as the canonical transcript.Data analyzed in this study was downloaded from the DDBJ and EMBL database. They can be accessed using the following accession numbers: Mouse WT tissues  ; Human tReads from human tissue samples were aligned to the hg38 genome (UCSC genome browser) using Hisat2 as described previously , 46. ReaP < 0.01. Using this method, 55 pairwise comparisons were performed between all mouse tissues. Afterwards, the intersect was taken of all the upregulated genes. Genes that were binned as upregulated in all pairwise comparisons, were classified as being part of that tissue’s UpSignature. Conversely, genes that were binned as downregulated in one tissue compared to all other tissues were classified as being part of that tissue’s DownSignature  and utilized for the splicing efficiency analysis in Fig. To detect AS across minor introns, we extracted all uniquely mapped spliced reads spanning the minor intron. Reads mapping with only one nucleotide on either side of the junction were excluded. The remaining spliced reads were then binned into one of 9 splicing categories Fig. a using BP-value< 0.05 for DAVID; FDR-corrected P-value< 0.05 for ToppGene; g:SCS threshold< 0.05 for g:Profiler) were isolated. Only GO-Terms that were significant according to all three functional annotation tools are reported in Fig. UpSignature lists for each tissue were submitted for functional enrichment analysis to DAVID, ToppGene and g:Profiler –16. SignFor all alternatively spliced MIGs, we determined the new exons of the canonical transcript depending on the specific AS event that was observed. If multiple AS events were observed around a minor intron, these were treated as separate events. Moreover, if the read filter was passed for an AS event, as result of multiple coordinates being employed, but no single coordinate passed the read filter separately, the event was excluded from this analysis. An example would be the use of a cryptic 5′SS in a minor intron (CAT6) which was supported by 20 reads, however this was the sum of reads all supporting different coordinates being employed as a cryptic 5′ SS. The transcripts of the remaining protein-coding genes were then translated in silico and the open reading frame was predicted. The location of the new stop codon was compared with the annotated stop codon to determine whether the AS event had resulted in a truncation or extension of the ORF. If the new stop codon was found > 50 nt upstream of an exon-exon junction, the alternatively spliced isoform was predicted to be degraded by NMD. If no stop codon was identified, then the transcript was predicted to be degraded by NSD. In all other cases, the transcript was predicted to give rise to a protein.To determine whether AS events across minor introns had been annotated, we downloaded the exon coordinates of all MIG transcripts from Ensembl (v.95). Exon coordinates of each transcript were then compared to the minor intron coordinates and transcripts were binned into one of four categories: 1) transcripts that supported constitutive splicing of the minor intron, 2) transcripts that were truncated before the minor intron coordinates, 3) transcripts with their first/last exon within the minor intron coordinates, and 4) transcripts with AS across the minor intron.To determine whether the median length of retained vs. spliced minor introns were statistically different, we performed a Kruskal-Wallis rank sum test, followed by post-hoc Dunn test. A one-way ANOVA, followed by post-hoc Tukey test was employed for determining dynamic levels of retention for individual minor introns across tissues. To determine whether minor introns were retained at significantly higher levels than their flanking major introns, we also performed one-way ANOVA, followed by post-hoc Tukey test. Finally, to determine whether there was a significant difference in the median length of alternatively spliced vs. canonically spliced minor introns, we performed a Mann Whitney U-test.N = 3) were obtained from The Jackson Laboratory and anesthetized using isoflurane, and perfused using 0.125 M NaCl as described previously [Eight week old C57/Bl6J male mice (eviously . All mouN = 3). Cortex, heart, kidney, liver, lung, and testis were homogenized in 1 mL TRIzol (#15596018); skin, spleen, thymus and tibialis anterior  were homogenized in 500 µL TRIzol. RNA was extracted using the DirectZOL RNA Miniprep kit , per the manufacturer’s instructions. 1μg of total RNA was then used for cDNA synthesis, as described previously [Ten tissues were dissected from 8-week-old C57/Bl6J perfused male mice  analyses. For AS analysis in pan tissue, cDNA from three replicates was pooled. MSI values were determined by quantification of band intensity by ImageJ.Additional file 1:Figure S1.SignatureCalc pipeline. Schematic describing the pipeline to determine the UpSignature and DownSignature for bone. This pipeline was then run to determine these signatures for the other ten mouse tissues. NonDE = non-differentially expressed. (TIF 599 kb)Additional file 2:Figure S2. Functional enrichment of DownSignatures. (A) Dendogram showing the hierarchical clustering of mouse tissues based on overall gene expression. (B) Venn diagram showing the number of uniquely downregulated  MIGs in each tissue. (TIF 3875 kb)Additional file 3:Figure S3. Bioinformatics strategy for detection of retention and AS of minor introns. (A) Schematic describing the pipeline to detect minor intron retention, including filtering criteria. (B) Schematic describing the pipeline to detect novel AS events across minor introns, including filtering criteria. Adopted from Madan et al., 2015 [l., 2015 . See alsAdditional file 4:Figure S4. Minor intron retention is not dependent on consensus sequence. Frequency plots of the annotated 5′SS, BPS and 3′SS of minor introns that are retained in 3 (top), 1 or 2 (middle), or 0 (bottom) replicates of at least one tissue. (TIF 5672 kb)Additional file 5:Figure S5. Minor intron retention is tissue-specific in human tissues. (A) Piechart with number of minor introns that show retention in number of replicates of at least one tissue. Boxplots reflect the 5th–95th percentile of minor intron length in each of the three categories. Significance was determined by Kruskal-Wallis rank sum test, followed by post-hoc multiple comparison using Dunn method. ** = P < 0.001; n.s. = not significant. (B) Histogram of the number of tissues in which minor introns were retained. (C) Venn diagram showing the overlap of retained minor introns across eight human tissues. Only minor introns that passed filtering criteria in at least three replicates of a tissue were included. (D) Stacked bargraph showing the number of retained minor introns in each tissue in mouse (blue), human (red), or both (grey). (TIF 10985 kb)Additional file 6:Figure S6. Location of minor introns within canonical transcript. Piechart showing the percentage of minor introns that have flanking major introns. (TIF 2247 kb)Additional file 7:Figure S7. Alternative splicing across minor introns is annotated in the Ensembl database. (A) Bar graph showing the number of annotated isoforms in the Ensembl database which resulted from AS across the minor intron. (B) Venn diagram showing the combined usage of AS events in annotated isoforms that are alternatively spliced across the minor introns. (C) Boxplots reflect the 5th–95th percentile of minor intron length in minor introns that are and are not alternatively spliced. Significance was determined by Mann Whitney U test. ** = P < 0.01. Transcr. = transcription. (TIF 11148 kb)Additional file 8:Figure S8. Alternative splicing across minor introns in human tissues is dynamic. (A) Bubbleplot reflecting AS usage across minor introns in 8 human tissues. Size of the circle represents the number of introns that passed the filtering criteria, the colour represents the type of AS. (B) Venn diagram revealing the overlap of MIGs that are alternatively spliced in at least one tissue between mouse and human. MSI = mis-splicing index. (TIF 6629 kb)Additional file 9:Table S1. Minor intron coordinates. Contains the coordinates of the identified minor introns in mouse and human, as reported on MIDB. (XLSX 85 kb)Additional file 10:Table S2. MIG orthologs. Contains information on MIG orthologs between mouse and human. (XLSX 81 kb)Additional file 11:Table S3. MIG expression. Contains the expression of MIGs across mouse and human tissues in TPM. (XLSX 208 kb)Additional file 12:Table S4. Functional annotation. Contains the functional annotations enriched for by the UpSignature and DownSignature of mouse tissues. (XLSX 15 kb)Additional file 13:Table S5. Minor intron retention. Contains the MSI values of retained minor introns across tissues. (XLSX 23 kb)Additional file 14:Table S6. Flanking major intron retention. Contains the MSI values of retained upstream and downstream major introns flanking the minor intron. (XLSX 53 kb)Additional file 15:Table S7. Alternative splicing around minor introns. Contains the MSI values of alternatively spliced minor introns in mouse and human. (XLSX 58 kb)Additional file 16:Table S8. RNAseq sample information. Contains information on sequencing depth and mapping percentage of all RNAseq samples used. (XLSX 13 kb)