The signaling of brain-derived neurotrophic factor (BDNF) has been suggested to be impaired in Alzheimer’s disease (AD), which may compromise the function of BDNF upon neuronal activity and survival. Accordingly, decreased levels of BDNF and its tropomyosin-receptor kinase B-full-length (TrkB-FL) have been detected in human brain samples of AD patients. We have previously found that neuronal exposure to amyloid-β (Aβ) peptide, a hallmark of AD, leads to calpain overactivation and subsequent TrkB-FL cleavage leading to decreased levels of TrkB-FL and the generation of two new fragments: a membrane-bound truncated receptor (TrkB-T′) and an intracellular fragment (TrkB-ICD). Importantly, we identified this TrkB-FL cleavage and TrkB-ICD presence in human brain samples, which indicates that this molecular mechanism contributes to the loss of BDNF signaling in humans. The exact role of this TrkB-ICD fragment is, however, unknown. Here, we used a human neuroglioma cell line and rat cortical primary neuronal cultures to track TrkB-ICD intracellularly. Our data show that TrkB-ICD is a relatively stable fragment that accumulates in the nucleus over time, through a phosphorylation-dependent process. We also found that TrkB-ICD has tyrosine kinase activity, inducing the phosphorylation of nuclear and axonal proteins. These findings suggest that TrkB-ICD may lead to a dysregulation of the activity of several proteins, including proteins in the nucleus, to where TrkB-ICD migrates. Since TrkB-ICD is formed by Aβ peptide-induced cleavage of TrkB-FL, the present data highlights a new mechanism that may have a role in AD pathophysiology. Alzheimer’s disease (AD) is a slow progressing neurodegenerative disease, leading to atrophy and neuronal loss of particular brain regions, in particular the hippocampus, therefore leading to cognitive impairments . After 16 h of transfection with TrkB-ICD vector, H4 cells were treated with cycloheximide , an inhibitor of protein biosynthesis, for 8 h and 24 h. TrkB-ICD levels were quantified at 0 h, 8 h and 24 h after CHX treatment; a time-dependent gradual decrease of TrkB-ICD expression levels was detected (p < 0.0001) towards near 50% of the value at time 0 , which codifies the nuclear import of proteins  revealed the presence of two bipartite NLS. In this algorithm, scores of 9/10 codify proteins exclusively present in nucleus, whereas scores of 1/2 indicate proteins only present in cytoplasm. In our case, both predicted sequences are characteristic of proteins that could be localized either in nucleus or in cytoplasm  that specifically detects phosphotyrosine-containing proteins and evaluated, through western-blotting and immunofluorescence assays, phosphotyrosine immunoreactivity of 24 h-transfected primary neurons. As shown in Considering that TrkB-ICD contains a TrkB-FL tyrosine kinase domain and also that phosphorylation is a central step for many biologic processes, we evaluated whether this fragment could We then evaluated if TrkB-ICD nuclear translocation was dependent on its kinase activity. To do so, immunofluorescence assays were performed using 24 h-transfected primary neurons incubated with K252a (200 nM), which inhibits Trk-FL kinase activity . These data indicate that PKA and PKC belong to the signaling cascade operated by TrkB-ICD that lead to protein phosphorylation  for 2 h, 8 h and 24 h, the overall levels of protein phosphorylation markedly decreased, being already nearly absent after 2 h of incubation with the tyrosine kinase inhibitor  is a relatively stable protein; (II) phosphorylates several proteins; and (III) accumulates in the nucleus .Previously, we described that TrkB-FL could be cleaved by Aβ peptide accumulation , having therefore a protective role. Although we do not exclude that hypothesis, it is unlikely that TrkB-ICD could have this putative protective role, since TrkB-ICD only has the anchorage site for PLCγ and we do not detect (results not shown) any activation of the three pathways. Furthermore, these mediators of TrkB-FL signaling are in close vicinity to the cytoplasmic membrane and our data suggest that TrkB-ICD is mainly located on nucleus.Calpain-mediated cleavage of several proteins, namely of p35, β-catenin or mGluR1α proteins, leads to the formation of intracellular fragments known to be involved in neuronal death, hyperphosphorylation of Tau protein, excitotoxicity, gene transcription modifications  and by the Ethical Comission of Centro Hospitalar de Lisboa Norte and Centro Académico de Medicina de Lisboa. In addition, this study was carried out in accordance with the recommendations of “Directive 2010/63/EU”.orcid.org/0000-0001-7915-3517) performed the experiments, analyzed the data and wrote the manuscript. AJ-S (orcid.org/0000-0002-9599-6597) performed the experiments and reviewed the manuscript. AL (orcid.org/0000-0003-4163-0044) and PC (orcid.org/0000-0002-1090-4631) collaborated in the experimental work. EC (orcid.org/0000-0002-1402-2791) and AS (orcid.org/0000-0001-9030-6115) discussed and reviewed the manuscript. MD (orcid.org/0000-0001-5486-6246) designed the experimental work, discussed the data and reviewed the manuscript.JF-G (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TM, was purified obtaining PVAm films with two degrees of purification: Low Grade (PVAm-LG) and High Grade (PVAm-HG). These two-grade purified PVAm were loaded with 3 wt% of graphene and graphene oxide to improve mechanical stability: indeed, pristine tested materials proved to be brittle when dry, while highly susceptible to swelling in humid conditions. Purification performances were assessed through FTIR-ATR spectroscopy, DSC and TGA analysis, which were carried out to characterize the pristine polymer and its nanocomposites. In addition, the membranes′ fracture surfaces were observed through SEM analysis to evaluate the degree of dispersion. Water sorption and gas permeation tests were performed at 35 °C at different relative humidity (RH), ranging from 50% to 95%. Overall, composite membranes showed improved mechanical stability at high humidity, and higher glass transition temperature (Tg) with respect to neat PVAm. Ideal CO2/N2 selectivity up to 80 was measured, paired with a CO2 permeability of 70 Barrer. The membranes’ increased mechanical stability against swelling, even at high RH, without the need of any crosslinking, represents an interesting result in view of possible further development of new types of facilitated transport composite membranes.In the present study, the separation performance of new self-standing polyvinylamine (PVAm) membranes loaded with few-layer graphene (G) and graphene oxide (GO) was evaluated, in view of their use in carbon capture applications. PVAm, provided by BASF as commercial product named Lupamin T. T−1 ref−1, which is quite reasonably associated to the neutralization of amine group from the R–NH3+ to the R–NH2 form, as also confirmed by the results obtained by Annenkov et al. [Concerning the polymer peaks, then, an interesting change in the different spectra is the shift of the peak related to N–H stretching before and after the resin treatment (PVAm-HG). This one moves from 3100 to 3300 cmv et al. . This neThermal and thermo-oxidative stability of Lupamin 9095 and purified PVAm was assessed through TGA analysis . The oveUntil 180 °C, TGA curves display weight loss due to absorbed water. The polymer degradation occurs in two main steps with onset temperature at 190–220 and 305–320 °C with only very minor differences among different materials, as visible in Sample weight is stable in the range 500–600 °C, indicating the end of the thermal degradation. Organic residues were oxidized by switching in air atmosphere at 600 °C and keeping the sample at that temperature for 30 min. After this treatment, purified films showed a significantly lower residue with respect to untreated Lupamin 9095. In particular, PVAm-LG and PVAm-HG showed residues in the order of 15% and 8% of the initial weight, respectively, confirming the better purification obtained when the ion exchange step was used after ethanol washing.No major modifications of the TGA curves of the polymer were observed upon graphene or graphene oxide addition; these nanofiller, therefore, did not seem to affect the thermo-oxydative stability of the matrix.g) of the films, as shown by DSC analyses reported in Interestingly, the degree of purification and nanofiller loading affected the glass transition temperature , which seem to act as a nanometric filler able to efficiently rigidify and reinforce the matrix through polar interactions with the polymeric chains. This hypothesis can explain the higher Tg in High Grade films (+20 °C), as in this case no rigid inclusions are present in the highly purified polymer, thus the nanofiller can properly act as reinforcement, hindering macromolecular motion.On the other hand, 3% in weight addition of both G and GO to PVAm-HG determined a sharp increase of TThrough SEM analysis, the fractured surfaces of produced films were examined to investigate the dispersion and the interaction of G and GO with the polymeric matrix.As shown in Differences in smoothness between the fracture surfaces may be considered as further proof of the achieved purification: the most purified materials presented fewer salt impurities on the surface.In the chart, it can be seen how Lupamin 9095, the commercial form, can absorb the largest amount of water, reaching 90 wt% of water mass gained at 60% water activity, as also shown in a previous work . It is fThe addition of a graphene-based phase presents different effects on the water sorption of the different materials. Indeed, the reduction of water uptake upon filler loading is negligible for PVAm-LG composites, while it results significantly higher for PVAm-HG based materials. In the latter case, even at relatively small amounts, the graphene-based nanofillers significantly decreases the water uptake of PVAm-HG based composites with respect to that of the non-loaded corresponding polymer. For example, pure PVAm-HG presents an uptake of 21 wt% at around 60% activity, while the addition of 3 wt% graphene and 3 wt% graphene oxide lower that value to, respectively, 15.1 and 7.8 wt%. This decrement can be interpreted as a positive interaction between the matrix and the filler, which is capable of stiffening the overall material structure, preventing an excessive swelling at high relative humidity. This result is more evident in the case of GO rather than G, likely due to the larger aspect ratio of the sheets and to the possible presence of electrostatic interaction among the carboxylic groups of GO and the primary amine groups of PVAm.Concerning the PVAm-LG + 3% GO behavior, it can be noted instead that it is in accordance with that discussed above regarding the materials glass transition temperature variation: the limited effect of the filler seems indeed to be related to the presence of finer dispersion of sodium formate crystals, which somewhat interact with the GO decreasing its ability to impact the properties of the polymeric matrix, and also to the cationic nature of low-grade samples, which impairs the possible interactions among the amine groups of the polymer and the GO’s carboxylic acid groups.2/N2 selectivity are also reported, when available. As quite common for hydrophilic membranes, permeability increased exponentially with the degree of humidification [The single gas permeation results for carbon dioxide for the different membranes tested are presented in fication  due to t2; in the case of nitrogen tests, no reliable data could be obtained, likely due to the longer experimental time required, which resulted in an excessive stress on the film. On the other hand, all films prepared using a graphene-based filler resulted mechanically stable and allowed extensive tests to be performed without any loss in permselective properties for several days. Apart from the general behavior and considering the different materials, as a first observation, it can be noted that for PVAm-HG a single point at intermediate humidity was obtained. This is due to the fact that PVAm in its pure form appeared to be quite unstable as a self-standing film, especially when purified. This led to the impossibility of running tests at higher humidity, due to the rupture of the film, verified on several specimens. For the case of PVAm-LG, the presence of residual sodium formate appeared to slightly increase the mechanical stability, allowing a full permeation curve to be acquired, at least for CO2 varies from 16.5 Barrer at 63% RH, up to 73.8 Barrer at 93% RH. For the same polymer, filled with 3 wt% of graphene oxide, values range from 1.7 Barrer at 53% RH to 71.0 Barrer at high humidity. PVAm-HG, loaded with the same quantity of graphene oxide, presented instead a permeability for carbon dioxide of 1.6 and 25.1 Barrer at 75% and 93% RH, respectively. When loaded with few-layer graphene, the same matrix presented values from 2.0 Barrer at 77% RH to 23.1 Barrer at 92% RH. From the experimental data, it can be seen how PVAm-HG presents a permeability of 4.2 Barrer at 56% RH, but no particular trend can be obviously inferred. For PVAm-LG, permeability of COThe non-loaded materials therefore present a higher permeability at the same relative humidity with respect to their counterparts containing a graphene-based nanophase. This is a somewhat expected result, since the platelets of graphene, due to their high aspect ratio, can easily increase the tortuosity of the diffusion pathway of the gases in the matrix. This effect appeared to be less pronounced in the case of PVAm-LG, possibly due to the polar interaction between graphene oxide and salt nanocrystal in these materials as already discussed by considering DSC and water sorption experimental results.2 permeability, as also shown by previous works [Regarding PVAm-HG, it is quite interesting to notice how no particular differences in gas permeability can be discerned between the materials obtained using two different fillers. This could be contradictory considering that GO usually presents a higher aspect ratio than few-layers graphene and that PVAm-HG + GO showed a lower water uptake than PVAm-HG + G. Usually, indeed, when no other factors are in play, a lower concentration of humidity in the matrix resulted in a lower COus works ,37,39,402 solubilization and facilitated transport execution, as previously shown in Equations (1)–(5) and As previously mentioned, nitrogen permeation tests were only possible on the nanocomposite membranes, due to their enhanced stability. For this reason, the ideal selectivity could be calculated only for these materials, using an exponential interpolation to report data from different gases at the same RH values. In the case of PVAm-LG+GO in particular, selectivity was shown to vary from 3.1 to 59.2 when humidity was raised from 60% to 95%. For PVAm-HG, instead, this value ranges from 3.0 at 75% RH to 80.7 at 95% RH for the graphene oxide sample and from 1.1 at 82% RH to 45.2 at 92% RH for the graphene one.2 permeability (≈70 Barrer), with lower CO2/N2 selectivity (≈60) compared to the high-grade one, which has higher selectivity (≈80) and lower permeability (≈35 Barrer). These differences are probably due to the swelling difference of the two composite materials, as already discussed, and shows a PVAm-LG + 3% GO more hydrophilic than the PVAm-HG-based composite. These results fit also with the chemical structure differences between the two materials: PVAm-HG has an almost totally neutral charge upon the polymeric chain, leading to a tighter packing of the chain itself, which cause the overall lower permeability, while the PVAm-LG material has much higher ionic repulsion [2/N2 selectivity.Analyzing PVAm-LG and -HG reinforced with graphene oxide, therefore, it resulted that the low-grade polymer has higher maximum COepulsion . Neverth2/N2 Robeson’s upper bound [Despite these differences, the two materials show a very similar behavior in terms of overall permselective performances as they lie very close, but are not able to overcome the COer bound , even atFrom this point of view, therefore, contrary to what expected, the further purification step seems not to give substantial advantages in terms of membrane performance. 2 permeabilities in the order of 50 Barrer and CO2/N2 selectivity lower than 10 [, as also reported in Among the many papers found in the literature about PVAm based system, it is very difficult to find references for a consistent comparison of the present results in order to give a more general picture of the overall potential of such membranes. Available data on FTM are usually measured at higher temperatures and/or different upstream pressures, and often refer to very complex systems also including mobile carriers. Among others, Zhao and coworkers, studying PVAm-based mixed-matrix membranes, tested PVAm thin films at 22 °C and 100% RH and reported CO than 10 . On the  than 10 , at 20%  than 10 , as also, however, is accompanied by a remarkable improvement of mechanical strength of the material: pristine PVAm sample indeed cannot be tested as pure polymer self-standing membrane and also literature data [This reduction of the separation performance with respect to the supported pure polymer from Kim et al. , howeverure data  were obt2 capture and purification. Graphene-based PVAm composite membranes were fabricated and tested to assess their potential in the field of COThe materials were produced by mixing polyvinylamine (PVAm) at different degrees of purification with few-layer graphene and graphene oxide, in an attempt to increase the mechanical stability of the water-soluble polymer without excessively reducing its transport properties.2 and N2 permeability at different relative humidity.Commercial PVAm, Lupamin 9095, was purified via a multistep procedure to obtain two grades of pure materials, PVAm-LG and PVAm-HG, which have, respectively, low and high purification grade. These samples were then loaded with 3 wt% of few-layer graphene or graphene oxide and were characterized through FTIR, SEM, TGA, and DSC techniques as well as by measuring water vapor sorption and COThe results obtained confirm the sodium formate removal during the purification steps and also the neutralization of amine groups on the polymeric chain in the purest product (PVAm-HG), which should improve the facilitated transport performance of the membrane. In addition, PVAm-HG also seems to be able to better disperse the nanofiller with respect to PVAm-LG; both DSC and water sorption results were greatly affected by the addition of the nanofiller in the highly purified polymer while remained substantially unchanged in the partially purified material. SEM images in this concern showed in the PVAm-LG materials the presence of sodium formate nanocrystal, which may interact with the polar graphenic nanofiller affecting its dispersion in the matrix. 2 permeability of almost 70 Barrer (PVAm-LG + 3% GO) and maximum CO2/N2 selectivity of about 81 (PVAm-HG + 3% GO). In general, both permeability and selectivity increased with the increasing of relative humidity and water content in the polymer. Moreover, PVAm-HG resulted to be more selective and less permeable than PVAm-LG-based materials and, among different nanofiller GO, resulted to give similar permeability but slightly higher selectivity with respect to graphene. Permeation tests, conducted at 35 °C, gave some interesting results at high relative humidity (95% RH), with a maximum COTherefore, the further purification step did not seem to remarkably modify the materials separation performances, which in any case resulted to be below the Robeson’s upper bound, for all the materials considered. This is likely because of the barrier effect performed by the high quantity of filler added to the system. Graphene-based nano-sheets, however, allowed achieving complete permeation tests on self-standing membranes, which was not possible in polymer without filler, due to film rupture at high relative humidity. In this way, graphene addition has confirmed the ability to act as a reinforcer for this type of polymers containing in side-chain amino groups, thus presenting some potential as an additive for FTM membranes applications. Further research, however, has to be performed to prevent excessive reduction of the pristine PVAm separation performances upon nanofiller addition.


The electronic band structure of bilayer graphene is studied systematically in the presence of substitutional B and/or N doping, using density functional theory with van der Waals correction. We show that introduction of B-N pairs into bilayer graphene can be used to create a substantial band gap, stable against thermal fluctuations at room temperature, but otherwise leaves the electronic band structure in the vicinity of the Fermi energy largely unaffected. Introduction of B-N pairs into B and/or N doped bilayer graphene likewise hardly modifies the band dispersions. In semiconducting systems (same amount of B and N dopants), however, the size of the band gap is effectively tuned in the presence of B-N pairs. Substitutional doping with foreign atoms is one of the main approaches to intrinsically alter the electronic properties5. In particular, graphene is often doped with B6 and N7, as these atoms have radii  similar to that of C (67 pm) and the planar geometry therefore is preserved9. Gradual transition from n-type to p-type character in contact with air can be neutralized by intentional overdoping15. Bilayer graphene early has been proposed as viable alternative to the monolayer16. In the case of AA stacking the material consists of two graphene layers located directly on top of each other, whereas in the case of AB stacking half of the C atoms from one layer sits on top of C atoms of the other layer and the other half sits on top of hollow sites. Both stackings have been observed experimentally18, AB stacking being 5 meV per C atom lower in energy than AA stacking19 and nowadays can be grown in high quality and with large area20. It has been demonstrated that both B and N doping requires less energy than in the monolayer21.Absence of a band gap limits applications of graphene in electronics, calling for band gap engineering22 both for the top and hollow sites (which refers here and in the following to the position of the dopant atom relative to the other layer) and similar electronic band structures have been found for the two sites in each case. B and N favor hollow sites over top sites by 11 meV and 16 meV, respectively21. In AB stacked bilayer graphene with one N atom in each layer the total energy declines significantly for increasing distance between the two dopant atoms, while for corresponding doping with B atoms hardly any dependence of the total energy on the distance is found23. Codoping of B and N in graphene (monolayer) is energetically favorable in the form of B-N pairs in ortho position, for example, by 20 meV as compared to next nearest neighbours24. Reference25 has studied bilayer graphene with one B atom in one layer and one N atom in the other, claiming that the electronic properties depend on the occupied sublattice rather than on the mutual distance. In the present work, we investigate the electronic properties of bilayer graphene in the more general case that B and N also can be codoped in the same layer. Interestingly, it turns out that the presence of such B-N pairs has only minor effects on the electronic band structure except for the position of the band edges in the case that the system is semiconducting. For this reason, our results indicate that intentional doping of B-N pairs is an effective way to control the size of the band gap.Introduction of one B or one N atom into AB stacked bilayer graphene has been studied in ref.26 and the generalized gradient approximation for the exchange-correlation potential (Perdew-Burke-Ernzerhof parameterization), taking into account a van der Waals correction with Becke-Johnson damping27. The plane wave energy cutoff is set to 500 eV. We employ a Γ-centered 2 × 2 × 1 k-mesh for the structure optimization and a Γ-centered 8 × 8 × 1 k-mesh for evaluating the electronic properties. Both for AA and AB stacking, a 5 × 5 supercell of bilayer graphene is constructed with a total of 100 C atoms (to consider doping effects) to which a 16 Å thick vacuum layer is attached along the c-axis in order to avoid artificial interaction between periodic images. The interlayer distance after structure optimization turns out to be 3.6 Å for AA stacking and 3.4 Å for AB stacking, in agreement with the experimental observations in refs29. These values are not modified by the presence of dopant atoms for the concentrations considered in this study. We obtain for both stackings an in-plane lattice parameter of 2.46 Å and a C-C bond length of 1.42 Å. As in no case atoms develop significant magnetic moments (≤0.01 μB), we present in the following spin-degenerate band structures.Spin-polarized first-principles calculations are performed using the Vienna ab-initio simulation package30. We next introduce one B atom into one layer and one N atom into the other, representing a dopant concentration of 2%, and examine different distances between them as well as doping at both the top and hollow sites. In each case we obtain minimal energy for minimal distance between the dopant atoms. The energetically favorable configuration with AB stacking has 467 meV (~5 meV per C atom) lower energy than that with AA stacking. In the case of AB stacking, when B sits at a top site and N at a hollow site the energy is 14 meV lower than when both sit at hollow sites, 75 meV lower than when N sits at a top site and B at a hollow site, and 65 meV lower than when both sit at top sites. The electronic band structure of the minimum energy configuration, see Fig. 25. There are two buried Dirac cones at the K point, which can be attributed to the layers doped with B (unoccupied cone) and N (occupied cone), and we obtain a band gap of 298 meV.For reference, we show the electronic band structure of AB stacked bilayer graphene in Fig. 31. Concerning the position of the B-N pair relative to the other layer we find that N occupying the hollow site gives an energy advantage of 22 meV, which agrees with the finding of ref.21 that N favors the hollow site in bilayer graphene more strongly than B (by 22 meV). It turns out that the total energy is 590 meV (~6 meV per C atom) lower for AB stacking than for AA stacking. The electronic band structure of the minimum energy configuration is shown in Fig. 32), with a band gap of 70 meV due to the presence of the B-N pair. As a remark, we note that the electronic properties depend on the sites  occupied by the dopant atoms rather than on the occupied sublattice as claimed in ref.25.Turning to the situation that both the B and N atoms are located in the same layer while the other layer is undoped, we confirm that for both AA and AB stacking the formation of a B-N pair is energetically favorable, as a strong covalent bond is formedWe next study, for different relative positions, one B-N pair in one layer and one N atom in the other. It turns out that the N atom of the B-N pair still favors the hollow site, as the coordination geometry is not modified. The numbers in Fig. Keeping one layer undoped, we now add one B atom to the layer containing the B-N pair. The numbers in Fig. We finally study, for different relative positions, one N atom in one layer and one B-N-B triple in the other. The numbers in Fig. In conclusion, the effects of B and/or N doping on bilayer graphene have been studied by first-principles calculations based on density functional theory. Special attention has been given to the modifications of the electronic band structure due to the presence of B-N pairs. We have demonstrated that such B-N pairs hardly modify the band dispersions in bilayer graphene, while opening a substantial band gap . Similarly, introduction of B-N pairs into N and/or B doped bilayer graphene largely maintains the electronic band structure (for any relative position between the dopant species). Interestingly, for semiconducting systems (same amount of B and N dopants) our results show that the presence of B-N pairs shifts the band edges  towards each other, i.e., the band gap shrinks. For this reason, introduction of B-N pairs into bilayer graphene can be used to tune the size of the band gap without critically affecting the band dispersions.


Background and objectives: Idiopathic pulmonary fibrosis (IPF) has a particularly poor prognosis, and most IPF-related deaths are due to acute exacerbation (AE) of this condition. Few reports about biomarkers to predict prognosis of AE-IPF have been published since the release of the new AE-IPF criteria in 2016. The present study investigated relationships between serological markers and in-hospital mortality after the onset of AE-IPF. Methods: Demographic, serological, and imaging data from patients hospitalized at the Maebashi Red Cross Hospital  between 1 January 2013, and 31 December 2017, were retrospectively reviewed. Subjects fulfilling the diagnostic criteria for AE-IPF were divided into those who survived or died; statistical analysis of risk factors was performed using data from these two groups. Results: Diagnostic criteria for AE-IPF were fulfilled by 84 patients ), with a median age of 78 years . IPF was diagnosed before hospitalization in 50 (59.5%) patients and 38 (45.2%) died in hospital. Among the serological markers at hospitalization in the deceased group, C-reactive protein (CRP) was significantly higher than in the survivor group (p = 0.002), while total serum protein (p = 0.031), albumin (p = 0.047) and total cholesterol (p = 0.039) were significantly lower. Cox hazard analysis of factors predicting mortality, corrected for age, sex and BMI, revealed the following: CRP (hazard ratio (HR) 1.080  1.022–1.141); p = 0.006), LDH (HR 1.003 (95% CI 1.000–1.006); p = 0.037), and total cholesterol (HR 0.985 (95% CI 0.972–0.997); p = 0.018). Conclusions: Our data suggest that CRP, LDH, and total cholesterol may be biomarkers predicting mortality in patients with AE-IPF. However, only prospective controlled studies can confirm or not our observation as a generalizable one. Acute exacerbation (AE) is the cause of most IPF-associated deaths: the mortality rate among patients with IPF increases to 40–80% once AE has occurred ,2,3. Ris2/FiO2) , and bei2/FiO2) . There h2/FiO2) , and rec2/FiO2) ; howeverThere have been numerous studies investigating the relationships between imaging and physiology tests and AE-IPF; however, reports describing the relationships between serological markers and AE-IPF have addressed only KL-6 and a small number of other markers. In addition, the number of published reports relating to the most recent AE-IPF diagnostic criteria, released in 2016 , is limiThe present study was a retrospective investigation of the relationships between serological/physiological markers and the onset of AE-IPF defined by the most recent diagnostic criteria , and an The present investigation was a single-center, retrospective study. Patients who were hospitalized at Maebashi Red Cross Hospital  between 1 January 2013, and 31 December 2017, were evaluated; those who fulfilled the diagnostic criteria for AE-IPF were included in the study. All participants in this study were confirmed to have met the 2016 AE-IPF diagnostic criteria at the enrollment of this study. We diagnosed IPF with a multidisciplinary approach. At least two respiratory specialists and a radiology specialist discussed and reviewed subjects. A histologist was also consulted when histological data were obtained.The AE-IPF diagnostic criteria, as described by Collard et al. in 2016 , were inThis study was reviewed and approved by the Ethics Committee of Maebashi Red Cross Hospital on 12 July 2018 (acceptance no. 30-19).2/FiO2 and alveolar-arterial oxygen difference (AaDO2) were calculated from arterial blood gas analysis results. In addition, the duration of hospitalization, and whether death occurred while in hospital, were obtained from the medical records.The following data were obtained from medical records: demographic information ; sociological information ; IPF treatment before admission; treatment in hospital; serological test results in hospitalization; arterial blood gas analysis results; imaging results; and treatment progression. Body mass index (BMI) and body surface area were calculated from data. PaOp < 0.05 was considered to be statistically significant.Statistical analysis was performed using R version 3.4.1 and EZR version 1.37. Continuous variables were analyzed and compared using the Mann–Whitney-U test and expressed as median (maximum and minimum). Nominal variables were analyzed and compared using the Fisher’s exact test and expressed as numbers and ratios; Cox hazard analysis was performed, and corresponding hazard ratio (HR) and 95% confidence interval (CI) were calculated. For parameters that demonstrated significant differences, factors previously demonstrated to be correlated with AE-IPF mortality were included. Receiver operating characteristic analysis was performed using continuous variables that were judged to be significant factors, and cut-off values were calculated.Of the patients who were hospitalized at the Maebashi Red Cross Hospital between 1 January 2013, and 31 December 2017, consecutive 84 patients met the diagnostic criteria for AE-IPF and were included in the present study . The medp = 0.071). No differences in the history of diabetes, dyslipidemia, myocardial infarction, cerebral infarction, malignant tumors, or bronchial asthma were found between the deceased and survivor groups. Regarding serological markers at hospitalization, c-reactive protein  was significantly higher in the deceased group (11.57 (95% CI 0.77 to 27.73) versus 6.69 (95% CI 0.1–21.84); p = 0.002), and serum total protein , albumin , and total cholesterol  were significantly lower (6.5 (5.2–8.4) versus 6.8 (5.5–9.4); p = 0.031), 2.75 (1.7–3.5) versus 2.95 (2–4.1); p = 0.047), and 133 (82–477) versus 159 (66–222); p = 0.039), respectively. Surfactant protein-A  tended to be higher in the deceased group (124.2 (30.4–251) versus 91.2 (27–270.7)); however, the difference was not significant (p = 0.067). No significant differences in KL-6 or surfactant protein D (SP-D) were found. Arterial blood gas analysis revealed no significant differences in PaO2/FiO2 ratio or AaDO2.The subjects were divided into two groups: those who died (deceased); and those who survived (survivor). Statistical analysis was performed using data from these groups. As shown in p = 0.059). No significant differences were found in the number of subjects who underwent steroid pulse therapy, cyclophosphamide pulse therapy, or recombinant thrombomodulin administration. The number of subjects administered sivelestat was significantly higher in the deceased group (7 (18.4%) versus 1 (2.2%); p = 0.02). We confirmed the presence or absence of malignant tumor from medical records. Fifteen and nine patients had malignancies as comorbidities and past tumors, respectively. The most frequent comorbid/past tumor was lung cancer, which was observed in 11 patients.No significant differences between the groups were found in the number of subjects who were admitted to the intensive care unit, or in whom tracheal intubation or nasal high-flow therapy were performed. The number of subjects in whom non-invasive positive-pressure ventilation was performed tended to be higher in the deceased group (8 (21.1%) versus 3 (6.5%)); however, the difference was not significant , and KL-6 and lactate dehydrogenase (LDH), which were already known to be correlated with mortality, were corrected for age, sex, and BMI, and Cox hazard analysis was performed (p = 0.006), LDH (HR 1.003 (95% CI 1.000–1.006); p = 0.037), and T-chol (HR 0.985 (95% CI 0.972–0.997); p = 0.018) were predictive factors for time until death. Receiver operating characteristic analysis was performed, and the cut-off values of the mortality-predicting continuous variables were calculated  has been low; new alveolar lesions have developed; and other causative diseases, such as infection and heart failure, could be ruled out.AE-IPF accounts for 15% to 40% of IPF-related deaths ,12. In tSingle-center studies by Kataoka et al. and Sakamoto et al. reported 44 cases and 80 cases of AE-IPF in five years and 10 years, respectively ,14. ThusThe new diagnostic criteria proposed in 2016 widened the range of AE-IPF , in thatIn addition, among the patients who had already been diagnosed, only four patients used antifibrotic drugs, and many patients remained untreated. It is plausible that primary care physicians did not introduce antifibrotic drugs into the treatment of elderly patients in consideration of the drug’s side effects and that non-specialists administered corticosteroids according to past recommendations, despite not corticosteroids but antifibrotic drugs having been recommended for IPF treatment. Moreover, while anti-fibrotic drugs suppress the incidence of AE, the corticosteroid administration group exhibited an increase in the incidence of AE; this may account for the limited prescription of antifibrotic drugs in this study.Previously, low forced vital capacity , elevateThe relationships between serological markers and AE-IPF were investigated in this study, and findings suggested that CRP, LDH, and T-chol might be predictive factors for in-hospital mortality after AE-IPF. KL-6, LDH, and CRP have been known to be mortality-predicting factors at AE-IPF onset ,3,14.CRP is a protein that is produced by hepatocytes in response to stimulation by cytokines such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6. During the chronic progression of interstitial pneumonia, it has been reported that a baseline CRP level associated with scleroderma was a long-term prognostic factor , and thaIt has been reported that elevation of serum LDH level in interstitial lung disease was a predictive factor for the onset of AE  and elevThere have been virtually no reports addressing the relationship between serum cholesterol level and AE-IPF. Correlations between low serum cholesterol level and mortality rate are known in several diseases, and it has been reported that hypocholesterolemia was an independent mortality-predicting factor with malignant tumors  and thatIt may be difficult to predict the prognosis of AE-IPF by using a single biomarker because various causes contribute to the development of AE-IPF. It has previously been reported that hypolipidemia was a risk factor for respiratory tract infection; it impairs immune responses to lung infection, resulting in the development of respiratory tract infection . Low totConsistent with this supposition, experimental and clinical investigations have revealed that high levels of cytokines, such as CRP, decreased circulating cholesterol levels during severe infection . Sepsis In addition to the presence of untriggered AE, this study included triggered AE caused by various factors, including infection and operation. Due to the heterogeneity among the pathologies of patients with AE-IPF, a single biomarker cannot reflect the various factors underlying the development of AE-IPF; hence, the AUC and specificity were relatively low.There are a few limitations in this study. Because this is a small, single-center retrospective study, the study population may not represent the entire AE-IPF patient population. In particular, a few patients in this study were treated with antifibrotic drugs. The administration of antifibrotic drugs is currently standard for the treatment of IPF; future analyses should thus be performed with populations featuring a greater proportion of patients treated with antifibrotic drugs. In addition, we were able to analyze serological markers only at admission and could not, therefore, record continuous changes in the levels of serological markers. This study cannot confirm serial changes of serological markers during AE. Therefore, the results in this study should be confirmed in a large size and in a multi-center study. In this study, we obtained spirometry data of only part of IPF patients before the onset of AE-IPF. Many of the 50 cases diagnosed with IPF before hospitalization were followed up at the local clinic when the IPF was at a stable condition and hence we could not obtain the data regarding spirometry performed at the clinic; alternatively in some other cases, a follow-up spirometry was not adequate at that time due to AE-IPF onset. Follow-up of spirometry in IPF patients is crucial as low forced vital capacity is associated with the onset of AE-IPF , and lowOur data suggest that CRP, LDH, and total cholesterol may be biomarkers predicting mortality in patients with AE-IPF. However, only prospective controlled studies can confirm or not our observation as a generalizable one.


In varicose veins, vascular smooth muscle cells (VSMCs) often shows phenotypic transition and abnormal proliferation and migration. Evidence suggests the FOXC2–Notch pathway may be involved in the pathogenesis of varicose veins. Here, this study aimed to explore the role of long non-coding RNA FOXC2-AS1 (FOXC2 antisense RNA 1) in phenotypic transition, proliferation, and migration of varicose vein-derived VSMCs and to explore whether the FOXC2-Notch pathway was involved in this process.The effect of FOXC2-AS1 on the proliferation and migration of human great saphenous vein smooth muscle cells (SV-SMCs) was analyzed using MTT assay and Transwell migration assay, respectively. The levels of contractile marker SM22α and synthetic marker osteopontin were measured by immunohistochemistry and Western blot to assess the phenotypic transition.The human varicose veins showed thickened intima, media and adventitia layers, increased synthetic VSMCs, as well as upregulated FOXC2-AS1 and FOXC2 expression. In vitro assays showed that FOXC2-AS1 overexpression promoted phenotypic transition, proliferation, and migration of SV-SMCs. However, the effect of FOXC2-AS1 overexpression could be abrogated by both FOXC2 silencing and the Notch signaling inhibitor FLI-06. Furthermore, FOXC2-AS1 overexpression activated the Notch pathway by upregulating FOXC2.FOXC2-AS1 overexpression promotes phenotypic transition, proliferation, and migration of SV-SMCs, at least partially, by activating the FOXC2-Notch pathway. Lower extremity varicose veins are a common disorder of venous dilation and tortuosity, and most varicose veins occur in the great saphenous vein , 2. The The forkhead box C2 (FOXC2) is a transcription factor of the human forkhead family involved in the metabolism of adipose cells, and is closely related to the occurrence and development of blood vessels and lymphatic vessels , 5. NotaLong non-coding RNAs (lncRNAs) have been shown aberrantly expressed in the primary great saphenous varicose veins, suggesting the potential involvement of lncRNAs in the pathogenesis of varicose veins . FOXC2 aIn this study, we first investigated the differences in morphology, VSMCs phenotypic transition, and FOXC2-AS1 expression between the normal veins and varicose veins. Subsequently, we investigated the role of FOXC2-AS1 in regulating phenotypic transition, proliferation, and migration of human great saphenous vein smooth muscle cells (SV-SMCs). Finally, we elucidated whether the mechanisms underlying the FOXC2-AS1-mediated effect was related to the regulation of FOXC2 and Notch signaling pathway.HE staining showed that the thickness of the intima, media, and adventitia of normal veins was normal. However, in the varicose veins, abnormal thickening of the intima was observed, and VSMCs proliferated in the media and adventitia of veins Fig. a. ImmunoThe qRT-PCR results showed that FOXC2-AS1 expression in the varicose veins was significantly higher than that in the normal veins .2O2 to quench endogenous peroxidase activity. Subsequently, the sections were rinsed with distilled water three times and then completely immersed in 0.01 mol/L citrate buffer, heated in a microwave oven, and washed twice with PBS. Following incubation with 5% BSA blocking solution, the sections were incubated with a primary rabbit anti-human SM22α  or rabbit anti-human OPN  at 37 °C for 90 min, followed by the biotin-labeled secondary antibodies at 37 °C for 20 min. The sections were incubated with the streptavidin–biotin–peroxidase complex (SABC) working solution at 37 °C for 20 min and then washed with PBS containing 0.1% TWEEN20. Then samples were stained with diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, and then embedded in paraffin. The yellowish-brown staining indicates a positive signal. The sections were observed under an Olympus BH-2 light microscope .Immunohistochemistry was used to observe the localization and expression of SM22α and OPN in varicose veins and normal veins. Briefly, the veins sections were dewaxed, hydrated, and then incubated in 3% H−ΔΔCt method and normalized to GAPDH. The primers were as follows: FOXC2-AS1 (forward) 5′-TTCATCGGCTGCGTATTCG-3′, FOXC2-AS1 (reward) 5′-TTGCCTTCTAGTCGCCTCC-3′; FOXC2 (forward) 5′-CGGCCCAGCAGCAAACTTTCC-3′, FOXC2 (reward) 5′-AGAGGCGGCGTGGATCTGTAG-3′; GAPDH (forward) 5′-CGCTGAGTACGTCGTGGAGT-3′, GAPDH (reward) 5′-CGTCAAAGGTGGAGGAGTGG-3′.qRT-PCR was performed to examine the expression of FOXC2-AS1 and FOXC2. Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen) and was reverse transcribed into cDNAs using the Reverse Transcription Kit (Takara). The cDNA template was synthesized through qRT-PCR using SYBR Green PCR Kit  by the ABI7900 system (Applied Biosystem). The relative mRNA expression levels were calculated by the 22 in a 25 mL glass culture flask, after which the flask was then inverted to stand upright and cultured at 37 °C. When the tissue block and the bottom of the bottle were observed to be firmly attached, the flask was placed flat, and then the complete medium  was added. After incubation for 12–14 days, the cells are crawled out from the edge of the tissue block, fused, and were routinely digested and passaged. The medium was changed once every 2 days.The human SV-SMCs were isolated from healthy human great saphenous vein. Briefly, the saphenous veins from the patients undergoing cardiac coronary artery bypass grafting surgery were collected and washed with PBS to remove blood. Then the extravascular connective tissues were carefully removed and the adventitia was exfoliated. The blood vessels were cut longitudinally and the inner membranes were bluntly scraped with the scalpel’s knife back. After being digested in 0.25% trypsin–EDTA solution for 5 min, the middle layer of the blood vessel was cut into a tissue block of about 1 mm × l mm × 1 mm in DMEM medium. Subsequently, the tissue block was evenly spread using an elbow pipette at a density of 3–5 pieces/cmThe SV-SMCs of the 3–5th generation were harvested and identified by α-SMA immunofluorescence. Briefly, when the cell confluence reached 75–85%, cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, and blocked with normal goat serum. Cells were then incubated with primary anti-α-SMA (1:110) overnight at 4 °C, followed by Alexa Fluor 568-labeled secondary antibody (1:60) at 37 °C for 1 h. Then cells were stained with Hoechst 33,342 and observed under a fluorescence microscope.® 2000 (Invitrogen) following the manufacturer’s protocol.To overexpress FOXC2-AS1, the full-length FOXC2-AS1 cDNA fragments were cloned into the pcDNA3.1 plasmid (Invitrogen), generating pcDNA3.1-FOXC2-AS1. An empty pcDNA3.1 vector was used as the control. To knock down FOXC2, FOXC2 siRNA (si-FOXC2) was designed and synthesized by GenePharma . A scramble siRNA was used as negative control (si-Ctrl). The SV-SMCs were transfected with these constructs using LipofectamineTotal protein from veins tissues and SV-SMCs was extracted using RIPA buffer . The protein concentrations were determined by BCA assay. Subsequently, equal protein from cell lysates was separated by 10% SDS-PAGE gels and transferred to PVDF membranes . After blocked with 5% skim milk, the membranes were incubated with the following primary antibodies against SM22α, OPN, FOXC2, Dll4, Notch1, Hey2, and EphrinB2  overnight at 4 °C, followed by the horseradish peroxidase (HRP)-conjugated secondary antibodies  at room temperature for 1 h. The protein was detected with an enhanced chemiluminescence kit  and the band intensity was quantified with Image J 14.0 software. GAPDH served as the loading control.3–5 × 103 cells/well. After 48 h of incubation, 20 μL MTT  was added into each well for 4 h of incubation at 37 °C. Then the medium was replaced with 150 μL DMSO (Sigma) for 10 min. Cellular viability was determined by measuring the optical density (OD) at 490 nm with averages from triplicate wells by an enzyme-labeled analyzer. Cellular viability was normalized to control well.Cell proliferation was analyzed by the MTT assay. Briefly, the SV-SMCs were harvested for 48 h after transfection and then seeded into 96-well plates at a density of 2 × 10Transwell assays were performed to assess cell migration. Briefly, the transfected cells were resuspended in serum-free medium, and 100 μL of the cell suspension was seeded in the upper chamber of Transwell inserts, while 600 μL full-serum medium containing DMEM with 10% FBS was added to lower chambers. After 24 h of incubation, non-migratory cells on the upper chamber of the inserts were scraped off with a cotton swab. Cells cling to the bottom side were fixed with methanol and stained with 0.1% crystal violet. The number of migratory cells was counted in six random fields under a microscope.t-test was used to analyze differences between two groups. One-way ANOVA was used to analyze differences among multiple groups. p < 0.05 was considered to indicate statistically significant.All statistical analyses were performed using SPSS 19.0. The data are presented as the mean ± standard deviation. The unpaired Student’s


E of the neat multimodal-HDPE significantly at the lowest carbon black content.The effect of carbon black (CB) and microwave-induced plasma graphene (g) on the crystallisation kinetics of the multimodal high-density polyethylene was studied under non-isothermal conditions. The non-isothermal crystallisation behaviour of the multimodal-high-density polyethylene (HDPE), containing up to 5 wt.% graphene, was compared with that of neat multimodal-HDPE and its carbon black based nanocomposites. The results suggested that the non-isothermal crystallisation behaviour of polyethylene (PE)-g nanocomposites relied significantly on both the graphene content and the cooling rate. The addition of graphene caused a change in the mechanism of the nucleation and the crystal growth of the multimodal-HDPE, while carbon black was shown to have little effect. Combined Avrami and Ozawa equations were shown to be effective in describing the non-isothermal crystallisation behaviour of the neat multimodal-HDPE and its nanocomposites. The mean activation energy barrier (ΔE), required for the transportation of the molecular chains from the melt state to the growing crystal surface, gradually diminished as the graphene content increased, which is attributable to the nucleating agent effect of graphene platelets. On the contrary, the synergistic effect resulting from the PE-CB nanocomposite decreased the Δ Multimodal high-density polyethylene (HDPE) is an engineered thermoplastic semi-crystalline polymer, which is widely used in films, pressure pipes, bottles, tubes and cables jacketing ,2,3,4,5.3, SiO2, TiO2, BaSO4, on the crystallisation kinetics of HDPE [Graphene has recently emerged as a very promising breakthrough material in the field of polymer nanocomposites and has attracted considerable scientific interest . A large of HDPE ,31,32,33 of HDPE . J. Kim  of HDPE . The mec of HDPE ,34,35,36 of HDPE . Also, a of HDPE . Moreove of HDPE . Polymer crystallisation is a process that essentially involves two consecutive steps, namely the nucleation and growth of the crystal nucleus ,34,35,363. Antioxidants masterbatch containing Irgafos 168 and Irganox 1010 were added to the polymers at 0.5 wt.% for optimum stabilisation during processing. Graphene powder was obtained from FVG Cambridge Nanosystems company , with ≥96% carbon purity, bulk density of 0.0266 g/mL, flake thickness < 1.0 nm with an average lateral size range of 150–500 nm. Carbon Black powder was provided by Orion Engineered Carbons GmbH , with ≥92 cc/100g oil absorption number, ash content of 0.10%, Sulphur content of 0.10%, tint strength of 103%, average primary particle size of 20 nm and a density of 1.7–1.9 g/cm3 at 20 °C.Unstabilised, high density polyethylene powders, produced with Ziegler Natta catalyst via a proprietary Borstar process , melt flow rate of 7.5 g/10 min , Mw = 280 kg/mole, Mn = 8.49 kg/mole and Mw/Mn = 33 and a density of 950 kg/mBoth multimodal-HDPE/graphene (PE-g) and multimodal-HDPE/carbon black (PE-CB) were prepared via melt-intercalation method using Coperion ZSK 18 twin extruder having a screw diameter of 18 mm and a barrel length of 720 mm (L/D = 40). The screw rotation speed was 600 rpm, barrel temperature profile was in the range of 170–240 °C and feed rate was between 1–2 kg/h. For more information, the reader is directed to our previous work [The nanofillers and a dry polyethylene powder were fed separately into the extruder via a spiral flow screw Brabender ISC-CM plus feeder. The nanofillers were fed at 0.1, 0.5, 1, 2 and 5 wt.% loadings. In order to prevent the polymer from degrading, an antioxidant masterbatch was simultaneously added through a side feeder, with the total loading of 0.5 wt.%. The extruded pellets were subsequently compression moulded to about 0.4 mm thickness, following ISO 293 under 5 MPa, at a temperature of 200 °C. This was undertaken via a compression moulding platen press , for an overall programming cycle of 32 min at a heating and cooling rate of 15 °C/min. The specimens were successively conditioned at 23 ± 2 °C and 50 ± 5%, for at least 48 h, prior to being tested. Polarised light microscopy (PLM) analyses were conducted on a ZEISS Axio scope.A1 HAL 100/HBO 100, operated with an AxioCam MRc 5 camera and an AxioVision software. Film samples were sectioned to a thickness of 15 μm, using a fully automated rotary microtome Leica RM2265 . Samples were cooled by DSC at 2.5 °C/min prior to being analysed under the polarised light. ImageJ software was used to calculate the mean particle size of the nanoparticle agglomerates and the %area fraction (200 × 200 µm2) upon the optical light microscopy and transmission electron microscopy (TEM) images. Transmission electron microscopy (TEM) was performed using Hitachi HT7700, at an accelerating voltage of 120 kV. Film samples were cryo-sectioned to a thickness of ~80 nm at −125 °C, using Leica EM UC7/FC7 Cryo-Ultra-microtome.Thermogravimetric analysis (TGA) was carried out with Q500 TGA  with a heating rate of 10 °C/min from room temperature to 1000 °C in a nitrogen atmosphere. High-Resolution (Hi-Res)-Dynamic mode was performed with sensitivity of 2.00 and a resolution of 4 °C. Rheological behaviour of the samples was studied using stress-controlled rotational rheometer (Anton Paar Physica MCR 301 with CTD450 heating unit) at 190 °C under a nitrogen atmosphere. The compression moulded sample, weighing 1.5 g and with measurements of 25 mm in diameter and 1.5 mm in thickness was conditioned at 40 °C for 48 h. The sample was then placed onto a 25 mm parallel plate fixture and trimmed to a thickness of 1.2 mm by slowly lowering the upper plate. The dynamic frequency sweep was conducted from 500 to 0.0154 rad/s at 5% strain. The reason for starting from the maximum frequency was to avoid sample degradation under high temperature and low angular frequency speed. The polydispersity index (PDI) was calculated as follows [G′ is the storage shear modulus, G” is the loss shear modulus, ω is the angular frequency and Differential scanning calorimetry (DSC) was performed in a N2 atmosphere (50 ± 5 mL/min), using approximately 5 mg of the sample, sealed in Al pans with TA Instruments Q2000. The samples were melted at 200 °C, then held for 10 min to eliminate the thermal history and subsequently cooled to 0 °C at cooling rates of 2.5, 5, 10 and 20 °C/min, respectively. The samples were again held at 0 °C for 10 min and subsequently heated to 180 °C at the same cooling rates as the prior cooling rate, in order to obtain the DSC exotherms. Heat flow, as a function of time and temperature, was recorded during both crystallisation and melting processes for consequent data analysis.Tc) was determined from the temperature at maximum heat flow of crystallisation peak. The peak crystallisation temperature (Xc) of the polymers, where ΔHv is the enthalpy of fusion determined from the experiment, ΔHv° is the enthalpy of fusion of the 100% crystalline polymer (ΔHv° = 290 J/g) and fW is the weight fraction of the filler content in HDPE [The following equation:%Xc=ΔHv were around 0.9 µm2 and 0.4 respectively, as shown in 2 and the %area fraction was less than 0.006. The %area fraction and mean particle size were calculated based on TEM and light microscopy analysis and graphene particles of less than 0.05 µm2 or 500 nm were excluded from the calculations. A decrease in the %area fraction means a better distribution and fewer agglomerates. The figure was higher with carbon black as the fused aggregate sizes are commonly bigger than graphene monolayers. As shown from the TEM images in The dispersion and distribution of graphene and carbon black were assessed by TEM and light microscopy, as shown in G′) and loss (G′′) moduli, as well as their frequency dependence [The dispersion and distribution of the nanofillers within the polymer matrix were further investigated by the rheological measurements ,46,47,48pendence ,46,47,48The presence of the nanofillers considerably increased the pseudoplasticity at a low shear rate. At the angular frequency of 0.0154 rad/s, the complex viscosity of the neat multimodal-HDPE increased from 0.19 MPa·s, to 0.34 MPa·s and 0.25 MPa·s at 1 wt.% loading of graphene and carbon black, respectively. Similarly, the loss and storage moduli of the neat polymer increased by a value of ~36% and ~32% with 1 wt.% loading of carbon black and by a value of ~99% and ~78% at 1 wt.% loading of graphene, respectively. The greater amount of storage and loss moduli of the reinforced polymer is attributed to the formation of a strong interfacial bonding between the polymer matrix and the high-modulus nanofillers, which has accordingly reduced the loss tangent, therefore the polymer became more elastic ,28,29,30GC) and crossover angular frequency point (ωC) of the reinforced polymers have shifted to a lower range. The addition of 1 wt.% of graphene decreased the GC and ωC of multimodal-HDPE by a value of 15% and 53%, respectively, while it decreased by a value of 13% and 46% with the 1 wt.% loading of the carbon black. These differences can be attributed to the specific surface area which each nanofiller can offer the polymer matrix through its specific dimensions [ωC to the lower region indicates that the reinforced polymers exhibited higher average molar mass and/or branched (entanglement) molecules [GC to the lower region upon the addition of nanofillers possibly arose also from exposing the neat polymer to a high temperature, under a combination of high shear and elongation forces, for a prolonged period of time. The weight average molar mass (Mw) and z-average molar mass (Mz) of the neat multimodal-HDPE powder decreased from 280 kg/mol and 2099 kg/mol, to 207 kg/mol and 1131 kg/mol, respectively, after being extruded in the conditions described in the experimental section  exhibited broader shape and shifted gradually to a lower temperature. The value of Tc increased from 114.8 °C to 119.8 °C for pristine multimodal-HDPE, when decreasing the cooling rate from 20 to 2.5 °C/min. In general, nucleation at a lower degree of undercooling tends to be sporadic and only a relatively small number of nuclei are obtained during the melt crystallisation [lisation . At highlisation ,11,36,51Hc) decreased where the cooling rate was elevated, as indicated in A rapid decrease in temperature is accompanied by an increase in the viscosity, thereby the transport of material to the growth point becomes more difficult and eventually reduces the growth rate . Hence, Hc at higher cooling rates, can also be attributed to thermal conductivity limitations, as well as lowered annealing effects. At a given cooling rate, Tc of multimodal-HDPE filled with graphene shifted to higher temperatures, as shown in Tc was clearly evident at the lowest graphene content. Afterward, the shift continued to gradually ascend as the concentration of graphene was increased, accompanied by a broadening of the peak  ,55,56,57peak see . For exar matrix . The forr is related to the time (t) and spherulite growth rate (v) through an equation in the form of r = vt, which is valid until the spherulites touch each other [A polarised light microscope was used to examine the spherulitic morphology of the polymers, as shown in ch other ,36,58,59ch other .T) as a function of crystallisation temperature can be calculated using the following equationdH/dT is the enthalpy of crystallisation evolved within an infinitesimal temperature range, T0 is the onset crystallisation temperature, T denotes the arbitrary crystallisation temperature and fT refers to the end temperature of crystallisation [T0 of the multimodal-HDPE filled with graphene shifted significantly to higher temperatures, as shown in T0 was clearly evident even at the lowest graphene content (0.1 wt.%) and continued to increase gradually as the concentration of graphene grew. For example, the value of T0, increased from 120.3 °C to 123.6 °C for PE-CB-1% and to 127.4 °C for PE-g-0.1%, at the cooling rate of 20 °C/min. However, the relative value of T0 remained almost the same for the PE-CB, regardless of the concentration of carbon black.The relative degree of crystallinity  is widely used as an indicator of spherulitic growth rate; the larger the difference, the lower the rate of growth in the spherulites [T0 − Tc) are listed in T0 − Tc) to increase. With a 1wt.% loading, it was increased by 144%, 121%, 105% and 84% at the cooling rate of 2.5, 5, 10, and 20 °C/min, respectively. The value of (T0 − Tc) gradually became larger with increasing graphene content. On the other hand, the relative value of (T0 − Tc) decreased at the lowest content of carbon black, especially at the cooling rate ≥ 10 °C/min and thereafter remained invariable at higher concentrations. These results are in concurrence with the PLM images, that is, the bigger the spherulite radius, the greater the rate of spherulitic growth (r = vt). In non-isothermal crystallisation, the arbitrary crystallisation temperature T is associated with the crystallisation time t through the following equation of the formThe difference between the onset and the peak crystallisation temperatures  extended significantly with the addition of graphene, especially at the cooling rate of ≤10 °C/min. As a further example, in PE-g-1%, the value of t0.5 increased from 0.5, 0.8, 1.2 and 2.1 to 0.8, 1.3, 2.3 and 4.1 min, at the cooling rate of 2.5, 5, 10, 20 °C/min, respectively. The marginal increase in the value of (t − t0.5), indicates that the time-lag occurring as a result of a longer incubation period. On the other hand, a slight decrease in t0.5 and t was generally observed with the addition of carbon black, as shown in t0.5 remained almost saturated when the carbon black content reached 1 wt.% and higher. These results are in agreement with the PLM images, that is, the spherulite radius is directly proportional to the rate of spherulitic growth (r = vt). This means that the crystallisation kinetics of PE-CB and PE-g were dominated by the nucleation process. As graphene has a huge surface area, it has introduced a large number of nucleation sites through which the crystallisation initiated and occurred at higher temperatures. The effect of graphene is consistent with most of the results reported in the literature on the effect of nucleating agents upon the behaviour of crystallisation in HDPE, with the exception of the declining rate of the crystallisation [T0 [The magnitude of parameters obtained from hene see c,d. Withlisation . Nonethelisation ,34,47,63lisation . Furtherlisation ,64. F. Ction [T0 ,64. ContCarbon black acting as a nucleating agent in polyethylene has never yet been reported according to the author’s knowledge. Many studies, such as those by S. Song et al. , A. Krum3, SiO2, TiO2 [n is a kinetic constant, m1/ refers to the cooling or heating rate required to achieve a certain relative degree of crystallinity at a unit crystallisation time and a is the ratio of Avrami exponent n to Ozawa exponent m, that is, a = n/m.Using an appropriate model to account for the varying temperatures and cooling rates allows better understanding of the crystallisation kinetics under typical non-isothermal conditions. Various scientists, such as Ziabicki, K. Nakamura et al., Jeziorny, Ozawa, R. M. Patel et al., Dietz, T. Liu et al., have proposed numerous models to describe the non-isothermal crystallisation kinetics of semicrystalline polymers ,22,23,38O2, TiO2 ,28,29,38a can be determined from the intercept and slope of the log φ versus log t plot respectively, as shown in 2 ≥ 0.998, was observed at a given relative degree of crystallinity, indicating that the T. Liu et al. model could aptly describe the non-isothermal crystallisation kinetics of the multimodal-HDPE, PE-g and PE-CB. The kinetic parameter TF as a function of relative degree of crystallinity and the nanofiller content, is shown in a were almost the same. The marginal changes in the values of a, ranged from 1 to 1.3, which also indicates that the combined Avrami-Ozawa equations suitably describes the non-isothermal crystallisation kinetics of the multimodal-HDPE, PE-g and PE-CB, as reported elsewhere [TF values of the neat HDPE and PE-CB were almost the same and increased in a similar pattern across the entire degree of relative crystallinity. A slight decrease in TF was observed when the polyethylene was loaded with ≥1 wt.% carbon black content, which is consistent with the results observed earlier in this study.Parameters lsewhere ,34,35,36TF parameter of PE-CB-1% decreased from 2.8, 7 and 69.5 °C/min to 2.4, 5.8 and 69.2 °C/min at Xt of 10%, 50% and 100%, respectively. This is a confirmation that carbon black did not form a continuous network structure, most probably because of its dimensionality. However, the relative increase in FT was clearly evident even at the lowest graphene content and continued to gradually rise as the concentration of graphene became larger, especially at Xt < 70%. The FT value of the multimodal-HDPE filled with 1 wt.% graphene, increased from 2.8, 7 and 69.5 °C/min to 7.8, 14.1 and 77.1 °C/min at Xt of 10%, 50% and 100%, respectively. This is indicative of a slower crystallisation process compared with the neat HDPE and/or HDPE filled with CB and it is related to the difficulty of crystallisation process to progress further due to the entangled-network structure induced by graphene platelets. These results are consistent with what was observed and discussed earlier in this study. Accordingly, knowledge of the conditions affecting the crystallisation kinetics is crucial for optimising the processing parameters, as this can affect the final properties of the product which includes warpage/shrinkage control, mechanical and optical clarity or an alteration of the cycle time in moulding or extrusion [The xtrusion . In pipextrusion . A replaE) required for the molecular chains to transport from the melt state to the growing crystal surface, in non-isothermal systems, was calculated in this study, using the following H. E. Kissinger equation [R is the universal gas constant. The higher the value of ΔE, the more difficult the transportation of macromolecular chains to the growing surface. These can generally result either from higher viscosity or restriction in the polymer chains as previously discussed in the present study. The activation energies obtained from the slope of the straight-lines in a E of the neat multimodal-HDPE, which was −535 kJ/mol. The value of ΔE decreased gradually to −556, −582, −591, −612 and −705 kJ/mol when increasing the concentration of graphene to 0.1, 0.5, 1, 2, 5 wt.%, respectively. However, the relative value of ΔE decreased significantly to −876 kJ/mol at the lowest carbon black content of 0.1 wt.% and remained almost saturated at higher concentration. In general, the decrease in the activation energy barrier is attributable to the role of the nanoparticles as a nucleating agent. The results indicate that graphene is likely to perform two functions in the multimodal-HDPE matrix. The platelets acted as nucleating agents and accelerated the primary nucleation, which was manifested by the gradual decrease in ΔE and the increase in T0 and Tc. However, the presence of the jammed network structure slowed the diffusion of the polymer chains into the crystalline lattice, thus lowering the overall crystallisation rate, as confirmed by the increase in FT, t0.5 and t. On the other hand, the change in crystallisation kinetics of the multimodal-HDPE, associated with the addition of carbon black, is suggested to be a result of the synergistic effects. This occurs where the longer macromolecular chains and broader MWD accelerated the crystallisation of PE-CB as confirmed from the values of ΔE, t0.5, Tc, FT, as well as rheological results.At a given cooling rate, the relative degree of crystallinity at the peak crystallisation temperature ranges between 11% and 30% for all the polymers. The mean activation energy barrier (Δequation ,36,38,39t0.5 increased significantly with the addition of graphene and continued to increase gradually with an increasing content of graphene. However, the spherulitic growth rate decreased at the lowest graphene content and continued its gradual descent with the increasing amount of graphene content. On the other hand, the non-isothermal crystallisation behaviour of the PE-CB nanocomposites relied on the cooling rate only and the effect of carbon black content as a nucleating agent was found to be marginal, with the exception of the peak crystallisation temperature which increased gradually in concurrence with the carbon black content. At a given cooling rate, the relative onset crystallisation temperature of PE-CB remained almost unchanged with the addition of the carbon black. Also, a slight increase in the spherulitic growth rate accompanied a relative decrease in t0.5 which was observed upon addition of 0.1% carbon black and these remained almost unchanged with further addition.The crystallisation kinetics of the multimodal-HDPE behaved differently with the addition of 0.1 to 5 wt.% carbon black or graphene, under non-isothermal conditions. The non-isothermal crystallisation behaviour of the PE-g nanocomposites relied heavily on both the graphene content and cooling rate. The relative peak crystallisation temperature of PE-g increased with decreasing the cooling rate for a given graphene content and increased gradually with an increase in the concentration of graphene at a given cooling rate. Incorporation of graphene caused a change in the mechanism of nucleation and crystal growth of multimodal-HDPE crystallites, with the effect being more evident at the lowest graphene content. At a given cooling rate, the nucleation initiated at a higher temperature and continued to increase with the graphene content. Similarly, the incubation period as well as the time required to reach 50% of relative crystallinity FT of the multimodal-HDPE filled with 1 wt.% graphene increased from 2.8, 7.0 and 69.5 °C/min to 7.8, 14.1 and 77.1 °C/min at Xt of 10%, 50% and 100%, respectively. However, a slight decrease in FT values was observed when the multimodal-HDPE loaded with ≥1 wt.% carbon black content. Using the H. E. Kissinger equation, the activation barrier energy of the multimodal-HDPE decreased gradually with an increasing the graphene concentration, while it rose by 64% at the lowest carbon black content. However, the value of ΔE remained almost unchanged as the content of carbon black increased. For PE-g, graphene platelets acted as nucleating agents and introduced foreign surfaces, which in turn reduced the barrier activation energy required for the crystallisation. However, the presence of the jammed network structure introduced by graphene platelets retarded the diffusion of the polymer chains into the crystalline lattice and thus slowing the primary crystallisation. Contrastingly, carbon black was shown to have little effect as a heterogeneous nucleating agent on the crystallisation kinetics of multimodal-polyethylene, possibly because of its surface properties.Combined Avrami-Ozawa equations proposed by Liu et al. was found to be effective in describing the non-isothermal crystallisation kinetics of the multimodal-HDPE, PE-CB and PE-g nanocomposites. The cooling rate required to achieve certain degree of crystallinity increased gradually as the graphene content increased. The relative kinetic parameter


In this work, we have prepared a series of polydimethylsiloxane (PDMS) composites containing various graphene flakes loadings (0.02–2 wt%), and their broadband optical properties are being investigated. We demonstrate the tunability and evolution of transmittance and reflection spectra of the composites in a wide spectral range (0.4–200 μm) as a function of graphene content. Using these data we derive the broadband wavelength-dependent absorption coefficient (α) values. Our results show that α is roughly constant in the visible and IR ranges, and, surprisingly, is approximately one order of magnitude lower in the terahertz regime, suggesting different terahertz radiation scattering mechanism in our composite. Our material could be useful for applications in optical communication, sensing or ultrafast photonics. The outstanding optical, electrical and mechanical properties of graphene flakes12. Examples are as follows: a flexible and/or transparent graphene thin films15; graphene based 3D structures - graphene foam17 or hydrogel20 filled with polymer; multi-stacked sandwiches22; and other nanocarbon/polymer composites24. Remarkably, regardless of the production method, it has been shown that even a small loading of a nanocarbon filler can transfer its extraordinary properties into a composite material. For instance, mechanical and electrochemical reinforcement28 or boost of electrical and thermal transport properties30 that enable increased thermal conductivity34 and stability35, gases impermeability37, increase of electrochemical reactivity and sensing39, or efficient electromagnetic interference shielding41.Literature provides numerous examples of graphene and other nanocarbon material based composites of different architecture and composition, prepared by a variety of methods1 in extremely broadband range. However, how this remarkable feature is transferred into graphene based composite material is still poorly studied and only several works have been written so far. For instance, graphene thin film (10 nm), composed of reduced graphene oxide sheets, deposited on polymer substrate exhibit a transmittance of 70%42. According to Bao et al.43 graphene load as small as 0.7 wt% in pristine polymer (PVAc) causes tenfold rise of the optical absorption of a composite in a UV-NIR range. Ultrathin graphene composite films can serve as an electrode for flexible electronics showing visible light transmittance in the range of 70–95% and can be tuned by graphene flake concentration44. Similar composites can be also applied as optical elements in fibre lasers, e.g. saturable absorbers46 or passive wave guides47.Carbon nanofillers such as graphene flakes offer a possibility of tuning the optical properties of the composite material. This is possible due to graphene’s particularly interesting optical features. It has been shown, for instance, that a single layer of graphene can absorb 2.3% of incident lightIn all above examples the tunability of optical properties is a key factor for progress in the application development. However, in literature the optical transmission/absorption of graphene based composites is investigated only in visible or near infrared range (still only few papers) and no systematic study on how the graphene content influence these properties can be found.In this paper, we demonstrate a systematic study of broadband optical properties of graphene flakes immersed with variable concentration (0.02 to 2 wt%) in PDMS polymer matrix. We show the graphene-content dependent scalability of the absorption, transmission and reflectance properties of the composite in the wavelength range of 0.4–200 µm. For instance, our study shows that less than 0.5% of graphene content is enough to totally block the incoming radiation. Additionally, the experimental transmittance of our samples was compared with a model for stacked graphene multilayer. Finally, we derive broadband absorption coefficient (α) of the composite, that exhibit significantly different behaviour for VIS-IR and THz range, and its values strongly differ from other carbon material.48 or styrene-butadiene rubber50. A specified amount of a few-layer graphene powder (Graphene Supermarket) with average thickness of several nanometers  supplied by Dow Corning Co. as a two-part liquid component kit (base and curing agent), since it is relatively transparent and has no colour, as compared to other commonly used polymer matrixes, i.e. poly methyl-vinyl siloxane rubber51.The impact of the graphene loading level on the optical properties of the composite is already visible with the naked eye, as illustrated in Fig. −1), G (1588 cm−1) and 2D (2680 cm−1) are characteristic for graphene materials, thus are not observed in the pristine PDMS sample. The spectra did not show any changes for different graphene loading . The Raman experiment was conducted using 633 nm excitation wavelength (low power) and 50x objective, at room temperature.To confirm the graphene presence and flakes deployment in the composite, we used Scanning Electron Microscope (Raith) and Raman spectroscope (Renishaw InVia). SEM image in Fig. n is the refractive index, κ is the extinction coefficient, T stands for transmittance, R is reflectance and The optical spectra were obtained using two different instruments depending on the wavelength range. For the VIS-NIR range 420–1700 nm) total reflectance (both specular and diffusive) and transmittance spectra were collected using a PV response analyser (PVE300 Bentham). For NIR-THz range (1.66–200 µm) only the transmittance measurements were conducted using a vacuum Fourier transform infrared spectrometer Bruker IFS-113v. The spectra were collected at the room temperature and ambient pressure (VIS-NIR range) or nitrogen pressure of about 6 mbr in the Bruker spectrometer. We then related obtained transmittance data to absorption coefficient 700 nm toFigure 3 stretch vibrations characteristic for PDMS material47. Intuitively, an addition of given amount of graphene to the composite lowers the transparency almost evenly, without changing the shape of the spectra. This behaviour reflects the property of graphene monolayer which is constant within the optical range24. Thus, we show that our material exhibits a similar feature to mono- or multi-stacked graphene sandwich sample in the UV-IR range.All optical spectra remain relatively flat, except for small peaks around 1.15 µm, which are related to CHet al.42, however our material is much easier to fabricate, requires no substrate. Moreover, it is easier for handling possible application.Clearly, the transmittance depends on the amount of graphene powder used in the process of fabrication. The more graphene is added to the composite, the less light is transmitted through the material. Interestingly, even a composite with the smallest graphene concentration (0.02 wt%) has significantly smaller transmittance (74% of incident light) as compared to the pristine PDMS sample (~94%). The transmittance level of a 0.02 wt% sample is comparable with a 10 nm thick layer of reduced graphene oxide on polymer reported by Wang The drop of the transmittance reaches a saturation point at 0.5 wt% graphene loading, where no light (VIS-NIR) is transmitted by the material, thus making it completely opaque in this range.52. For samples with higher graphene load the reflectance drops below this value, which might be related to a very high absorption level of those samples. This suggests that main contribution to the reflection comes from the polymer matrix, while graphene barely participate in the process or even cause the decrease of the reflection. The reflectance values of our composites are significantly lower than those of bulk graphite53 and approaching the values of mono- to a few layer graphene samples that show reflectance of roughly 1 to 2%2.The optical reflectance level of all studied composites for VIS-NIR range, is kept in 4–8% range see Fig. . On the 55. However, we note that due to the fact that the size of the flakes used in our material is roughly the same size as the wavelength of the incident light in the VIS-NIR spectral range and the non-zero value of absorption of the polymer matrix, radiation primarily scattered in the material would be absorbed before leaving the medium. Thus, it is likely that the mechanism based on internal multiple reflection is negligible. All this provides two conclusions: first, the main mechanism responsible for blocking the light in the studied range is the absorption and secondly, only small fraction of graphene in the material is needed to fully tune its optical properties.Both transmittance and reflectance data are then used to derive the absorption values of our composite for the VIS-NIR spectra range see Fig. . As only47, while the values and their changes depend only on graphene loading. Although there are regions where PDMS transmittance signal is immeasurable, which may be due to relatively high thickness of the composite, we concentrate on “transmittance windows”, where the measurements of the influence of graphene flakes content is possible. In the IR region, again all composites with 0.5 wt% graphene concentration and higher show no measurable transmittance. Remarkably, this is not reproduced in the THz region, (for the wavelengths greater than 44 µm) our material is much more transparent with respect to the graphene content (e.g. sample with 1.5 wt% has ~10% transmittance at 3 T Hz).Next we discuss the transmittance spectra beyond VIS-NIR range, up to terahertz region (1.66–200 µm). Corresponding data are shown Fig. In order to analyse the correlation between graphene loading and broadband transmittance of the fabricated composites in respect to the wavelength, the spectral range was divided into three parts – I: 400–1700 nm; II: 1, 66-6 μm and III: 44–200 μm. For each range a set of wavelengths was selected. Collected values of transmittance were then normalized according to the transmittance of pure PDMS, averaged within their ranges and presented in Table 56. We used this model to fit our data and match the obtained values of transmittance with corresponding number of graphene layer, in order to find a correlation between those two factors . The decrease slope of the transmittance is less steep as compared to first and second stages and values of T(wt%) reach zero only for the composite with 2 wt% load of graphene flakes. This demonstrates that the mechanisms behind the optical transmission of our composites exhibit significantly different behaviour for VIS-IR and THz range.62. In the first and second range the Those differences are even more apparent if we take into account the absorption coefficient  dispersion absorption coefficient63 and requires more thorough study.Calculated 62 are 3 orders of magnitude higher as compared to our materials, similarly if we compare 49 and our composite.Interestingly, it is striking that the values of 43. It appears 0.5 wt% graphene concentration is sufficient to receive a material of almost 100% absorption of incoming radiation with the wavelength between 420 nm and 44 µm, while graphene load of 2 wt% creates a material that is nontransparent in the whole studied spectral range – up to 200 μm.Graphene flakes used as a nanofiller in a polymer composite have direct influence on the material transmittance. Filler concentration as small as 0.02 wt% affects the received material absorption, raising it 30 times compared to the pristine PDMS samples. That influence surpasses other works, which report absorption enhancement by 10 times with 0.07 wt% graphene loadingThe influence graphene loading has on the transmittance of a composite can be compared with transmittance of stacked graphene multilayer. The 0.5 wt% of graphene flakes corresponds to a 90–150 nm thick multilayer graphene, but is much easier and more effective to produce in a form of a PDMS/graphene composite than through multiple delamination, or mechanical exfoliation, thus making the composite more desirable for possible applications.Although the transmittance of pure graphene is known to be independent of wavelength, we show that when graphene is used as a nanofiller in a polymer composite, the transmittance changes as a function of the wavelength change due to different attenuation mechanisms. Calculated absorption coefficients also vary due to the wavelength. This change is most visible in the third spectral range (FIR), where absorption has significantly smaller value – The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.