Irradiation dynamics of a single graphene sheet bombarded by hydrogen atoms is studied in the incident energy range of 0.1 to 200 eV. Results for reflection, transmission, and adsorption probabilities, as well as effects of a single adsorbed atom to the electronic properties of graphene, are obtained by the quantum-classical Monte Carlo molecular dynamics within a self-consistent-charge-density functional tight binding formalism We compare these results with those, distinctly different, obtained by the classical molecular dynamics.PACS: 61.80.Az, 61.48.Gh, 61.80.Jh, 34.50.Dy. Graphen storage ,6. These storage ,8. The r storage ,9. For eIn this paper, we study the perpendicular impact of hydrogen on a single graphene sheet over more than three decades of impact energies (0.1 to 200 eV) using methods of quantum-classical Monte Carlo molecular dynamics. Our approach is described in detail in the second section entitled 'Methods'. The irradiated target was an infinite graphene sheet obtained by applying 2-D periodic boundary conditions to a graphene cell of size 29.12 × 28.53 Å (336 C atoms). The graphene was prepared at a temperature of 300 K by a Nose-Hoover thermostat and left free during each collision event, which lasted 200 to 500 fs, depending on the impact energy. The irradiation was performed by more than 1,000 independent trajectories for each impact energy, with randomly chosen position of emission of an atom above the surface of the graphene cell. In this method, the total electronic energy of the system is solved quantum-mechanically at the beginning of each time step (on the order of a femtosecond), maintaining fixed atom positions; after incorporating the nucleus-nucleus interaction into the total electronic energy, forces on each atom are updated, and the atoms are moved classically within the time step. The electronic structure is solved here by the self-consistent-charge-density functional tight binding (SCC-DFTB) method -12. To al-hE, in the difference, l-hE, of the (discrete) lowest unoccupied molecular orbital and highest occupied molecular orbital energies in response to the hydrogen adsorption. These changes are indicative of possible changes in the graphene sheet conductance. They are, surprisingly, on the order of 1 eV and depend on the vibrational energy of the adsorbed hydrogen. Adsorption occurs only for the low-energy impacts (< 1 eV). This confirms some predictions in literature on the extreme sensitivity of the highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) gap and transport properties of graphene and SWCNT to the adsorption of hydrogen and other atoms and molecules [Results for reflection and transmission probabilities, angular distributions, and adsorption probabilities at low energies (0.1 to 1 eV) are shown and analyzed in the first part of the 'Results and discussion' section, entitled 'Irradiation dynamics and effects on electronic structure'. Additionally, changes in the molecular orbital levels close to the Fermi energy, which influence the non-equilibrium ballistic electron transport properties  of the system, are calculated and characterized by the changes, ∆olecules -19.In the second part of the 'Results and discussion' section entitled 'Comparison with classical molecular dynamics', we perform classical molecular dynamics (CMD) calculations with two state-of-the-art bond order hydrocarbon potentials, reactive empirical bond-order (REBO)  and adapTel = 1,000 K, which has a similar effect to averaging over many electronic states near the Fermi level.To simulate effects of irradiation on graphene, one can apply direct molecular dynamics methods in which electronic structure is treated explicitly using quantum mechanics, while the motion of the nuclei is described by the means of the classical dynamics. This allows one to accurately describe bond breaking and formation as well as the interatomic potentials. Such an approach is, however, computationally very expensive, which greatly limits the system sizes, timescales, and choice of quantum mechanics-based methods. To mimic the dynamics observed by experiment, we apply a Monte Carlo approach to the trajectories, i.e., using a large number of trajectories, randomly varying 'impact parameters' to obtain acceptable statistics of the collision events. Even using this approach, we must use a less expensive and more approximate quantum-mechanical approach. Here, we use the SCC-DFTB method, an approximate density functional theory (DFT) method in which only valence electron interactions are considered. Although a full DFT treatment would be ideal, this is currently too expensive computationally, even for a handful of trajectories. In SCC-DFTB, the total electronic densities and energies are expressed by solution of the Schrodinger equation in the Kohn-Sham form, using predetermined Hamiltonian and overlap integrals as well as repulsive splines fit to reference systems . The tight binding methods applied to the large (solid-state) systems have a long history. Here, we use a self-consistent charge version developed by Bremen Group  -12. SCC To safely allow for high-energy bombardment simulations (in our case 200 eV), we use a refitted version of the original DFTB PBC-0-3  parametez-position above the graphene/coronene plane. The coronene potentials show bonding that is roughly 1 eV weaker and a potential barrier at the hexagon center that is 1 eV higher, reflecting the changes in electronic structure between hydrogen-terminated and periodic sp2 carbon. Despite these differences, the forms of the H-graphene and H-coronene interactions are very similar. Thus, the agreement of SCC-DFTB with DFT calculations of the coronene molecule in Figure Figure Notably, there are two bonding regions in the H-graphene potential. For incidence directly upon a lattice carbon, the potential minimum occurs at approximately 1.1 Å, while incidence upon a C-C bond center shows a shallower potential with minimum close to 1.0 Å. Indeed, there are many potential wells in the 3-D multibody potential that are responsible for capturing impinging hydrogen atoms; these will later be shown to have an effect on the electronic structure of resultant H-graphene surfaces. There are repulsive barriers at the bond center and hexagon center of heights 17 and 2.5 eV, respectively. Notably, hydrogen encounters no barrier before entering the potential well when incident directly on a lattice carbon.z = 0 plane and periodically extended in the xy coordinate plane. To simulate the bombardment in a real-world environment, the sample is thermostated (via Nose-Hoover scheme) to 300 K before bombardment and left to evolve freely during approximately 0.1 to 1 ps (depending on incident energy) simulation time. The impinging hydrogen atom is released from a random  position in the z = 10 Å plane, with velocity perpendicular to the graphene sheet.About ten per decade incident kinetic energies ranging from 0.1 to 200 eV are considered for the impinging hydrogen atom. While cumulative bombardment is not investigated, 1,008 single impact simulations are performed for each incident energy; this is achieved using 1,008 processors, one for each trajectory, on the Kraken Cray XT5 supercomputer . The target graphene surface described in the 'Background' is situated in the Three outcomes of the bombardment are observed: reflection, transmission, and adsorption of the incident hydrogen atom; no sputtering of any type was observed in our quantum-classical approach. Figure Reflection of the incident hydrogen can occur at all points in the graphene lattice. As can be seen in Figure x-y positions for adsorption. Adsorbed atoms are clustered around the carbon atoms, often showing some lateral vibration.By examining the position within the hexagon where incident atoms are reflected, transmitted, or adsorbed, one can infer the form of the many-body potential at nonsymmetrical parts of the lattice. Figure Reflection is distributed evenly around the perimeter of the hexagon, indicating that incident atoms are deflected away from the hexagon center due to the relatively low force experienced here. Also due to the weak interaction at the hexagon center, it is the most probable location for transmission to occur. Thus, atoms incident upon or deflected toward this position are both able to penetrate. These results agree with those from a previous study , which fThe scattering of incident noble gas atoms has been investigated at high energies (keV), where transmitted particles were found to have very little angular deflection while leaving the graphene relatively unaffected . Here, sNmax normalizes the distribution, and the differential solid angle dΩ becomes 2π sin θ dθ due to the azimultal symmetry of the problem. N(θ ± Δθ/2) is the number of atoms scattered into a bin of width Δθ centered at polar angle θ.Here, 1/x- or y-components of an atom's linear momentum are much more visible for low incident energies, where these changes can be comparable to the initial momentum. In the SCC-DFTB simulations, atoms with such low incident energy tend to reflect when not adsorbed, and the reflected angular distribution shows much more scattering. Transmitting hydrogen atoms in these simulations tend to have higher incident energies, so the small x- or y-forces don't produce a significant angular displacement of their momenta. While atoms incident at 5 and 10 eV have a wider distribution than at the higher energies, they tend to penetrate only near the center, where the H-C interactions are weakest.Small changes in the l-h Equantity of the affected graphene. However, roughly a third of the incident atoms are found to bond to the surface after initially being reflected at a large angle relative to their initial momenta. These 'wandering' hydrogen atoms, primarily seen at 0.5 eV incidence, generally drift above the graphene surface at a distance of about 3 Å for 2 to 5 fs before falling toward a lattice carbon and adsorbing. Roughly 10% of these 'wanderers' do not bond to a carbon within the simulation time. Therefore, while they are counted as adsorbed in Figure The dominance of adsorption in SCC-DFTB simulations at impact energies below 1 eV provides enough statistical weight for an investigation of the effects of H-adsorption on the l-h Eby subtracting the energy of the highest occupied orbital from that of the lowest unoccupied orbital. The 1,000-K electronic temperature used creates a 'smearing' of the orbital occupations near the Fermi level. We use occupations of 1.8 for h  and 0.2 for l . This allows us to accomplish significant statistics while accounting for the different sites of adsorption and variety of vibrational states in which atom is adsorbed. The system is a 336-atom supercell, equivalent to an 18 × 18 × 1 k-point grid.The graphene band gap is often computed using a band structure or density of states calculation. However, the graphene system studied here is subject to thermal motion as well as bombardment, and the impinging particle should not be included in Brillouin zone integration. As discussed earlier, we simply define a quantity l-h Eover a number of time steps at the end of the simulation. When doing this time-averaging, it is important to avoid time steps at which some of the hydrogen atoms have not yet bound to the graphene surface. Figure z-position distribution averaged over 1, 4, 12, and 24 fs of simulation time. In all cases, the mean value is within a single standard deviation of 1.2 Å.Figure z-positions. As shown in Figure l-h Eon average.One can see that the standard deviation, representative of the distribution's width, is higher for low averaging times. Additionally, atoms incident with higher kinetic energy are adsorbed with greater vibrational energy, so they display a wider distribution of l-h Eis 171.5 meV for 0.1 eV incidence, 165.1 meV for 0.2 eV incidence, and 157.7 meV for 0.5 eV incidence  and high-energy (0.5 eV incidence) oscillations are 0.1 and 0.3 Å, respectively. The change in l-h Edecreases with increasing adsorbate distance, and the flattening observed below 1 Å causes the minimum position in oscillation to affect the gap less than the maximum position. Thus, larger oscillation amplitudes produce, on average, a smaller change in the l-h Equantity. These averages are shown as thick dashed lines and have a difference of 30 meV, which is on the order of the 14 meV difference between average changes induced by 0.1 and 0.5 eV bombardments.Figure In the classical molecular dynamics approach, the physical accuracy of the simulation is determined mainly by the quality of the interatomic potentials. Like its predecessor, the REBO potential, AIREBO is a member of the classical bond-order family of potentials ,21 of thcluster, i.e., no periodic boundary conditions. All three potentials display a well near 1.2 Å for incidence upon a lattice carbon, and the subsequent repulsive barriers agree well as they are all fit to ZBL [z = 0 for AIREBO and REBO, only 2 eV higher than the corresponding SCC-DFTB curve. REBO is consistently 2 to 5 eV more repulsive than DFTB, but qualitatively very similar. The most distinctive difference between these three potentials is their treatment of the graphene π-orbitals. Clearly, the AIREBO Lennard-Jones interactions coming from the six adjacent carbons produce a potential barrier at the graphene hexagon center that is more than 60 eV (525%) higher than the potential in its predecessor, which is in turn roughly 10 eV (380%) higher than DFTB. The REBO potentials clearly agree much more with the DFTB calculations than those of AIREBO, which indicates that the Lennard-Jones interactions which produce the observed potential barriers likely overestimate the hydrogen-graphene interaction.Figure t to ZBL . HoweverIto et al. ,30, Nakad Eis a carbon atom displacement energy from the rapheme, then the kinetic energy of the impact atom in the head-on binary collision is Another result of the increased repulsiveness of AIREBO is the occurrence of physical carbon sputtering upon impact of hydrogen. Figure x- or y-momentum, resulting in a cos(θ-θo) distribution with maximum at roughly 37°. Transmitting atoms also interact with the potential barriers on the opposite side of the surface, which are responsible for deflecting these atoms and producing the much wider distribution than observed in the SCC-DFTB calculations.Lastly, the CMD calculations result in larger angular scattering effects than SCC-DFTB, as can be seen in Figure l-h Equantity, analogous to the HOMO-LUMO gap in clusters, were investigated, predicting a notable change of the graphene electrical conductivity for even one H-atom chemisorbed. Adsorption was found to be the dominant process below 1 eV, with transmission dominating above 20 eV and reflection dominating at the intermediate energies. Reflection was found to have a more significant scattering effect than transmission.Understanding the effects of irradiation is paramount in developing graphene-based nanosensors and nanoelectronics. Thus, in this work, simulations of single-layer graphene bombarded by hydrogen atoms for a wide range of incident energies were carried out using quantum-classical molecular dynamics based on the self-consistent-charge-density functional tight binding method for treatment of the electron dynamics, combined with classical dynamics of the nuclei. The effects of this bombardment on the graphene sheet and the scattered particle distributions were analyzed in terms of reflection, transmission, and adsorption probabilities and angular distributions. Particularly significant effects of adsorption on the graphene A comparison between results of the SCC-DFTB simulations and classical MD simulations employing the AIREBO potential was made, showing a significant difference in the calculated probabilities and chemistry, mainly caused by differences in the multibody potentials. The AIREBO H-graphene potential overestimates (in comparison to SCC-DFTB) the interaction at the hexagon center  and C-C bond center  lattice positions. A comparison of REBO and AIREBO showed that the overestimate is a result of the Lennard-Jones terms in AIREBO. The effect of this added repulsiveness permeated all of the dynamics, producing wider scattering, a much smaller adsorption probability, and nonzero sputtering yields. Refitting of these terms may significantly improve the accuracy of AIREBO.l-h Equantity, qualitatively associated to the H-L gap and electric conductance of graphene, were found to depend on incident atom energy. Using an averaging time of 32 fs, in addition to averaging over all adsorbed trajectories, the adsorption effect on the l-h Ediffered by roughly 10 meV between incident energies. By virtue of higher vibrational energy, larger incident kinetic energies are found to have a smaller effect on the band gap, as shown in Figure l-h Echanges and/or adsorbed vibrational modes could support the application of graphene in a hypersensitive slow single-particle detector in agreement with the sensitivity to a single biomolecule being coupled to a graphene sheet [l-h Equantity to hydrogen adsorption indicates that the functionality of graphene-based nanoelectronics could be adversely affected by the irradiation by light, chemically reactive species.Changes in the graphene ne sheet . This hyThe authors declare that they have no competing interests.RCE carried out computations, and analyzed results with PSK and JD. RCE and PSK prepared the manuscript, which was finalized together with JD, JJ, and PRCK. JJ provided help in using DFTB. All authors read and approved the final manuscript.


The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure. 2+ ions in isolation ► Nucleotide substitution in the G•A pairs of the k-turn prevent folding in isolation, but some can be stabilized in the context of the riboswitch, allowing binding of SAM ligand ► The crystal structure of a SAM-I riboswitch with a variant k-turn provides the first example of a k-turn with an A•A pair at the 2b•2n position► The folded conformation of a kink turn is stabilized by long-range tertiary interactions in RNA ► The SAM-I riboswitch contains a k-turn, that folds in the presence of Mg Both subunits of the ribosome contain multiple k-turns , and k-tg/kturn/ .trans sugar edge-Hoogsteen edge base pairs that direct the minor groove edges of the two adenine bases toward the minor groove of the C helix. Intimate A-minor interactions and a number of specific hydrogen bonds , and on its 5′ side by regular base pairing (the C helix) A and 1B.en bonds  stabiliz2+ ions induces folding into the structure observed in the crystal in a noncooperative two-state process with a midpoint of the order of 100 μM  have beIn principle, a third process might lead to stabilization of the kinked conformation of the k-turn. The sharp change in helical trajectory at the turn is frequently used to create long-range architecture in large RNA molecules. Conversely, tertiary interactions in such species might stabilize the kinked geometry of the k-turn, so that in a sense the RNA structure forces the k-turn to adopt its folded conformation. We therefore set out to investigate this possibility, and for this purpose we chose the SAM riboswitch as a model system.cis-acting elements occurring in mRNA of principally prokaryotic organisms, that bind small molecules in a manner that leads to metabolic autoregulation      are supee: 3cc2) , with ane: 3npb)  and T. t2+-induced folding of the k-turn of the SAM-I riboswitch as an isolated species. For this purpose, we constructed an RNA duplex of 25 bp with a central sequence corresponding to the riboswitch k-turn. In order to study this by fluorescence resonance energy transfer (FRET), fluorescein (donor) and Cy3 (acceptor) fluorophores were attached to the 5′ termini (We have studied the possible Mg termini A. FoldinEFRET) was measured by steady-state fluorimetry, using the acceptor normalization method  resulted in no detectable evolution of heat on addition SAM B. EvidenKd = 0.31 ± 0.06 μM is similar to that of the natural riboswitch. The conclusion that emerges is that the G2nA substitution that prevented the folding of the isolated k-turn is tolerated in the context of the riboswitch, indicating that the riboswitch structure has stabilized the variant k-turn that was not capable of folding in isolation. The riboswitch carrying a G3nU substitution in its k-turn also binds SAM exothermically . The same modification was introduced into the complete riboswitch, which was titrated with SAM C. In conrmically D, with trmically . Thus thG° = −33 ± 0.4 kJ mol-1 , with an rmsd of 0.53 Å B. Thus, n region C shows te k-turn  are pres network . The for network .We have also solved the crystal structure for the SAM-I riboswitch in which the k-turn contains the double G2nU, G3nU substitution. As with the G2nA, this intrinsically unstable k-turn is stabilized in the context of the riboswitch. The crystals diffracted to 2.9 Å, but due to low completeness between 2.9 and 3.0 Å the data set is regarded as 3 Å resolution. The overall fold and structure of the riboswitch is again maintained A, with a2+ ions in free solution, but folding is prevented by sequence substitutions that disrupt the G⋅A pairs at the 1b⋅1n and 2b⋅2n positions. In all respects, the behavior of the SAM-I riboswitch k-turn is closely similar to that of a standard k-turn such as H. marismortui Kt-7.The SAM-I riboswitch contains a near-standard k-turn that plays an important role in the architecture of the functional riboswitch. The axial kink of the k-turn generates the correct trajectory of helix P2b that permits its terminal hairpin loop to make a loop-loop interaction with an acceptor in helix P4. Inserted into a simple helix, the k-turn folds normally on addition of MgWe observed that the substitutions G2nA  and G2nU, G3nU (disrupting both the 2b⋅2n and 3b⋅3n pairs) prevented significant folding into the kinked structure in the isolated helical RNA. Yet, those same substitutions within the SAM-I riboswitch did not prevent SAM binding. This suggested that in the context of the more complex RNA structure, the k-turn could fold despite the disruption of the 2b⋅2n and 3b⋅3n pairs. This was confirmed more directly by solving the X-ray crystal structures of the two variant riboswitches, revealing that the modified k-turn adopts a standard k-turn fold in both cases. Thus sequence substitutions that prevent metal ion-induced folding of the isolated RNA do not prevent folding within the larger structure of the riboswitch. Evidently, the tertiary interactions in the riboswitch “force” the impaired k-turns to fold. There is likely to be some overall stabilization resulting from binding the SAM ligand to the riboswitch. But since this occurs at a site that is remote from the k-turn or loop-loop interaction the stabilization of the k-turn must be mediated through the tertiary structure of the RNA. The overall free energy of riboswitch folding is sufficient to stabilize some compromised k-turn structures. By contrast, the A1nC- or A1nU-substituted riboswitches did not bind SAM, indicating that these variants could not be induced to fold by the overall RNA structure. Disruption of the 1b⋅1n is clearly more destabilizing, such that the free energy of riboswitch folding is insufficient to overcome the destabilization of the k-turn in this case. Further support for the stabilization of the k-turn in the context of the riboswitch is provided by NAIM experiments of H. marismortui Kt-7. Unfortunately the resolution of the G2nU, G3nU riboswitch is insufficient to be confident about the interactions stabilizing the core of that structure. However, the positions of all the nucleobases of the G2nA riboswitch are very well defined in that structure, and so the hydrogen bonding can be identified with good confidence. All the standard, well-conserved hydrogen bonds are present, and the adenine nucleobases at the 1n and 2b positions are placed correctly so that A-minor interactions between the minor groove edges of the C and NC helices are formed. The variant sequence forms an A⋅A pair at the 2b⋅2n position, held in place by a number of hydrogen bonds, and this study provides the first crystallographic structure of a standard k-turn with an A⋅A base pair at this position. Thus the A⋅A pair is evidently not sufficient to permit the stable formation of this k-turn in isolation, yet the k-turn can fold when stabilized by the tertiary interactions. Interestingly, an A⋅A pair occurs naturally at the 2b⋅2n position of the sequence of T. solenopsae Kt-23 in the small ribosomal subunit, and can be induced to fold by the binding of L7Ae protein. At present there is no structure available for this k-turn, but a crystallographic study of this RNA is underway in this laboratory, and it will be interesting to see if the structure formed in Kt-23 is the same as that observed here. In principle, an A⋅U pair could form at the 2b⋅2n position of the G2nU, G3nU variant, but we cannot be sure that it does so on the basis of the present structure for that riboswitch. In some natural k-turn sequences, the usual G at the 2n position is replaced by U, exemplified by Kt-23 of the T. thermophilus 16S rRNA. In that case, U2n forms a reverse Hoogsteen A⋅U pair with the A2b  were measured using the acceptor normalization method  at 4°C using an SLM-Aminco 8100 fluorimeter. Values of FRET efficiency  as described by 3gx5  and BM-14 (G2nA) at the European Synchrotron Radiation Facility in Grenoble, France. The structure of G2nU, G3nU was solved by performing a rigid body refinement using REFMAC5  with the3gx5  as a preFuller experimental details are presented in the


Idiopathic pulmonary fibrosis (IPF), the most prevalent idiopathic interstitial pneumonia, is associated with a poor prognosis. An accurate diagnosis of IPF is essential for optimal management. The recent ATS/ERS/JRS/ALAT recommendations on the diagnosis and management of IPF were developed from a systematic review of the published literature. High-resolution computed tomography (HRCT) scanning has a central role in the IPF diagnostic pathway with formal designation of criteria for an HRCT pattern of UIP. In the correct clinical context, a UIP pattern on HRCT is indicative of a definite diagnosis of IPF without the need for a surgical lung biopsy. However, although the 2011 ATS/ERS/JRS/ALAT statement is a major advance, the application of guideline recommendations by clinicians has identified limitations that need to be addressed in future statements. Key problems include: 1) the lack of management recommendations for the highly prevalent clinical scenarios of probable and possible IPF; 2) the ongoing confusion about the diagnostic role of bronchoalveolar lavage (reflecting ambiguity in the current recommendation); 3) HRCT misdiagnosis by less experienced radiologists, increasingly recognised as a major problem; and 4) the lack of integration of clinical data, including the treated course of disease, in the designation of the diagnostic likelihood of IPF. Idiopathic Interstitial Pneumonias (IIPs) make up a heterogeneous group of diseases, which are collectively included in the umbrella term “Interstitial Lung Diseases (ILDs)” . In 2002IPF is defined as a specific form of chronic, progressively fibrosing IIP, occurring primarily in older adults, limited to the lungs, and associated with the histopathological and/or radiologic pattern of unspecified interstitial pneumonia (UIP) . IPF is An IPF diagnosis is based the absence of a known cause of lung fibrosis, computed tomography (CT) findings and, in cases with CT abnormalities that are not classical for IPF, the use of pathological criteria. Diagnostic uncertainties, partly due to the multiple different ways in which physicians approach IPF  along with the variability in the natural history of disease and in HRCT appearances; and the lack of a validated algorithm for excluding known causes of lung fibrosis all contribute to the inherent confusion surrounding this topic.The lack of a known cause of lung fibrosis represents a key factor in the diagnostic process. Even when a surgical lung biopsy (SLB) reveals a histopathological pattern of UIP, a definitive diagnosis required the exclusion of other known causes of ILD, including collagen vascular disease, drug toxicity, and various environmental exposures Table  1,5]. I,5. I1,5]The updated evidence-based guidelines for diagnosis and management of IPF were devised in 2011 by the collaborative effort of the American Thoracic Society, the European Respiratory Society, the Japanese Respiratory Society, and the Latin American Thoracic Association. The main objective was to provide recommendations on the diagnosis and management of IPF, based on a thorough review of the evidence published to date, and the use of expert opinion only when the evidence base is inadequate.The diagnostic criteria from 2000 indicate that in the correct clinical context, the finding of a UIP pattern on a high resolution computed tomography (HRCT) scan may be sufficient to diagnose IPF, without the need to perform SLB . This coThe ATS/ERS/JRS/ALAT 2011 guidelines clearly state the precise HRCT features that meet the criteria for ‘‘UIP’’, ‘‘possible UIP’’ and ‘‘inconsistent with UIP’’ patterns Tables  &4 [2]. A multidisciplinary, dynamic approach, with the input of clinicians, radiologists, and pathologists has been shown to improve diagnostic accuracy ,8 and isA key problem in applying the updated guidelines on management is related to the designation of IPF as “definite”, “probable” or “possible” Tables  &4 2]. . 2]. On The value of bronchoalveolar lavage (BAL) in the diagnosis of IPF is contentious. In patients with suspected IPF, the most important application of BAL is to increase the index of suspicion for alternative disorders, including HP and NSIP, by the demonstration of a lymphocytosis >30%. The ATS/ERS/JRS/ALAT 2011 guidelines make a weak negative recommendation that BAL cellular analysis should not be performed in the diagnostic evaluation of IPF in the majority of patients, but may be appropriate in a minority of patients [However, feedback from European practitioners indicates that this recommendation has caused a good deal of confusion as it can be applied to either of two very separate scenarios. At the time of BAL, a HRCT scan has almost always been performed. If HRCT features are classical for IPF, and the clinical context is correct, the patient does not have “suspected IPF” but “definite IPF”. If the HRCT features are not classical for IPF, but IPF is suspected, the diagnostic overlap includes IPF/NSIP, IPF/HP, IPF/sarcoidosis and IPF/unclassifiable. It is not helpful to combine these two scenarios in a single recommendation. Two independent statements are required. Thus, some clinicians take the recommendation to apply to patients, with clinical and HRCT features typical of IPF on HRCT (“definite IPF”). Others assume that the recommendation does indeed apply to “suspected IPF”, in which, by definition, the likelihood of IPF is probable or possible but realistic differential diagnoses exist, usually of HP or NSIP. These two scenarios differ radically.Regarding the first scenario , it is likely that most clinicians would agree that an additional diagnostic test is not required. As occasional patients have uncertain exposures, which might or might not be relevant to a diagnosis of HP, a weak negative recommendation for the use of BAL, reserved for those patients, seems appropriate. However, even this apparently logical conclusion can be questioned. In a study by Ohshimo et al., of the diagnostic significance of a BAL lymphocytosis when the clinical and HRCT presentation is typical of IPF , a BAL lBy contrast, when the diagnosis of IPF is “suspected” and, thus, by definition, uncertain, most European clinicians would favour the performance of BAL, with findings expected to radically alter the likelihood of alternative diagnoses. This view was endorsed by formal voting in a large European-wide meeting of clinicians with expertise in IPF and flies directly in the face of the ATS/ERS/JRS/ALAT 2011 recommendation. This is likely to reflect the fact that the ATS/ERS/JRS/ALAT recommendation covered two such dissimilar clinical scenarios with a single recommendation, although cultural differences between Europe and the USA in the performance of BAL might also have played an important role. However, irrespective of the source of confusion, there is a perceived need for separate recommendations to address the two distinct clinical scenarios, as in the British Thoracic Society (BTS) guidelines on the diagnosis and management of interstitial lung disease. In the BTS guidelines, the diagnostic use of BAL was reserved for patients in whom the diagnosis is uncertain after clinical assessment and HRCT scanning, with the case by case exclusion of patients with severe disease or because a biopsy will ultimately be performed [Some radiologists continue to perform sub-optimal CT scans, using historical thick section protocols (rather than high resolution CT), a practice that needs to be eradicated in the diagnostic evaluation of interstitial lung disease. This issue aside, expert HRCT  interpretation is central to the diagnosis of IPF and requires the identification of honeycombing. In this regard, there are two issues of major concern: the distinction between honeycombing and traction bronchiectasis, and the distinction between honeycombing and the admixture of emphysema and fibrosis. Both distinctions can be difficult, even for an experienced observer. The accuracy of a trained observer in discriminating between ILDs appears to be around 80-90% -14. HoweThe under-diagnosis of IPF, albeit a less prevalent problem, is also important as a confident diagnosis of IPF is made by HRCT evaluation in, at most, two thirds of IPF patients ,15. In aClearly, training in applying guideline recommendations in the diagnosis of ILD/IPF should be directed not only to radiologists but also to pathologists and clinicians. Inter-obsever variation is a significant problem in histopathologic diagnosis. Multidisciplinary diagnosis is a relatively recent phenomenon in interstitial lung disease and there is a need for clinician training in integrating and reconciling data to distil a final accurate clinical/radiological/histologic diagnosis.The principle of integrating HRCT and histopathologic data in assigning a diagnostic likelihood of IPF is an admirable feature of the 2011 ATS/ERS/JRS/ALAT statement, which may well be helpful in the majority of cases and sets an important precedent for future statements. However, a problem arises in an important subgroup of patients. Sometimes, HRCT appearances are apparently incompatible with IPF but the treated disease course is classical for IPF, with inexorable progression despite therapy. Often, in this setting, a SLB reveals histologic abnormalities that are absolutely typical of IPF. Using the schema advanced in the ATS/ERS/JRS/ALAT 2011 statement, such patients cannot be diagnosed as having definite or probable IPF but must be classified as having possible IPF. This is a deeply unsatisfactory state of affairs. A SLB is often performed in these cases because the treated course of disease is typical of IPF [A deficiency common to all guideline statements is the absence of pragmatic guidance on the level of risk of SLB in individual patients. A clearer separation between patients who can undergo a SLB with an acceptably low risk and those in whom SLB should be avoided would be helpful to clinicians. The presence of major co-morbidity is an obvious contraindication to SLB but the level of severity of IPF associated with a significant increase in post-operative mortality has not been sufficiently addressed. It is acknowledged that in the absence of definitive data, broad statements based on indirect evidence might be required. However, guidance on this point, even based solely on expert opinion, would be extremely useful as a means of facilitating patient participation in decisions based on “risk-benefit” thinking.The evidence-based ATS/ERS/JRS/ALAT 2011 guideline recommendations have assisted clinicians in making a diagnosis and subsequent treatment decisions for the clinical management of IPF. The guidelines are a major improvement on the previous statements of the committee that were published in the early 2000s, with an impressive review of the literature and a disciplined approach to a previously undisciplined field. However, in common with all such statements, guidance mainly focuses on definite IPF, most often diagnosed from typical HRCT appearances. The definition of probable and possible IPF is also an advance, but the failure to provide “intention to manage” guidance on these highly prevalent clinical scenarios is a major limitation.Other aspects of the diagnostic guidelines would merit review, based on problems that have become increasingly apparent as recommendations have been applied. The BAL recommendation has caused confusion due to the use of a single recommendation to cover two radically different clinical scenarios. The over-diagnosis of IPF on HRCT appears to be widespread, with urgent work needed on the standardisation of HRCT diagnosis by expert practitioners. The integration of HRCT and histologic data in the assigning of diagnostic likelihood is more often helpful than not, but does not meet the needs of some patients in whom a multidisciplinary diagnosis of definite IPF should be made, based on the treated course of disease and typical biopsy appearances, even when HRCT appearances are non-diagnostic or suggestive of an alternative diagnosis. As with any other disease, these issues highlight the continued need for updating the guidelines, including diagnostic and therapeutic aspects, based upon the limitations that become evident as recommendations are applied.Pending revision by expert groups, awareness of the flaws in the current IPF guidelines should alert clinicians to the need to reject or modify guideline recommendations in individual patients, provided that these decisions can be defended at multidisciplinary review.Athol U. Wells has received consultancy fees from Actelion, Centricorps, Gilead and Intermune and has also received fees for participating as a speaker in symposia sponsored by Actelion and Intermune.The author thanks C. Trenam, I. Mandic and M. Smith of IntraMed Communications for editorial assistance in the preparation of the manuscript. Development of this article was supported by InterMune AG.


One of the variables which interfere in the maintenance of sterility in the materials is the storage. There are many official and not-official recommendations for temperature and air humidity control in the storage area, however without any theoretical or experimental basis. Some hospitals do not have a system that makes the control of temperature and humidity possible, and, in most cases, the sterilizing chambers are next to the storage area, constantly releasing heat and water vapour. Considering that one of the functions of the wraps is to maintain the sterility of the content, even under adverse conditions, the recommendations to have an environmental control in the storage area, a priori, would have a secondary importance. For these reason the doubt about the real importance of the temperature and humidity in the contamination of the materials stored after the autoclave sterilization arise.Serratia marcescens 106and stored in an environment with temperature around 35°C and air humidity around 75%. This group was compared with another group, the negative control, stored in temperature around 20 °C and air humidity around 60%. After a period of 30 days of storage, the carriers were removed from the boxes and incubated.Therefore, an experiment was developed in which boxes with surgical instruments and cylinders carriers were packed in cotton sheets, crepe paper, SMS and surgical-grade paper, sterilized, and intentionally contaminated externally with No bacterial growth was detected in any of the samples.The experiment results allowed concluding that the high temperature and high air humidity do not interfere in the barrier efficiency of the packs.None declared.


Graphene grown on crystalline metal surfaces is a good candidate to act as a buffer layer between the metal and organic molecules that are deposited on top, because it offers the possibility to control the interaction between the substrate and the molecules. High-resolution angular-resolved ultraviolet photo electron spectroscopy (ARPES) is used to determine the interaction states of iron phthalocyanine molecules that are adsorbed onto graphene on Ni(111). The iron phthalocyanine deposition induces a quenching of the Ni d surface minority band and the appearance of an interface state on graphene/Ni(111). The results have been compared to the deposition of iron phthalocyanine on graphene/Ir(111), for which a higher decoupling of the organic molecule from the underlying metal is exerted by the graphene buffer layer. The interest in the preparation of ordered layers of organic molecules is rapidly growing, because of the possibility to realize advanced electronic- and spin-based devices –3. TransThe magnetic and electronic properties of the adsorbed molecules may be strongly influenced by the interface and can be potentially tuned by using an appropriate buffer layer. Graphene (Gr), thanks to its unique electronic properties  and to t−10 mbar range at the LOTUS laboratory of the Università La Sapienza (Roma). The Ni(111) single crystal was cleaned by several sputtering–annealing cycles . Graphene was obtained by exposing the sample, which was kept slightly below 600 °C to 6000 L of ethylene (1 L = 10−6 torr·s). The formation of graphene on the Ni(111) surface is complicated by the segregation of carbon from the bulk, because of the high solubility of carbon in Ni [+ for 30 min, 1200 °C for 60 s). The preparation of graphene was done by several 120-seconds long exposures to ethylene while flash-heating the sample up to 1100 °C.Experiments were performed in situ in ultra-high-vacuum (UHV) chambers with base pressures in the low 10on in Ni –20. The The deposition of FePc was carried out by using a custom-made quartz crucible and the molecular deposition was controlled by using a quartz microbalance. One single-layer (SL) is defined as the molecular density of flat molecules fully covering the graphene layer, and it corresponds to a nominal thickness of about 3.4 Å.α (21.218 eV) and HeIIα (40.814 eV), was provided by a SCIENTA VUV-5050 monochromatic source.Low energy electron diffraction (LEED) was used to check the symmetry of both clean and Gr-covered surfaces. LEED was performed in the energy range of the primary beam of 90–140 eV. High-resolution angular-resolved photoelectron spectroscopy data was carried out by using a SCIENTA SES-200 analyzer with an angular acceptance of ±8° and a resolution of 16 meV. All spectra have been taken along the ΓK direction. The UV radiation, HeIThe ARPES band structure of the Gr/Ni(111) and Gr/Ir(111) systems along the ΓK direction of the two-dimensional (2D) Brillouin Zone (BZ) is presented in * states with the Ni d bands [The presence of a distinct single π band for both Gr sheets (on Ni and on Ir) reveals the single-layer nature of graphene. For Gr/Ir111), the Dirac point is localized on a projected bulk band gap and the graphene π band looks very similar to the band of free-standing graphene, the linear dispersion of the π band is preserved close to the K point with only a slight p-doping, which is in agreement with the literature –13. The 1, the Di d bands .The photoemission data in the low binding energy region for the iron phthalocyanine molecules deposited on the Gr/Ni surface, and on Gr/Ir for comparison, as a function of the thickness of the FePc layer is shown in In order to better understand the nature of the interaction, we also analyze data at the K point of the BZ, as shown in When used as a buffer layer between an organic molecule and a metal surface, graphene plays a different role in the molecule–metal interaction that depends on the interaction of graphene with the metal substrate. Graphene on Ni(111) reveals a strong interaction with the substrate and strong alteration of the ideal graphene π band. After deposition of small amounts of FePc molecules, by means of high-resolution UV photoemission we give direct experimental evidence of an interaction of the molecule with Ni through graphene, as shown by the emerging of an interface state at about 0.3 eV binding energy in normal emission and by the quenching of the Gr–Ni π−d hybrid state at the K point of the BZ.


Use of Hirshfeld surfaces calculated from crystal structure determinations on various transition metal ion complexes of three terpyridine ligands carrying trimethoxyphenyl substituents has enabled an assessment of the contribution made by the ligand components to the interactions determining the lattice structures, interactions expected also to be present in metallomesogens derived from similar ligands. The form of the link joining the trimethoxyphenyl substituent to the 4′ position of 2,2′;6′,2″-terpyridine is of some importance. In the case of the Co(II) complexes of two of the ligands, their spin-crossover characteristics can be rationalised in terms of the different interactions seen in their lattices. Thus, there is again minimal evidence for a significant role of aromatic–aromatic, specifically phenyl–pyridyl, interactions in defining the lattice of these complexes, although the formation of bridges via π-type interactions between acetonitrile pairs and pyridyl rings is a feature of all three structures . Examination of the three structures using CrystalExplorer shows that the role of the trimethoxyphenyl groups in lattice construction is largely associated with the methoxy groups, principally through O…π interactions and H-bond acceptance by the oxygen atoms and H-bond donation, including CH…π, by the methyl groups, some examples of these interactions being shown for [Fe(L1)2](ClO4)2·CH3CN in Given that the metal ions are encased in a large ligand sheath and that perchlorate and tetrafluoroborate are tetrahedral anions of similar size, it is unsurprising that [Fe(L2 produces a lattice of [Co(L2)2](BF4)2·H2O which is very similar to those just described for L1 complexes. The space group is again P-1 and the shortest Co…Co separation of 9.177(3) Å, significantly shorter than that of [Co(L1)2](BF4)2·CH3CN·0.5H2O, is again found in pairs (isolated in this case) linked by aromatic-CH…FBF…HC-aromatic bridges, here coupled with aromatic-CH…O(water)…FBF…HC-aromatic bridges, with no “terpyridine embrace” evident. Viewed down a, the lattice has a very similar appearance to that of [Co(L1)2](BF4)2·CH3CN·0.5H2O in that sheets can be seen edge-on, sheets in which cation chains lie side by side, very much as seen in various complexes of terpyridine with simple polyphenyl substituents [L2)2](BF4)2·H2O, these sheets lie parallel to the (0 1 2) plane and the chains  within these sheets (L2)2](BF4)2·H2O, where heating and cooling cycles produce continuous changes (L1)2](BF4)2·CH3CN·0.5H2O (ClO4)2·CH3CN·Et2O has a somewhat different composition to its Co(II) analog, it also crystallises in space group P-1 and the lattice is very similar to that just described, except that the cation sheets lie parallel to the (0 1 −1) plane. The shortest Cd…Cd separation is 9.73(2) Å, but there are other pairs with a separation of 9.90(2) Å and in fact all the cation pairs here can be considered part of a chain with alternating links of the slightly different distances resulting from bridging by differently oriented perchlorate anions. In the sheets parallel to (0 1 −1), it is again possible to discern chains of cations with overlapping aromatic units, here with Cd…Cd alternating between 15.51(3) and 15.56(3) Å, but what is striking is that there is now no indication of stacking interactions involving the triple-bond carbon atoms (their separations being ~0.15 Å greater than in the Co(II) complex). There are various contacts indicative of π-type interactions but only some of these involve the facing aromatics (While the complex [Cd(romatics  and onceL3)2](ClO4)2·CH3CN, once again belonging to space group P-1, can be considered as composed of slightly undulating sheets of cations lying parallel to the bc plane and separated by layers containing the acetonitrile molecules and perchlorate anions. 2](ClO4)2·CH3CN and [Ni(L1)2](ClO4)2·CH3CN, with no indication of any of the π-type interactions seen in those cases (and in the lattice of [Co(L1)2](BF4)2·CH3CN·0.5H2O), confirming thus their secondary importance. A possibly significant feature of the hydroxymethylene link between the aromatic units of L3 is that it appears to allow not only bending of the mean plane of the ligand, as seen for one ligand of the two on a metal ion in all the present cases (an asymmetry noted in various related systems [L3)2](ClO4)2·CH3CN that this additional flexibility may explain is the retention of a vestigial “embrace” array (The lattice of [Cu( anions.  Viewed dL3)2]ClO2·CH3CN,  systems ) but alse” array , althougL3)2](BF4)2 was determined at two temperatures (100(2) and 294(2) K) corresponding, on the basis of magnetic susceptibility measurements (see ahead), to low- and high-spin species, respectively. The same space group, P-1, was retained at both temperatures, so the spin state change is not complicated by phase changes. The change in size of the Co(II) centre due to the change in spin state is accommodated in bond length changes (L3)2](BF4)2 all major cation–anion interactions via aromatic-CH…F bonding are retained and it is essentially all π-type interactions (including O…π) which are lost or weakened. Thus, the only C…C interaction apparent is one still involving C43 but now with C14′ (C43…C14′ 3.32(6) Å) for cations where Co…Co is 10.22(3) Å. The rings involved are not parallel.The crystal structure of [Co2](BF4)2 proved to be rather different to those of [Co(L1)2](BF4)2·CH3CN·0.5H2O, with a spin state change occurring quite abruptly, though without any hysteresis (L3)2](BF4)2 may, as we have suggested for various related systems, [L3 compared to L1 noted above and also the lack of the rather unusual specific interactions involving lattice acetonitrile seen in [Co(L1)2](BF4)2·CH3CN·0.5H2O. The fact that there are numerous points of interaction possible for cation and anion and thus that stabilisation can be achieved in multiple arrangements may be a general factor influencing spin state changes in all the Co(II) complexes presently considered.Somewhat unexpectedly, the magnetic properties of terpyridine-4′-ol , 5-(bromomethyl)-1,2,3-trimethoxybenzene , and K2CO3  in dry DMF (50 mL) was heated at 100 °C for 12 h. After cooling to room temperature, the precipitate was filtered out and the solvent was removed under reduced pressure. The residue was subjected to chromatography on silica using ethylacetate/hexane  eluent to give the desired product as a pale yellow powder. Yield, 0.32 g, 37%. 1H NMR : δ = 8.74 , 8.68 , 8.18 , 7.92 , 7.40 , 7.37 , 7.06 , 5.36 , 3.95 , 3.93 .Ligand L1)2](BF4)2·CH3CN·0.5H2O [L1 only.All metal ion complexes were prepared and crystallised for structure determinations by methods essentially identical to those used for [Co(L1)2](ClO4)2·CH3CN: L1  was dissolved in hot CH3CN (10 mL) and Fe(ClO4)2·H2O  in CH3CN (5 mL) slowly added. The mixture was stirred for 2 h at 60 °C, then evaporated to dryness under reduced pressure. The purple residue was washed with cold MeOH and dried in air. Yield: 0.11 g. Vapour diffusion of diethyl ether into an acetonitrile solution of the product gave purple crystals suitable for an X-ray structure determination. 1H NMR : δ = 9.18 , 8.71 , 7.97 , 7.58 , 7.23 , 7.14 , 7.12 , 4.15 , 3.95 . Anal. Calcd. for C50H45Cl2FeN7O14: C, 54.86; H, 4.14; N, 8.96. Found: C, 54.80; H, 4.16; N, 9.01%.[Fe(L1)2](ClO4)2·CH3CN: Substitution of Ni(ClO4)2.6H2O  for Fe(ClO4)2·H2O in the above procedure gave a pale yellow powder. Yield: 0.10 g. Anal. Calcd. for C50H45Cl2NiN7O14: C, 54.72; H, 4.13; N, 8.93. Found: C, 54.76; H, 4.12; N, 8.90%.[Ni(L2)2](BF4)2·H2O: Use of L2  and Co(BF4)2.6H2O  gave a deep orange powder, Yield: 0.12 g. Anal. Calcd. for C52H44B2CoF8N6O7: C, 56.91; H, 4.04; N, 7.66. Found: C, 56.95; H, 4.01; N, 7.60%.[Co(L2)2](ClO4)2·CH3CN·Et2O: Use of L2  and Cd(ClO4)2.H2O  gave a pale yellow powder. Yield: 0.09 g. 1H NMR : δ = 8.82 , 8.65 , 8.27 , 8.13 , 7.55 , 7.53 , 7.08 , 3.94 , 3.83 . Anal. Calcd. for C58H55CdCl2N7O15: C, 54.71; H, 4.35; N, 7.70. Found: C, 54.80; H, 4.26; N, 7.75%.[Cd(L3)2](BF4)2: Use of L3  and Co(BF4)2·6H2O  gave an orange powder. Yield: 0.11 g. Anal. Calcd. for C52H48B2CoF8N4O8: C, 57.32; H, 4.44; N, 5.14. Found: C, 56.98; H, 4.41; N, 5.10%.[Co(L3)2](ClO4)2·CH3CN: Use of L3  and Cu(ClO4)2·6H2O  gave a green powder. Yield: 0.12 g. Anal. Calcd. for C54H51Cl2CuN5O16: C, 55.89; H, 4.43; N, 6.03. Found: C, 56.01; H, 4.39; N, 6.11%.[Cu(3.3.L1)2](ClO4)2·CH3CN, crystals were coated with Paratone-N oil and the diffraction data measured at temperatures between 100 and 294 K depending on the particular species with synchrotron radiation  on an ADSC Quantum-210 detector at 2D SMC with a silicon (111) double crystal monochromator (DCM) at the Pohang Accelerator Laboratory, Korea. The ADSC Q210 ADX program [L1)2](ClO4)2·CH3CN, data were collected at 173(2) K using graphite-monochromated MoKα radiation with a CCD area detector on a Bruker APEX2 diffractometer [F2 using SHELXL-97 [viawww.ccdc.cam.ac.uk/data_request/cif by citing CCDC 955464–955470.For all complexes other than [Fe( program  was used program  was usedctometer . The strctometer  and refiHELXL-97 . The pos4.viz., interactions of the aromatic–CH…base type, should generally be considered more important influences upon the lattice structures than those arising from any face-to-face contacts of aromatic groups within separate cations. This is consistent with known theoretical calculations for systems even where there are such contacts giving rise to the terpyridine embrace [The use of Hirshfeld surfaces calculated from single-crystal X-ray structure determinations is a convenient means of identifying labile interactions in metal complexes of functionalised terpyridine ligands. It shows that peripheral interactions of aromatic groups in cationic complexes with their counteranions,  embrace ,9. It is


The intercalated and the derived exfoliated OMMT have been deeply characterized, mainly by X-ray diffraction analyses. Monolithic composite aerogels, with large amounts of both intercalated and exfoliated OMMT and including the nanoporous-crystalline δ form of syndiotactic polystyrene (s-PS), have been prepared, by scCO2 extractions of s-PS-based gels. Also for high OMMT content, the gel and aerogel preparation procedures occur without re-aggregation of the exfoliated clay, which is instead observed for other kinds of polymer processing. Aerogels with the exfoliated OMMT have more even dispersion of the clay layers, higher elastic modulus and larger surface area than aerogels with the intercalated OMMT. Extremely light materials with relevant transport properties could be prepared. Moreover, s-PS-based aerogels with exfoliated OMMT could be helpful for the handling of exfoliated clay minerals.Supercritical carbon dioxide (scCO Over the last decades, clay polymer nanocomposites (CPN)  constitute effective ways to increase dispersion and delamination in polymer/clay nanocomposites  can be achieved by scCO2 treatments on pure organoclays, where alkali counterions have been exchanged with long-chain alkylammoniums  -dimethylammonium (2HT) was purchased from Laviosa Chimica Mineraria S.p.A.13C nuclear magnetic resonance characterization showed that the content of syndiotactic triads , used for clay dispersions, was purchased from Aldrich and used without further purification.2 at 40°C and 200 bar, for 16 and 32 h under quiescent conditions, in order to promote the diffusion of the supercritical fluid into the clay interlayer space. The system was then rapidly depressurized (1 min) to atmospheric pressure and the expansion of the scCO2 between the layers caused the clay exfoliation.Exfoliated clay samples were obtained by using a SFX 200 supercritical carbon dioxide extractor (ISCO Inc.). Organically modified clays  were processed in scCODispersions of the clays in DCB were obtained with both as received and exfoliated samples. A clay dispersion was initially prepared by adding the appropriate clay amount in 5 ml of DCB. The mixtures were homogenized for 1 h under magnetic stirring and sonicated in a 5000 mL batch bath ultrasound (Badelin Sonorex RK 1028 H) for 1 h.sPS/clay gels were prepared, in hermetically sealed test tubes, by heating the clay dispersions above the boiling point of the solvent until complete dissolution of the polymer and the appearance of a transparent and homogeneous solution had occurred. The hot solution was then cooled to room temperature, where gelation occurred. For instance, 655 mg of sPS and 5 mL of 2 wt % clay dispersion were mixed to obtain clay/polymer gels. The overall amount of polymer and clay in the gels was generally fixed to 10 wt %.T = 40°C, P = 200 bar, extraction time t = 6 h. The prepared s-PS/clay aerogels present a weight composition ranging between 96/4 and 50/50. The aerogels, as prepared from gels with an overall polymer-clay content of 10 wt %, present a porosity close to 90%.Aerogels were obtained by treating sPS/clay gels with a SFX 200 supercritical carbon dioxide extractor (ISCO Inc.) using the following conditions: P of the aerogel samples can be expressed as:For monolithic aerogels with a regular cylindrical shape, the total porosity can be estimated from the mass/volume ratio of the aerogel. Then, the percentage of porosity pol is the density of the polymer matrix and ρapp is the aerogel apparent density calculated from the mass/volume ratio of the monolithic aerogels.where ρDhkl correlation length of crystals was determined applying the Scherrer equation:Wide-angle X-ray diffraction (WAXD) patterns with nickel filtered Cu-Kα radiation were obtained, with an automatic Bruker D8 Advance diffractometer, in reflection. The intensities of the WAXD patterns were not corrected for polarization and Lorentz factors, to allow an easier comparison with most literature data. The K is the Scherrer constant, λ is the wavelength of the irradiating beam , βhkl is the width at half height, and θhkl is the diffraction angle. The instrumental broadening, b, was determined by obtaining a WAXD pattern of a standard silicon powder 325 mesh (purity >99%), under the same experimental conditions. For each observed reflection with βhkl < 1°, the width at half height was evaluated by subtracting the unavoidable instrumental broadening of the closest silicon reflection from the experimental width at half height, βhkl, using the following relationship:where: The internal morphology of the aerogels was characterized by means of a scanning electron microscope . Samples were prepared by fracturing small pieces of the monoliths in order to gain access to the internal part of the specimen. In fact, the external lateral surfaces of most samples were found to be flat and free of porosity. Low energy was used (5 keV) to obtain the highest possible surface resolution. Before imaging, all specimens were coated with gold using a VCR high resolution indirect ion-beam sputtering system. The samples were coated depositing approximately 20 nm of gold. The coating procedure was necessary to prevent surface charging during measurement and to increase the image resolution.Nitrogen adsorption at liquid nitrogen temperature (77 K) was used to measure surface areas of OMMT and polymer/OMMT aerogels with a Nova Quantachrome 4200e instrument. Before the adsorption measurement, OMMT powders were degassed at 100°C under vacuum for 24 h, while polymer/clay aerogels were degassed at 40°C, in the same conditions. The surface area values were determined by using 11-point BET analysis.The differential scanning calorimetry (DSC) of OMMT powders and polymer/OMMT aerogels was carried out under nitrogen from 0 to 300°C at a heating rate of 10°C/min on a TA instruments DSC 2920.E′ was obtained, as a function of temperature, at a frequency of 1 Hz and an amplitude of 0.03 mm. The heating rate was 2°C/min in the range of 0, +100°C.Dynamic-mechanical properties were studied using a Triton dynamic-mechanical thermal analyzer. The spectra were recorded in the three-point bending mode, on samples with the following dimensions: length 15 mm, width 10 mm, and thickness 2 mm. The modulus l reflections (up to l = 12) that indicate a high degree of order perpendicular to the clay layers and an interlayer spacing of 3.5 nm, the pattern shows well defined weak peaks, corresponding to the typical 020, 210, and 060 in-plane MMT periodicities   pattern in the 2\upvartheta range 2–80° of the OMMT, with 40 wt % of 2HT, is reported in Figure 2 are shown in Figures l peaks as well as of the rotator order peak (100r) are strongly reduced , not yet reported in the literature, assures the maintenance of a long-range order in the clay layers. In this respect, it is worth adding that the half-height width of the 060 reflection, after exfoliation, remains equal to 0.45° indicating a correlation length D060 = 28 nm.In summary, the described long-term scCO2-treated OMMT, can be obtained by DSC scans . An additional reason for this choice is the easy obtainment of aerogels exhibiting s-PS nanoporous-crystalline δ  patterns of s-PS based aerogels, containing intercalated and exfoliated OMMT, are shown in Figures l reflections of the OMMT are not detected for the aerogels with low clay content (4 and 8 wt %) while for higher clay contents (20 and 50 wt %) a narrow and intense 001 reflection is present, while the 002 and 003 reflections of the starting clay have disappeared. Moreover, the 001 reflection is markedly shifted with respect to its original position (from 2θ = 2.6° up to 2θ = 3.8°), indicating a decrease of the interlayer spacing from d = 3.5 nm down to d = 2.3 nm.All patterns of Figure 2 extraction, for low OMMT content, could lead to clay exfoliation, as already observed for scCO2 treatment of the neat OMMT in Figure The results of Figures r ≈10 J/g ≈20 J/gOMMT).Additional information on the structural organization in the s-PS/OMMT aerogels comes from DSC analyses. In particular, DSC heating scan of a 50/50 by wt. s-PS/as-received-clay aerogel is shown in Figure d001) from 3.5 nm down to 2.3 nm, with only partial loss of the hydrocarbon rotator order in the interlayer space.The combined information of the X-ray diffraction patterns of Figures l clay reflections, while show the isolated weak 060 in-plane clay reflection . However, aerogels with the exfoliated clay Figure  are muchAlso the SEM images of the two aerogels are completely different. In fact, the SEM of the aerogel including the intercalated OMMT is dominated by the micrometric OMMT particles Figure  while thThe results of the SEM analyses suggest that also the large difference in the visual appearance between the two aerogels of Figure DMA analyses indicate that aerogels based on the exfoliated clay present an elastic modulus definitely higher than for those based on intercalated OMMT (36 MPa vs. 15 MPa).SBET, as obtained by N2 adsorption data at 77 K, for the intercalated and exfoliated OMMT, as well as those of the corresponding aerogels, are compared in Table SBET of the neat s-PS aerogel presenting the same porosity is shown in the last row of Table SBET of the OMMT is rather low and is substantially increased for the exfoliated OMMT (SBET = 18 m2/g). The s-PS/exfoliated-clay aerogels present values of SBET (281 m2g−1) much higher than those of the s-PS/intercalated-clay aerogels (166 m2g−1) and not far from those observed for pure s-PS aerogels (312 m2g−1). This indicates that, also for this high OMMT content (20 wt %), the exfoliated clay not only does not disturb the formation of the nanoporous crystalline phase but also does not alter the amorphous aerogel porosity.Surface areas A schematic representation of the sPS/exfoliated-OMMT aerogels is shown in Figure 2-induced exfoliation of OMMTs has been conducted mainly by X-ray diffraction and DSC characterization techniques. The starting material is a MMT intercalated with ammonium cations bearing two long hydrocarbon tails. Suitable scCO2 treatments led to exfoliation of the OMMT and also led to a complete loss of long-range order in the packing of the hydrocarbon tails of the cationic surfactant. The maintenance of hk0 reflections (mainly of the isolated 060 reflection), not yet reported in the literature, assures the maintenance of a long-range order in the clay layers. DSC scans of the intercalated OMMT present a reversible transition that corresponds to the loss of rotator order of the hydrocarbon chains in the interlayer spacing while those of the exfoliated OMMT do not present any thermal transition up to 100°C. This confirms the absence of any 3-D order for the exfoliated clay.A thorough investigation of scCOd001) from 3.5 nm down to 2.3 nm, with only partial loss of the hydrocarbon rotator order in the interlayer space. For aerogels with high content of the exfoliated OMMT, the gel and aerogel preparation procedures allow to maintain clay exfoliation without re-aggregation, as instead observed for other common polymer processing.Monolithic composite aerogels, filled with large amounts of both intercalated and exfoliated OMMT, have been prepared, starting from s-PS-based gels. In particular, for aerogels with high content of the intercalated OMMT, the preparation procedure brings to a reduction of the basal spacing (2g−1) much higher than those of the s-PS/intercalated-clay aerogels (166 m2g−1) and not far from those observed for pure s-PS aerogels (312 m2g−1). This indicates that, also for high content, the exfoliated clay does not alter the aerogel porosity.A strict comparison between s-PS monolithic aerogels with a porosity of 90% and a OMMT content of 20 wt %, as obtained by using intercalated or exfoliated OMMT, has been also reported. Although the two aerogels present similar degree of polymer crystallinity (≈40%) as well as the same polymer crystalline form , aerogels with the exfoliated OMMT are much more homogeneous than aerogels with the intercalated OMMT. This difference, clearly apparent both on visual inspection as well as on SEM analysis, is due to micrometric and nanometric size of intercalated and exfoliated clays, respectively. Aerogels based on the exfoliated clay also present elastic modulus definitely higher than those based on intercalated OMMT. Moreover, s-PS/exfoliated-clay aerogels present values of surface area , could be helpful to an easier handling of exfoliated OMMT, removing the risks connected with inhalable nanoparticles.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.


Although some preliminary work has revealed the potential transcriptional regulatory function of the introns in eukaryotes, additional evidences are needed to support this conjecture. In this study, we perform systemic analyses of the sequence characteristics of human introns. The results show that the first introns are generally longer and C, G and their dinucleotide compositions are over-represented relative to other introns, which are consistent with the previous findings. In addition, some new phenomena concerned with transcriptional regulation are found: i) the first introns are enriched in CpG islands; and ii) the percentages of the first introns containing TATA, CAAT and GC boxes are relatively higher than other position introns. The similar features of introns are observed in tissue-specific genes. The results further support that the first introns of human genes are likely to be involved in transcriptional regulation, and give an insight into the transcriptional regulatory regions of genes. Protein coding genes in eukaryotes contain multiple introns that are cut off in RNA splicing process The characteristics of human introns have attracted considerable attention The CpG island is also known to be involved in transcriptional regulation of genes. Generally, unmethylated CpG islands are the binding sites of some important transcription factors (TFs), such as Sp1 Transcriptional regulatory elements in many genes consist of several essential motifs In this paper, we focus on investigating the intron features associated with transcriptional regulation in human genes. In order to systematically obtain the rational results, we analyze intron features not only in the entire sample genes, but also in introns of tissue-specific (TSP) genes, additionally. Firstly, we compare the intron length in various ways and find that the first introns are generally longer than other introns, consistent with the previous studies A C++ script was written to extract data from NCBI Genbank database (builder 36.2). Only genes with transcript and coding sequence (CDS) features were utilized. Intron lengths were inferred according to the mRNA location information. Since some genes have multiple transcripts , we only retained one transcript feature to avoid duplicate identification of introns for the identical genes. Thus a set of about 185,000 introns were obtained from 19770 intron-containing genes. In order to compare the intron features better, sampled introns were classified by two ways. The first classified pattern is ‘first introns’ which contain all the first introns and ‘non-first introns’ in which all other introns are classified into the same group. The second way is to classify introns based on their position specificity in genes .We retrieved the TSP genes from the HugeIndex database The characteristic length  between all sampled first introns and non-first introns were compared. For the genes containing two or more introns, we also compared the length of the first intron with the average intron length  of the same gene. Due to the large sample size and unknown distributions of the intron lengths, two-sample Kolmogorov-Smirnov (KS) tests were used to assess the significance of the length difference between the first introns and non-first introns.Nucleotide contents, especially high C+G and CG dinucleotide contents are usually thought to be related to transcriptional regulation of genes However, for large sample sizes, the exact computation may take too much time. Based on center limit theorem in statistics, the binomial distribution has an asymptotic standard normal distribution as Namely, Choosing significant level The similar method and significant level were used to examine the difference in occurrence frequency of dinucleotides between the first introns and non-first introns.We searched potential CpG island regions in introns using the method described by Gardiner-Garden and Frommer (1987) The frequencies of introns harboring potential TATA, CAAT and GC boxes were assessed, respectively. The corresponding consensus sequences for the three motifs were TATAWAW (W represents A or T), CCAAT and GGGCGG.The average intron lengths of the entire samples, HK genes and TSP genes are listed in About 2/3 of the first introns are longer than the expected length of the same gene. Except breast and colon-specific genes, the genes with the first introns longer than the expected length are more than the genes with the first introns shorter than the expected length . The rese longer . We can The contents of nucleotides and dinucleotides in introns are shown in  introns , the res overall . We founThe u -value analysis to investigate usages bias of nucleotides and dinucleotides in the first introns relative to position-specific non-first introns are located in the introns, we calculated the frequencies of introns that contain TATA, CAAT and GC boxes. The results show that, of all the first introns, 56.4% contain TATA boxes; 66.2% and 56.2% contain CAAT and GC boxes, respectively. However the percentages of the non-first introns that contain TATA, CAAT and GC boxes are 49.9%, 52.6% and 20.6%, respectively. This indicates that the three TFBSs occur more frequently in the first introns than in the non-first introns. In particular, the occurrence frequency of the GC box in the first introns is nearly 36% higher than that in the non-first introns, in accordance with previous finding that the first introns contain a larger degree of GC dinucleotide and CpG island than the non-first introns. The frequencies of TATA and CAAT boxes in the first introns are only 6.5% and 13.6% higher than those in the non-first introns. Nevertheless, since nucleotides A and T and their dinucleotide compositions and the CA dinucleotide are under-represented in the first introns, it is reasonable to think that the contents of TATA and CAAT boxes in the first introns are richer than those in the non-first introns.Similar results are also found in introns of the TSP genes . In all Our study also reveals that there are differences in frequencies of TATA, CAAT and GC boxes with respect to the intron position . The notIt is noted that, we determined the first intron of a gene only based on one transcript of the gene. However, most genes have alternative transcription start sites which maybe affect what is annotated as the ‘first’ intron and the characteristics of the first introns. To counter these problems, we random chose two groups of alternative transcripts from sampled genes and performed the similar analysis for the features of introns. Excitingly, the scores of intron features have no significant difference between two collections of transcripts (data not shown). For instance, the Using the introns derived from nearly 20000 intron-containing genes, we analyze the sequence features associated with transcriptional regulation. The various characteristics of the first introns within these genes suggest that the first introns are most likely to be involved in transcriptional regulation. Consistent with previous observations Another feature is that C, G and their dinucleotide compositions are over-represented in the first introns, whereas A, T and their dinucleotides are under-represented. In addition, nearly 70% of the first introns contain the CpG island, not only higher than the other introns but also higher than the estimated percentage in entire human genes (40∼50%) Obvious differences in the distributions of TATA, CAAT and GC boxes between the first introns and the non-first introns are also observed. Generally, the frequencies of these three TFBSs are higher in the first introns than in the non-first introns, suggesting the potential for the first introns to bind TFs and participate in gene expression regulation.One additional finding of this study is the differences in sequence features between introns within HK genes and TSP genes. The HK genes have shorter introns than the TSP genes. In addition, the frequencies of CpG islands and GC boxes are higher in the HK genes than those in the TSP genes, whereas the HK genes contain fewer TATA and CAAT boxes than the TSP genes. The results hint that there are the different mechanisms of transcriptional regulation between the HK and TSP genes. For example, one possibility is that there are more CG-rich regulatory elements in the HK genes but more AT-rich elements in the TSP genes since the differences of contents of CpG island and TATA box between introns of HK and TSP genes. Besides, there are also differences among the introns within different kinds of TSP genes, indicating the laws of transcriptional regulation between these groups of TSP genes are not the same. Since the differences between HK genes and TSP genes, our next plan is to detect the transcriptional regulatory elements located in the first introns of HK genes and each group of TSP genes and explore the transcriptional regulation mechanism concerned with introns in these genes.Our results support the hypothesis that the first introns may contain transcriptional regulatory elements and provide intriguing hints into the regulatory potential of the first introns. Therefore, the promoter regions might be extended to at least the first introns while studying the transcriptional regulations of genes.Table S1List of houskeeping and tissue-specific genes. (XLS)(XLS)Click here for additional data file.Figure S1Characteristic lengths of introns in housekeeping and tissue-specific genes. (TIF)(TIF)Click here for additional data file.Figure S2Frequencies of introns overlapped with at least one CpG island in housekeeping and tissue-specific genes. (TIF)(TIF)Click here for additional data file.Figure S3TATA, CAAT and GC boxes frequencies of introns in housekeeping and tissue-specific genes. (A) Frequency of introns with TATA boxes. (B) Frequency of introns with CAAT boxes. (C) Frequency of introns with GC boxes. (TIF)(TIF)Click here for additional data file.


The 2D peak variation originates in that the three effective sub-modes correlate with the light polarization through different relations. We develop a method to quantitatively analyze the positions, intensities, and polarization dependences of the three sub-peaks. The analysis reflects the local strain, which changes with detected area of the graphene film. The measurement can be extended to detect the strain distribution of the film and, thus, is a promising technology on graphene characterization.We report the strain effect of suspended graphene prepared by micromechanical method. Under a fixed measurement orientation of scattered light, the position of the 2D peaks changes with incident polarization directions. This phenomenon is explained by a proposed mode in which the peak is effectively contributed by an unstrained and two uniaxial-strained sub-areas. The two axes are tensile strain. Compared to the unstrained sub-mode frequency of 2,672 cm Recenologists-19. The ologists-22. In aologists-25. Receologists-30.2 are defined by photolithography on an oxidized silicon wafer with oxide thickness of 300 nm. Reactive ion etching is then used to etch the squares to a depth of 150 nm. Micromechanical cleavage of HOPG with scotch tape is then used to deposit the suspended graphene flakes over the indents, as shown in the schematic of FigureSuspended graphene are fabricated by mechanical exfoliation of graphene flakes onto the oxidized silicon wafer. First, ordered squares with areas of 6 μmΦ) are shown in FigureΦ is defined as the included angle between the incident polarization direction and the analyzer. The clear anti-symmetric spectra with different polarization angle can be observed in the spectra. Fitting of the 2D peaks is done using a double-Lorentzian function for Φ of (a) 0° and (b) 90°.Polarized Raman spectra of 2D modes under incident lights with different polarization angle (−) and 2,660 (2D+) cm−1, while that for Figure−) and 2,662 (2D+) cm−1. To systematically analyze the sub-peak of the 2D modes, the spectra of 2D modes with different polarization angle can be fitted by double-Lorentz function, and the 2,647 and 2,660 cm−1 by average of all the peak positions of 2D+ and 2D− modes with different polarization angles. The 2D0 showed the peak position at 2,672 cm−1 as an original 2D peak in the unstrained graphene. The peak positions of 2D+ and 2D− modes compared with an original 2D peak both red shifts. The results showed that the 2D+ and 2D− modes are both tensile strains on suspended graphene. To understand the strain effect of the suspended graphene, fitting the 2D peak of graphene by a triple-Lorentzian function whose three peaks correspond to the sub-modes of 2D+, 2D−, and 2D0 with Φ = 40°, which is just an example of all the spectra, is shown in FigureThe 2D peak originates from four-step Stokes-Stokes double-resonance Raman scattering. AccordiI(2D+), I(2D−), and I(2D0) as functions of Φ is shown in Figure+ and 2D− sub-bands which have the peak positions of 2,647 and 2,660 cm−1, respectively, showed a prominent sinusoidal intensity modulation with a period of 180°. Both the modulation of the 2D+ and 2D− bands can be fitted by a function of cos2(θA − θP), where θA and θP are the polarization angles of the analyzer and polarizer, respectively. Both the intensities of 2D+ and 2D− modes are the maximum when Φ = 0° and minimum when Φ = 90°. This result of suspended graphene is very different from those of the supported graphene by previous research[+ and 2D− modes can be obtained.To systematically analyze the intensities of the fitting sub-peaks, the plots of research. Compare+ and 2D− can be calculated by employing the Grüneisen parameter[0 is the 2D peak position at zero strain and Δω is the shift caused by the strain of ε. For the uniaxial strain,+ and 2D− are estimated as 0.44% and 0.93%, respectively. Based on these results, the distribution of strain on the suspended graphene can be obtained through our analysis.The strains of the 2Darameter (γ) for graphene. In addigraphene. Hence t+ and 2D− sub-bands having the peak positions of 2,651 and 2,661 cm−1, respectively, showed a prominent sinusoidal intensity modulation with a period of 180°. Both the modulation of the 2D+ and 2D− bands can be fitted by a function of cos2(θA − θP), where θA and θP are the polarization angles of the analyzer and polarizer, respectively. Both the intensities of 2D+ and 2D− modes are the maximum when Φ = 1° and minimum when Φ = 91°. Using the same calculation, the stains of+ and 2D− are estimated as 0.44% and 0.93%, respectively. The result in FigureAnother interesting phenomenon measured from our sample can be observed in FigureWe have explored the suspended graphene by polarized Raman spectroscopy. In the exploration, the polarization direction of the incident and scattered light are variable and fixed, respectively. The position and intensity of the graphene's 2D peak is modified by the incident polarization, and the modification is explained by a proposed biaxial-strained model. In this model, the 2D peak is contributed by three effective areas related to unstrained and two tensile-strained graphene, respectively. The two strains are uniaxial and in the same directions. The strength of the strains is quantified through our analysis. This analytical method can be used to probe strain and help us understand the situation of suspended graphene. Hence, this method provides great application potential on graphene-based electrical and optical devices, whose performance usually relies on strain.The authors declare that they have no competing interests.CWH and BJL carried the experimental parts: the acquisition, analysis, and interpretation of data. CWH also had been involved in drafting the manuscript. HYL and CHH performed the analysis and interpretation of data. They also had been involved in revising the manuscript. FYS and WHW  prepared the samples, suspended the graphene using micromechanical method, and captured the OM and AFM images. CYL has made substantial contributions to the conception and design of the study, and the critical revision of the manuscript for important intellectual content. HCC, the corresponding author, had made substantial contributions to the conception and design of the study, and had been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.CWH received his BS degree in Electronic Engineering from the National University of Kaohsiung, Kaohsiung, Taiwan, in 2008. He studied his MS degree in 2008 and PhD degree immediately in 2009. Currently, he is a PhD candidate in the Department of Photonics, National Cheng Kung University, Tainan, Taiwan. He focuses on the property of graphene and surface plasmon resonance of nanoparticles.BJL received his BS degree in Physics from the National Chung-Hsing University, Taichung, Taiwan, in 2010. He received his MS degree in Photonics from the National Cheng Kung University, Tainan, Taiwan, in 2012. His research interests mainly include Raman measurement of graphene. He is in compulsory military service now.


This study aims to quantify the spontaneous proliferation of PBMCs using flow cytometry. PBMCs were cultured for 24 to 120 hours in the presence of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). For comparison, PBMCs were also cultured with [3H]thymidine. The cutoff values for spontaneous proliferation were >0.06 for the division index and >5.8% for the percentage of divided cells. Sixty-two percent of HTLV-1-infected individuals presented spontaneous proliferation of PBMCs, which was detected in the first 24 hours. Moreover, proliferation was detected in CD4+ and CD8+ T-lymphocyte subsets. A positive correlation was found between the division index and [3H]thymidine incorporation. This method may prove useful to better understand the phenomenon of spontaneous proliferation of PBMC of patients infected with HTLV-1.The spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) is a hallmark of the human T-lymphotropic virus (HTLV) type-1. Cell proliferation is usually measured using a [ A proliferation assay based on cell staining with 5,6-carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry analysis has been described as an alternative approach thymidine . After this period, the content of the plate was harvested to determine the [3H]thymidine incorporation using a Beta Radiation Counter . Results were expressed as mean counts per minute.Additionally, cells from 11 HTLV-1-infected individuals were cultured for 5 days and pulsed overnight with 1 P value of less than 0.05 denoted a statistically significant difference. Graphad Prism 5.0 software was used for all statistical analyses.Data were expressed as percentages, means, standard deviations, medians, and 25% and 75% percentiles. Groups were compared using the Mann-Whitney test and Kruskal-Wallis test. Correlations were evaluated by Spearman correlation. A The cutoff values to define spontaneous proliferation were >0.06 for the cell division index and >5.8% for the percentage of divided cells, which represents three times the division index mean and three times the percentage of divided cells in PBMCs from uninfected controls. Under these criteria, 62% of HTLV-1-infected individuals presented spontaneous proliferation in PBMCs as measured by the cell division index, while 47% presented proliferation using the percentage of divided cells .P = 0.9) (Cell proliferation was detected in the first 24 h and remained constant over 120 h (P = 0.9) .+ and CD8+ T-lymphocyte subsets were similar to those defined in the PBMC quantification: >0.06 for the cell division index of cells and >5.2% for the percentage of divided cells. Under these criteria, 50% of HTLV-1-infected individuals presented spontaneous proliferation of CD4+ T lymphocytes, while 30% presented spontaneous proliferation of CD8+ T lymphocytes , as well as between the percentage of divided cells and [3H]thymidine incorporation  (A positive correlation was found between the division index of cells and [= 0.004) .3H]thymidine incorporation assay. The cell division index (average number of cell divisions) showed a better correlation than percentage of divided cells, since it may determine more accurately the number of cell divisions occurring during culture [This study determined the parameters to quantify the cell proliferation of PBMC from patients infected with HTLV-1 by flow cytometry. The results obtained indicated that there was a strong correlation between the intensity of spontaneous proliferation measured by the cell surface stain CFSE and the [ culture .3H]thymidine incorporation assay, which requires three to four days of culture. The intensity of proliferation remained stable over a culture period of 120 hours. Sixty-two percent of the individuals infected with HTLV-1 had spontaneous proliferation of PBMCs. This proportion was similar to that found by other authors using the [3H]thymidine incorporation assay, which described that 50% to 67% of individuals infected with HTLV-1 has spontaneous proliferation of PBMC thymidine incorporation assay, classically used to evaluate proliferation in the context of the HTLV-1 infection, the assay using cell staining with CFSE provided a greater number of parameters for statistical analysis is less laborious and expensive process and safer, since it does not require the use of radioactive substances [Compared with [bstances –24. ThisThe spontaneous proliferation of PBMC is assessable using flow cytometry. This method may prove useful to better understand the phenomenon of spontaneous proliferation of PBMC of patients infected with HTLV-1.The authors declare that there is no conflict of interests.