Patients with idiopathic pulmonary fibrosis (IPF) frequently develop a dry, irritating cough which often proves refractory to anti-tussive therapies. The precise pathogenetic mechanisms responsible for this cough are unknown. We hypothesised that changes in nerves modulating mechanical sensitivity in areas of interstitial fibrosis might lead to enhanced cough response to mechanical stimulation of the chest in IPF.We studied 27 non-smoking subjects with IPF , mean (SD) age 71.7 (7) years and 30 healthy non-smokers. Quality of life (Leicester Cough Questionnaire), cough symptom scores and cough severity scores  were recorded. Percussion stimulation was applied over the posterior lung base, upper anterior chest and manubrium sternum at sequential frequencies (20 Hertz (Hz), 40 Hz and 60 Hz) for up to 60 seconds and repeated twice at two minute intervals. The number of subjects achieving two and five-cough responses, total cough counts and cough latency were recorded. In separate experiments, the effect of mechanical stimulation on the pattern of breathing was determined in eight IPF subjects and five control subjects.In patients with IPF, we demonstrated strong correlations between subjective cough measurements, particularly the cough symptom score and Leicester Cough Questionnaire . Mechanical percussion induced a true cough reflex in 23/27 (85%) IPF subjects, but only 5/30 (17%) controls (p < 0.001). More patients with IPF reached the two-cough response at a lower frequency (20 Hz) posteriorly than at other positions. Highest mean cough totals were seen with stimulation at or above 40 Hz. Mechanical stimulation had no effect on respiratory rate but increased tidal volume in four (50%) subjects with IPF, particularly at higher frequencies. It was associated with increased urge to cough followed by a true cough reflex.This study demonstrates that patients with IPF show enhanced cough reflex sensitivity to mechanical stimulation of the chest wall whilst normal individuals show little or no response. The observation that low frequency stimulation over the lung base, where fibrosis is most extensive, induces cough in more patients than at other sites supports the hypothesis that lung distortion contributes to the pathogenesis of cough in IPF. Idiopathic pulmonary fibrosis (IPF) is a disease characterised by lung parenchymal distortion by fibroblastic proliferation with extracellular matrix deposition and an inflammatory cell infiltration. Patients typically present with progressive breathlessness but the majority develop an irritating cough during the course of the disease,2. This The majority of respiratory diseases associated with cough, such as chronic bronchitis, asthma and acute viral infections, predominantly affect the airways or upper respiratory tract where sensory innervation is dense. By contrast, pathological changes in IPF principally affect the lung parenchyma and alveoli, where innervation is sparse. It is therefore surprising that cough is so common in this disorder. The mechanisms which cause cough in IPF are unknown but several theories have been proposed. These iCough reflex sensitivity to chemical stimulation from inhaled capsaicin and substance P has been shown to be increased in patients with IPF, suggesting functional upregulation of pulmonary c-fibres,6. HowevCrystal et al.  reportedMechanical stimulation of the throat and trachea has been shown to induce cough in patients with upper respiratory tract infection but little or no cough in healthy subjects,10. In oIn this context, the present study was devised with the following aims:• To examine whether mechanical stimulation of the chest wall can induce cough in patients with IPF and if so, whether this response is reliable and reproducible.• To assess whether varying the frequency of vibration and the site of stimulation induces different patterns of cough in patients with IPF.• To correlate measures of any cough induced by mechanical stimulation with subjective measures of cough assessed by validated questionnaires.• To determine whether mechanical stimulation of the chest wall has any effects on the pattern of breathing in patients with IPF or controls.Twenty seven patients fulfilling the American Thoracic Society/European Respiratory Society criteria for the diagnosis of IPF were recruited. All patThree separate measures of cough were undertaken. All subjects were asked to grade their cough severity from 0 (no cough) to 100 (worst cough ever) using a 100 mm linear visual analogue scale (VAS). The impSubjects underwent mechanical stimulation of the chest wall using a percussor . A soft sponge rubber applicator  was used to apply the percussor to the chest wall. Use of a right angle directional stroking adaptor converted the oscillatory effect produced by the face plate to an optimal percussive effect with stimulation frequencies from 20 to 60 Hz , was recorded as the six minute cough frequency. The number of subjects who achieved a two-cough and a five-cough response were recorded as C2 and C5 cough thresholds respectively. For determination of C2 and C5 responses, only coughs occurring during or within 15 seconds of cessation of mechanical stimulation were counted, in accordance with guidelines for other cough challenges. Cough lTo determine any effects of mechanical stimulation on the pattern of breathing, the above protocol was repeated in eight patients with IPF  and five normal volunteers using a portable recording device designed for the diagnostic assessment of cardio-respiratory sleep disorders . This equipment provides measurement of: airflow from a nasal pressure cannula and oral thermistor; movements of abdominal and chest wall from effort belts, oxygen saturation and pulse using pulse oximetry. A built-in microphone also records associated sounds simultaneously. At the end of the recording period, all subjects were asked to describe subjective feelings of urge to cough, change in pattern of breathing or feelings of breathlessness.t test was also used to compare baseline variables between groups. Non-parametric data are expressed as median (IQR) and compared by the Mann-Whitney U test and Wilcoxon's signed rank test. When analysing multiple comparisons of mean cough counts at different stimulation frequencies within individuals, a repeated measures one-way analysis of variance (ANOVA) was applied to normally distributed data with Tukey's pairwise analysis for determining true differences. Fisher's exact test was used to analyse categorical data and Spearman's rank correlation coefficient to assess association between variables[Total cough counts during six minutes of stimulation are expressed as mean (SEM) and compared by the unpaired t-test. The unpaired variables. p valuevariables. All datBaseline demographic and pulmonary function data for patients and control subjects is shown in table Compared with controls, IPF patients had significantly greater median cough symptom scores than control subjects as assessed by VAS  and CSS  and lower median LCQ scores . In patients with IPF, there were strong negative correlations between the LCQ score and both the CSS and VAS scores, with a further strong positive correlation between the VAS and CSS  exhibited no cough at any frequency at any site of stimulation. By comparison, 23 out of 27 patients (80%) with IPF coughed on percussive stimulation (p < 0.0001). Of the four patients with IPF who did not cough at all, three were receiving prednisolone.Of the IPF patients, 17 out of 27 reached a two-cough response during an individual period of mechanical stimulation. This was detected in eleven subjects at 20 Hz, four at 40 Hz and two at 60 Hz Figure . A five-Nineteen subjects with IPF achieved a two-cough response. This was achieved by nine subjects at 20 Hz, six at 40 Hz and four at 60 Hz Figure . In fiftOf the IPF group, 13 subjects demonstrated a two-cough response. This occurred in five subjects at 20 Hz, seven at 40 Hz and one at 60 Hz Figure . In eighThe mean total cough counts (corrected for background cough) for the total three stimulation episodes at different sites of stimulation and different frequencies are shown in Figure In IPF subjects who coughed in response to mechanical stimulation, the mean time to first cough (cough latency) ranged from 32.7 to 57.3 seconds at each stimulation frequency in each location tested. No clear pattern emerged in relation to site of chest wall stimulated, frequency of stimulation or subjective measures of cough (data not shown).1, FVC, TLC or DLCO).The correlation between individual IPF patients' total cough counts at different sites and frequencies of percussion and the three subjective cough symptom scores are shown in table Repeat experiments under identical conditions in six patients with IPF studied at least one week apart suggested there was some variability in mean total cough counts and C2 cough threshold Figure . HoweverIn healthy subjects, there was no change in rate or depth of respiration at any stimulation frequency. Similarly, in patients with IPF, no change in respiratory rate was observed. However, an increased tidal volume was clearly demonstrated in four patients (50%) in response to mechanical stimulation and this became more pronounced at higher frequencies Figure . It was This is the first study to investigate the effects of mechanical chest wall percussion on the cough reflex and patterns of breathing in patients with IPF. The technique appears to induce a true cough rather than an expiratory reflex in the majority of subjects with IPF, but has little or no effect on healthy controls in whom any cough was minor and short-lived. The latter observation is consistent with previous studies in healthy humans,19,20. IRARs terminate in the intrapulmonary airways of all mammalian species and are exquisitely sensitive to mechanical stimulation-23. TheyIn this study, we excluded patients with chest wall deformities in order to minimise this as a confounding factor. However, stimulation over different areas of the chest wall may result in variability in the percussive stimulus exerted on the underlying lung due to differences in chest wall anatomy or thickness. As a group, the Body Mass Index of our patients with IPF was significantly greater than controls. Thus, any reduced transmission of the vibration due to increased chest wall thickness would favour a null hypothesis.We cannot be certain that direct stimulation of RARs by mechanical vibration contributed to the cough response seen in patients with IPF. Indeed, the precise distribution and structure of RAR terminations in the intrapulmonary airways of patients with this condition are unknown ,22. FurtThis implies the mechanical vibration applied in the subsequent experiments was a true stimulatory impulse.In contrast to the C2 response, fewer patients with IPF achieved the C5 threshold at any frequency. These results are similar to those of a previous study which used chemical stimulation with capsaicin to induce cough in IPF patients. This obOur measurements of chest and abdominal wall movement and airflow at the mouth together with audio-visual recordings demonstrated that the cough we induced by vibration was indeed a true cough and not an expiratory reflex. Interestingly, it appears to be preceded by an increase in tidal volume but not rate of respiration and then followed by an urge to cough. The explanation as to why this should occur is uncertain but may relate to variation in density or sensitivity of RARs between individuals. In a previous study on chest wall vibration in five patients with asthma, the subjects described a sensation of breathlessness similar to an acute exacerbation of asthma but no cough was reported . To our It is noteworthy that a previous study reported an increased cough threshold to citric acid in healthy volunteers during simultaneous vibratory stimulation of the chest wall anteriorly in the right second intercostal space. The autThe total cough response (six minute cough count) in patients with IPF was greater when higher frequency stimulation 40 and 60 Hz) was applied to all three areas of the chest wall, whilst again normal controls showed little or no cough response. This may indicate there is a threshold frequency for induction of cough which is lowered in patients with IPF. Interestingly, the total cough count correlated with some subjective assessments of cough severity, particularly the CSS. A recent study demonstrated strong correlations between objective 24 hour cough frequency and both the VAS and LCQ scores in patients with IPF. These f0 Hz was In the present study, patients were asked to provide three different subjective measure of cough severity, all of which have been previously validated. The median VAS score of 38 mm was similar to results from previous studies,6,28,29.Previous studies in IPF have been unable to demonstrate a correlation between the VAS cough score and measures of disease severity assessed by pulmonary function,6. The pIn summary, this study demonstrates that patients with IPF show an enhanced cough reflex sensitivity to mechanical stimulation of the chest wall. It also shows there is a good correlation between cough induced by mechanical stimulation and subjective measures of cough severity, particularly the CSS. The observation that low frequency stimulation of the postero-basal lung base, where fibrosis is often most extensive, induces cough in more patients than at other sites is consistent with the hypothesis that lung distortion is a contributory factor to the pathogenesis of cough in IPF, possibly by activating RARs or destruction of inhibitory c-fibres. The use of chest wall percussion to induce a true cough reflex may prove a useful additional method for assessing novel anti-tussive therapies for cough in patients with IPF.The authors declare that they have no competing interests.RMJ contributed to the design of the study, data collection, analysis and interpretation and drafting/revising of the manuscript. SH was also involved in the data collection. RMJ, NKH and RE were involved in drafting and revising the manuscript. NKH, SH, BDMHG and RE were all involved in the study design and conception and provided substantial input into the analysis and interpretation of the data. All authors have read and approved the final version.


Conventionally, SPPs have been studied in noble-metal structures, where the SPPs are intrinsically bound to a 2D metal-dielectric interface. Meanwhile, recent advances in the growth of hybrid 2D crystals, which comprise laterally connected domains of distinct atomically thin materials, provide the first realistic platform on which a 2D metal-dielectric system with a truly 1D metal-dielectric interface can be achieved. Here we show for the first time that 1D metal-dielectric interfaces support a fundamental 1D plasmonic mode (1DSPP) which exhibits cutoff behavior that provides dramatically improved light confinement in 2D systems. The 1DSPP constitutes a new basic category of plasmon as the missing 1D member of the plasmon family: 3D bulk plasmon, 2DSPP,  Low-dimensional collective electron excitations at metal surfaces, the so-called surface plasmon-polaritons (SPPs), provide a route towards tremendous electric field enhancement4567891011121314151617181920568910111214816Meanwhile, at the cutting edge of materials science are hybrid 2D crystals222324222324231DSPP, and 0D localised SP).In this study, we show that 1DMD interfaces in general 2D metal-dielectric systems support a fundamental and unique 1D plasmonic mode (1DSPP). Through an illustrative example on a hybrid graphene/graphene platform, we show how unique cutoff behaviour of 1DSPPs could allow for a dramatic improvement in the electric field confinement of plasmons in 2D systems, exceeding that of previously predicted quasi-1D and 1D plasmonic excitations in graphene891011121314y = 0 plane consisting of two semi-infinite domains that are laterally connected along the z axis with sheet conductivities of σ(L) = σ(L)′ + iσ(L)″ (x < 0) and σ(R) = σ(R)′ + iσ(R)″ (x > 0) ″ > 0) and dielectric (σ(R)″ < 0) domains, respectively. At this point, to simplify our discussion, we omit ohmic and interband losses in both domains ′ = 0; the lossy case is considered below and in the 26z = 0) in the quasi-static limit can be shown to be (see Methods): σ(R)″ = Kσ(L)″ (K < 0), the normalised effective index N = n/n2D  is the effective index of the 1D interface plasmon with wavenumber q normalised to that of the planar 2D transverse magnetic (TM) plasmons of region L, n2D = 2cεε0/σ(L)″  system in the L)″ Ref. , and m iN(K) is plotted in N is purely real) exist strictly in the window 0 ≤ −K < 1. At K = 0, the plasmon wavenumber takes its minimum value as that of the bare edge plasmon21K| → 1, that is, when the electric susceptibilities on the two sides of the interface become equal (|σ(R)″| → σ(L)″), the plasmon wave number diverges. Interestingly, this divergent behaviour reveals a strong connection to the conventional 2D surface plasmon polaritons (SPPs) localised to bulk metal-dielectric interfacesqSPP = nSPPk0) diverges at the surface plasmon (SP) frequency ωSP given by the non-retarded SP conditionεd + εm(ωSP) = 0; εd > 0 and εm < 0 are the relative permittivities of the dielectric and the metal, respectively. Likewise, at the 1DMD interface, we note the cutoff condition K = −1, or σ(R)″ + σ(L)″ = 0 ′ = 0). This reveals that the plasmonic mode of a 1DMD interface is a 1D manifestation of SPPsin 2D systems (1DSPP). An important consequence of the reduced dimensionality can be observed in the respective divergence behaviours: the SPP behaviour takes the form ε = εd − |εm|), which diverges much less rapidly than that of the 1DSPP, n~1/Δσ (Δσ = σ(L)″ − |σ(R)″|).While σ ″ > 0 i.e., where both domains (L and R) are metallic. In that case, while there is no solution to N, there is a solution for complex N which corresponds to a so-called inter-edge plasmon that is damped as it leaks into the extended 2D plasmons supported by the domain with the smaller (positive) imaginary part of conductivityK < 1) is entirely non-leaky, and completely bound to the 1DMD interface. It is also important to note a clear distinction between the 1DSPP, and the recently predicted quasi-1D plasmonic excitations of graphene p-n junctions1414σ″), much like the multiple modes of a graded-index waveguide, and the graphene is everywhere metallic. In contrast, the 1DSPP manifests exclusively as a consequence of the metallic-to-dielectric transition across the 1DMD interface, either side of which the conductivity is spatially uniform. In this regard, we predict that the IR edge wave observed graphically in Vakil et al. ′, σ(R)′ ≠ 0) is readily obtained on introducing the complex parameter K defined in terms of the complex conductivities K = σ(R)/σ(L) = K′ + iK″, and solving for the complex normalised effective index N = n/n2D = N′ + iN″ (where n2D = i20cε/σ(L) – Ref. σ(L)′ = 0, σ(R)′ ≠ 0), we obtain the convenient and meaningful expressions K′ = σ(R)″/σ(L)″ , and K″ = −σ(R)′/σ(L)″. Thus we may imagine that fixing K′ and simultaneously increasing |K″| corresponds to fixing of the imaginary part of conductivity of the L and R domains, and linearly increasing the loss (σ(R)′) in the dielectric domain. Conversely, fixing |K″| and increasing K′ is equivalent to fixing σ(R)′ and linearly increasing σ(R)″. Meanwhile, the quantity N′/(2πN″) reduces to the propagation length defined as the number of optical cycles within one exponential decay length (l = Re(q)/[2πIm(q)]). We note that this physical scenario is particularly relevant to a hybrid graphene/graphene metal-dielectric system at finite temperature . To illustrate and quantify the onset of 1DSPP damping, the main window of K″ dependence of the 1DSPP propagation length at several fixed values of K′. At a given value of K″, the propagation length decreases with an increase in K′, which is typical of plasmons as their wave number increases . We note that, at each value of K′, the curve is cutoff at the x-axis corresponding to the propagation length of just one wavelength . It is illustrative to compare the K″ cutoff with a particular K′ e.g., when K′ = −0.6, the cutoff is K″ ~ −0.06, corresponding to K″/K′ = −σ(R)′/σ(R)″ ~ 0.1. Obviously, the loss is more forgiving for smaller K′, e.g. when K′ = −0.2, the cutoff is K″ ~ −0.12, corresponding to −σ(R)′/σ(R)″ ~ 0.6.The green and red curves in the inset of μ is the Fermi energy of domain L or R, respectively, and details at finite temperatures are given in the σ″ at the interface  provides convenient closed-form expressions for the optical conductivity in both domains567face see , so hybre see 222432.n2D) is completely determined by the dimensionless conductivity K, this behaviour suggests a flexible tunability of n through the variation of either σ(R)″ (μ(R)) or σ(L)″ (μ(L)) with the doping of either graphene domain. We demonstrate this tunability and simultaneously verify our quasi-static analytical results by employing a mode-solver tool in the numerical finite element method (FEM) package COMSOL, which includes retardation effects. In n on μ(R) for several fixed values of μ(L) (see respective curves) and the spatial evolution of the 1DSPP electric field components xE and yE. Note the normalisation of μ in terms of f = 20 THz and 80 THz. One can observe excellent agreement between the dispersion analytically predicted by n as μ(L) decreases (at fixed μ(R)) can be readily understood in terms of the corresponding decrease in σ(L)″and thus Δσ . Each curve diverges asymptotically towards a cutoff value of μ(R) given by the condition σ(L)(μ(L)) + σ(R)(μ(R)) = 0. The cutoff value of μ(R) approaches μ(L) increases. When the plasmon energy μ(L) are of approximately the same order of magnitude , the cutoff is very close to 0.5 because of the relative strengths of the interband and intraband terms in the graphene conductivity (see Methods).The conductivities on either side of the interface are completely determined by the respective normalised frequenciesσ(R) = 0) demonstrate the nature of the cutoff dynamics at the 1DMD interface. The net power flow parallel to the interface is given by the integral of zS = xHy*E − yHx*E over the xy plane. zS is symmetric about the x axis, although the terms xHy*E and yHx*E both exhibit odd sign parity about the y axis; thus, cumulative integration on either side of the interface leads to their respective partial cancellation. This cancellation is weak in the case of GEPs because of the strong field asymmetry, and therefore, the net power flow is significant. Conversely, as the cutoff condition (σ(R)″ → −σ(L)″) is approached, the symmetrisation of the field components caused by equal charge screening on either side of the interface ; a two-order-of-magnitude improvement over the graphene bare edge plasmon .K. 1DMD interfaces in graphene/graphene hybrid 2D systems at finite temperature are intrinsically lossy because of the presence of interband transitions, which are essential to achieving the dielectric character in one domain ″ < 0; K < 0), and further exhibit a temperature-dependent upper limit on |σ(R)″| (and |K|). Nevertheless, we predict the propagation lengths of 1DSPPs on hybrid graphene systems to be several plasmon wavelengths at room temperature (T = 300 K) and ~100 plasmon wavelengths at the temperature of liquid nitrogen (T = 80 K); for example, at a free space wavelength λ = 1.8 μm we find l ~100nm (μ(L) = 1 eV, μ(R) = 0.42 eV, T = 300 K) and l ~ 2.6 μm (μ(L) = 1 eV, μ(R) = 0.40 eV, T = 80 K), respectively . The effective index of 1DSPPs diverges asymptotically towards a cutoff as the magnitudes of the susceptibilities on the two sides of the interface become equal, in striking analogy to conventional SPPs at bulk metal-dielectric interfaces. On a sample 2D metal-dielectric graphene/graphene platform, highly sensitive tunability of the 1DSPP dispersion was demonstrated via the doping of either graphene domain, which further allowed for the achievement of spatial confinement of electric-field intensity to regions orders of magnitude smaller than that of the plasmonic excitations of a bare graphene edge (GEP). The unique and tuneable cutoff behaviour of 1DSPPs thus presents a means for dramatic enhancement of light confinement in 2D systems over other 1D or quasi-1D excitations, such as GEPs or plasmonic excitations of graphene p-n junctions. At a frequency of 50 THz, we predicted tremendous localisation of the electric-field intensity of the 1DSPP to a modal area more than one million times smaller than the diffraction limit. As a new member of the existing plasmon family of bulk plasmons, surface plasmons, localised plasmons, etc., we foresee a new field of low-dimensional plasmonics based on 1DSPPs, particularly branching out into various multi-material 2D systems222324y = 0 plane comprising two semi-infinite domains laterally connected along the z axis with sheet conductivities of σ(L) = σ(L)′ + iσ(L)″ (x < 0) and σ(R) = σ(R)′ + iσ(R)″ (x > 0) (see inset in ε. In the quasi-static theory of Volkov and Mikhailov26φ(r) = φ0exp(iqzz−iωt), the dispersion relation of plasmons propagating along a 1D junction between two adjoining 2D electron gases (2DEG) is given byWe consider a hybrid 2D system in the βγδσ = σ(R)βγ − σ(L)βγ, where β and γ are tensor indices , and R,Lε are the effective dielectric permittivities of the left (L) and right (R) 2DEGε0 is the permittivity of free space. For electrically isotropic 2DEGs in the absence of an external magnetic field, we have σxz = 0 and δσxz = 0. Taking δσxx = δσ = σ(R) − σ(L), we obtain δσ ≠ 0, the dispersion relation reduces to when the hyperbolic cosine of the argument in the above equation is zero. To simplify the analysis we start by setting σ′ = 0, which neglects ohmic and interband losses −1/2, we arrive at f(KN) − f(N), where C, its numerical solution combined with its asymptotic behaviour is revealing. We find that Im[f(C)] = 0 when C ≤ 0, Im[f(C)] = π2/2 when C ≥ 1, and Im[f(C)] increases monotonically as C increases within the range 0 < C < 1  when K > 0  because 0 < f(KN), f(N) ≤ π2/2, which constrains −π2/2 < Im[f(KN) − f(N)] < π2/2, so that the imaginary part of m. Indeed, this was earlier pointed out by Mikhailovσ″).Here, K < 0; σ(L)″ > 0, σ(R)″ < 0)]. This case has not been previously considered26σ″ in conventional 2DEGs. New opportunities to realise 1DMD interfaces in hybrid 2D systems prompted us to investigate fundamental solutions to K<0 when N > 1 (and m = 0), provided that Re[f(−|K|N)] = Re[f(N)] ] = 0 for NK < 0 , and Im[f(N)] = π2/2 for N > 1, thus satisfying the imaginary parts of f(C), we empirically find the relation Re[f(C + Δ)] ≈ Re[f(−C)] (C > 0) (see the inset of C = |K|N leads to Re[f(|K|N + Δ)] ≈ Re[f(−|K|N)], and recalling the solution condition Re[f(−|K|N)] = Re[f(N)], we must have (comparing the positive arguments of the function Re[f(C)] which monotonically increases for C > 1, and noting Δ > 1) |K|N + Δ = N. This indicates a dispersion relation of the form N = eN/(1 − |K|), where we have identified Δ = eN as the bare edge plasmon dispersion , or recalling the definitions of N and K and takingN,N|K| ≫ 1; writing f(−|K|N)−f(N) and Taylor-expanding the respective arguments to the first order, we find K| → 1 of N ≈ (2/π)(1 + |K|)/(1 − |K|), which is consistent with the (1 − |K|)−1 dependence in the empirical relation obtained above. Solution to m = 0 naturally precludes the existence of higher-order, multipolar modes because of the step-like transition of the conductivity across the junction; this result is expected, considering that multipolar modes of a bare edge plasmon manifest only when the conductivity decreases monotonically from a constant to zero over a non-zero length a beyond some cutoff26K = 0, given by the root of eN = N ≈ 1.217…N(K) as determined from the numerical solution of K| > 0.5 .Now, we turn to the pertinent case of the 1D metal-dielectric (1DMD) interface [www.comsol.com). In our example of the graphene/graphene hybrid 2D system, the conductivities on either side of the junction are calculated using the random phase approximation (RPA) in the local and zero-temperature limitδ = 0.2 nm, an effective relative dielectric permittivityε = 1+iσ/ωε0δ, and σ given by δ → 0 limit (at δ = 0.2 nm).The 1DSPP dispersion was obtained using the mode analysis tool in the COMSOL RF module (x > 0) as (Ω(R))−1 >0.6, thus confirming the previous assertionK > 0. At values of (Ω(R))−1 < 0.5, the dispersion must be solved for a complex value of N because of the introduction of non-zero σ(R)′; nevertheless, considering that we would likely have σ(R)′ > |σ(R)″| ″| see , the 1DSD. M. and S. M. contributed equally to this work. D. M. and S. M proposed and verified the existence of the 1DSPP. D. M. and S. M. performed theoretical and numerical calculations, S. Y. assisted in data interpretation, and N. P. supervised the work and added physical interpretations of the results. All authors participated in discussions and in writing the manuscript.1DSPP Supplementary Information


Valid sarcoidosis incidence assessment is contingent on access to medical care, thoroughness of reportage, assiduity of radiographic interpretation, employment and health care screening policies, misclassification, and population ethnicity. To diminish ambiguity and foster inter-population comparison, the term “sarcoidosis incidence” must be modified to convey the methodology employed in compiling the numerator. In age-delimited cohorts, valid comparison to population incidence requires age adjustment due to the age-dependency of incidence. The “true incidence” of sarcoidosis is a notional concept: more than 90% of cases are subclinical and radiographically inevident. Occupational causal inference based on incidence differential vs. populations has been undermined by methodological differences in ascertainment and computation. Assessment of sarcoidosis incidence requires dividing the annual number of newly identified cases (numerator) by the number in the source population (denominator). This apparent simplicity is illusory, for the numerator is a function of ascertainment methodology and ethnic composition.Ascertainment depends on access to medical care and thoroughness of reportage. Additionally, it reflects acuteness of recognition, which is influenced by the experience and assiduity of the interpreting radiologist. Sensitivity to subtle hilar adenopathy and pulmonary shadowing, easily overlooked in mass population screening, is enhanced (“ascertainment bias”) by awareness of a putative germane exposure e.g., among World Trade Center disaster responders. Increasingly frequent performance of chest CT and transbronchial mediastinal biopsy may increase ascertainment .Methodological differences are a major impediment to obtaining comparable incidence data. This is illustrated by differential incidence ascertainment in observable (see below) cases. To formalize its meaning, the term “incidence” must be preceded by an adjective defining the operation by which the numerator is acquired, thereby facilitating valid inter-population comparison. Observable cases are divisible into three operationally defined classes, each subsuming the prior class: Cases identified because of symptoms or an incidental chest radiograph are designated “clinically identified.” Addition of cases identified by health or employment screening chest radiographs  defines “clinically recognized.” Addition of cases identified by population screening defines “clinically ascertainable.” This classification scheme can be visualized as concentric circles in a Venn diagram.The incidence of clinically identified cases peaks at ages 20–49. It varies widely between ethnic and geographic populations; it is far higher in Blacks and Northern Europeans. The annual incidence of clinically identified sarcoidosis in Whites in the U.K is 3.4 . The latIn summary, among US Whites, 3 is a plausible provisional estimate of clinically identified sarcoidosis incidence; 4 may be considered an upper bound. The figure is slightly higher in the UK. The clinically identified incidence in Scandinavian populations is 5. The clinically ascertainable incidence is three-fold the clinically identified incidence.The clinically identified sarcoidosis incidence in Blacks in the two reporting, ethnically mixed, US health maintenance organizations were 36.4 and 35.5 . BecauseA requirement for histological verification is not typically stated in sarcoidosis incidence compendia. Clinical experience has verified the accuracy of clinical diagnosis in stage I; Hillerdal et al. accepted a clinical diagnosis of stage II .Individuals exhibiting a local or regional granulomatous response to a neoplasm are not infrequently classified erroneously as having sarcoidosis. The proportion of unascertained chronic berylliosis in sarcoidosis registries is unknown. A phenocopy of sarcoidosis, it can escape detection unless careful inquiry into industrial exposure, supported by beryllium-lymphocyte proliferation testing, has been undertaken .Lacking a diagnostic gold standard, autopsy prevalence is imprecise. Enumeration is contingent, in part, on the pathologist’s decision whether pulmonary involvement suffices, whether one or more than one organ demonstrating epithelioid granulomatous changes are required for confirmation, and whether historical or pathologic evidence of potentially confounding, co-existent diseases or exposures are present. Hägerstrand and Linell in a Swedish autopsy series of 6,706 cases reported a prevalence of “certain” (pulmonary or multi-organ epithelioid granulomas) of 640, while the prevalence of uncertain cases was 210. The 640 figure was 10-fold the point prevalence  sarcoidosis estimated by mass radiographic screening in a Stockholm population . Reid reEmploying as the denominator the number of persons at risk (rather than the population from which they are derived) in computing the incidence results in a spuriously elevated figure if the peak incidence is concentrated in the age range of the at risk cohort. This consideration is directly applicable to the occupational incidence of sarcoidosis because ~ 85% of cases are identified in the occupational age group, 20–49.Several investigators have inferred a causal connection between sarcoidosis and environmental or occupational exposures based on a marked incidence differential vs. population based, clinically identified figures. This inference is weakened by ascertainment bias, lack of an identified putative antigen, the incidence-trebling effect of cohort radiographic surveillance, and the employment of the surveyed, working age cohort as the denominator in the computation .1. Methodological identity is required to ensure valid inter-population incidence comparison.2. The clinically identified incidence of sarcoidosis in US Whites is 3; in UK Whites, 3.4; in persons of Scandinavian descent, 5. Population screening of Whites triples that figure. The clinically identified incidence in US Blacks is 36.3. “The” incidence of sarcoidosis is indeterminable. It is a notional concept due to a combination of ascertainment and methodological contingencies and the preponderance of unobservable cases.4. Judging from autopsy prevalence, sarcoidosis is common; clinically identified cases constitute a small minority of those that are histologically verifiable.5. Causal inference based on incidence differential is confounded by differences in case acquisition and incidence computation.The author declares that he has no competing interests.The Author has had an active interest in this subject for a number of yearsAm J Med 1985, 78:61–67.1. Reich JM, Johnson RE: Course and prognosis of sarcoidosis in a nonreferral setting: analysis of 86 patients observed for ten years. Cancer 1985, 55:366–369.2. Reich JM: Acute myeloblastic leukemia and sarcoidosis: implications for pathogenesis [case report]. J Roy Soc Med 1992, 85:306.3. Reich JM: Sarcoidosis and acute leukemia [letter]. Sarcoidosis 1993, 10:4–8.4. Reich JM: Acute myeloblastic leukemia and sarcoidosis [editorial]. Chest 1995, 107:605–613.5. Reich JM, Mullooly JP, Johnson RE: Linkage analysis of malignancy-associated sarcoidosis. J Intern Med 1994, 235:179–182.6. Reich JM: Sarcoidosis and agnogenic myeloid metaplasia [case report]. Chest 1995, 105:1474–1475.7. Reich JM, Johnson RE: Sarcoidosis and malignancy [letter]. Sarcoidosis Vasc Diffuse Lung Dis 1996, 13:173–177.8. Reich JM, Johnson RE: Estimated incidence of clinically identified sarcoidosis in a Northwest United States population. Am J Med 1997, 102:131.9. Reich JM: Sarcoidosis mortality in the United States [letter]. Thorax 1997, 52:102.10. Reich JM: BTS sarcoidosis study [letter]. Chest 1998, 113(1):147–153.11. Reich JM, Brouns MC, O’Connor EA, Edwards MJ: Mediastinoscopy in patients with presumptive stage I sarcoidosis: a risk/benefit, cost/benefit analysis. Am J Epidemiol 1998, 148(1):100–101.12. Reich JM: Racial differences in sarcoidosis incidence: a 5-year study in a health maintenance organization [letter]. Chest 1998, 113:559–160.13. Reich JM: Corticosteroid therapy and relapse in sarcoidosis [letter]. Chest 1998, 113:1143.14. Reich JM: Deciphering histoplasmosis, systemic noncaseating granuloma, and sarcoidosis in the literature [letter]. Thorax 1998, 53(7):625–626.15. Reich JM: Malignant neoplasms in pulmonary sarcoidosis [letter]. Sarcoidosis Vasc Diffuse Lung Dis 1999, 16:108.16. Reich JM: How common is sarcoidosis? [letter]. Eur Respir J 1999, 14(2) 482–483.17. Reich JM: Sarcoidosis and cancer revisited [letter]. Chest 2000, 118(6):1838.18. Reich JM: Clinical diagnosis of stage I sarcoidosis [letter]. Eur Respir J 2001, 17:833.19. Reich JM: Course and prognosis of sarcoidosis in African-Americans vs. Caucasians [letter]. Chest 2002, 121:32–39.20. Reich JM: Mortality of intrathoracicsarcoidosis in referral vs. population-based settings: influence of stage, ethnicity, and corticosteroid therapy. Chest 2003, 124:367–371.21. Reich JM: What is sarcoidosis? Sarcoidosis Vasc Diffuse Lung Dis 2003, 20(3):227–234.22. Reich JM: Adverse long-term effect of corticosteroid therapy in recent-onset sarcoidosis. Chest 2004, 125(3):1172–1173.23. Reich JM: Sarcoidosis cycle [letter]. Eur J Intern Med 2004, 15(5):269–273.24. Reich JM: Eight fundamental unsolved problems in sarcoidosis. Chest 2005, 27:1864–1866.25. Reich JM: Treatment outcome in M.avium pulmonary disease: a correction and comment [letter re. M. avium and sarcoidosis]. Eur J Intern Med 2006, 17(2):81–87.26. Reich JM: Neoplasia in the etiology of sarcoidosis. Chest 2006 April 12.27. Reich JM: Investigation of Löfgren’s syndrome [E-letter]. Sarcoidosis Vasc Diffuse Lung Dis 2007, 24:77.28. Reich JM: A requirement for histologic confirmation of stage I sarcoidosis is far more likely to prove harmful than helpful [letter]. Cytopathology 2007, 18:392–397.29. Reich JM: Tissue verification of stage I sarcoidosis [letter]. Chest 2008, 133(6);1529–1530.30. Reich JM, Patterson J, Asaph J, Brouns M: Tissue verification of stage I sarcoidosis: the question is if, not how [letter]. Chest 2009; 136:943.31. Reich JM: Concurrent sarcoidosis and lung cancer [letter]. BMJ Nov 10, 2009.32. Reich JM: Comments on Sarcoidosis (review) [Owen JD et al.] Rapid response Chest 2010, 137:235–236.33. Reich JM: Endobronchial Ultrasonography-Guided Transbronchial Needle Aspiration in Patients With Suspected Sarcoidosis. Respir Med 2010, 104:1778–1781 and 1782–1783. [Pro: Culver DA. Complete debate: 1775–1783.]34. Reich JM: Con: The treatment of the granulomatous response is beneficial in acute sarcoidosis [Debate]. Am J Ind Med 2011, 54(9):696.35. Reich JM: “Sarcoid like" granulomatous pulmonary disease in World Trade Center disaster responders: Influence of incidence computation methodology in inferring airborne dust causation [letter]. Eur J Intern Med 2012, 23:105–109. doi:10.1016/j.ejim.2011.09.01136. Reich JM: On the nature of sarcoidosis. J Occup Environ Med 2012, 54(1):2.37. Reich JM: Sarcoidosis and World Trade Center Disaster [letter]. Am J Respir Crit Care Med 2012, 185(4):461–462.38. Reich JM: Sarcoidosis mortality [letter—in response to Swigris and Baughman]. Am J Ind Med 2013, 56:496–500. doi:10.1002/ajim.22083.39. Reich JM: Shortfalls in imputing sarcoidosis to occupational exposures [editorial]. Am J Med 2013, 126(1):e17. doi:10.1016/j.amjmed.2012.05.03140. Reich JM: Neoplasia in the etiology of sarcoidosis [letter]. Am J Ind Med 2013, 56(4):503–504. doi:10.1002/ajim.2214941. Reich JM: Response to Dr. Michael Hodgson’s letter re. “Shortfalls in imputing sarcoidosis to occupational exposures.” J Bronchol Intervent Pulmonol 2013 (in press)42. Reich JM: Tissue confirmation of presumptive stage I sarcoidosis [editorial].


The humidity sensor consists of a sensing capacitor and a sensing film. The sensing capacitor is constructed from spiral interdigital electrodes that can enhance the sensitivity of the sensor. The sensing film of the sensor is polypyrrole, which is prepared by the chemical polymerization method, and the film has a porous structure. The sensor needs a post-CMOS process to coat the sensing film. The post-CMOS process uses a wet etching to etch the sacrificial layers, and then the polypyrrole is coated on the sensing capacitor. The sensor generates a change in capacitance when the sensing film absorbs or desorbs vapor. The ring oscillator circuit converts the capacitance variation of the sensor into the oscillation frequency output. Experimental results show that the sensitivity of the humidity sensor is about 99 kHz/%RH at 25 °C.This study presents the design and fabrication of a capacitive micro humidity sensor integrated with a five-stage ring oscillator circuit on chip using the complimentary metal oxide semiconductor (CMOS) process. The area of the humidity sensor chip is about 1 mm The humidity sensor was a capacitive type, and the polyimide was locally cured at a temperature over 350 °C for 1 hour by the MEMS micro hotplate. The humidity sensor exhibited a sensitivity of 0.77 pF/%RH and a hysteresis of 0.61 %RH. Miyoshi et al. [et al. [et al. [et al. [Humidity sensors that are used to measure and monitor environmental humidity are important devices in many industrial and biomedical fields. The micro electromechanical system (MEMS) technique has become popular for the miniaturization of sensors. Humidity sensors fabricated by the MEMS technique have the advantages of small size, high performance, easy mass-production and low cost . Severalet al.  presente [et al.  was fabrIn this study, we investigate the fabrication of a micro humidity sensor integrated with a ring oscillator circuit on chip using the CMOS-MEMS technique. The sensing material of the sensor is polypyrrole. The post-process uses wet etching to etch the sacrificial layers and the polypyrrole is coated on the humidity sensor. The sensor produces a change in capacitance when the sensing film absorbs or desorbs vapor. The ring oscillator circuit converts the capacitance of the humidity sensor into the oscillation frequency output.2.sensor represents the capacitance of the humidity sensor and Cload is the load capacitance. The ring oscillator circuit is used to convert the capacitance variation of the sensing capacitor into the frequency output. As shown in A five-stage ring oscillator is adopted as the sensing circuit owing to the fact its phase noise is smaller than that of three-stage ring oscillator, and it has a stable output signal . Figure sensor of the ring oscillator circuit is given by [inv is the delay time associated with the inverters, τsensor the delay time associated with the humidity sensor, Cload the load capacitance, Csensor the humidity sensor capacitance, and ΔV and Iave are the threshold voltage and average current, respectively. According to The ring oscillator circuit connects with odd inverters does not have a stable operating point. Therefore, the circuit will oscillate when any of the odd inverter input or output voltage separates from the unstable operating point. The oscillation frequency fgiven by (1)fsensThe professional circuit simulation software, HSPICE, was used to analyze the characteristics of the ring oscillator circuit. 3.The commercial 0.35 μm CMOS process of Taiwan Semiconductor Manufacturing Company (TSMC) was utilized to manufacture the integrated humidity sensor chip. The chip requires a post-process to etch the sacrificial layers and to coat the polypyrrole film after completion of the CMOS process. 4H5N) is mixed with 50 mL deionized water and stirred continuously; (2) 10 g anhydrous ferric chloride (FeCl3) is mixed with 30 mL deionized water and stirred continuously; (3) the solution of FeCl3 is added into the pyrrole solution with stirring for 48 h. The resulting product is filtered, dropped on the humidity sensor chip. Then, the chip is followed by calcination in air at 120 °C for 3 h.4.The performance of the humidity sensor was measured by a power supply, a LCR meter, a spectrum analyzer and a test chamber . The test chamber could supply a temperature range of 0–100 °C and a humidity range of 25–95 %RH. Temperature and humidity in the test chamber can be tuned separately and kept at constant levels. The spectrum analyzer was utilized to record the signal response of the sensor to humidity changes. The LCR meter was adopted to detect the capacitance of the sensor. The humidity sensor was set in the test chamber. The power supply provided a bias voltage of 3.3 V to the sensor. In order to characterize the capacitance variation of the sensor to humidity, the sensor without the ring oscillator circuit was tested. The test chamber supplied different humidity to the humidity sensor. When the humidity rose or dropped, the humidity sensor changed in capacitance. The capacitance variation of the humidity sensor was measured by the LCR meter. In order to understand the influence of temperature, the output frequency of the humidity sensor was tested at different temperatures. S represents the sensitivity of the humidity sensor and T is the temperature. According to Dai  proposed5.This humidity sensor integrated with a five-stage ring oscillator circuit has been implemented using the commercial 0.35 μm CMOS process and the post-process. The humidity sensor was composed of a sensing film and a sensing capacitor. The sensing film was polypyrrole, that was synthesized by the chemical polymerization method. The electrodes of the sensing capacitor were designed with a spiral interdigital shape that could increase the sensor capacitance. The post-process used wet etching to etch the sacrificial layers, and the polypyrrole was coated on the sensing capacitor. The ring oscillator circuit converted the capacitance variation of the sensor into the frequency output. The experiments showed that the ring oscillator circuit was normally operation after the wet etching post-process. Thereby, the post-process was compatible with the commercial CMOS process. The measured results depicted that the humidity sensor had a sensitivity of 99 kHz/%RH at 25 °C, and its sensitivity exceeded that of Dai . The hum


To the best of our knowledge, this is the first report of a case of conjunctival sarcoidosis that presented as limbal conjunctival hypertrophy and was controlled with topical cyclosporine.A 70-year-old Asian woman presented with painless limbal conjunctival hypertrophy. An incisional biopsy of the hypertrophic lesion showed noncaseating granulomas that consisted of multinucleated giant cells and epithelioid cells. The results of a systemic evaluation were normal except for a slight increase in serum angiotensin-converting enzyme to 58.9IU/L . Twice-daily 0.05% topical cyclosporine was prescribed for the treatment of remaining lesions. After two months, the lesions had completely resolved and her serum angiotensin-converting enzyme level had normalized.Sarcoidosis should be considered in the differential diagnosis of conjunctival hypertrophy. Topical cyclosporine can be useful in conjunctival sarcoidosis. Sarcoidosis is a multisystemic inflammatory process characterized by noncaseating granulomatous infiltration . Ocular We present the case of a patient with isolated conjunctival sarcoidosis presenting as limbal conjunctival hypertrophy who was successfully treated with 0.05% topical cyclosporine.A 70-year-old Asian woman was referred to our hospital for an evaluation of limbal conjunctival hypertrophy in her left eye. She reported mild discomfort but had no other symptoms, such as pain or itching. She had chronic angle closure glaucoma in her left eye and was on 0.1% brimonidine twice a day and 0.005% latanoprost four times a day. Her past medical history was unremarkable except for hypertension and hyperlipidemia. A slit-lamp examination showed multiple yellowish conjunctival nodules in both eyes and hypertrophy around the superior limbal area in her left eye Figure , and herAlthough conjunctival sarcoidosis mostly presents as multiple nodules resembling follicular conjunctivitis, other conjunctival manifestations including conjunctival deposit, large conjunctival tumor and multilobular limbal nodules have been reported -3,5-7. TCyclosporine is known to be useful in various ocular surface inflammatory diseases, such as ocular rosacea, dry eye disease, atopic keratoconjunctivitis, graft-versus-host disease and herpetic stromal keratitis, due to its anti-inflammatory and immunomodulatory action . AlthougOur experience suggests that sarcoidosis can manifest as limbal conjunctival hypertrophy, and should therefore be included in the differential diagnosis of sarcoidosis. Topical cyclosporine can be a useful option in the treatment of conjunctival sarcoidosis, especially when there are concerns about steroid side effects.Written informed consent was obtained from the patient for publication of this manuscript and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ACE: angiotensin-converting enzyme; CD: cluster of differentiation.The authors declare that they have no competing interests.SBH and HKY acquired, analyzed and interpreted the patient data regarding the sarcoidosis. JYH and WRW performed the histological examination of the conjunctival tissue sample. SBH and HKY were major contributors in writing the manuscript, and JYH and WRW revised it critically. All authors read and approved the final manuscript.SBH, JYH and WRW work in the corneal and refractive surgery division at Seoul National University College of Medicine, and HKY works in the strabismus and pediatric ophthalmology division at Seoul National University College of Medicine.


Schizosaccharomyces pombe, is an important model species with a low intron density. Previous studies showed extensive intron losses during its evolution. To test the models of intron loss and gain in fission yeasts, we conducted a comparative genomic analysis in four Schizosaccharomyces species. Both intronization and de-intronization were observed, although both were at a low frequency. A de-intronization event was caused by a degenerative mutation in the branch site. Four cases of imprecise intron losses were identified, indicating that genomic deletion is not a negligible mechanism of intron loss. Most intron losses were precise deletions of introns, and were significantly biased to the 3′ sides of genes. Adjacent introns tended to be lost simultaneously. These observations indicated that the main force shaping the exon-intron structures of fission yeasts was precise intron losses mediated by reverse transcriptase. We found two cases of intron gains caused by tandem genomic duplication, but failed to identify the mechanisms for the majority of the intron gain events observed. In addition, we found that intron-lost and intron-gained genes had certain similar features, such as similar Gene Ontology categories and expression levels.The fission yeast,  Spliceosomal intron densities vary greatly among different organisms Saccharomyces cerevisiaeDictyostelium discoideum, Schizosaccharomyces pombe, Mycosphaerella, Cryptococcus, Caenorhabditis elegans, Anopheles, and mammals Fusarium graminearum, Magnaporthe grisea or rice Neurospora crassa and Aspergillus, the biased position of intron loss has been observed in a comparative analysis among distantly related species Arabidopsis and Drosophila, conflicting results have been reported Cryptococcus, Fusarium, Aspergillus, Drosophila and mammals Caenorhabditis, Plasmodium or ArabidopsisDrosophila, and Muridae ArabidopsisDrosophilaThree models of intron loss have been proposed: the reverse transcription (RT) model, the genomic deletion model, and the model of non-homologous end joining (NHEJ) repair of double strand breaks Daphnia and AspergillusOikopleura and MycosphaerellaDrosophila and ArabidopsisCladosporium and Dothistroma, introner-like elements (ILEs) were found to contribute to new introns Chironomus, Aspergillus and MycosphaerellaCompared with intron loss, the possible mechanisms underlying intron gain are much more diverse. At least six mechanisms have been proposed for intron gain: intron transposition, transposon insertion, group II intron insertion, tandem genomic duplication, intron transfer and NHEJ-mediated intron gain events Cryptococcus, Fusarium, Caenorhabditis, mammals and PopulusA broad definition of intron loss and gain could include de-intronization and intronization, which are conversions of intron sequences to exon sequences, and vice versa, by mutations Schizosaccharomyces pombe, has a low intron density S. pombe with distantly related organisms, such as vertebrates and plants. Most of the evolutionary traces of intron loss and gain could not be retained over such a long evolutionary history. For this reason, we compared four closely related species; Schizosaccharomyces cryophilus, Schizosaccharomyces octosporus, S. pombe and Schizosaccharomyces japonicus with six outgroup fungus species  from the Broad Institute . Data for Saitoella complicata, Aspergillus niger, Nectria haematococca, Sporobolomyces roseus, Cryptococcus neoformans and Phycomyces blakesleeanus were obtained from JGI . Saitoella complicata was selected because it is closely related to the Schizosaccharomyces genus and the other five outgroup species were selected because of their relative abundance of introns. The phylogenetic relationship between the Schizosaccharomyces species and the outgroup species is shown in We downloaded the genome sequences and gene annotations of four fission yeast species , 1 (has an intron) or ? (no orthologous regions). This data list was imported into the Dollop (Dollo and Polymorphism Parsimony) program in the PHYLIP 3.6 Schizosaccharomyces group. Finally, considering the limitation of Dollop . The transcripts were mapped onto the corresponding genomic sequences using BLAT 34 http://www.broadinstitute.org/annotation/genome/schizosaccharomyces_group/FeatureSearch.html) to get additional transcripts that are only available on the webpages.In order to exclude the possibility that the unique intron positions, including intron losses/gains and intronizations/de-intronizations, were gene structure annotation artifacts instead of real intron changes, it is necessary to use transcriptome data to identify related gene structures. The RNAseq/Inchworm/PASA assembly sequences of the four fission yeast species were downloaded from the Broad Institute  created the two splicing sites  could produce alternative products Multiple signals are required for efficient splicing The identification of intron loss or gain events were initially based on the gene structure annotations of the four fission yeast species. However, some of these annotations might be erroneous, which could be seen from the fact that most putative intronization or de-intronization events were annotation errors. For intron losses and gains, gene annotation errors could also lead to false-positive results. If an exonic segment was mis-annotated as an intron in orthologous genes, a simple deletion of it would lead to a false-positive case of intron loss. Similarly, a simple insertion of exonic sequence mis-annotated as an intron would lead to a false-positive case of intron gain. Therefore, the transcriptome data were also required to support the target intron loss or gain events.In our datasets, we found that most intron loss and gain positions were supported by transcriptome data –S3. In oSchizosaccharomyces group was negatively correlated with the intron loss rates ; therefore, we suggest that the genomic deletions that have actually occurred in evolution should be more frequent than the imprecise intron deletions we observed.In addition, the frequency of genomic deletion might also been underestimated for methodological reasons. In previous studies S. cryophilus and S. octosporus, 17 in S. pombe, seven in the ancestor of S. cryophilus, S. octosporus and S. pombe and eight in S. japonicus. Referring to the method of Roy and Gilbert −7) to deny the null hypothesis. Furthermore, we observed a preferential loss of introns at the 3′ side of genes. As shown in Similarly to previous studies in other taxa P>0.10 in all species and nodes). Thus, the NHEJ-mediated model was not supported by the intron losses in fission yeast.Meanwhile, we also tested the NHEJ-mediated model of intron loss by surveying the micro-homology between 5′ and 3′ splice sites of lost introns SPOG_01682 gained its intron at position 576-0  and its ortholog SOCG_00815 gained its intron at another position 612-0. Both of the introns comprised tandem repeats . Therefore, the NHEJ model of intron gain was not supported in fission yeasts.We also attempted to test the NHEJ model of intron gain by surveying the frequency of direct repeats near intron-exon boundaries S. pombe GO annotation from the Gene Ontology website (http://www.geneontology.org). Compared with the whole gene sets, IL genes were more likely to participate in metabolism, molecular transportation and enzyme activity regulation  genes and intron-gained (IG) genes are clustered in certain special features. BiNGO 2.44 gulation . Interesgulation . We alsogulation . These rAs discussed above, intron losses were mainly mediated by reverse transcriptase. In the current models of intron gain, only the intron transposition model shares a similar mechanism (i.e. a requirement for reverse transcription) with intron loss. However, the intron transposition model requires similarities between newly gained introns and other extant introns, which was not observed in our study. As the selective constraint is much lower for introns than for coding regions, accumulated mutations in gained intron sequences might have resulted in them being significantly divergent from the source sequences. It is also possible that some of the identified intron gains might be intron losses in other taxa, considering the high intron loss rates in fission yeasts. False-positive results of intron gains would cause the IG genes share some false-positive similarities with IL genes.In fission yeasts, we found a higher frequency of intron loss than intron gain. Although a moderate number of putative intronization and de-intronization events were observed, most cases were filtered out using transcriptome data. Careful examination of the confident cases of intronization and de-intronization revealed them to be caused by mutations in splice sites and branch sites. Although at a low frequency, imprecise intron deletions were observed, supporting the genomic deletion model of intron loss. The characteristics of most intron losses are consistent with the RT model. Similar to previous studies Table S1Conserved introns across the four fission yeast species.(XLS)Click here for additional data file.Table S2Lost introns across the four fission yeast species.(XLS)Click here for additional data file.Table S3Gained introns across the four fission yeast species.(XLS)Click here for additional data file.Table S4Intron-lost genes and intron-gained genes in fission yeasts have higher expression levels.(DOC)Click here for additional data file.Figure S1Number of extant introns and intron loss rate are negatively correlated. The intron loss rate and the number of extant introns are negatively correlated. S. cryophilus has the largest number of extant introns and has experienced the lowest intron loss rate, while S. pombe has the fewest introns and had the highest intron loss rate. Species name abbreviations: S. cryophilus (Scry), S. octosporus (Soct), S. pombe (Spom), and S. japonicus (Sjap).(TIF)Click here for additional data file.Figure S2Large indels neighboring unique intron positions in fission yeasts. The alignments of DNA sequences around unique intron regions are shown. Exon sequences are shown in upper case while intron sequences are shown in lower case. Exonic sequence indels accompanying intron loss are marked in red. Species name abbreviations: S. cryophilus (Scry), S. octosporus (Soct), S. pombe (Spom), and S. japonicus (Sjap).(TIF)Click here for additional data file.Text S1The limitation of the Dollop program.(DOC)Click here for additional data file.


In recent years a number of studies have shown that whole grain products in particular have strong antioxidant activity. Primary cultures of rat hepatocytes were used to investigate whether and how a fermented powder of wheat (Lisosan G) is able to modulate antioxidant and detoxifying enzymes, and whether or not it can activate Nrf2 transcription factor or inhibit NF-kB activation. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by 0.7 mg/ml Lisosan G treatment. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different degrees, at the transcriptional, protein and/or activity levels by the treatment. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus after 1 h of Lisosan G treatment was revealed by immunoblotting. Lisosan G was also observed to decrease HTaken together, these results show that this powder of wheat is an effective inducer of ARE/Nrf2-regulated antioxidant and detoxifying genes and has the potential to inhibit the translocation of NF-kB into the nucleus. In vivo, this vitamin acts as scavenger of oxygen radicals and also as competitive inhibitor of nitrosamine synthesis from nitrite and amines in vivoHumans are constantly exposed to factors causing oxidative stress including pollutants, radiation and oxidized food Several studies have shown that some phytochemicals can modulate antioxidant and phase II enzymes through the activation of nuclear factor E2-related protein (Nrf2) In recent years, many authors have suggested the existence of cross-talk between Nrf2/ARE and the nuclear factor-kappa B (NF-kB) signaling pathways in response to inflammation Triticum Sativum (wheat) and it is registered with the Italian Ministry of Health as a nutritional supplement. In the production process, the wholegrain is first ground to a rough powder. From this intermediary product, the bran and germ are separated and collected for further treatment which consists in the following: water is added to moisten the mix, then selected microbic starting cultures are inoculated to initiate fermentation. The starting cultures typically consist of a mix of lacto-bacillus and natural yeast strains. Once the product is sufficiently fermented, it is dried. The resulting dry powder is now Lisosan G, which is widely used in food production thanks to its rich nutritional content.Lisosan G is a powder obtained from In vivo, Lisosan G protects against cisplatin induced toxicity It contains vitamins, minerals and polyunsatured fatty acids as well as having significant antioxidant activity 2O2) were all supplied by Sigma-Aldrich . Rabbit polyclonal anti-Nrf2 (sc-13032), anti-heme oxygenase-1 (sc-10789), NFkB (sc-7178), β-actin (sc-130657), PARP-1 (sc-25780) and goat anti-rabbit (1∶2000 or 1∶5000) were from Santa Cruz Biotechnology Inc. . Collagen (type I) was prepared by the method of Beken et al. (1998).Lisosan G is registered as nutritional supplement by the Italian Minister of Health and was supplied by Agrisan Company, Larciano (PT), Italy. Collagenase; dexamethasone; insulin; glucagon; penicillin/streptomycin; ampicillin/kanamycin; fetal bovine serum; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); Tween 20; phenylmethylsulfonyl fluoride (PMSF); leupeptin; apoprotein; pepstatin; tunicamycin; Williams E medium; bovine serum albumin (BSA); epidermal growth factor (EGF); β-nicotinamide adenine dinucleotide reduced (NADPH); ethylenediaminetetraacetic acid (EDTA); ethylene glycol tetraacetic acid (EGTA); glutathione (GSH); Glutathione disulfide (GSSG); and hydrogen peroxide  and then subjected to midline laparotomy in order to exteriorize the liver and isolate the portal vein. A needle was inserted into the portal vein and then the liver was perfused as described previously 6 cells/8 ml on 100 mm cell culture dish pre-coated with 3 ml of collagen (type I) solution (1 mg/ml). The cultures were maintained at 37°C in 5% CO2 in a humidifier incubator. After 5 h, the medium was replaced with serum-free Williams E medium supplemented with 2% BSA, 7.5 µg/ml hydrocortisone 21-hemisuccinate sodium salt and EGF 20 ng/ml. Cultures were maintained in this medium at 37°C and 5% CO2 for 24 h. After this period, the medium was removed and a second layer of type I collagen was added to create a collagen-gel sandwich culture 2O2 200 µM and in the last group, the H2O2 200 µM was added after 1 h pre-treatment with Lisosan G 0.7 mg/ml (Lis+H2O2). We have used different concentrations of H2O2 (10–500 µM) and we chose 200 µM (good toxicity); we also used for Lisosan G different concentrations from 0.1 to 2.8 mg/ml and 0.7 mg/ml was the best concentration in terms of maximum protective effect and without toxicity. The cytotoxic effects compared with the vehicle-only controls, was also measured by lactate dehydrogenase assay (data not shown). We have also performed experiments in function of time of treatments of H2O2 and Lisosan G and we chose the time of treatment on the basis of the best results.After filtration and centrifugation, the cell viability was determined by trypan blue exclusion. The cells were dispersed in Williams E medium containing 39 ng/ml dexamethasone, 0.5 U/ml insulin, 0.007 µg/ml glucagon, 5 µg/ml penicillin and streptomycin, 5 µg/ml ampicillin and kanamycin and 10% fetal bovine serum. The cells were plated at a density of 4.5×102O2 decomposition at 240 nm, as described by Cao and Li After 24 h the end of treatment, the medium was removed and a collagenase solution was added. After 30 minutes, recovered cells were centrifuged (400× g) for 3 minutes at 4°C. The cell pellet was sonicated and used for the microsomal preparation Total cellular RNA was extracted from primary rat hepatocytes 4 h after ITC treatment, using the RNeasy Mini Kit , following the supplied protocol. RNA was quantified using NanoDrop ; its purity and integrity were evaluated by checking the absorbance ratio at 260–280 nm and assessing the sharpness of 18S and 28S ribosomal RNA bands on agarose gel stained with ethidium bromide. Genomic DNA elimination and reverse transcription of total RNA were performed using QuantiTect Reverse Transcription Kit (Qiagen).Two microliters of cDNA were added to a PCR Master Mix  for the amplification reaction (various cycles) performed using for each transcript 400 nM of forward–reverse primers for heme oxygenase-1 (GenBank accession no. NM_012580.2), NQO1 (GenBank accession no. NM_017000.3), β-actin, as housekeeping gene, (GenBank accession no. NM_031144.2) and the annealing temperature indicated in Nuclear and cytosolic extracts were prepared by previously established methods Nuclear and microsomal proteins from primary rat hepatocytes were separated according to Laemmli Results are reported as means ± SD. Statistical significance was determined by Student's t-test for comparison between control and treated groups or the one-way ANOVA and the Dunnet tests. The statistical program used was Graphpad Prism 4. P value<0.05 was considered to be significant.2O2 caused a decrease in reduced glutathione levels, whereas pretreatment with 0.7 mg/ml Lisosan G followed by H2O2, raised the amount of intracellular GSH to levels above control cells values. Lisosan G alone was able to increase reduced glutathione levels in hepatocytes treated for 24 h.To verify the ability of Lisosan G to protect hepatocytes from damage caused by oxidative stress, we treated a first group of cells with hydrogen peroxide, while a second group was pretreated with Lisosan G prior to exposure to hydrogen peroxide. As shown in 2O2 which had been pretreated for 1 h with Lisosan G. 2O2-induced lipid peroxidation. Exposure of cells to H2O2 for 24 h significantly increased lipid peroxidation while preincubation of cells for 1 h prevented this from occurring. Also, Lisosan G on its own reduced lipid peroxidation levels compared to control (CTR) cells.As a biomarker for lipid peroxidation, the concentration of malondialdehyde (MDA) in microsomes was measured in cells treated with H2O2 treatment reduced NQO1 activity, but NQO1 activity remained high when a 1 hour pre-treatment with . H2O2 also caused a decrease in HO-1 activity compared to control cells, but pretreatment with Lisosan G before H2O2 treatment elevated the activity above the level of CTR. HO-1 activity was significantly induced by Lisosan G treatment   and catalase. Hent with  Lisosan l value) . As for l values . This phl values , followi2O2 for 4 h. As shown in 2O2 didn't cause any significant change in NQO1 expression, but both the treatment with Lisosan G only and Lis+H2O2 increased its expression compared to the control levels.NQO1 and HO-1 were chosen to analysis of the response to Lisosan G at the transcriptional level. The expression of their transcripts in primary rat hepatocytes were analyzed by semi-quantitative RT-PCR, using the sets of primers listed in 2O2 caused a rise in HO-1 expression but in this case, H2O2 alone also induced the gene.A similar trend was found for HO-1 expression, although to different extents . For thi2O2 no effect was noticed after 24 h treatment. On the contrary, the pre-treatment with Lisosan G before H2O2, raised the HO-1 protein level 1.6 fold above the CTR value, in agreement with results from the activity assays and transcription analysis.The effect of Lisosan G on heme oxygenase-1 was also assessed at the protein level by western blot . In micr2O2 nuclei. On the contrary, Nrf2 was clearly visible in the nuclear extracts of hepatocytes treated with Lisosan G and with Lisosan G+H2O2. The results have been normalized to PARP-1 levels.Since a major component of cellular defense against oxidative or electrophilic stress is the activation of the Nrf2/ARE signaling pathway 2O2 treated hepatocytes. The signal in control cells was similar to that of cells treated with Lisosan G, both very weak. The signal from nuclear extracts of hepatocytes treated with Lisosan G+H2O2 is fainter than that obtained in hydrogen peroxide treated cells. The results have been normalized to β-actin levels.As cross talk between Nrf2 and NF-kB is an area of intense interest, we investigated whether Lisosan G treatment would prevent the NF-kB translocation to the nucleus, caused by hydrogen peroxide. We analyzed the nuclear fraction by western blot . NF-kB w2O2 induced oxidative stress and whether it was able to modulate phase 2 enzymes by activating the Nrf2 protein and causing its translocation into the nucleus. We also analyzed its ability to prevent NF-kB nuclear translocation. We used sandwich cultures of primary rat hepatocytes, a unique in vitro system that preserves hepatic cytomorphology, as well as its drug metabolism, deposition and toxicity, to allow close resemblance with in vivo parameters In the present study, we investigated whether a fermentated wheat powder, Lisosan G, was able to protect against H2O2, and increased NQO1, HO-1, GST and catalase activity, suggesting that Lisosan G has antioxidant properties and confirming earlier in vivo studies which had demonstrated the ability of Lisosan G to elevate GST, NQO1, catalase and GSH peroxidase activity 2O2 induced oxidative stress. Several classes of phytochemicals such as phenols, flavonoids, isothiocyanates, organosulfurs, and indoles can induce detoxifying enzymes such as NQO1 and HO-1 2O2. GSH is a multifunctional intracellular non-enzymatic antioxidant and it is considered to be the major thiol-disulphide redox buffer of the cell Lisosan G defended cells against damage induced by HNQO1, HO-1 and GST gene expression is regulated by several key transcriptional factors located in the upstream region It is important to note that the Nrf2 and NF-kB signaling pathways interface at several points to control the transcription or function of downstream target proteins. We therefore tried to understand if Lisosan G was able to regulate NF-kB. It is known that released NF-kB translocates into the nucleus where it regulates the transcription of genes for chemokines, cytokines, immunoreceptors, cell-adhesion molecules, growth factors, tumor necrosis factor α (TNFα), inducible NOS (iNOS), interleukin-1 (IL-1), interleukin-6 (IL-6) and cyclooxygenase (COX-2) The ability of Lisosan G to induce phase II enzymes via Nrf2 and inhibit NF-kB activation, may be linked to its composition Lisosan G also contains minerals such as iron, zinc, copper and it is rich in vitamins . Vitamin E is an important natural antioxidant, and its most common and biologically active form is α-tocopherol. Unpublished results have shown that α-lipoic acid (ALA) is among the various components in Lisosan G, and that it is present in a concentration of 66 mg/kg.in vitroALA is a thiol antioxidant found in vegetables, including broccoli, spinach and tomatoes 2O2-induced translocation of NF-kB to the nucleus. It seems likely that the beneficial effects of Lisosan G derive from its high phytochemical and vitamin content.This study has established for the first time in primary rat hepatocytes that Lisosan G can modulate phase 2 enzymes through the activation of Nrf2 pathway. We have demonstrated the Lisosan G decreases the H


Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8+ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8 Programmed death-ligand 1  is a coinhibitory molecule that has been associated with impairment of the T cell response. PD-L1 is one of the ligands that interact with the inhibitory PD-1 receptor, which is expressed on activated T cells , and cytInfluenza virus infection may trigger an exacerbated immune response, which has been correlated with illness severity and sometimes death –14. Lymp+ T cell-mediated immune response against influenza plays a role in limiting the severity of infection in the absence of previous antibodies [Cellular immunity, may contribute to virus clearance, reduction of symptoms and prevention of secondary infections , 23. Thetibodies . Howevertibodies . TherefoThirteen patients from two hospitals from the Mexican Social Security Institute (IMSS) with RT-PCR-confirmed pandemic influenza infection (pH1N1+), 12 PCR negative patients with influenza-like illness (ILI) (pH1N1−), and 10 healthy controls (HC) were included in this report. Patients were recruited during the first and second pandemic waves in Mexico City. Informed consent was obtained from participants. Study approval was obtained from the IMSS through the National Commission of Scientific Research, which comprises the Scientific, Ethics, and Biosafety Committees, in accordance with Good Clinical Practice. The project's ethics authorization number is: CNIC 2010-785-002.Blood samples from patients and controls were collected in EDTA tubes. Peripheral blood mononuclear cells (PBMCs) were separated by gradient centrifugation using Lymphoprep  and cryopreserved until use. PBMCs from buffy coats were obtained from healthy volunteer donors according to institutional guidelines.6) from buffy coats were placed in 24-well plates  with RPMI-1640 . They were stimulated with 10 pgmL−1 staphylococcal enterotoxin B , 10 µgmL−1 of the TLR7 synthetic agonist CL264 , 80 HAUmL−1 (hemagglutination units) of live and UV-inactivated influenza A/Mexico/4482/2009(H1N1) virus and A/Panama/2007/1999(H3N2) virus provided by the Instituto Nacional de Referencia Epidemiológica (INDRE), or 10 μgmL−1of recombinant A(H1N1)pdm09 virus hemagglutinin (HA), kindly provided by Dr. Clara Espitia from the Instituto de Investigaciones Biomédicas, UNAM. The PBMCs were incubated for 18 h, 3 or 7 days at 37°C/5% CO2 prior to flow cytometry analysis of PD-1 and PD-L1 expression on DCs and T cells, respectively. For de novo protein synthesis analysis, PBMCs were stimulated with A(H1N1)pdm09 for 2 h, then cycloheximide  was added to the culture for another 16 h.PBMCs (1 × 107) from buffy coats were incubated in supplemented RPMI-1640 at 37°C/5% CO2 for 1.5 h, in Petri dishes . Nonadherent cells were removed, washed, and quantified. T cells were then isolated by negative selection using a cocktail of PE-conjugated anti-CD19, anti-CD14 , anti-CD56, and anti-HLA-DR antibodies , with anti-PE magnetic microbeads in a MidiMACS system with LD columns . Dendritic cells were isolated the same way, but instead of anti-HLA-DR, PE-conjugated anti-CD3 (eBioscience) was used.PBMCs (2 × 105 cells/well) were placed into 48-well plates (Corning) with supplemented RPMI-1640 and stimulated with 10 pgmL−1 SEB and 80 HAU/mL A(H1N1)pdm09 virus and incubated for 48 h at 37°C/5% CO2. Enriched DCs (1.5 × 106) were placed in 24-well plates (Corning) with supplemented RPMI-1640 and stimulated with CL264 or A(H1N1)pdm09 virus. The cells were incubated for 18 h at 37°C/5% CO2, collected, and labeled for flow cytometric analysis.The enriched T cells (5 × 106 cells/well) were left untreated or stimulated with influenza virus and incubated for 18 h. Next, the cells were labeled with CFSE , and 5 × 105 cells/well were seeded into plates. These cells were left untreated or treated with 25 μgmL−1 anti-PD-L1 antibody 29E.2A3  or an isotype control MPC-11 (BioLegend) on days 0, 3, and 5, or with SEB (10 pgmL−1) on day 0. The cells were incubated for 7 days at 37°C/5% CO2, collected, and proliferation was measured by flow cytometry. The proportion of apoptotic cells was detected by flow cytometry with Annexin V/Pacific Blue and 7-AAD staining (both from BioLegend).Buffy coat PBMCs (5 × 10Supernatants from the T cell proliferation culture were cryopreserved until use. Cytokine levels were measured using the human Th1/Th2/Th17 cytometric bead array kit (CBA) according to the manufacturer's instructions (BD Biosciences).+ T cells (Tm) were isolated with the human memory CD4+ T cell isolation kit (Miltenyi Biotec). For co-culture assays, a 1 : 3 ratio of cDCs : T cells (1.5 × 104 cDCs and 4.5 × 104 T cells) were placed in 96-well plates with A(H1N1)pdm09 virus and incubated for 7 days at 37°C/5% CO2, with or without PD-L1 blocking; the supernatant was collected to conduct CBA's analysis. Representative plots of cDCs and CD4+ T cell purity are shown in Figure S1 .Sorting of cDCs from enriched DCs was performed in a FACSAria cell sorter (BD Biosciences). After the preenrichment previously described, the negative cell fraction was labeled with anti-CD123-/PE-Cy5 and anti-HLA-DR/APC-Cy7 (BioLegend), to identify the cDC population; this population was isolated with a purity of about 90%. Memory CD46 cells were evaluated for PD-L1 expression on both DCs and T cells. When the number of cells was inferior, only DCs or T cells were analyzed. Hence, PD-L1 expression was analyzed on T cells of 9/13 pH1N1+ patients and 6/12 pH1N1− patients. In the case of DCs, PD-L1 expression was evaluated in 11/13 pH1N1+ patients and 10/12 in the pH1N1− group. The PBMCs from patients were labeled with the fluorochrome-conjugated antibodies, PD-L1/PE-Cy7, CD8/APC-Cy7, HLA-DR/APC-H7, CD4/PE-Cy7, CD123/PE-Cy5, and the lineage cocktail  (BD Biosciences) and fixed with paraformaldehyde (4%). The T cells and DCs from buffy coats used for in vitro PD-L1 expression, proliferation, and cell death assays were labeled with all of the former antibodies, in addition to PD-1/FITC, CD4/APC-Cy7, CD2/PE, (BD Biosciences), and CD8/APC (Invitrogen). The cells were fixed with FACS Lysing Solution 1x (BD Biosciences) and cell viability was determined with Hoechst 33258 staining (Invitrogen). All samples were analyzed in either a FACSAria II or a FACSCanto II (BD Biosciences) using FlowJo (version 8.7) software . A minimum of 1 × 104 CD2+ events were collected for T cell samples and 5 × 104 Lin-events for DCs samples.The cryopreserved PBMCs from patients and controls were thawed and counted. Only the samples with total PBMCs above 1 × 10t-test with a two-tailed P value was performed. In the case of patients, a nonparametric Student's t-test was performed (Mann-Whitney test). Correlations were established with Spearman's test. Statistical significance was established at P < 0.05.Statistics were calculated with Prism, version 5.0, from GraphPad Software . To test for significant differences in PD-L1 expression between treatments, MFI values were normalized and a parametric Student's P < 0.01) and plasmacytoid dendritic cells (pDCs) (P < 0.001) (+ (P < 0.001) and CD8+ T cells (P < 0.001) similar to SEB (+ and CD8+ T cells was detected after 18 h and decreased over time (P < 0.001) (+ T cells and after 7 days in CD8+ T cells (P < 0.05) (+ cell-depleted T cells with A(H1N1)pdm09. We found that PD-L1 expression on DCs was induced after interaction with the virus and was dependent on de novo protein synthesis pdm09 later in time.To test whether A(H1N1)pdm09 could induce PD-L1 and PD-1 expression on DCs and T cells, we stimulated human PBMCs with A(H1N1)pdm09. After 18 h of contact with the virus, we detected PD-L1 expression on conventional (cDCs) (< 0.001) . This war to SEB . We did r to SEB , althoug< 0.001) . In the  < 0.05) . To eluc Figures ; however Figures . When we Figures . These r+ 7-AAD−) CD8+ T cells, 7 days after exposure to the H1N1 virus . CD4+ T cells did not show any significant differences in apoptosis after PD-L1 blocking  of the A(H1N1)pdm09 virus and measured cytokine production. We observed lower levels of IFN-γ, IL-10, and TNF when the whole A(H1N1)pdm09 virus was added, compared to HA stimulation. Moreover, when PD-L1 signaling was blocked, the cytokine levels induced by the virus were higher than those induced by the hemagglutinin. HA cytokine production was not affected by PD-L1 blockade, suggesting that this effect is A(H1N1)pdm09 virus-dependent . To evaluate if IFN-γ, IL-10, and TNF were mainly expressed by CD4+ T cells, we co-cultured isolated memory CD4+ T cells with sorted cDCs with or without PD-L1 blocking, and found that cytokine production by CD4+ T cells was dependent on the presence of cDCs and increased when PD-L1 was blocked . Neither in bulk PBMCs nor in co-cultures of purified cells did we observe an effect of PD-L1 blocking on IL-4, IL-17A, or IL-6 production (data not shown). In addition, we found that after 7 days of culture, enriched cDCs still expressed PD-L1 after stimulation with A(H1N1)pdm09 virus, in contrast, high expression of PD-L1 was observed in memory CD4+ T cells even in the absence of virus stimulation . Together, these results indicate that blocking PD-L1 on PBMCs had no effect on T cell proliferation but significantly decreased CD8+ T cell apoptosis and increased IFN-γ, IL-10, and TNF production by CD4+ T cells.We analyzed if PD-L1 expression induced by A(H1N1)pdm09 could impair the T cell response against the virus. We blocked PD-L1 signaling during virus-induced T cell activation and established that blocking PD-L1 did not compromise T cell proliferation induced by the A(H1N1)pdm09 virus Figures . Howeverblocking . We also3) and in half of the pH1N1− patients (1913.0 ± 1243.49 cells/mm3). We evaluated the frequencies and phenotype of DCs and T cells by flow cytometry; the gating strategy and representative plots are shown in Figure S4.We evaluated PD-L1 expression in A(H1N1)pdm09 infection. We analyzed PD-L1 expression on T cells and DCs from cryopreserved PBMCs collected from patients during the 2009 influenza pandemic. Our study included 25 patients with influenza-like illness and 10 HCs, as shown in P < 0.05). No differences in the pDCs proportions between both groups were detected . CD4+ T cells proportion tended to decrease in both groups of patients compared to that of HCs . Relative PD-L1 expression was increased on CD4+ T cells in both groups of patients compared to that in HCs , while in CD8+ T cells, it was only increased in pH1N1+ patients (P < 0.05).We found that pH1N1+ patients had a lower proportion of cDCs compared to the HCs ; we did not find a significant correlation between PD-L1 expression and cell proportion in pH1N1− subjects or in DCs subsets (data not shown). As a whole, these results suggest that PD-L1 expression on T cells could be one of the factors mediating the decrease in the T cell proportion in pHN1+ patients.Finally, to establish if PD-L1 expression in PBMCs from patients could be associated with the T cell proportion during infection, we performed a series of correlations of DCs and T cell proportions and determined PD-L1 expression in pH1N1+ and pH1N1− subjects. We detected an inverse correlation between PD-L1 expression on CD8in vitro. We also suggest that PD-L1 expression could have implications during the acute natural infection.PD-L1 expression plays a critical role in chronic infections by impairing T cell function . We repode novo protein synthesis. Additionally, we show that H3N2 seasonal virus failed to induce PD-L1 expression on either DCs or T cells; considering UV-inactivated pH1N1 induced PD-L1, it is possible that the expression of PD-L1 observed is independent of the infection capacity of the viruses. According to these results, we conclude that in vitro, there is an important difference between the pandemic and the seasonal influenza viruses in terms of their ability to induce PD-L1 expression. Furthermore, we found that PD-1 up regulation was detected at late time points in CD4+ T and CD8+ T cells cultures, after contact with the A(H1N1)pdm09 virus. This result is concordant with those observed in a recent mouse infection model, in which cognate viral antigen was necessary and sufficient to induce PD-1 expression on T cells, and that PD-1 was expressed by lymphocytes in the lower airways during acute influenza infection in humans [A(H1N1)pdm09 was able to induce PD-L1 expression on DCs in a similar manner to a TLR7 ligand. It has been documented that TLR7 and retinoid-induced gene receptor 1 (RIG-1) mediate the recognition of influenza virus in DCs . Therefon humans .in vitro and in vivo models [+ T cells, whereas cytokine production was increased; however, no impact was observed on T cell proliferation. One explanation for these results could be the late up regulation of PD-1 expression on T cells. Therefore, T cell proliferation may not be affected by the PD-L1 blockade because PD-1 expression was not apparent until day 3; however, the effects that we observed on CD4+ T cell differentiation (cytokine production) when PD-L1 signaling was blocked, could be attributed to their expression of PD-1 until day 3. In addition, PD-L1 expression is maintained over time (7 days) in A(H1N1)pdm09 stimulated cDCs and is highly expressed on memory CD4+ T cells, indicating that these cells could be a source of PD-L1 during the late phase of T cell differentiation. Moreover, PD-1 is expressed on CD8+ T cells 7 days after A(H1N1)pdm09 stimulation, which correlates with decreased T cell death. There are previous reports suggesting that after direct virus exposure, human CD8+ T cells are more susceptible to apoptosis than CD4+ T cells [+ but not CD4+ T cell death.PD-L1 expression has been associated with T cell exhaustion and dysfunction during chronic viral infections and in some acute infections in both o models , 27. We  T cells . In agreγ, and TNF that may be associated with impairment of T cell differentiation induced by the virus, because when we stimulated cells with HA and blocked the PD-1/PD-L1 interaction, we did not observe any effects on cytokine production. The A(H1N1)pdm09 virus has also been reported to induce a decrease in cytokine levels in human DCs when compared with seasonal viruses in vitro [+ T cells. It has been established that T cells from patients infected with A(H1N1)pdm09 cannot differentiate into effector cells, do not respond to mitogens, and highly express CD95 (Fas), suggesting an apoptosis-related mechanism for the lymphopenia reported in A(H1N1)pdm09 infection [Interestingly, we also observed that blocking PD-L1 caused an increase in the production of IL-10, IFN-in vitro . Our stunfection . Furthernfection .+ T cells in influenza-infected patients; thus, CD8+ T cells may also have been redistributed to the lungs. However, we found an increase in PD-L1 expression in DCs and T cells of pH1N1+ patients; it is possible that PD-1/PD-L1 signaling enhanced CD8+ T cell apoptosis as reflected in the decreased T cell proportion and as we showed in the in vitro assays results. We observed low frequency of cDCs in the blood of patients infected with A(H1N1)pdm09 during the first and second pandemic waves in Mexico City. It has been reported that in influenza-infected patients, DCs are recruited in the lung, suggesting that the low proportions that we observed may be caused by the redistribution of the DC population from the blood to the lung . Moreovein vitro results showed that unlike the A(H1N1)pdm09 virus, the H3N2 virus did not induce PD-L1 expression either in DCs or T cells; however, in addition to the seasonal H3N2 virus, seasonal H1N1 viruses were also circulating at that time in Mexico, so we cannot rule out that seasonal H1N1 virus could have also induced PD-L1 expression. Considering that our ex vivo results showed that PD-L1 up regulation may not be strain specific, and that in the natural infection additional immune mediators may contribute to PD-L1 up regulation, we do not discard the possibility that different types of influenza A virus could induce PD-L1 expression on DCs and T cells during acute infection. Our PD-1 and PD-L1 have been recently reported to be expressed in the lungs of A(H1N1)pdm09 patients . Since w+ T cells is inversely correlated with CD4+ and CD8+ T cell proportions, but this correlation was only observed in pH1N1+ patients; this finding could be explained by the fact that the lymphopenia induced by A(H1N1)pdm09 has been reported to be more severe and refractory than that associated with seasonal infection, which is modest during the first days and resolves earlier [+ T cells and the proportion of T cells observed only in pH1N1+ patients [In A(H1N1)pdm09 infected patients, PD-L1 expression on CD8 earlier . Since wpatients , 33.Our data suggest that viral infection may impair the induction of an efficient adaptive immune response in the early stages of infection by promoting PD-L1 expression on DCs and T cells; this could be a mechanism of immune evasion by the A(H1N1)pdm09 virus, similar to that reported in chronic and acute viral infections , 34–36. The 2009 pandemic influenza A(H1N1) virus is able to impair T cell responses through PD-L1 expression, suggesting that the virus could modulate host immune responses during infection by this mechanism.+ T cells (Tm). (a) Gating strategy reflecting the purity of isolated memory CD4+T cells (Tm) characterized by the CD3+CD4+CD45RO+CD45RA− phenotype (purity of 99.6%). (b) Gating strategy for sorting cDCs. (c) Gating strategy to phenotype cDCs, purity of 90.0% of lineage negative cDC population , HLA-DR+, and CD123dim.Figure S1. Representative plots of sorted conventional dendritic cells (cDCs) and isolated memory CD4+ and CD8+ T cells after 18 h of stimulus. Enriched (HLA-DR+ cell-depleted)T cells and DCs (b) were stimulated with pH1N1, SEB or CL264; PD-L1 expression in DCs and T cells was analyzed by flow cytometry and representative histograms are shown. M: medium.Figure S2. The A(H1N1)pdm09 virus does not induce PD-1 expression;whereas PD-L1 expression on DCs is induced directly by the A(H1N1)pdm09 virus whilst PD-L1 expression on T cells is dependent on the presence of antigen-presenting cells. (a) PBMCs were stimulated with A(H1N1)pdm09 virus (pH1N1), seasonal influenza virus (H3N2), staphylococcal enterotoxin B or synthetic TLR7 agonist (CL264); PD-1 expression in DCs and T cells was analyzed by flow cytometry. Fold increase in PD-1 expression in cDCs and pDCs, CD4+ T cells after 5 and 7 days of culture with A(H1N1)pdm09. (a) PD-L1 expression on isolated memory CD4+ T cells, 7 days after co-culture with sorted cDCs in the presence (blue) or absence (red) of pH1N1 virus. (b) PD-L1 expression on cDCs cultured for 5 days in the presence (blue) or absence (red) of pH1N1.Figure S3. PD-L1 is expressed in cDCs and memory CD4−HLA-DR+CD123dim) and pDCs . Gating strategy and representative histograms of PD-L1 expression in CD4+ T cells  and CD8+ T cells . The shaded histogram represents PD-L1expression in a healthy control, whereas the blue and red histograms are representative of two pH1N1+patients.Figure S4. Gating strategy and representative plots of analyzed dendritic (DCs) and T cells from patients and healthy controls. Gating strategy and representative histograms of PD-L1 expression in cDCs (a, LinClick here for additional data file.