P < 0.05). in all groups and had higher stimulatory effect in lymphocytes from day 10 than day 0 of estrous cycle. Progesterone inhibited lymphocyte proliferation, and inhibition was directly related to the dose, in all groups of either sex. Testosterone did not inhibit proliferation at any dose level and did not show any consistent and lucid effects on lymphocyte proliferation. Present study revealed that buffalo lymphocytes produce appreciable amounts of NO in culture system after treatment with estradiol. Significantly high levels of NO in culture supernatant were found in prepubertal buffalo calves and least in post pubertal buffaloes, which had an inverse relation with lymphocyte proliferation in presence of estradiol. NO in culture supernatant was high at the lowest dose of progesterone which was proportional to the lymphocyte proliferation when treated with progesterone. No significant difference in NO culture supernatant was observed between different concentrations of testosterone treatment.Present study was carried out on forty four apparently healthy Murrah buffaloes of different age groups of both sexes to investigate the effects of sex steroids on cell mediated immunity in vitro. Estrogen inhibited proliferation in mitogen-stimulated lymphocytes from prepubertal but not post pubertal buffaloes of either sex. Estrogen at 100 pg/mL concentration stimulating the proliferation significantly ( The sex steroids are in use for the last more than a millennium to correct reproductive disorders and augment reproductive efficiency in animals. However, it has been increasingly apparent that the effects of sex hormones extend far beyond their predominant role in sexual differentiation and reproduction and are now being realised as integral signalling modulators of mammalian immune system. Sexual dimorphism in disease incidence and host response in wide range of animal species provides an indirect evidence of role of sexual hormones in immune modulation –3. ImmunForty-four apparently healthy Murrah buffaloes of three age groups of both sexes  were selected from NDRI herd for conducting present experiments. Eighteen male buffaloes were divided into 3 groups of 6 buffaloes on the basis of their age. Group-I included buffaloes with age ranging between 11 and 13 months; group-II consisted of buffaloes with age ranging between 22 and 24 months. Group-III had postpubertal buffaloes 34 and 36 months of age. Similarly 18 female buffaloes were grouped exactly the same way as in males except that female post pubertal group had cyclic heifers at day 8–10 of estrous cycle. Eight cyclic heifers were selected and studied on day 0 and day 10 of estrous cycle. The estrum (day 0) was confirmed through rectal palpation by an expert and concerned veterinarian at the farm. All these Murrah buffaloes were maintained under routine management and nutritional practices as followed in the herd at the institute. The animal experiments were acceptable to the ethical standards of the National Dairy Research Institute, Karnal, India vide IAEC no. 23/09-21/11.The lymphocytes from peripheral blood were isolated as per the method followed by Young et al.  with som6 cells per culture well. The culture medium used in present study was RPMI-1640 from Sigma Chemical Co., St. Louis, MI, USA, supplemented with L-glutamine, sodium bicarbonate, sodium pyruvate, and antibiotics , besides 10% fetal calf serum. 20 μL of phytohemagglutinin (PHA-P) was added per well to yield a concentration of 5 μg/mL of culture medium. 20 μL of steroid hormone (working concentration) was added to each well. The final volume in each well after adding mitogen and or sex steroid hormone was made to 200 μL with culture medium. Blank wells contained only 200 μL of culture medium with no cells, whereas S–M and S + M wells contained no mitogen and mitogen, respectively, besides lymphocytes. Every observation was made in triplicate wells. The cells were cultured for 36 hours at 37°C in a humidified CO2 incubator (5% CO2). After 36 hours of incubation, 100 μL of culture supernatant was harvested in sterile 1.5 mL eppendorf tubes and stored at −20°C for estimation of nitric oxide. Equal volume of culture media with supplements, preincubated at 37°C, was added into the wells. 20 μL of the MTT solution (5 mg/mL dissolved in DPBS and filtered through 0.22 μm Millex-GV filter unit) was added to each well. The plates were again incubated for 4 hr at 37°C in humidified CO2 incubator. Thereafter, the supernatant was pipetted out completely without disturbing formazan crystal layer and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. After incubating the plate at room temperature for 15 minutes, it was shaken on a microplate shaker and the optical density was read using ELISA reader (Microscan MS-5608A) in dual wavelength measuring system, at a wavelength of 570 nm and a reference wavelength of 630 nm. Lymphocyte blastogenic response was expressed as Proliferation Index (PI) and was calculated as follows:Trypan blue dye exclusion method was used to determine the proportion of viable cells in the separated lymphocytes. When the lymphocyte viability was above 95%, the lymphocytes were processed further for culture. Culture of lymphocytes was carried out in 96-well, tissue culture, flat bottom, sterile microplates with lid from Greiner Bio-One, Germany. The cells were adjusted to 1 × 10μg/mL. 10 μL of it was used for every 90 μL culture medium in each culture well to yield concentration of PHA 5 μg/mL. Stock solution of estradiol, progesterone, and testosterone was prepared in ethanol which was diluted with culture medium to working concentrations of 10 ng/mL, 5 ng/mL, 1 ng/mL, and 100 pg/mL for estrogen, 10 μg, 5 μg, 1 μg, and 100 ng for progesterone, and 1000 ng, 100 ng, 10 ng, and 1 ng/mL for testosterone. 10 μL of working steroid solutions was added to every 90 μL culture medium in culture well yielding final concentration of steroids as 1 ng/mL, 500 pg/mL, 100 pg/mL, and 10 pg/mL for estrogen, 1000 ng/mL, 500 ng/mL, 100 ng/mL and 10 ng/mL for progesterone, and 100 ng/mL, 10 ng/mL, 1 ng/mL, and 0.1 ng/mL for testosterone. All the steroid hormones, β-Estradiol, Progesterone (Δ4-pregnen-3-20-dione) and Testosterone (Δ4-androsten-17β-ol-3 one), Histopaque 1077, Phytohemagglutinin (PHA-P), Trypan blue, and Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Sigma Chemical Co., St. Louis, MI, USA. Dulbecco's Phosphate Buffered Saline (DPBS) was purchased from Himedia Laboratories Pvt. Ltd. India. Phytohemagglutinin used as a mitogen for proliferation of T-lymphocytes was made in culture medium at 50 x) using modified Griess reaction as described by Miranda et al. [μL of test culture supernatant and standards  prepared in culture medium was pipetted out into a 96-well microtitre plate and blank wells contained culture media only. The tests, blanks, and standards were run in triplicates. 50 μL Griess I (1% sulphanilamide) was added to each well and held at room temperature for 5 minutes. 50 μL Griess II was then added to each well and plates were incubated at 37°C for 30 min. After incubation, the absorbance was read at 540 nm wavelength in ELISA plate reader (Microscan MS-5608A). The concentration was calculated from the standard curve using linear regression equation. Detection limit of NO was 1.56 μM. Inter-assay and intra assay difference was 3.4% and 4.62%, respectively.Plasma nitric oxide was estimated as total nitrite . Analysis of variance of the data was performed using three-way ANOVA with variables age, sex, and concentration included in the model as fixed effects and Tukey's honestly significant difference test was employed. Values are presented as mean ± S.E. Graphs and charts were prepared in Microsoft Excel 2007.) on lymphocyte proliferation response that was calculated as proliferation index (PI), in buffalo lymphocytes cultured in vitro, is presented in P < 0.001) variation between age groups and treatment levels was observed in the present study. Estrogen was found inhibiting proliferation in group-I and group-II buffaloes by 5% as compared to group-III post pubertal buffaloes where it stimulated the proliferation to the level of 4%. Among all the groups, post pubertal female buffaloes registered the highest (P < 0.05) PI (1.09 ± 0.03) with estrogen treatment. Among the four concentrations of estrogen, 100 pg/mL registered significant (P < 0.001) stimulatory effect on PI (1.05 ± 0.02) to the level of 5% as compared to 1000 pg/mL (0.93 ± 0.02) and 10 pg/mL (0.95 ± 0.01) that instead inhibited lymphocyte proliferation by 7% and 5% respectively.The effect of four different levels of estradiol  higher stimulatory effect on PI to as high as 10% (1.10 ± 0.03) as compared to 3% (1.03 ± 0.01) on day 0 of estrous cycle. Among the estrogen concentrations, 1000, 500, and 100 pg/mL stimulated the lymphocyte proliferation  significantly (P < 0.05) to the level of 12%, 10%, and 7%, respectively, whereas low level (10 pg/mL) inhibited proliferation (0.99 ± 0.03) up to 1% in cyclic buffaloes. Thus, in cycling females only low level of estrogen was found to be inhibitory to lymphocyte proliferation while all other concentrations were stimulatory.Estrogen treatment of the lymphocytes collected from cyclic buffaloes on day 10 registered significantly  in both males and females, the inhibitory effect was severe in males (0.90 ± 0.01) to the level of 10% as compared to females (0.97 ± 0.01) only 3%. There was no significant difference in the effects of progesterone between different age groups of buffaloes. Progesterone inhibitory effects were significantly (P < 0.001) dose-dependent and PI was inversely proportional to the progesterone concentration. The highest concentration of progesterone (1000 ng/mL) inhibited proliferation (0.81 ± 0.02) by 19% whereas the lowest (10 ng/mL) registered stimulatory effect (1.058 ± 0.02) of 6%. Among all groups, group-III post pubertal female buffaloes registered highest (P < 0.01) proliferation (1.065 ± 0.02) and group-III post pubertal males the lowest (0.86 ± 0.03) when treated with progesterone.Though progesterone inhibited lymphocyte proliferation significantly (P < 0.01) with highest concentration 1000 ng/mL progesterone inhibiting proliferation (0.976 ± 0.02) by 2% and 10 ng/mL stimulated proliferation (1.103 ± 0.02) by 10%. The trend in lymphocyte proliferation with decreased dose of progesterone was linear.The progesterone effects were not significantly different between the lymphocytes from the day 0 and day 10 of reproductive cycle. However, dose dependent effect of progesterone on proliferation was significant (P < 0.01) inhibited proliferation by 4% in group-II males but stimulated by 13% in group-I males, irrespective of testosterone concentration. Contrary to this, testosterone significantly (P < 0.01) inhibited proliferation (0.958 ± 0.01) by 5% in group-I females but stimulated (1.101 ± 0.03) by 10% in group-III post pubertal females.The effects of testosterone treatment on in vitro proliferation of buffalo lymphocytes of different sex and age are presented in  There was no significant effect of testosterone on lymphocyte proliferation index in cycling buffaloes. However, proliferation was comparatively low in lymphocytes from day 0 (1.049 ± 0.03) as compared to day 10 (1.104 ± 0.03) of estrous cycle when treated with testosterone. The testosterone in all its concentrations stimulated proliferation to the level of 10% (1.104 ± 0.04) at 10 ng/mL and 5% (1.049 ± 0.04) at 0.01 ng/mL concentration in cyclic buffaloes.P < 0.001) higher (8.52 ± 0.39 μM/L) NO in culture than females (6.13 ± 0.39 μM/L). The difference was also significant (P < 0.05) among the three groups with lowest in group-III (6.23 ± 0.48 μM/L), followed by group-II (8.07 ± 0.48 μM/L) and group-I (7.68 ± 0.48 μM/L). Among all groups, group-II male buffaloes registered the highest (9.89 ± 1.18 μM/L) and group-III female buffaloes the lowest (4.87 ± 0.42 μM/L) levels of nitric oxide in culture supernatant. In cyclic buffaloes the nitric oxide concentration in culture supernatant did not differ significantly with the stage of estrous cycle and was 5.20 ± 0.29 and 4.88 ± 0.41 μM/L on day 0 (estrus) and day 10 (diestrus), respectively  higher levels of nitric oxide were recorded in culture supernatant of males (9.73 ± 0.30 μM/L) than females (6.75 ± 0.30 μM/L) after 36 hours of culture supplemented with estradiol. The NO levels in culture supernatants supplemented with estradiol were significantly (P < 0.001) different between age groups with group-I buffalo lymphocytes registering NO as high as 11.36 ± 0.37 μM/L followed by group-II and group-III (μM/L) in culture supernatant (P < 0.05) effect of different concentrations of estrogen on nitric oxide levels in culture supernatant; however, NO levels were maximum (8.77 ± 0.74 μM/L) at 100 pg/mL concentration of estrogen and minimum at 10 pg/mL level ( Significantly (roup-III . Among ternatant . There wmL level .μM/L) significantly (P < 0.001) higher than (3.47 ± 0.38 μM/L) day 0 (estrous) lymphocytes. Similarly different concentrations of estrogen in culture system had significant (P < 0.001) effect on NO in culture supernatant in cyclic buffaloes. Significantly higher levels of NO (5.82 ± 0.39 μM/L and 5.51 ± 0.39 μM/L) were recorded at 100 pg/mL and 500 pg/mL estradiol, respectively, as compared to (3.43 ± 0.93 and 2.79 ± 0.39 μM/L) at 1000 pg/mL and 10 pg/mL estradiol, respectively, in cyclic buffaloes  lymphocytes was  difference in levels of nitric oxide in culture supernatant supplemented with different concentrations of progesterone was registered between the sexes  and between the age groups . Among the groups, group-II males registered highest (P < 0.05) levels of NO in culture supernatant where as group-I females the lowest (P < 0.01) between various concentrations of progesterone treatment ( Significant (e lowest . Similarreatment .P < 0.01) higher (5.68 ± 0.40 μM/L) NO in culture supernatant as compared to day 0 (3.98 ± 0.40 μM/L) (P < 0.01) decline in NO production with increasing concentrations of progesterone supplementation with highest NO at 10 ng/mL progesterone followed by 100, 500, and 1000 ng/mL progesterone respectively in cyclic buffaloes  . There wuffaloes .μM/L NO, respectively. Further, NO in culture supernatant was significantly (P < 0.05) high (7.98 ± 0.29 μM/L) in group-II buffaloes as compared to group-III (7.08 ± 0.29) and group-I (6.96 ± 0.29 μM/L). Among the all groups, group-II male buffaloes recorded the highest (P < 0.01) NO whereas group-I males the lowest (μM/L) with 10 ng/mL supplementation.Analysis of variance revealed significant influence of sex on nitric oxide production in culture supernatant supplemented with testosterone and male and female buffaloes on an average, which produced 7.13 ± 0.23 and 7.54 ± 0.23 e lowest . The leve lowest . HoweverμM/L in day 10 and day 0 lymphocyte culture, respectively  as compared to females (3%). This sex-dependent variability in progesterone effects may probably be due to the adaptation of lymphocytes to progesterone because of being exposed to it in females. There was an inverse relationship between the dose and proliferation index. The highest concentration of progesterone (1000 ng/mL) inhibited proliferation by 19% whereas the lowest (10 ng/mL) registered stimulatory effect of 6%. These findings are in partial agreement with those reported by Marco et al.  reportedIn current investigation, testosterone treatment stimulated lymphocyte proliferation in both males and females to the tune of 2 and 5%, respectively, with no significant dose-dependent effect. Testosterone treatment in lymphocyte culture did not yield any dependable and lucid effects either at any particular dose or in any age group. In group-I males it stimulated proliferation at all concentrations of testosterone with maximum at 0.1 ng/mL but in case of group-I female it inhibited proliferation at all concentrations of testosterone. In group-II males contrary to group-I males, testosterone inhibited proliferation except at 1 ng/mL concentration but at the same time in group-II females it stimulated proliferation at all concentrations of testosterone with maximum at 1 ng/mL. Whereas in group-III post pubertal males picture was same as that of group-II males but in group-III females, testosterone stimulated proliferation at all concentrations with maximum at 100 ng/mL. With these varied effects of testosterone, it is hard to arrive at any definitive conclusion. It may be possible that the lymphocytes possess the capability to convert testosterone into estrogen, which may govern the proliferation in adult buffaloes. This option may be working since we got similar observations with testosterone and estrogen in post pubertal female lymphocytes. Benten et al.  have dem Sex and age bias in NO production by lymphocytes in culture was evident in present study. Significantly high levels of NO were found in culture supernatant of males as compared to females treated with estrogen. Among the groups, group-I buffaloes had more NO in culture as compared to group-II and III. However, production rate was not affected by increasing dose of estrogen in culture. The proliferation was higher in group-III where NO was lowest suggesting inverse relationship between PI and nitric oxide production in culture and probably estrogen affected proliferation through NO levels in lymphocyte culture. However, the NO in culture supernatant of day 10 lymphocytes was significantly higher than day 0 while proliferation was also significantly high at diestrous than estrous phase. NO has been reported to be secreted by lymphocytes , 35 and  Nitric oxide in lymphocyte culture treated with different concentrations of progesterone was significantly high in males as compared to females. Among the groups, significantly high levels of NO in culture supernatant were found in group-III and least in group-I buffaloes. In cyclic buffaloes, diestrous phase lymphocytes produced more NO than estrous phase, treated with progesterone. Similarly the nitric oxide levels in supernatant differed between the concentrations of progesterone with lowest NO at highest progesterone concentration. The same trend was also observed in lymphocyte proliferation. This suggested that progesterone probably has no direct effect on NO production by lymphocytes and as such more proliferation is accompanied with more NO.μM/L NO, respectively, in culture. This indicated that the lymphocytes obtained from female animals were equally responsive to testosterone as males. However, levels tended to be significantly (P < 0.05) high in group-II buffaloes as compared to group-I and III. In general, low levels of proliferation were associated with less production of NO in culture in different age groups of buffaloes. Differences were not significant during different stages of estrous cycle which suggested that variations in the immune responses during estrous cycle were not related to testosterone as was evident through PI also. NO in lymphocyte culture supernatant at any level of testosterone did not show any significant difference between sexes with male and female buffaloes on an average, producing 7.13 ± 0.23 and 7.54 ± 0.23 Since it happens to be the first report to register NO in culture supernatant of lymphocytes from buffaloes of different sex and age with sex steroid treatment, it needs further investigation before arriving at any final conclusion on relationship of sex steroids, NO, and lymphocyte proliferation.Effect of female sex steroids on lymphocyte proliferation in vitro was found to be existing. Estradiol effects were dose dependent too, and high as well as too low levels of estradiol were inhibitory to lymphocyte proliferation. Progesterone was found to be inhibiting lymphocyte proliferation and there was inverse relationship between progesterone dose and lymphocyte proliferation. Testosterone did not show any consistent and lucid effects on lymphocyte proliferation, nor did it inhibit proliferation at any dose level. The proliferation was higher in group-III where NO was lowest suggesting inverse relationship between PI and nitric oxide production in culture and probably estrogen affected proliferation through NO levels in lymphocyte culture. NO was not the candidate mediator of effects of all sex steroids in lymphocyte proliferation. NO levels in supernatant differed between the concentrations of progesterone with lowest NO at highest progesterone concentration and lowest proliferation. This suggested that progesterone probably has no direct effect on NO production by lymphocytes and as such more proliferation is accompanied with more NO. No significant difference in NO culture supernatant was observed at different concentrations of estradiol or testosterone.


I2D/IG ratios, and enhancements of G and 2D bands with suspended and supported graphene flakes were measured and analyzed. The peak shifts of G and 2D bands between the Raman and SERS signals demonstrate the doping effect induced by silver nanoparticles by n-doping. The I2D/IG ratio can provide a more sensitive method to carry out the doping effect on the graphene surface than the peak shifts of G and 2D bands. The enhancements of 2D band of suspended and supported graphenes reached 138, and those of G band reached at least 169. Their good enhancements are helpful to measure the optical properties of graphene. The different substrates that covered the graphene surface with doping effect are more sensitive to the enhancements of G band with respect to 2D band. It provides us a new method to distinguish the substrate and doping effect on graphene. The interactions between phonons and electrons induced by the dopants or the substrate of graphene in spectroscopic investigation reveal a rich source of interesting physics. Raman spectra and surface-enhanced Raman spectra of supported and suspended monolayer graphenes were measured and analyzed systemically with different approaches. The weak Raman signals are greatly enhanced by the ability of surface-enhanced Raman spectroscopy which has attracted considerable interests. The technique is regarded as wonderful and useful tool, but the dopants that are produced by depositing metallic nanoparticles may affect the electron scattering processes of graphene. Therefore, the doping and substrate influences on graphene are also important issues to be investigated. In this work, the peak positions of G peak and 2D peak, the 78.67.Wj ; 74.25.nd ; 63.22.Rc (phonons in graphene) With our analysis, details about the effects of charged impurities and substrate can be realized. The peak shift of G and 2D bands and the I2D/IG ratio are useful to demonstrate the dopants and substrate effects on the graphene. The well-enhanced G and 2D bands are obtained to enhance the weak Raman signals. Moreover, the enhancements of G band with respect to 2D band are found to be more sensitive to various substrate influences on the graphene surface. This paper provides a new approach to investigate the substrate and doping effect on graphene.Graphene, the thinnest operties -3. The woperties -9. The poperties -12. Ramaoperties -18, and operties -24. The operties ,26. Theroperties ,28. The 2 were first defined by photolithography on an oxidized silicon wafer with an oxide thickness of 300 nm. Reactive ion etching was then used to etch the squares to a depth of 150 nm. Highly ordered pyrolytic graphite was consequently cleaved with the protection of scotch tape to enable the suspended graphene flakes to be deposited over the indents. To study the SERS, silver nanoparticles were deposited on the graphene flake at a deposition rate of 0.5 nm/min by a thermal deposition system. A 5-nm-thick layer of silver nanoparticles on the graphene flake was thus formed. To measure the graphene flake, a micro-Raman microscope  was utilized to obtain the Raman and SERS signals of monolayer graphene. The monolayer graphene was identified through optical observation with various color contrast and by Raman spectroscopy with the different shape bandwidths and peak positions of 2D band under different graphene layers. During spectroscopic measurement, a 632-nm He-Ne laser was used as the excitation source; the power was monitored and controlled under 0.5 mW to avoid the heating of the graphene surface.Suspended graphene was fabricated by mechanical exfoliation of graphene flakes onto an oxidized silicon wafer. The optical image of suspended and supported graphenes and the illustration of their coverage by silver nanoparticles are shown in Figure To explore the SERS on graphene, the interactions between metallic nanoparticles and graphene surface has to be presumably understood. This is because the plasmonic resonances of nanoparticles with different shapes and sizes can affect the interactions between them, and then change the SERS signals ,29-33. TI2D/IG ratios of G and 2D bands were important indicators of doping effect on graphene [I2D/IG ratio is particularly more sensitive than the peak shifts to the doping effect. A lower I2D/IG ratio is related to more charged impurities in graphene. The Raman and SERS signals of the suspended and the supported graphenes are shown in Figure According to previous work, the peak positions and graphene -40, in wI2D/IG ratio of suspended graphene under Raman signals is 4.1 ± 0.1 and larger than supported graphene which is 3.6 ± 0.5, while the I2D/IG ratio of suspended graphene on the SERS signals is around 2.9 ± 0.1 and smaller than supported graphene which is 3.0 ± 0.2. The result disclosed the substrate effect on the supported graphene is stronger than the suspended graphene. To our understanding, this above effect can be reasonable because SiO2/Si substrate has a direct contact on the graphene surface so the silver nanoparticles deposition causes stronger doping effect on the suspended graphene than supported graphene.In order to minimize the random errors, each Raman spectra data point was obtained by five-time repetitions. As presented in Figure To understand the Raman and SERS signals, the enhancements of G and 2D bands with the suspended and supported graphenes are shown in Figure I2D/IG ratios were varied. The enhancement effect of suspended and supported graphenes was calculated and analyzed. The peak shifts of G and 2D bands and their Raman spectra and I2D/IG of SERS signals are found very useful in the investigation of the substrate and doping effect on the optical properties of graphene. The enhancements of G and 2D bands have been found to cause the great improvement of weak Raman signals. Otherwise, the more sensitive enhancements of G band with respect to 2D band are related to the doping effect with various substrates that covered the graphene surface. The optical emission spectra of suspended and supported graphenes have provided us a with new identification approach to understand the substrate and doping effect on graphene.In our work, Raman and SERS signals of supported and suspended monolayer graphenes were measured systematically. The peak positions of G and 2D bands and the The authors declare that they have no competing interests.C-H and B-JL carried on the experimental parts: the acquisition of data and analysis and interpretation of data. C-H also had been involved in drafting the manuscript. H-YL and C-HH analyzed and interpreted the data. They also had been involved in revising the manuscript. F-YS and W-HW  prepared the samples, suspended graphene using by micromechanical method, and captured the OM and AFM images. C-YL have made substantial contributions to the conception and design of the study and revising it critically for important intellectual content. H-CC, the corresponding author, had made substantial contributions to the conception and design of the study and had been involved in drafting the manuscript and revised it critically for important intellectual content. All authors read and approved the final manuscript.


Sarcoidosis is a multisystem granulomatous disease with an unknown etiology. It most commonly affects young and middle-aged females. It can affect any organ, but mostly lung, skin, and eyes. Up to half of patients are asymptomatic and the disease is often detected incidentally on abnormal chest radiography. We report the case of a 31-year-old male with bilateral recurrent dacryostenosis. The nasolacrimal obstruction was the initial manifestation of systemic sarcoidosis. Sarcoidosis is a multisystem granulomatous inflammation of unknown etiology . It typiThis is a 31-year-old male diagnosed with nasolacrimal sarcoidosis. He first presented complaining of bilateral epiphora worse on the left. There is no history of endonasal surgery nor facial trauma. He complained of a chronic nasal obstruction and had been treated for sinusitis in the past. He was otherwise asymptomatic and in good general health.Exam revealed complete dacryostenosis of the left and partial obstruction of the right ducts. He was initially presumed to have idiopathic obstruction and underwent external left dacryocystorhinostomy. On followup one year later, he presented with complete obstruction on the right and bilateral chronic maxillary sinusitis as seen on sinus CT . We perfHistologic evaluation of ethmoidal tissues revealed granulomatous inflammation compatible with sarcoidosis. Pulmonary CT demonstrated bilateral hilar adenopathy and parenchymatous infiltration consistent with stage 2 sarcoidosis . AngioteOn follow-up exam one year later, we noted right endonasal synechia . The patSarcoidosis affects primarily the respiratory tract. Although periorbital involvement is uncommon, any part of of the eye, orbit, and adnexal structures may be involved. The lacrimal gland seem to be the most affected, followed by the orbit, eyelids and the lacrimal sac . There aDemirci and Christianson  report aAlthough there is some nasolacrimal involvement cases reported in literature, most of them presented with known sarcoidosis. Fergie et al. reported 4 patients with nasolacrimal involvement, 3 of whom did not show evidence of systemic disease . VasquezMoreover, the utility of routine histopathologic examination of tissue obtained during DCR is controversial. While most nasolacrimal obstructions are idiopathic, some authors recommend histopathologic analysis in young patients, as we report here, and in the context of atypical clinical presentation. Heindl et al. performed biopsy of the lacrimal sac in 19 of 500 cases which were found to be atypical. 3 out of 19 biopsies performed were positive for sarcoidosis . Tucker et al. obtained 162 lacrimal sac biopsy specimens in 150 consecutive patients undergoing dacryocystorhinostomy for clinical primary-acquired nasolacrimal duct obstruction . BiopsieNasolacrimal obstruction as an initial presentation of sarcoidosis is rare. Although uncommon and unrelated to specific symptoms, this condition should be considered in the differential diagnosis of epiphora and sinus complaints. This case emphasizes the clinical purpose of routine histopathological analysis of the lacrimal sac when performing DCR.


Climate variation has long been studied in relation to human health. The aim of the present work was to evaluate the relationship between environmental humidity, and air temperature with the prevalence of diabetes, among elderly islanders.During 2005–2011, 1959 elderly (aged 65 to 100 years) individuals from 13 Mediterranean islands were enrolled. Socio-demographic, clinical and lifestyle factors were assessed using standard procedures. Diabetes was defined as fasting blood glucose levels > 125 mg/dl. Relative environmental humidity was measured as a percentage of air moisture and mean daily temperature in degrees Celsius.For the present analysis 713 men (74 ± 7 years) and 596 women (73 ± 7 years) with complete data were studied; 27% of both men and women had diabetes. The prevalence of diabetes was 42% in the elders living in high relative humidity areas  as compared with 24% among those living at low relative humidity residential areas (p < 0.001). After adjusting for age, sex and mean temperature, an increase in the area’s relative humidity by 1 degree, increased the likelihood of having diabetes by 12% . No significant association was observed between mean temperature and diabetes .A considerable proportion of elderly, especially those living in high relative humidity areas, had diabetes. Further research is needed to confirm this observation and to understand the underlying mechanisms. Worldwide 347 million of people have diabetes . There aClimate variation -15 and aDiabetes is a clinical condition, often asymptomatic and with high prevalence in older adults. Given the lack of data regarding the effect of environmental factors, and particularly climatological, on the prevalence of diabetes, the aim of the present work was to evaluate the association between relative humidity and mean air temperature, with the prevalence of diabetes, among elders (> 65 years old) that are lifelong inhabitants of Mediterranean islands.This work is under the context of the MEDIS Study . During nd WMA General Assembly, Edinburgh, Scotland, October 2000). The Institutional Ethics Board of Harokopio University approved the study design (16/19-12-2006). Participants were informed about the aims and procedures of the study and gave their consent prior to being interviewed.The study followed the ethical considerations provided by the World Medical Association  during the past 5 years before sampling, were recorded using data from the National Meteorological Service . Areas wAs already has been presented in previous reports from the MEDIS study ,23 the dContinuous variables were presented as mean ± standard deviation (SD), and categorical variables as frequencies. Comparisons of normally distributed continuous variables  between groups were performed using Student’s t-test. Spearman rho coefficient was applied to evaluate the correlation between mean relative humidity, mean temperature and physical activity, the MedDietScore of the participants. Normality was tested using P-P plots. Independence between categorical variables was tested using the chi-square criterion. Additive logistic regression models were used to evaluate the association between participants’ characteristics  and presence of diabetes. Results are expressed as odds ratios and the 95% confidence intervals. Deviance residuals and Hosmer-Lemeshow criterion evaluated models’ goodness-of-fit. P-value <0.05 was considered to be statistically significant. SPSS software (version 18) was used for all calculations .The prevalence of diabetes was 27% for the entire cohort. No significant variations were observed between islands. When comparing data from participants living in high humidity areas vs. participants living in low humidity ones, the prevalence of diabetes was 42% and 23%, respectively. Specifically, participants 65 to 80 years old living in high humidity areas, had almost double prevalence of diabetes compared with those living in low humidity areas . In the next age group  the percentage increased for participants living in high humidity areas while for those living in low humidity areas remained at the same levels . Demographic, behavioral and lifestyle characteristics of the sample, by relative humidity, are summarized in Table The clinical characteristics and the prevalence of diabetes, based on humidity conditions, are presented in Table Furthermore, an inverse correlation was observed between high humidity areas of living and the physical activity level . An inverse association was also observed between humidity and adherence to the Mediterranean dietary pattern . Both findings suggest that people living in high humidity areas tend to be less physically active and away from the Mediterranean dietary pattern.models 1, 2 and 3), even after adjusting for several confounders, such as physical activity, adherence to the Mediterranean diet, smoking habits, living conditions (model 2), hypertension, hypercholesterolemia and obesity (model 3).Age, sex and mean air temperature adjusted logistic regression analysis showed that people living in high humidity areas were 3.1-times more likely to have diabetes, compared to those living in low humidity areas (95% CI 1.65 to 5.94). Specifically, when the relative humidity in an area increases by one degree, the likelihood of an elder having diabetes increases by 12% . However, residual confounding may exists, and therefore multiple logistic analyses were further applied. Results have been displayed in Table n = 341), and those above 80 years (n = 76), it was found that the positive association of relative humidity with diabetes was significant only in the group of very old people , while among those 65–80 years no significant association was found . Moreover, when the analysis was stratified by the level of adherence to the Mediterranean diet according to the MedDietScore cutoffs, it was observed that relative humidity was associated with higher likelihood of diabetes, only for the low adherence group . For the moderate/high adherence group, relative humidity was not associated with diabetes . Moreover, no association was observed between mean temperature and diabetes among those <80 or those >80 years, as well as those close or away to the Mediterranean dietary pattern.In addition, when data analysis was repeated separately for those between 65–80 years old  was similar to other recent studies conducted in European countries  and in GTo further explore the aforementioned finding, additional analysis of clinical and lifestyle components and the area’s relative humidity was performed. It was also observed higher prevalence of hypertension, central fat, along with lower physical activity in the elders living in high humidity areas, compared those living in low humidity ones. These factors could play a moderating role in the investigated research hypothesis. Despite the lack of previous studies on relative humidity and prevalence of diabetes and the other aforementioned clinical conditions, a number of studies have already investigated the effect of climate variations  on human health -20,22. SAccording to Köppen’s climate classification the Mediterranean climate is a particular variety of the subtropical climate found around the Mediterranean Sea. The climate in this area is characterized by relatively warm to hot, dry summers, mild to cool, rainy winters and extended sunshine almost all the year. The islands included in the MEDIS study have Mediterranean climate temperatures, with high air relative humidity and long, hot summers. In the mainland, the mean humidity is around 65% while in the islands is around 75% . High aiMoreover, several studies have proposed that higher rates of relative humidity are associated with lower mood scores ,34. DiscAdditional analysis showed a consistent association between relative humidity and the likelihood of an elder having diabetes, only in the group of 80 years old and above, and only in elders with low adherence to the Mediterranean diet. The first could be attribute to the fact that prolonged age accumulate several harmful factors, including the additive effect of lifelong living in an environment with high humidity. Regarding the second finding, recent studies have associated high adherence to the Mediterranean diet with better glucose homeostasis in the human body. The Mediterranean dietary pattern emphasizes on a consumption of high monounsaturated fats, mainly from olive oil, and encourages consumption of fruits, vegetables, tree nuts, legumes, whole grains, fish and poultry in low to moderate amounts, a relatively low consumption of red meat, as well as a moderate consumption of alcohol. The proposed mechanism was through regulations in plasma glucose and insulin levels . This efPhysical activity and adherence to the Mediterranean diet have been associated with insulin resistance and the development of diabetes in many previous studies ,37. The The present study has several strengths. It is one of the first to evaluate the effect of climate variations in the health status of a large sample of “healthy”, free-living elderly people in the Mediterranean islands. The limitation of the study is mainly caused by its cross-sectional design and therefore the lack of causal relationships. Also, measuring climate condition only by environmental humidity (and not indoor) can be considered as a study limitation.The prevalence of diabetes was high in the studied elderly and free of cardiovascular disease, insular population, and elders living in high humidity areas had almost double prevalence of diabetes, as compared with those living in low humidity residences. The latter attempted to be partially explained by the accumulation of cardiovascular disease factors and by the modifications in lifestyle habits, this population appeared to have made. However, further research is needed to confirm this observation and to understand the underlying mechanisms.The authors declare that there are not any financial or non-financial competing interests to declare in relation to this manuscript.ST, DP, CH, CL, EP conceived of the study, carried out its designing and implementation, drafted the manuscript, and performed the statistical analysis. MM, KK, NT, AZ, EG, GM, VB participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.


Graphene has received significant attention due to its excellent mechanical properties, which has resulted in the emergence of graphene-based nano-electro-mechanical system such as nanoresonators. The nonlinear vibration of a graphene resonator and its application to mass sensing (based on nonlinear oscillation) have been poorly studied, although a graphene resonator is able to easily reach the nonlinear vibration. In this work, we have studied the nonlinear vibration of a graphene resonator driven by a geometric nonlinear effect due to an edge-clamped boundary condition using a continuum elastic model such as a plate model. We have shown that an in-plane tension can play a role in modulating the nonlinearity of a resonance for a graphene. It has been found that the detection sensitivity of a graphene resonator can be improved by using nonlinear vibration induced by an actuation force-driven geometric nonlinear effect. It is also shown that an in-plane tension can control the detection sensitivity of a graphene resonator that operates both harmonic and nonlinear oscillation regimes. Our study suggests the design principles of a graphene resonator as a mass sensor for developing a novel detection scheme using graphene-based nonlinear oscillators. Graphene has recently been attracting the scientific community due to its excellent electrical -3 and/orTo gain a detailed insight into the underlying mechanism of the vibration of a graphene resonator, an atomistic simulation such as molecular dynamics (MD) simulation has been widely utilized. For instance, Park and coworkers ,23 have The computational limitation of atomistic simulations in depicting the underpinning principles of experimentally observed mechanics of graphene resonators has led researchers ,25-27 toIn this work, we have studied the nonlinear vibration of a graphene resonator using a continuum elastic model, i.e., plate model. We have found that nonlinear oscillation is a useful avenue for improving the detection sensitivity of a graphene resonator and that the detection sensitivity of a graphene-based nonlinear oscillator is governed by both the actuation force (which determines the nonlinearity of vibration) and the size of a graphene resonator. It is shown that the nonlinearity of vibration for a graphene resonator can be tuned by an in-plane tension and that such in-plane tension can modulate the detection sensitivity of a graphene resonator that operates in both harmonic and nonlinear oscillations. In particular, an in-plane tension improves the dynamic frequency range and detection sensitivity of a graphene resonator that operated in harmonic oscillation, while an in-plane tension deteriorates the dynamic frequency range and sensing performance of a graphene-based nonlinear oscillator. Our study sheds light on a continuum elastic model for gaining insight into not only the underlying mechanisms of nonlinear vibration-based enhancement of the dynamic frequencies and sensing performance of a graphene resonator, but also the role of an in-plane tension in modulating the nonlinearity of a graphene resonator.UB and stretching energy US represented in the form [Graphene can be modeled as a plate whose mechanical deformation is attributed to the strain energy composed of bending energy the form κESh, and ν represent the bending rigidity, axial stretching modulus, thickness, and Poisson's ratio of a graphene, respectively, w(xyt) indicates the out-of-plane deflection of a graphene, x and y are the coordinates along the in-plane direction of a graphene, respectively, N0 is a constant axial tension (due to pre-strain) applied to a graphene, and the symbol Ω in an integrand indicates the surface integral. The strain energy can be related to a potential field prescribed to the atomic structure of a graphene, as has been elucidated in Cauchy-Born model [where rn model -34, suchi-th carbon atom for a graphene, r is the atomic coordinates of a graphene, and N is the total number of carbon atoms for a graphene. Here, it should be noted that when the elastic constants of a graphene (i.e. κ and ES) are determined from Equation 2, an axial tension N0 is assumed to be zero . As described in a literature [κ = 1.5 eV and ESh = 2,000 eV/nm2.Here, terature , the forT for a vibrating graphene. The kinetic energy T can be written asIn order to obtain the equation of motion, we need to know the kinetic energy ρ0 is the mass density of a graphene; the mass density ρ0 can be straightforwardly determined from a relation of ρ0 = NmC/S, where mC is the atomic mass of a carbon atom, and S is the surface area of a graphene. The equation of motion for a vibrant graphene can be obtained from the minimization of a Hamiltonian H defined as H = U + T − W, where W is the work done by an external force field such as actuation force. The variation of the Hamiltonian, δH, can be obtained as [where ained as ,35f is an actuation force per unit area for a graphene, a symbol δ indicates a variation, δw is a virtual out-of-plane deflection of a graphene, a Greek index indicates the coordinates, i.e., α = x (for α = 1) or y (for α = 2), and a repeated Greek symbol represents Einstein's summation rule. The equation of motion is therefore given bywhere w(xt). In this work, for theoretical convenience, we assume that axial force N0 is the biaxial loading represented in the form of N0 = N0(ex + ey), where ex and ey indicate the directional unit vectors in the x and y directions, respectively. Furthermore, the actuation force f is assumed to be in the form of f = f0cosΩt, where f0 is the amplitude of an actuation force, and Ω is a driving frequency. For solving the equation of motion given by Equation 5, we assume that the out-of-plane displacement, i.e., w(xyt), can be decomposed in the following form [Here, it should be noted that in-plane displacements are ignored in the governing equation given by Equation 5 since in-plane displacements are small in comparison with out-of-plane displacement ing form ,36-38z(t) indicates a time-dependent amplitude, and ψ(xy) represents the deflection eigenmode for a vibrant graphene. In our work, we presume that a monolayer graphene exhibits a rectangular shape and that all edges of a graphene are clamped. The deflection eigenmode that satisfies the clamped boundary conditions is represented in the formHere, a and b are the lengths of the graphene edges, respectively  as shown in Figure μ is given byFor a case in which atoms are adsorbed onto a graphene resonator, as shown in Figure δ(x) is the Dirac delta function, and Δm is the total mass of adsorbed atoms. For the case in which atoms are uniformly adsorbed onto a graphene , the inertia term μ is written aswhere m0 is the mass of atoms adsorbed onto the unit area of a graphene, while Δm is the total mass of adsorbed atoms onto the entire surface of a graphene. Based on inertia terms for both mass-adsorbed graphene (as described in Equation 10) and a bare graphene (represented in Equation 9.a), it is straightforward to compute the resonant frequency shift, Δω, of a graphene resonator due to the mass adsorption; Δω = ω(m + Δm) – ω(m), where ω(m) is the resonant frequency of a bare graphene resonator whose effective mass is given as m (with m = ρab), and ω(m + Δm) is the resonant frequency of a graphene resonator onto which the mass adsorption with the amount of Δm occurs. This frequency shift due to mass adsorption is typically negative since mass adsorption increases the overall mass of a resonator and, consequently, reduces the resonant frequency of a resonator with respect to that of a bare resonator.Here, Δp0 = 0.001 aN (where 1 aN = 10−18 N) in order to induce the harmonic oscillation of a graphene resonator. Moreover, we have taken into account the case in which an in-plane tension N0 is driven by pre-strain, such as N0 = EShϵ0/(1 − ν), where ESh, and ν indicate the stretching modulus, thickness, and Poisson's ratio of a graphene resonator, respectively, and ϵ0 is the pre-strain applied to a graphene resonator. With ϵ0 = 4 × 10−5, the resonant frequency of a graphene undergoing a harmonic oscillation is predicted as ω0 = 20.63 MHz, which is consistent with the experimentally measured resonant frequency of 19.8 MHz  = ∇ψ(xy) = 0 along all edges; this boundary condition is referred to as “fully clamped” boundary condition. In order to understand the effect of boundary condition on the resonant frequency of a graphene resonator, we have taken into account the deflection eigenmode in the form of ψ(xy) = sin(πx/a)⋅sin(πy/b), which satisfies the boundary condition as follows: ψ(xy) = 0 at all edges, but ∇ψ(xy) ≠ 0 along all edges. This boundary condition is referred to as “weakly clamped” boundary condition. As shown in Figure As described above, the resonant frequency of a graphene resonator predicted from a continuum elastic model is overestimated in comparison with that measured from experiment. This may be attributed to the boundary condition that we used in our simulation; in our simulation, we have utilized the fully clamped boundary conditions such as D = 250 nm, to which pre-strain with the amount of ϵ0 = 10−5 is applied. It is shown that when the amplitude of an actuation force is in the order of 0.01 fN, the graphene resonator undergoes harmonic oscillation. On the other hand, when a graphene resonator is actuated by the amplitude of an actuation force in the order of >0.05 fN, the graphene resonator experiences nonlinear vibration /Ω0, where ω is the resonance of a nonlinearly oscillating graphene, and Ω0 is the harmonic resonance of a graphene defined as Ω0 = (α/μ)1/2. Here, it should be noted that the dimensionless parameter θ represents the degree of nonlinearity for the resonance of a graphene. Figure θ) as a function of the amplitude of an actuation force. It is found that when a graphene resonator bears a pre-strain with the amount of 10−5, the amplitude of an actuation force in the order of 0.5 fN results in θ = 0.15, which indicates that the resonance behavior at an amplitude of 0.5 fN is close to the harmonic oscillation. As the amplitude increases, the dimensionless parameter θ significantly increases, indicating that the nonlinearity of graphene resonance can be induced by a large amplitude of actuation force. In particular, when the graphene resonator is actuated with an actuation amplitude of 5 fN, the parameter θ becomes θ = 0.75, indicating that the resonance behavior is highly nonlinear. This indicates that the nonlinear vibration of a graphene resonator can be easily observed even when the actuation amplitude is applied in the order of 1 fN. The nonlinear vibration of a graphene resonator actuated with an actuation amplitude p0 even in the order of 1 fN is attributed to the fact that the deflection amplitude of a graphene resonator is typically much larger than the thickness of a graphene resonator.Now, we have taken into account the nonlinear oscillation of a squared graphene resonator with a size of n Figure a. This iEven though graphene resonator has recently been extensively considered as a NEMS device for its applications in actuation, it has been barely employed as a mass sensor that enables highly sensitive atomic detection . The high detection sensitivity of a graphene resonator is attributed to the high-frequency dynamic range that is achieved due to high elastic stiffness and low mass density of a graphene resonator. In this study, we have scrutinized the resonant responses of graphene resonators, which undergo not only harmonic oscillation but also nonlinear vibration, to atomic adsorption onto the surface of a graphene resonator. Our study on the nonlinear response of a graphene resonator to mass adsorption is ascribed to our previous finding ,20,21 thD = 250 nm without any applied pre-strain . Figure p0 = 1 aN). On the other hand, when a graphene resonator is excited by a large amplitude of actuation force , the frequency shift due to mass adsorption is no longer proportional to the adsorbed mass. It is also found that as the amplitude of actuation force increases, the resonant frequency shift due to mass adsorption significantly increases, which indicates that nonlinear oscillation increases the detection sensitivity of a graphene resonator as anticipated. In order to gain a deep insight into the effect of nonlinear oscillation on the detection sensitivity of a graphene resonator, we have taken into account the resonant response of a graphene resonator to atomic adsorption with the amount of Δm = 10 ag. As shown in Figure m = 10 ag is critically dependent on the amplitude of actuation force. In addition, we have shown the aforementioned dimensionless parameter θ as a function of the amplitude of actuation force , the frequency shift of a graphene resonator with a size of D = 250 nm (without any pre-strain) due to atomic adsorption with the amount of 10 ag is in the order of 0.1 GHz. On the other hand, when an actuation amplitude is increased to 5 fN, which corresponds to highly nonlinear oscillation , the frequency shift due to mass adsorption (with the amount of 10 ag) increases by about sixfold . This clearly elucidates that the nonlinear vibration is useful in increasing the frequency shift of a graphene resonator due to mass adsorption, which highlights the nonlinear vibration that improves the detection sensitivity of a graphene resonator. We have considered a squared graphene resonator whose size is e Figure b. It is D) on the frequency shift of a graphene resonator due to mass adsorption, Δω, with respect to the actuation amplitude (p0). It is found that for a graphene resonator whose size is D ≥ 150 nm, the increase of actuation amplitude enhances the frequency shift of a graphene resonator due to mass adsorption, which is consistent with our conjecture that nonlinear vibration improves the detection sensitivity of a graphene resonator. On the other hand, for a graphene resonator whose size is D ≤ 100 nm, an increase in the actuation amplitude does not significantly amplify the frequency shift of a graphene resonator due to mass adsorption in comparison to that of a large-scale graphene resonator . This indicates that even though large actuation amplitude induces the nonlinear oscillation of a small-scale graphene resonator , the nonlinear vibration does not remarkably increase the frequency shift due to mass adsorption. This result suggests that the length scale of a graphene resonator plays a central role in not only the dynamic frequency range of a graphene resonator but also in the sensing performance of graphene-based nonlinear oscillators.We have also studied the effect of graphene size  as a function of actuation amplitude and in-plane tension. When the actuation amplitude is in the order of 0.01 fN, the vibration behavior of a graphene becomes almost harmonic oscillation regardless of in-plane tension, as anticipated. For a graphene resonator actuated by an actuation amplitude of 1 fN, the dimensionless parameter for a bare graphene resonator (i.e. N0 = 0) is in the order of 10, which indicates that the vibration behavior of a bare graphene is almost nonlinear oscillation. On the other hand, when an in-plane tension with the amount of 10 pN/nm is applied to a graphene resonator actuated by the amplitude of 1 fN, the dimensionless parameter is in the order of 0.5, which indicates that an in-plane tension reduces the nonlinearity of the resonance of a graphene. As in-plane tension increases, the nonlinearity is significantly reduced even up to the order of 10−1, indicating that the resonance behavior is almost harmonic oscillation. Our result suggests that an in-plane tension plays a central role not only in increasing the dynamic frequency range of a graphene resonator but also in inducing the transition from nonlinear vibration to harmonic oscillation.In order to validate our conjecture that an in-plane tension can play a role in the transition from nonlinear vibration to harmonic oscillation, as shown in Figure D = 200 nm, the resonance behavior of a graphene actuated by an amplitude of ≤0.3 fN is almost close to harmonic oscillation. On the other hand, the vibrational behavior of a 350-nm graphene resonator driven by even an amplitude of 0.1 fN becomes nonlinear oscillation. This indicates that the size of a graphene resonator determines the actuation amplitude that is required to induce the nonlinear vibration of a graphene resonator. Moreover, it is shown that the smaller the graphene resonator is, the smaller is the amount of an in-plane tension that can induce the transition from nonlinear oscillation to harmonic resonance. This suggests that an in-plane tension-driven transition from nonlinear vibration to harmonic oscillation is determined by the size of a graphene resonator.Figure m = 20 ag) as a function of the size of graphene as well as in-plane tension. It is interestingly found that for graphene resonators operating in both nonlinear oscillation and harmonic vibration, an in-plane tension-induced improvement of detection sensitivity of a graphene resonator is significantly dependent on the size of a graphene such that an in-plane tension is useful in increasing the detection sensitivity of a graphene resonator whose size is D ≈ 100 nm, whereas an in-plane tension is ineffective in enhancing the sensing performance of a graphene with a size of D > 300 nm in comparison with the detection sensitivity of a graphene resonator with a size of D = 100 nm. Our study sheds light on the important role of an in-plane tension on modulating not only the resonance behavior of a graphene resonator but also the detection sensitivity of a graphene resonator.Now, we have studied the role of an in-plane tension on the detection sensitivity of a graphene resonator that experiences both nonlinear vibration and harmonic oscillation Figure . For a gIn this work, we have studied the vibrational behaviors of graphene resonators as well as their sensing performance based on continuum elastic model such as plate model. It is shown that nonlinear vibration is useful in improving the detection sensitivity of a graphene resonator and that an in-plane tension is able to tune both the nonlinearity of the vibrating graphene resonators and their detection sensitivity. It should be noted that, in this work, our continuum model is only applicable to a monolayer graphene resonator. For modeling the multilayered graphene resonator, the interactions between graphene sheets have to be considered in the continuum modeling , which wThe authors declare that they have no competing interests.KE and C-WK designed the research. MDD performed the simulation. MDD, C-WK, and KE analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.Figure S1. The dependence of frequency shift due to mass adsorption of a graphene on the location at which atomic mass was adsorbed.Click here for file


We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219–414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an  Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease that commonly strikes people between 40 and 60 years of age. During the disease, motor neurons in the spinal cord and brain degenerate, normally leading to death in 1–5 years. The progressive deterioration of patients with ALS results in enormous healthcare and social costs, yet little is known about the disease process and no long-term effective treatments exist. Frontotemporal lobar degeneration (FTLD) is a collective term for a group of neurodegenerative diseases associated with degeneration in the frontal and temporal lobes of the brain The majority of ALS cases are sporadic but ∼5% of patients have a familial mutation. In 2006, TAR DNA binding protein 43 (TDP-43) was identified as a major protein constituent within ubiquitinated neuronal inclusions in a large proportion of ALS and FTLD cases in vivo and how this may contribute to the disease process.TDP-43 has been found to associate with cytosolic RNA stress granule (SG) proteins. This may be an essential early step in pathological accumulation of TDP-43 It is well known that protein kinases control movement and accumulation of SG proteins including hnRNPs and HuR in vivoWhether additional kinases have a critical role in controlling TDP-43 nuclear to cytosolic trafficking and subsequent accumulation in the cytosol is not clear. Glycogen synthase kinase 3 (GSK3) has a central role in neurodegeneration due to its modulation of the microtubule-associated protein, tau In the present study we examined whether additional kinases are involved in accumulation of TDP-43 in SGs in cell culture. Using our established model of mitochondrial inhibition to induce TDP-43-positive SGs in SH-SY5Y cells, we screened 80 kinase inhibitors covering 35 different kinases (Tocriscreen kinase inhibitor toolbox) to determine the effect of kinase inhibition on TDP-43 localization to SGs.kinase inhibitor toolbox was from Tocris Bioscience .4′,6′ Diamino-2-phenylindole dihydrochloride (DAPI) was obtained from Invitrogen . N,N′-dimethyl-4,4′-bipyridinium dichloride (paraquat) and sodium arsenite, were from Sigma Aldrich . Tocriscreen Polyclonal TDP-43 antisera were purchased from Proteintech Group . Polyclonal antisera to hnRNP K were purchased from Abcam . Monoclonal antisera to hnRNP A1 were from Merck . TIA-1-related (TIAR) polyclonal antisera were from Cell Signaling Technology . Monoclonal antisera to HuR were obtained from Invitrogen .2 at 37°C.The cell lines used in this study were human neuroblastoma SH-SY5Y cell line and the human epithelial HeLa cell line. Cells were passaged and maintained in DMEM plus 5% FBS (HeLa cells) or DMEM/F12 plus 10% FBS (SH-SY5Y cells). To induce differentiation, SH-SY5Y cells were treated with 10 µM retinoic acid for 7 days. Differentiation was confirmed by morphological changes (neurite extension) and up-regulated expression of synaptophysin, tyrosine hydroxylase and VMAT2 2O and added at indicated concentrations and the medium was briefly mixed by aspiration. Incubations were performed for periods stated in individual experiments. Where indicated, cells were co-treated with kinase inhibitors at 10 µM from Tocriscreen kinase inhibitor toolbox stock solutions prepared at 10 mM in DMSO. See Undifferentiated cells were grown in 24 or 6-well plates or on 12 mm coverslips (for immunofluorescence) for 2–3 days before experiments (∼80% confluent). Where indicated, retinoic acid-treated SH-SY5Y cells were cultured for 7 days before experiments. Paraquat or sodium arsenite was prepared in dHCell lysates prepared in Phosphosafe Extraction Buffer at equal protein concentration were mixed with electrophoresis SDS sample buffer and separated on 12% SDS-PAGE Tris-Glycine gels. Proteins were transferred to PVDF membranes and blocked with 4% skim milk solution in PBST before immunoblotting. For detection of total TDP-43, membranes were probed with polyclonal antisera  against TDP-43. For detection of total and phospho-forms of extracellular signal regulated protein (ERK), CDK2 and p38, polyclonal antisera from Cell Signaling Technology was used at 1∶5,000. Secondary antiserum was rabbit-HRP at 1∶5,000 dilution. Blots were developed using GE Healthcare ECL Advance Chemiluminescence  and imaged on a Fujifilm LAS3000 imager . Expression of GAPDH was determined using antisera at 1∶5,000 for protein loading controls where necessary. Densitometric analysis of TDP-43 expression was performed using NIH Image J quantification of protein bands from at least three separate cultures.SH-SY5Y cells, or HeLa cells, were grown on 12 mm diameter coverslips and treated with stress inducers and kinase inhibitors as indicated. Cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After blocking for 1 h with 10% normal goat serum, cells were incubated with primary antibody for total TDP-43 , HuR (1∶50), TIAR (1∶40), hnRNP A1 (1∶200) or hnRNP K (1∶200) for 2 h at room temperature or overnight at 4°C. This was followed by labeling with secondary AlexaFluor or FITC goat anti-mouse or anti-rabbit antisera at 1∶500 for 2 h at room temperature or overnight at 4°C. After washing, the coverslips were incubated with DAPI at 0.5 µg/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Coverslips were examined blinded to the inhibitor treatment and the numbers of TDP-43, HuR, hnRNP K or TIAR-positive stress granules were manually counted in pre-determined multiple fields of view of two to three coverslips for each treatment. Where inhibitors increased or decreased stress granules, treatments were repeated twice. Stress granules were determined as positive if they were distinctly brighter than the surrounding cytoplasmic or nuclear immunofluorescence and/or were clearly separated from surrounding immunofluorescence to allow positive identification of a discrete structure. Results are presented as a percentage change compared to the number of stress-granule-positive cells from paraquat treatment alone. Images shown are representative of multiple fields and replicate or triplicate coverslips per experiment.Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer’s instructions. DNA was quantified and TDP-43 inserts were identified positively by digestion with BamHI and XhoI.Plasmid DNA corresponding to GFP-tagged full-length wild-type (WT) TDP-43 (pEGFP-TDP WT) or C-terminal fragments of TDP-43, (pEGFP-TDP 219–414) or empty expression vector pEGFP-C1 were prepared as described by Nonaka et. al. 5 cells per well in 24 well–plates on coverslips. Non-retinoic acid-treated cells were transfected 24 h after seeding with the pEGFP-C1 empty vector, pEGFP-TDP WT, and pEGFP-TDP 219–414 using Attractene (Qiagen) according to manufacturer’s instructions. Kinase inhibitors were added at 10 or 1 µM after 24 h. After a further 24 h incubation, cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min. and permeabilized with 90% chilled methanol for 5 min. After washing, the coverslips were incubated with DAPI at 0.5 µg/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Expression of TDP-43 was determined by the EGFP-tagged construct SH-SY5Y cells were seeded at 2×10All data described in graphical representations are mean ± standard error of the mean (SEM) unless stated. Results were analysed using a two-way ANOVA and Dunnett post-hoc test.To examine the role of different kinase pathways in stress-induced accumulation of TDP-43, we exposed non-differentiated SH-SY5Y neurons to 1 mM paraquat overnight in the presence or absence of 80 different kinases inhibitors covering 35 separate kinases (Tocriscreen kinase inhibitor toolbox). Each inhibitor was used at a concentration of 10 µM, except where indicated, and coverslips were examined for the number of cells containing TDP-43- and/or HuR-positive stress granules. Paraquat-treated cells revealed robust formation of TDP-43 and/or HuR-positive stress cytosolic granules consistent with our previous studies . Stress Subsequently, we examined the effects of additional kinase inhibitors. The phosphoinositol-3-kinase (PI3K) inhibitor, LY294002 (#7) significantly reduced TDP-43-positive stress granule formation with no significant change to HuR-positive stress granules . InhibitMany additional kinase inhibitors either decreased or increased the number of both TDP-43 and HuR-positive stress granules. For example the MEK inhibitor, U0126 (#8) ; Raf inhFrom this screen, we identified that multiple inhibitors of particular kinases or known kinase pathways induced consistent effects on TDP-43-positive stress granule formation. As shown in To determine if the action of some key kinase inhibitors reflected the ability of the inhibitor to abrogate phosphorylation of its target kinase, we performed a dose-response analysis on TDP-43-positive stress granule accumulation compared to kinase phosphorylation. As shown in Inhibitors of some kinases showed a great deal of variation. Six inhibitors of EGFR were examined with a range of effects including no inhibition , and inhibition of TDP-43-positive stress granules only (#6 and #43) . SummariThe inhibition of TDP-43 stress granule formation by various kinase inhibitors could potentially reflect an inhibition of TDP-43 expression rather than inhibition of TDP-43 cytosolic accumulation. To investigate this, we measured the effect of selected inhibitors on TDP-43 expression in SH-SY5Y cells by western blot. Our initial broad kinase inhibitor screen  identifiNext we examined if selected inhibitors identified in our initial screen of SH-SY5Y cells treated with paraquat induced similar effects on TDP-43 in additional models of stress. This was performed to determine if the effects of the inhibitors were consistent in alternate forms of stress. Initially we examined the effect of these inhibitors in retinoic acid-treated neurons We previously reported that while JNK and ERK inhibitors were able to block formation of TDP-43-positive stress granules when added at the time of insult Finally, to obtain a further insight into the action of the selected kinase inhibitors on TDP-43 cytosolic accumulation, we examined the effect of the inhibitors on C-terminal TDP-43 219-414-GFP (CTF-TDP-43). C-terminal TDP-43 is the major accumulating form of TDP-43 in TDP-43 proteinopathies. We have previously shown that kinases may, at least partially, control C-terminal TDP-43 accumulation. Therefore, in this study we examined this further. As previously reported, non-differentiated SH-SY5Y cells transfected with CTF-TDP-43 revealed localized accumulation of TDP-43 within the cytosol without additional stress induction. Upon treatment with selected kinase inhibitors, we observed the inhibition of TDP-43 accumulation with LY294002 ; olomoucine ; GW5074 ; SB 203580 ; SP600125 ; SB 415286 ; SB 239063 ; and CGP 53353  . InteresThe accumulation of stress granule proteins such as hnRNPs, HuR and TIA-1 or TIAR are controlled by the action of protein kinases Due to the fact that most kinase inhibitors reveal only selectivity for particular kinase targets and are not specific for only one kinase, it can be problematic to identify a kinase associated with inhibition of TDP-43 accumulation based on a single inhibitor. However, for many of the kinase targets, there were multiple inhibitors tested and this can increase the likelihood that a specific target kinase is associated with TDP-43 accumulation. Using this approach we identified inhibitors of CDKs as a potentially important control point for TDP-43 cytoplasmic accumulation. We found that four inhibitors  blocked formation of TDP-43-positive stress granules without effect on HuR-positive stress granule formation. Two additional CDK inhibitors  blocked formation of both TDP-43 and HuR-positive stress granule formation while two  had no effect at the concentrations tested. Notably, CDK inhibitors also reversed the accumulation of TDP-43-positive stress granules when added for the final 6 h of incubation. This strongly suggests that while JNK, other MAPKs and GSK3 are associated with formation of TDP-43-positive stress granules, CDKs may be essential in maintaining their formation once established.The involvement of CDKs in modulating TDP-43 and additional stress granule protein accumulation is not well understood. Lian and Gallouzi reported that decreased levels of cyclin kinase inhibitor p21 in senescent cells was associated with increased stress granule formation Drosophila model. Microtubular integrity is also essential for stress granule dynamics GSK3 was also identified as a key kinase associated with formation of TDP-43-positive stress granules. Three inhibitors of GSK3 all reduced formation of TDP-43-positive stress granules and two of the three (SB 216763 and SB 415286) were specific for TDP-43 effects. GSK3 has an essential role in neurodegenerative disease and has long been a potential therapeutic target for treatment of Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia and ALS One of the intriguing aspects of kinase control of TDP-43 is that the latter has no known consensus sites for phosphorylation by these kinases. Hasegawa et al. in vitro model of TDP-43-positive stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We have found that while a number of kinase inhibitors, particularly of the MAPK pathways, modulated both TDP-43 and the global stress granule marker, HuR, multiple inhibitors also more specifically targeted TDP-43 accumulation, including inhibitors of CDKs and GSK3. Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR while differential effects were often observed on HuR accumulation. Further examination of selected inhibitors also revealed a high level of conservation of TDP-43-controlling kinase pathways in different cell types and stresses. Moreover, CDK inhibitors were found to reverse pre-formed TDP-43-positive stress granules rather than just preventing their initial formation. Inhibition of CTF-TDP-43 accumulation in transfected cells with CDK and GSK3 inhibitors suggests a role for modulation of C-terminal TDP-43 trafficking by the identified kinase groups. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partners, hnRNP K and/or TIAR. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation that is a hallmark of TDP-43 proteinopathies such as ALS and FTLD and may herald new opportunities for kinase modulation-based therapeutic intervention in these diseases.In summary, we have used an Figure S1Effect of selected kinase inhibitors on TDP-43 and HuR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; 10 µM U0126 ; 10 µM olomoucine ; 10 µM SB 415286 ; or 10 µM SB 239063 . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules common to both TDP-43 and HuR images. Closed arrowheads indicate HuR-specific stress granules. Open arrowheads indicate cytosolic diffuse TDP-43. Bar = 10 µm.(TIF)Click here for additional data file.Figure S2Effect of additional selected kinase inhibitors on TDP-43 and HuR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM PD98059 ; 10 µM GW5074 ; 10 µM SB 203580 ; or 10 µM Arctigenin . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules common to both TDP-43 and HuR images. Bar = 10 µm.(TIF)Click here for additional data file.Figure S3Effect of additional selected kinase inhibitors on TDP-43 and HuR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 1 µM aminopuvalonol A ; 10 µM HA 1100 ; 10 µM CGP 533353 ; 10 µM arcyriaflavin A ; or 10 µM ZM 447439 . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules common to both TDP-43 and HuR images. Closed arrowheads indicate HuR-specific stress granules. Bar = 10 µm.(TIF)Click here for additional data file.Figure S4Effect of selected kinase inhibitors on TDP-43 expression. SH-SY5Y cells were treated with paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; olomoucine ; ZM 449829 ; GW 5074 ; SB 203580 ; SB 415286 ; arctigenin ; SB 239063 ; (1 µM) aminopurvalanol A ; TBB ; HA 1100 ; BIBX 1382 ; CGP 53353 ; arcyriaflavin A . Western blot analysis of TDP-43 expression was determined compared to GAPDH control. Representative image from three experiments. Dotted lines indicate removal of unrelated lanes.(TIF)Click here for additional data file.Figure S5Effect of selected kinase inhibitors on TDP-43 and hnRNP K-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; 10 µM U0126 ;10 µM olomoucine ; 10 µM SB 415286 ; or 10 µM SB 299063 . Green = TDP-43, red = hnRNP K, blue = DAPI. Righthand column shows merged images of TDP-43 and hnRNP K. Arrows indicate stress granules common to both TDP-43 and hnRNP K images. Bar = 10 µm.(TIF)Click here for additional data file.Figure S6Effect of selected kinase inhibitors on TDP-43 and TIAR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; 10 µM olomoucine ; or 10 µM SB 415286 . Green = TDP-43, red = TIAR, blue = DAPI. Righthand column shows merged images of TDP-43 and TIAR. Arrows indicate stress granules common to both TDP-43 and TIAR images. Bar = 10 µm.(TIF)Click here for additional data file.Figure S7Effect of CDK inhibitors on hnRNP A1 accumulation in paraquat-treated cells. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight with or without 10 µM of the CDK inhibitors olomoucine (#12) or arcyriaflavin A (#45). Green = hnRNP A1, blue = DAPI. Righthand column shows merged images of hnRNP A1 and DAPI. Bar = 10 µm.(TIF)Click here for additional data file.Figure S8Stress granule inhibition by olomoucine (#12) CDK inhibitor in retinoic acid-treated and non-treated SH-SY5Y cells. SH-SY5Y cells were treated with retinoic acid or left un-treated as described in Methods. Cells were then treated overnight with 1 mM paraquat (PQ) in the presence or absence of 10 µM olomoucine. Green = TDP-43, blue = DAPI. Arrows indicate TDP-43-positive stress granules. Bar = 10 µm.(TIF)Click here for additional data file.Figure S9Stress granule inhibition by olomoucine (#12) CDK inhibitor in HeLa cells. HeLa cells were treated overnight with 50 µM sodium arsenite overnight in the presence or absence of 10 µM olomoucine. Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate TDP-43-positive stress granules. Arrowhead indicates HuR-specific stress granule. Bar = 10 µm.(TIF)Click here for additional data file.Figure S10Effect of selected kinase inhibitors on reversal of pre-formed TDP-43 and HuR-positive stress granules. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight and exposed to selected inhibitors for the final 6 h of incubation ; or 10 µM arcyriaflavin A . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules. Bar = 10 µm.(TIF)Click here for additional data file.Table S1List of kinase abbreviations and names.(DOCX)Click here for additional data file.Table S2Effect of kinase inhibitors on TDP-43 and HuR-positive stress granule formation induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S3List of kinase classes associated with formation of TDP-43 and/or HuR-positive stress granules induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S4Dose-response effect of representative kinase inhibitors on TDP-43-positive stress granules and inhibition of target kinase phosphorylation.(DOCX)Click here for additional data file.Table S5Effect of kinase inhibitors on TDP-43, hnRNP K, TIAR and HuR-positive stress granule formation induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S6Comparison of kinases associated with formation of TDP-43, hnRNP K, TIAR and/or HuR-positive stress granules induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S7retinoic-acid treated compared to non-treated SH-SY5Y cells.Effect of kinase inhibitors on formation of TDP-43-positive stress granules induced by paraquat treatment in (DOCX)Click here for additional data file.Table S8Effect of kinase inhibitors on formation of TDP-43 and HuR-positive stress granules induced by sodium arsenite treatment in HeLa epithelial cells.(DOCX)Click here for additional data file.Table S9reverse TDP-43 and/or HuR-positive stress granule formation induced by paraquat treatment when for the final 6 hr of 24 hr paraquat treatment (SH-SY5Y cells).Ability of kinase inhibitors to (DOCX)Click here for additional data file.


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An estimated 1500–3000 invasive Endometrial Stromal Sarcomas (ESS) cases annually occur worldwide. Before 2003, ESS was divided as low and high grade ESS based on mitotic activity. In 2003 the WHO changed the names, excluded mitoses and made nuclear atypia and necrosis the essential diagnostic criteria to distinguish ESS, Low Grade  and Undifferentiated Endometrial Sarcoma . We have evaluated in WHO2003 defined ESS-LG whether proliferation biomarkers predict recurrence. Using survival analysis, the prognostic value of classical mitosis counts  in haematoxyllin-eosin (H&E) sections, and immunohistochemical proliferation biomarkers (Ki-67 and PhosphoHistone-3 (PPH3)) were examined in 24 invasive endometrial stromal sarcomas. Three of 24 (12.5%) ESS-LG recurred. The MAI, PPH3 and Ki-67 were all prognostic . MAI values were >3 in the recurrent cases, but never exceeded 10 . Non-recurrent cases had 0≤MAI≤3. PPH3 and Ki67 counts can be easier to perform than MAI and therefore helpful in the diagnosis of ESS, Low Grade. In conclusion, in this small study of WHO2003 defined ESS-LG, high levels of proliferation as measured by MAI, PPH3 and Ki-67 are predictive of recurrence. Larger studies are required to confirm these results. Invasive endometrial stromal neoplasms are rare uterine tumors, accounting for 0.2 to 0.7% of all uterine malignancies and 15% of all uterine sarcomas During the past decade, the rules for what constitutes an Endometrial Stromal Sarcoma (ESS) and what not have changed. Before 2003, invasive endometrial stroma tumors were classified according to their mitotic count per 10 high power fields (HPF) as low (<10 per 10 HPF) or high grade (≥10 per 10 HPF) A number of tumors, whose mitosis counts were more than 10 but without nuclear atypia and necrosis, previously classified as high grade ESS, are now ESS-LG. On the other hand, some previously low grade ESSs are now UES. Because of changes in definition, studies on biologic and prognostic features before 2003 might have biases. Moreover, many studies since 2003 have still used the old classification. Thus, results of prognostic features in ESS-LG need to be thoroughly interpreted.Due to the rarity of ESS-LG and UES, therapies used vary widely from observation without additional treatment after different surgery, to hormone therapy, chemotherapy and radiation therapy, either alone or in varying combinations JAZF1/JJAZ1 and other fusions are not commercially available, it is important to have easy and widely available methods allowing pathologists to assess which patients with an ESS are at high recurrence risk.Regarding the etiology, several studies suggested that ESS-LG and UES have distinct cytogenetic profiles. Two zinc finger genes, JAZF1 and JJAZ1, at the sites of the 7p15 and 17q21 breakpoints were the first translocations identified in endometrial stromal nodules and low grade ESS. The presence of JAZF1-JJAZ1 markedly inhibited apoptosis and induced proliferation rates While WHO2003 ESS-LG tumors in general have a good prognosis and behave in a relatively indolent manner, late recurrences and distal metastases do occasionally occur. This has in recent years fostered a rebirth of studies of nuclear proliferation markers. The proliferation biomarker Cyclin D1 can be used as a simple immuno-histochemical surrogate biomarker for YWHAE-FAM22 ESS To further study this hypothesis, we have investigated WHO2003 defined ESS-LG with reasonably long term follow-up  This study was approved by the Institutional Review Board of the Obstetrics and Gynecology Hospital of Fudan University (FUOGH), Shanghai, China, where the patients were diagnosed and treated, and more recently by Regional Ethics Committee of Norway . Details for the patients have been described elsewhere Mitotic activity index (MAI) was assessed in H&E stained 4 µm thick paraffin sections. Following Good Laboratory Practice criteria, the Standard Operating Procedure for the assessment of the MAI was the same as described in details elsewhere for breast cancer Immunohistochemistry (IHC), antigen retrieval and antibody dilution were optimized prior to the study onset. To ensure uniformity, all sections were processed simultaneously. Four micrometer paraffin sections adjacent to the H&E sections used for histologic assessment were mounted onto Superfrost Plus slides , for PPH3 dried overnight at 60°C and for Ki-67 dried for one hour. Sections were deparaffinized in xylene and rehydrated in decreasing concentrations of alcohol. The Ki-67 and PPH3 antibodies have been described in detail before SPSS version 18  was used for the statistical analyses. For evaluating the variables' prognostic significance, Kaplan-Meier survival analysis was performed. Recurrence free survival and overall disease related survival were endpoints . For age, MAI, Ki-67 and PPH3 which are continuous variables, the optimal prognostic threshold of sensitivity and specificity were detected by Receiver Operating Curve (ROC) analysis using the MedCalc® program .The clinico-pathologic features for the subset of 24 patients for whom the tissue blocks were suitable for the current study were not statistically different from that of the full cohort of 68 ESS-LG patients presented elsewhere Median follow-up time for the 24 patients was 53 (range: 23–84) months. Three (12.5%) patients developed recurrences at 4, 23 and 35 months. The mean age of patients without and with recurrence was not different . 17 of the tumors were stage 1, 6 were stage 2, and 1 was stage 3.Two patients with stage 1 and one with stage 3 disease recurred and stage was prognostically not significant.Like for the full cohort of 68 patients, the variable in the current cohort of 24 patients that correlated strongest with recurrence was the Mitotic Activity Index with a threshold of 0–3 versus 4 or more per 10 HPF (these 10 HPF represent 1.59 mm2 at specimen level) (P = 0.001), PPH3 (P = 0.002) and Ki-67 (P = 0.03) showed comparable results and correlated with recurrence, as is also clear from a mitotic figure”, how and where to select fields of vision for mitosis counts and the wide variation of the area at specimen level of “10 high power fields of vision”. In spite of these influencing factors, mitoses can be well reproducible as was shown in the in the nationwide Dutch MMMCP protocol comparing mitosis counts in nearly 3500 consecutive breast cancers in 34 different pathology laboratories over a three years period. In that study the pathologists all used the same protocol and microscopic objective, resulting in a total measurement area of 1.59 mm2 at specimen level. However, other studies stating to have used “10 HPFs” have used objectives with a much larger diameter, resulting in a total measurement area of nearly 2.8 mm2Before 2003, Endometrial Stromal Sarcomas were traditionally divided as low and high grade, based on the mitotic counts (<10 versus ≥10 per 10 High Power Fields). The change by the WHO2003 of the definition and classification and nomenclature of “low and high grade ESS” into ESS, Low Grade and UES has a long history, going back to Evans in 1982 Nevertheless, about 10% of the WHO2003 ESS-LG still recurs and it would be of clinical significance to identify which cases do and which do not recur. Aided with the exact protocol available for assessment of Mitotic Activity Index and the immunohistochemical proliferation biomarkers Ki-67 and PPH3, a “diagnostic proliferation add-on” consisting of the MAI, Ki-67 and PPH3 could be of diagnostic value.Due to the rarity of ESS-LG and UES, the number of studies since 2003 on ESS-LG and UES is limited The purpose of this study therefore was to identify whether proliferation biomarkers of Endometrial Stromal Sarcomas, Low Grade can help to predict recurrence. All three proliferation biomarkers  significantly predicted recurrence, thereby strengthening the historically known fact (from before the WHO2003 definition) that high proliferation is associated with an increased risk on recurrence.The fact that all three proliferation features are prognostic is interesting as they represent different parts of the cell cycle and their expressions are only partly overlapping. This greatly strengthens the findings and underlines that their increased values in recurrent ESS-LG indeed reflect biological increased growth speed in the recurrent cases. It makes it also very unlikely that the increased proliferation detected is due to chance. Cells in their resting phase (G0-phase) show no activity for any of the three proliferation markers. Cells in the cell-division cycle or “cycling cells” are the cells preparing to divide. These cells all go through a sequence of identical phases  before they divide to form two identical daughter cells. The duration for the entire cell-division cycle process to go to completion varies greatly from one tissue to another, but generally takes 18–24 hours (sometimes shorter or longer). The M(itosis) phase, well known to pathologists as metaphases are visible during this phase, varies greatly but roughly accounts for approximately 4% of the cell cycle duration and typically is confined to the last hour of the cycle. Nearly all phases of cycling cells  express Ki-67. The PPH3 antigen is expressed nearly exclusively in the cells late G2-phase  and M-phase (mitotic figures). Therefore, the frequency of PPH3 and Ki-67 positive cells understandably is higher than that seen for the MAI. Yet, all three proliferation markers tell the same story: increased proliferation in ESS-LG is associated with a significantly increased chance on recurrence.In the past, the prognostic threshold for low and high grade ESS was 10, but we found that 4 or more mitoses per 10 HPF  were significant in ESS-LG. This is very similar to the one found by Abeler et al The cell proliferation index assessed by using MIB-1 antibody against the Ki-67 antigen is widely used and accepted because of its crisp contrast-rich staining pattern and being sympathetic to less than stellar managing of tissue acquisition and tissue handling. Many studies have found it to be diagnostically and prognostically useful and in a small series of 11 low grade ESS it successfully predicted the 2 patients who developed recurrent disease Other studies also found a relation between high-risk on recurrence and increased proliferation in ESS-LG Of course, the therapeutic consequences of increased recurrence rate in ESS-LG with elevated proliferation are a matter of further studies. Li In spite of these promising and biologically understandable results, the number of cases was small . On the other hand, the differences were highly significant (P<0.0001) in 2 of the 3 markers and the three different proliferation biomarkers all point into the same direction. The likelihood that the differences are due to chance, when there are no real differences, are therefore very low. Based on the national incidence rates of ESS in the whole of Norway, with less than 90 cases of ESS and UES in more than 30 years, the chances to soon get a large enough series of WHO2003defined ESS-LG, with long enough follow-up and rather homogeneous treatment, are slim. We call for international multicenter collaborative studies to validate the current results. The quality of the immuno-histochemical stainings should be carefully controlled, preferably by means of an external international Quality Control and Assurance system, such as NordiCQ In conclusion, in FIGO2009, WHO2003 defined ESS-LG, elevated levels of proliferation as measured by MAI, PPH3 and Ki-67 seem predictive of tumors that will recur. Larger independent studies are required to confirm these results.