Photo-current is produced upon illumination due to presence of the build-in potential between dissimilar metal electrodes on graphene as a result of charge transfer. The sensitivity of the photo-detector is ∼1.1 μA/W when irradiated with 515 and 1030 nm wavelengths; a smaller separation between the metal contacts favors gradient formation of the built-in electric field and increases the efficiency of charge separation. This optically-thin graphene-on-membrane photodetector and its interdigitated counterpart has the potential to be used within 3D optical elements, such as photonic crystals, sensors, and wearable electronics applications where there is a need to minimise optical losses introduced by the detector.A broadband graphene-on-Si Graphene—a two dimensional 2D) crystal—has become the subject of intense research for a variety of applications including optoelectronics due to its exceptional electronic ,2,3, optD crystalDespite the fact that graphene is a gapless material, it demonstrates strong interband absorption from a broad range of wavelengths between visible and near-infrared (near-IR). Graphene for use in photodetectors rely on one or more photo current generation principles. These are: photo-conductive, photo-thermoelectric (via Seebeck effect), and bolometric photo-voltage generation ,13,14,153N4 membranes are good candidates due to their low mass and excellent optical properties  . Dependi0 [cm−1] .3N4 membrane was tested earlier with a 4.5 μm gap width and dissimilar metal  electrodes [The miniaturisation of the initial photodetector on a micro-Siectrodes  and was ectrodes .3N4-membrane and fabricated using magnetron sputtering of Pd and electron beam evaporation of Ag. The deposited layers were 35 nm thick. By alignment of the second pattern, the gap between the dissimilar metal electrodes could be made as required during the contact step  is placed below the graphene instead of the Si-current . The eleincrease . Hence, graphene . The gragraphene ,38, and graphene .3N4 membrane transfer method with jet-printing and magnetron sputtering for contact deposition. Sensitivity of the photodetector was ∼1.1 μA/W at both 1030 and 515 nm wavelengths when the Si3N4 membrane was 1 μm-thick. In addition, standard photolithography was used to define photodetector contacts on a 100-nm-thick Si3N4 membrane showing further miniaturization feasibility for visible and near-IR radiation detection at the nanoscale.We demonstrate a photodetector made by lithography-free methods combining a simple graphene-to-SiThe two demonstrated modalities of photodetector fabrication via (1) lithography-free and direct-write of metal electrodes and (2) two-step photolithography on sub-micrometer-thick membranes opens new possibilities for the miniaturisation and integration of photodetectors. The new capability to embed such spectrally broad photodetectors into integrated systems, including those that can be 3D laser printed, is a promising direction . The senThe proposed photodetector was inferior to the current mature silicon photodetector technology in terms of sensitivity as it was not designed for maximum absorbance. However, for the particular function of detecting light inside 3D photonic structures without perturbation, the low sensitivity (invisibility) is the required property.The proposed simple fabrication of photodetectors with sub-wavelength thickness can find applications in wearable opto-electronics and monitoring sensor applications.


Inflammatory bowel disease (IBD) and sarcoidosis, primarily considered distinct entities, share commonalties in pathophysiology and clinical manifestations. This study aimed to examine the in‐hospital outcomes of patients with concurrent IBD and sarcoidosis.The National Inpatient Sample was used to identify hospitalized adult patients with IBD and sarcoidosis from 2010 to 2014. Primary outcomes were in‐hospital mortality, rates of septic shock, acute renal failure, respiratory failure, length of stay, and total hospitalization charges. Secondary outcomes were IBD‐specific complications and surgery interventions.P < 0.0001) and colectomy . Subgroup analysis demonstrated lower rates of colectomy when comparing CD/sarcoidosis (P < 0.05) and UC/sarcoidosis (P = 0.0003) versus CD or UC alone. There was no difference in mortality.A total of 3995 patients with IBD and coexisting sarcoidosis (IBD/sarcoidosis), of which 2500 patients had Crohn's disease with coexisting sarcoidosis (Crohn's disease [CD]/sarcoidosis) and 1495 patients had ulcerative colitis with coexisting sarcoidosis (ulcerative colitis [UC]/sarcoidosis), were included. Patients with IBD/sarcoidosis had a lower risk of penetrating disease (adjusted odds ratio [aOR] 0.3, 95% confidence interval [CI] 0.16–0.55, IBD/sarcoidosis is associated with lower risks of penetrating disease and colectomy when compared to patients with IBD alone. Sarcoidosis is associated with a lower rate of penetrating disease, leading to less colectomy in patients hospitalized with Crohn's disease and a higher rate of respiratory failure but a lower rate of colectomy in patients hospitalized with ulcerative colitis. Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn's disease (CD), and sarcoidosis are multifactorial disorders resulting from a complex interplay between genetic predisposition and environmental influence.In fact, increasing amounts of immunological, bacteriological, and genetic data have supported a link between IBD and sarcoidosis.Despite the tremendous progress in our understanding of the link or potential overlap of IBD and sarcoidosis, little is known regarding their clinical correlation. To date, there have been no large nationally representative studies to clarify the clinical outcomes in patients with coexisting IBD and sarcoidosis. In this nationwide study, we aimed to examine the outcomes  for patients admitted with IBD who had coexisting sarcoidosis. Specifically, we investigated the disease‐/hospitalization‐related complications and their interventions, which can represent IBD severityWe obtained data from the Nationwide Inpatient Sample (NIS) database, which is the largest all‐payer inpatient care database in the United States. It is designed to approximate a 20% sample of the U.S. community hospitals. Yearly sampling weights are applied to generate national estimates.versus CD alone, as well as coexisting UC and sarcoidosis versus UC alone. Demographic and clinical characteristics were collected (Table Our study was a retrospective cohort study of adult (18–90 years old) patients hospitalized with coexisting diagnosis codes for IBD  and sarcoidosis (ICD‐9 CM code: 135) in the United States from 2010 to 2014. The control group was all IBD patients without a diagnosis of sarcoidosis . Subgroup analysis was performed to compare coexisting CD and sarcoidosis The primary outcomes were in‐hospital outcomes, including mortality, septic shock, acute renal failure, respiratory failure, LOS, and total hospitalization charges. Our secondary outcomes included the IBD‐specific outcomes, including known complications of IBD such as malnutrition, penetrating disease, stricturing disease/bowel obstruction, and surgical interventions such as colectomy and ileostomy.SAS Survey Procedures  was used for all statistical analyses. The propensity scoring methodt‐test was used to analyze the normally distributed continuous variables. Variables that are not normally distributed were tested by the Wilcoxon rank‐sum tests.The national estimates were calculated after accounting for the sample design elements  provided by the NIS. Continuous variables were reported as weighted means ± standard errors (SEs), and categorical variables were reported as weighted numbers (N) and percentages (%). The SEs of weighted means were estimated by using the Taylor linearization method that incorporates the sample design. The Rao‐Scott‐modified chi‐square test was used to test the difference in distribution for categorical variables, while a weighted Student's A multivariate logistic regression was used to estimate the odds ratio of in‐hospital mortality, septic shock, acute renal failure, respiratory failure, IBD complications, and surgical interventions. Confounders included in the regression analysis were patient demographics, hospital bed size, hospital location/teaching status, insurance type, median household income, and ECI score. In addition, a multivariate linear regression was used to estimate the average change in LOS and total hospital charges after adjusting for the same covariates described above.In this study, a total of 302 105 patients were admitted for IBD, of which 192 290 patients had CD and 109 815 patients had UC. A total of 3995 IBD patients were diagnosed with coexisting sarcoidosis (IBD/sarcoidosis), of which 2500 patients had CD coexisting with sarcoidosis (CD/sarcoidosis) and 1495 patients had UC coexisting with sarcoidosis (UC/sarcoidosis). After matching, the IBD/sarcoidosis, CD/sarcoidosis, and UC/sarcoidosis groups were compared to equal numbers of patients with IBD, CD, and UC without coexisting sarcoidosis, respectively. IBD/sarcoidosis patients were an average of 54.5 years old, of which 64.8% were female, 60.6% were Caucasian, and the patients were mostly admitted to large (63.5%) and urban teaching (58.8%) hospitals with an average ECI score of 5. No statistical difference was found between the IBD/sarcoidosis and IBD alone groups in terms of patient demographics , hospital characteristics (bed size and teaching status), and ECI score  and colectomy  in the IBD/sarcoidosis group compared with the IBD alone group. Not surprisingly, IBD/sarcoidosis was associated with a higher rate of respiratory failure . Although we observed lower rates of hospital mortality , malnutrition , stricturing disease/bowel obstruction , and ileostomy  in the IBD/sarcoidosis patient cohort, these results were not statistically significant. In addition, there was no difference in the rates of septic shock, acute renal failure, and hospitalization burden in terms of LOS or total hospitalization charges found between the IBD/sarcoidosis and IBD alone groups. Upon multivariate analysis (Table P < 0.001) compared to patients with IBD alone. In terms of IBD‐specific complications, IBD/sarcoidosis was independently associated with a significantly lower rate of penetrating disease  compared to IBD alone. In addition, IBD/sarcoidosis was also associated with a lower rate of colectomy  when compared to IBD alone. Interestingly, after adjusting for confounders, we found higher total hospitalization charges  in the IBD/sarcoidosis group when compared to the IBD alone group.Interestingly, our analysis revealed lower rates of penetrating disease  and colectomy  in the CD/sarcoidosis group compared to the CD alone group. After adjusting for confounders, this result remained significant for less penetrating disease  and colectomy  in patients with CD/sarcoidosis.  compared to UC alone. After adjusting for confounders (Table P = 0.0031) when compared to UC alone. Multivariate regression analysis also showed a lower rate of colectomy  in the UC/sarcoidosis group when compared to the UC alone group.There was no significant difference with regard to age, gender, race, hospital type, hospital teaching status, and ECI score between the UC/sarcoidosis and UC alone groups Table . In the rs Table , UC/sarcversus CD alone and UC/sarcoidosis versus UC alone. The major findings were that IBD/sarcoidosis was associated with a significantly lower rate of penetrating disease, fewer colectomies, and a higher rate of respiratory failure compared to IBD alone. Subgroup analysis further specified that both CD/sarcoidosis and UC/sarcoidosis demonstrated lower risk of colectomy when compared to CD and UC alone, respectively. UC/sarcoidosis was particularly associated with a higher rate of respiratory failure.This nationwide study examined the in‐hospital outcomes of patients with IBD/sarcoidosis compared to IBD alone. Subsequent subgroup analysis further compared CD/sarcoidosis The association of sarcoidosis with less penetrating disease and colectomy in IBD patients is incompletely understood and not previously reported. Here, we propose several hypotheses to explain this finding. First, this finding may suggest that the coexistence of sarcoidosis impacts disease subphenotype in IBD. It has been accepted that the location of IBD is clearly associated with different risks of complications.versus colonic CD has been extensively studied. ATG16L risk alleles, IL‐23/Th17 axis, αE+ T‐cells, and microbial community profiles in different key pathways have been shown to be significantly different between ileal and colonic CD.The pathogenesis of ileal Second, aside from dominant location subphenotypes of IBD, the phenotype of endoscopic lesions (i.e. the size and depth of ulcers) has also likely been of important clinical significanceIn addition, a changing pattern of disease behavior has been described in IBD.As expected, our study revealed a high rate of respiratory failure in patients with IBD/sarcoidosis compared to the IBD alone group. It is well accepted that more than 90% of patients with sarcoidosis have evidence of intrathoracic involvement.This study has several strengths. As a nationwide sample, the NIS database provided a large sample size to study the IBD/sarcoidosis population. Therefore, our results are based on a high‐power study and provide a national review of the disease. To the best of our knowledge, this is a leading study to evaluate the clinical outcomes of patients with coexisting IBD and sarcoidosis. Identification of significant clinical outcomes associated with the immunopathological and genetic association of IBD and sarcoidosis may aid in making decisions regarding diagnosis and treatment. Our study also has limitations. All diagnoses are dependent on the accuracy of ICD‐9 codes, for which validation is routinely performed by the Agency for Healthcare Research and Quality, although coding errors may compromise the quality of the data. This study's main diagnostic ICD‐9‐CM codes for IBD and sarcoidosis have been previously used and validated in different studies.In conclusion, this national study used a large dataset from the United States to examine the in‐hospital outcomes of IBD/sarcoidosis. Our results first identified that sarcoidosis is associated with unique clinical features when coexisting with IBD, including lower risk of penetrating disease and colectomy compared to IBD alone. Furthermore, we strengthened the previous understanding of pulmonary sarcoidosis, leading to significant respiratory complications for hospitalized patients. These results may suggest a specific clinical and pathological subphenotype of IBD when coexisting with sarcoidosis. Prospective studies at the genetic and clinicopathological levels will assist in gaining a better understanding of the mechanism of the interaction between IBD and sarcoidosis. Ultimately, more diagnostic and therapeutic strategies are expected.Table S1 ICD‐9‐CM codes.Click here for additional data file.


We theoretically investigate multiple Fano resonances in an asymmetric hybrid graphene–metal metamaterial. The multiple Fano resonances emerge from the coupling of the plasmonic narrow bonding and antibonding modes supported by an in-plane graphene nanoribbon dimer with the broad magnetic resonance mode supported by a gold split-ring resonator. It is found that the Fano resonant mode with its corresponding dark mode of the antibonding mode in the in-plane graphene nanoribbon dimer is only achieved by structural symmetry breaking. The multiple Fano resonances can be tailored by tuning the structural parameters and Fermi levels. Active control of the multiple Fano resonances enables the proposed metamaterial to be widely applied in optoelectronic devices such as tunable sensors, switches, and filters. Optical Fano resonance (FR) is a typical destructive interference effect resulting from the strong coupling between spectrally overlapping narrow discrete and broad excitation modes, which shows a sharp and asymmetric line shape and produces a large electromagnetic field congregation ,2. In reMonolayer graphene has become a new focus of research in active control of FRs effects thanks to its exceptional electrical and optical properties, such as its ultrahigh electron mobility and tunable conductivity for flexible plasmon excitations ,21,22. SIn this work, we demonstrate that a properly designed graphene–metal system with decreased structural symmetry can exhibit plasmonic multiple FRs with narrow linewidths. We adopt a commonly used hybrid structure composed of a gold split-ring resonator (SRR) and an in-plane graphene nanoribbon dimer (GNRD), where the magnetic dipole resonance of the gold SRR acts as a broad mode and the plasmonic hybridized in-phase and out-of-phase bonding resonant modes as well as the antibonding resonant mode excited in the GNRD act as the three narrow modes. Interestingly, it is found that one Fano resonance with its corresponding narrow dark mode being the antibonding modes of the GNRD is only excited via the introduction of structural symmetry breaking in the graphene–metal metamaterial. By decreasing the vertical coupling distance between the GNRD and SRR, extra Fano transmission peaks can be further obtained. The active control of the multiple transparency windows is demonstrated by tuning graphene’s Fermi energy and electronic mobility of the GNRD. The proposed hybrid metamaterial can be realized without changing the structure, suggesting potential applications such as tunable sensors, switchers, and filters.y direction and a gold SRR. The electric field Ein, magnetic field Hin, and wave vector Kin of the incident light are along the x, y, and z directions, respectively. The gap size d1 between the two in-plane graphene nanoribbons, the vertical distance d2 between the GNRD and the gold SRR, and the offset d3 between the geometrical centers of the constituent hybrid metamaterial in the x axis are also shown. The arm length l, arm width w, and thickness t of the SRR are fixed at 3 μm, 0.75 μm, and 30 nm, respectively. The width wg of the graphene nanoribbon (GNR) is fixed at 0.65 μm, while the length of the GNR is infinite along the y direction. The periodicity P along both x and y directions is set as 6 μm. The surrounding media are assumed to be air for simplicity (εd = 1).ωp = 1.37 × 1016 s−1 and a collision frequency of ωc = 4.08 ×1013 s−1. The thickness (tg) of monolayer graphene was set as 1 nm. The graphene surface conductivity (σg) including the intraband and interband transition contribution was calculated by the local limit of random-phase approximation [Ef and electron mobility are 0.7 eV and 10,000 cm2/Vs for graphene. In this work, all transmission spectra and electric field distributions were calculated by the commercial software package “EastFDTD, version 5.0”, which is based on the well-known finite difference time domain method for the numerical simulation of the propagation of electromagnetic waves. For the details of the commercial software package, please visit the website (https://www.eastfdtd.com). In the z axis direction, two perfectly matched layers (PML) were set to avoid the scattering of electromagnetic waves at the boundary of the calculated region. In the x and y axis directions, two periodic boundary conditions were applied. A Gauss pulse was set as the light source, and the transmission spectra were obtained by Fourier transform. The electric field distributions on a plane can be recorded directly. In the regions of graphene and SRR, the minimum mesh size was set to be 0.05 and 5 nm. For the other region, the homogeneous mesh size was set to be Δs = 20 nm, and the corresponding time step Δt = Δs/2c, where c is light speed in vacuum. Our designed hybrid structure can be fabricated by the following process: The gold SRR on the silica substrate is firstly fabricated by electron beam lithography. Then, a dielectric spacer with thickness of hundreds of nanometers is deposited on top of the array of the gold SRR structure by the electronic beam evaporation method. After that, a monolayer graphene is transferred onto the dielectric spacer. Finally, the GNRD is obtained by the second electron beam lithography or by the ultraviolet lithography with high precision alignment.The dielectric properties of gold were calculated by using the Drude model  with a pximation . The dieθ) of 0° and 45°. Here, the width of the GNR and the gap size between the in-plane GNRD are 0.65 and 0.2 μm. For a normal incident TM wave, an electric dipole resonant mode of a single GNR is located at 15 μm, shown by the black solid curve in θ = 45°), two new plasmonic dipole bonding and antibonding [Beginning with the design of the dark elements, we numerically calculated the plasmon hybridization of the GNRD. The simulated transmittance spectra of a single GNR and the in-plane GNRD are shown in ibonding ,27 modesibonding .d1, d2, and d3 are set as 0.4, 0.6, and 0.4 μm, respectively. The Femi energy Ef and the graphene’s electron mobility are fixed at 0.7 eV and 10,000 cm2/Vs. The multiple narrow modes shown in the red curve in Ez and surface charge distribution from the front view (in xz plane) for the two narrow modes (I and II) in Ez and surface charge distribution shown in d3 = 0.4 μm), which is further discussed in wg) of the GNR increases. d1 between the GNRD being tuned from 0.2 to 0.5 μm. The resonant positions of the two Fano peaks (I and II) blueshift as d1 increases, while the resonant position of the Fano peak III slightly redshifts as d1 increases, which shows the same variation tendency observed in the pure GNRD structure shown in d2 between the GNRD and gold SRR is presented in d2 decreases from 0.8 to 0.6 μm, the three transparency windows become more prominent. More interestingly, when d2 further decreases to 0.4 μm, more transmission peaks emerge in the same structure due to the enhancement of the near-field coupling between the GNRD and the gold SRR. Such similar multiple FRs in hybrid metal–graphene structures have also been reported in a previously published paper [d3) between the geometrical centers of the GNRD and the gold SRR along the x axis. d3 increases from 0 to 0.4 μm, indicating the enhancement of the degree of the structural symmetry breaking, the Fano peak III emerges and gradually becomes clear at around 14.5 μm.We further demonstrate the influence on the multiple FRs by varying the geometric parameters of the proposed hybrid structure. ed paper . We furtμ) from 10,000 to 500 cm2/Vs while keeping other parameters unchanged. μ decreases from 10,000 to 5000 cm2/Vs. When μ further decreases to 2500 cm2/Vs, only one Fano peak with the corresponding narrow mode being the dipole bonding mode is observed. As μ becomes further smaller than 500 cm2/Vs, all three FRs disappear. This is due to the energy loss becoming dominant for the graphene with a small μ.The ability to actively control the optical properties of graphene by tuning its Fermi level is significant for dynamic optoelectronic devices. In summary, we have theoretically studied the tunable multiple Fano resonances in a hybrid metamaterial with decreased structural symmetry composed of an in-plane graphene nanoribbon dimer and gold split-ring resonator. The multiple Fano resonances are obtained by the near-field coupling between two hybridized narrow electric dipole bonding modes as well as a narrow dark electric antibonding mode of the graphene nanoribbon dimer and the broad fundamental magnetic resonance of the gold split-ring resonator. Interestingly, the Fano resonance with its corresponding dark mode of the antibonding modes in the graphene nanoribbon dimer is only excited by structural symmetry breaking. The multiple Fano modes can be tailored by controlling the structural parameters and Fermi levels. In addition, compared with the graphene nanopatch array, our designed hybrid nanostructure with a graphene nanoribbon dimer keeps continuous form with the benefit of preserving the high mobility of graphene and may simplify the possible fabrication processes. The dynamic multiple Fano resonances are achieved by varying the Fermi energy of graphene, which makes our proposed hybrid graphene–metal device more active than the metallic ones.


FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants  compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death. FOXC2 gene (MIM *602402) are linked to lymphedema-distichiasis (LD) syndrome (MIM #153400) and cause pleiotropic effects in different types of tissue [The forkhead transcription factor C2 (FOXC2) plays a key role in regulation of lymphatic endothelial cell differentiation, formation of smooth muscle cell layers and morphogenesis of lymphatic valves during embryogenesis. Dominant mutations in the ome MIM #53400 andene MIM *02402 aref tissue ,8,9,10. f tissue ,12,13,14ome MIM #3400 and f tissue . DespiteThe FOXC2 gene at 16q24.3 produces a transcript from a 1.5-kb single exon coding region. The 501 amino acid FOXC2 protein contains: (i) a transactivation domain 1 (AD-1) extending from the first amino acid to the forkhead DNA binding domain ; (ii) the FHD (amino acids 71 to 162) with nuclear localization signal 1  ; (iii) awww.hgmd.cf.ac.uk) [The majority of FOXC2 mutations linked to lymphedema-distichiasis syndrome are insertion/deletion (73%) and nonsense mutations (10%) B 2,21,22,23. In f.ac.uk) . A few hf.ac.uk) ,27. Thosf.ac.uk) ,26, whilIt may be supposed that the pathogenesis of LD is mostly associated with FOXC2 haploinsufficiency. However in a restricted number of patients, a gain in FOXC2 function has been observed and demonstrated to be equally harmful ,27. The In this study, we performed the functional characterization of different types of mutations identified in six unrelated families with LD to obtain insights into structure-function relationships of FOXC2 transcription factor. We also explored the involvement of some FOXC2 mutant proteins in cell death.The clinical features of the patients included in our study have already been reported . The maiTo determine whether the different mutations affected FOXC2 function, altering nuclear localization or impairing transactivation activity, we subcloned wild type FOXC2 cDNA into a pcDNA3.1/NT-GFP-TOPO expression vector and performed specific site-directed mutagenesis. Then we transfected HeLa cells with FOXC2-GFP mutant plasmids and performed Western blot, immunofluorescence and transactivation analysis. pFOXC2-GFP) and missense mutant plasmids (pFOXC2(L80F)-GFP; pFOXC2(I213V)-GFP; pFOXC2(V228M)-GFP) -GFP) A. Unlike8M)-GFP) A, lane 48M)-GFP) A lane 4.8M)-GFP) A, lane 6We then investigated whether the subcellular localization of the FOXC2 mutant proteins was affected. Immunofluorescence analysis revealed a homogeneous nuclear distribution of wild type FOXC2 protein B. None op = 0.04, p = 0.045, p = 0.047). These mutations impaired more than 50% of FOXC2 activity. The p.V228M mutation also decreased transcription activity, but only by 30% with respect to FOXC2 wild type (p = 0.11). Instead, the I213V mutation showed transcription activity slightly higher than that of the FOXC2 control protein (p = 0.9). The p.Y109* mutant protein was excluded from this set of experiments since it was previously demonstrated as unable to localize in the nucleus . These mutant proteins formed nuclear aggregates that were clearly visible in all the transfected cells. p.R121H was also included since it previously produced nonhomogeneous nuclear staining  [Observation of abnormal nuclear aggregation of some FOXC2 mutant proteins led us to speculate that it could induce cell death. Therefore, wild type and FOXC2 mutant plasmids were used to perform HeLa transfection experiments and cell death was monitored by trypan blue staining at various times  after transfection. As shown in = 0.000) . On the At the same time, we evaluated the effect of p.L80F, p.H199Pfs*264 and p.I213Tfs*18 mutant proteins on the mitotic process by immunofluorescence analysis 24 and 72 h after transfection of HeLa cells. Wild type and p.H199Pfs*264 FOXC2 plasmids showed a similar rate of transfection B. Wild tTo verify whether the c.595dupC (p.H199Pfs*264) and c.638delT (p.I213Tfs*18) mutations cause nonsense-mediated decay of mutant mRNAs in vivo, we analyzed total FOXC2 mRNA expression in peripheral blood of LD patients. Comparative RT-PCR analysis detected mRNA in similar amounts in three LD patients carrying frameshift mutations and in control subjects A. RT-PCRThe FOXC2 RT-PCR products obtained from LD patients were then sequenced. FOXC2 mutant mRNAs were detected in the lymphocytes of patients (data not shown). The mutations analyzed in this study were localized in the forkhead domain and in the central region of the FOXC2 protein A. To furhttp://genetics.bwh.harvard.edu/pph2) and SIFT (http://sift.jcvi.org) supported the hypothesis that p.L80F alters FOXC2 activity. Indeed, both programs predicted a deleterious effect of this mutation. p.V228M was only predicted to be damaging by Polyphen-2, while p.I213V was classified as benign or tolerated by both bioinformatic tools . The mutation p.Y109* is located in the forkhead domain and was predicted to produce a short, truncated protein. Immunofluorescence and immunoblot results clearly showed that p.Y109* was not able to localize into the nucleus, although it still contained the first nuclear localization signal. Therefore, even if it is stably synthesized in vivo, it cannot carry out its transcription factor function. The mutation p.L80F is the first mutation identified in NLS1 of FOXC2. Bioinformatic prediction tools suggested that p.L80F is damaging (Sift) and probably damaging (Polyphen-2). On the basis of homology data comparison, the mutation is in a highly conserved region of the FOXC2 protein. Three-dimensional structure prediction showed that it modifies the folding of the FHD, decreasing its ability to link DNA. In agreement with the bioinformatic data, our functional studies revealed that p.L80F strongly decreased the protein’s capacity to activate the promoter of the reporter gene, although it correctly localized in the nucleus. Indeed, I-Tasser software analysis did not show alterations in NLS1 secondary structure but about twenty amino acids beyond, in the FHD. Immunofluorescence analysis of p.L80F mutant proteins shows that they segregate into nuclear aggregates, like other FHD missense mutations already demonstrated to dramatically impair DNA binding activity ,27,34. TFinally, we performed a functional evaluation of two frameshift mutations, p.H199Pfs*264 and p.I213Tfs*18, localized in the central region of FOXC2. They lead to the formation of truncated proteins, lacking more than 50% of the native sequence and with an extra-long stretch of 264 and 18 amino acids, respectively. Although these mutant proteins showed a relevant modification of 3D structure, they conserved the ability to localize to the nucleus and to bind DNA. However, both caused a significant loss of protein function and the formation of nonhomogeneous nuclear aggregates. Indeed, the p.L80F and p.R121H mutations also show nuclear protein aggregates, but they never colocalize with DNA ,26,27. SWhen we investigated the consequence of p.H199Pfs*264 and p.I213Tfs*18 protein expression in transfected cells, we found that they strongly induced cell death. Similar results were obtained for missense mutations in the FHD, p.L80F and p.R121H. Conversely, the other missense mutations outside the FHD (p.I213V and p.V228M) did not show protein nuclear aggregation, nor did they alter cell viability. Cells transfected with mutant proteins that cause nuclear aggregation are unable to promote mitotic entry. At different periods after transfection , their number did not increase and most of them died. On the contrary, frequent mitotic events can be observed in cells transfected with FOXC2 native sequence, demonstrating that the transcription factor is involved in cell proliferation. This result agrees with a great deal of previously reported evidence attesting the key role of FOXC2 in promoting the proliferation of tumor cells in vitro and in vivo ,37,38. IIn conclusion, our functional studies further highlighted the different molecular consequences of some FOXC2 mutations identified in families with lymphedema-distichiasis. We also provided the first evidence that some inactivating mutations cause strong inhibition of cell proliferation. Larger functional and clinical studies are needed to elucidate a possible correlation between different FOXC2 mutations and the broad phenotypic heterogeneity observed in LD patients: heterogeneity that is not yet supported by biochemical or genetic evidence.FOXC2 cDNA was previously cloned into pcDNA3.1/NT-GFP-TOPO to produce NT-GFP-FOXC2, expressing FOXC2 with GFP at the N-terminus . Point mHeLa cells were transiently transfected with NT-GFP-FOXC2 recombinant plasmids using the Lipofectamine 2000 transfection reagent . After 48 h of transfection, cytosol and nucleus subcellular protein fractions were prepared from confluent HeLa cells. After extensive washing with Dulbecco’s phosphate-buffered saline (DPBS), subcellular protein fractions were extracted using FractionPrep cell fractionation kit . The protein concentration of cell extracts was quantified by Coomassie (Bradford) Protein Assay Kit . Proteins (25 μg/well) were separated by electrophoresis on 10% SDS-polyacrylamide gel , transferred to a nitrocellulose membrane  and then immunoblotted using a mouse monoclonal antibody (dil. 1:2000) raised against Green Fluorescent Protein . Signals were detected using the SuperSignal West Pico Complete Mouse Detection Kit  containing ImmunoPure peroxidase conjugated goat antimouse IgG .For transient transfections, HeLa cells were cultured on glass coverslips in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and allowed to adhere overnight. The next day, the cells were transiently transfected with recombinant NT-GFP-FOXC2 plasmids using the Lipofectamine 2000 transfection reagent . After 24 h, cells were fixed, stained with DAPI , and examined with a Leica MB5000B microscope equipped with 40× and 100× Fluorart oil immersion objectives .v/v) Triton X-100 (PBS-X), blocked with 5% (w/v) bovine serum albumin in PBS-X and incubated with antiFOXC2 polyclonal antibody  (1:500) for 1 h at room temperature. After extensive washing in PBS-X, the cells were incubated with a FITC-conjugated goat secondary antibody at a dilution of 1:500 . Cells were stained with DAPI, mounted on slides and examined by microscope as previously described.Cells transfected with expression vectors containing FOXC2 without tag were permeabilized with phosphate-buffered saline containing 0.1%  was calculated after correction for transfection efficiency. This was obtained as a measure of GFP fluorescence in the cells transfected with NT-GFP-FOXC2 recombinant plasmids [Photinus pyralis luciferase (reporter vector) and Renilla reniformis luciferase (control vector). Luminescence detection for all transfected plasmids was performed using the Glomax luminometer . Reactions were replicated three times, using the Promega Dual Luciferase Assay kit .HeLa cells were cotransfected with 20 ng FOXC1 luciferase reporter  and 100 plasmids . The sam2 atmosphere for 24, 48 and 72 h. The cells were then suspended in DPBS containing trypan blue and examined to determine the percentage of cells with clear cytoplasm (viable cells) versus cells with blue cytoplasm (nonviable cells) under a light microscopic. Each sample was performed in triplicate. The experiment was repeated twice.HeLa cells were transfected with wild type and FOXC2 mutant plasmids and incubated at 37 °C in a 95% air/5% COTotal RNA was isolated from lymphocytes of LD and control subjects with TRIzol . All subjects gave their informed consent before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki. One microgram of RNA was converted to cDNA by RT-PCR using random hexamers (0.5 µmg), 400 units of MMLV-RT(Moloney murine leukemia virus), 1.6 mM total dNTPs, 20 units of Rnasin and 0.4 mM dithiothreitol in a 50 µL reaction solution containing 10× RT buffer. Moreover, for each biological sample, 1 µg RNA was incubated with all the reagents reported above with the exception of MMLV-RT.Before reverse transcription, RNA was treated with DNase I in order to eliminate DNA contamination. RT-PCR reactions were optimized for FOXC2 and GAPDH genes in order to avoid saturation. Regression curves assaying different amounts of cDNAs (corresponding to different mRNA concentrations) and different numbers of amplification cycles were performed (data not shown). Fifty nanograms of cDNA were used to perform PCR amplification with primers designed to produce an 89 bp fragment of the FOXC2 transcript. PCR conditions were as follows: denaturation at 95 °C for 90 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s in the first round, denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s for 24 cycles; denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and terminal extension at 72 °C for 60 s in the last cycle. Analysis of FOXC2 expression was carried out on +RT and –RT cDNA samples in order to exclude the presence of traces of DNA. Negative control samples were also added to each RT-PCR experiment. The PCR products were electrophoresed on a 2% agarose gel containing GelGreen Nucleic Acid Gel Stain . The concentration of FOXC2 gene was normalized to the constant level of GAPDH in each sample. Images of gels were acquired  and scanned using Quantity One Analysis software . This software provides the mean of each band  and the concentration of PCR products, calculated from standards included in each gel.Finally, the RT-PCR products of LD patients were purified  and sequenced on a 3730 DNA analyzer by the BigDye Terminator V1.1 Cycle sequencing kit .t-test. A p-value ≤ 0.05 was considered statistically significant.Statistical analysis of quantitative RT-PCR and luciferase assay data was performed using SPSS v.19 package . Values were compared using Student’s https://www.ebi.ac.uk/Tools/msa/clustalw2/), SIFT (http://sift.jcvi.org) and PolyPhen (http://genetics.bwh.harvard.edu/pph2) software. A multiple sequence alignment of mammalian FOXC2 proteins was used as input for ClustalW. The NCBI reference sequence (FOXC2 protein NCBI accession number: NP_005242.1.) of human FOXC2 protein was used as input for SIFT and PolyPhen, with default query options. Investigation of secondary and tertiary structure was performed using i-Tasser bioinformatic tool (https://zhanglab.ccmb.med.umich.edu/I-TASSER/).The effect of amino acid substitutions on protein function was predicted using ClustalW (


In this paper ultra clean monolayer and bilayer Chemical Vapor Deposited (CVD) graphene membranes with diameters up to 500 µm and 750 µm, respectively have been fabricated using Inverted Floating Method (IFM) followed by thermal annealing in vacuum. The yield decreases with size but we show the importance of choosing a good graphene raw material. Dynamic mechanical properties of the membranes at room temperature in different diameters are measured before and after annealing. The quality factor ranges from 200 to 2000 and shows no clear dependence on the size. The resonance frequency is inversely proportional to the diameter of the membranes. We observe a reduction of the effective intrinsic stress in the graphene, as well as of the relative error in the determination of said stress after thermal annealing. These measurements show that it is possible to produce graphene membranes with reproducible and excellent mechanical properties. Most of the studies on graphene are usually performed on supporting substrates such as SiO2/Si. However, substrate induced carrier scattering, dopants and phonon leakage obscure the intrinsic properties of graphene significantly. Suspended graphene has revealed superior properties in certain aspects, and thus it becomes an ultimate platform to exploit the capabilities of the material15. From a mechanical standpoint, since graphene is formed by strong sp2 covalent carbon-carbon bonds, it presents the largest Young’s modulus in nature (together with carbon nanotubes) with 1 TPa1. In addition, when very small areas are probed, it behaves extremely close to an ideally brittle solid, showing an incredibly high yield strength1. But this is when we can probe a single grain, with no dislocation. Polycrystalline graphene, on the other hand, contains randomly distributed topological defects such as dislocations and grain boundaries18. These defects affect the effective properties measured in the devices, both electrically19, mechanically20, and thermally21. In particular, it has been shown that grain boundaries reduce graphene strength substantially, even with small misorientation angles22. Therefore, despite of its ultrahigh theoretical strength, polycrystallinity threatens the practical applications of graphene as a mechanical material.Graphene films have raised lot of attention in the past 15 years due to their outstanding material properties. In particular, given the combination of mechanical and electrical properties, these films are very promising for micro- and nano-electromechanical systems (MEMS & NEMS) and their applications23, DNA sequencing25, pressure sensors26, temperature sensors27, gas sensors28, bolometers29. For most of these applications, having extreme aspect ratio membranes, i.e. a large area and extremely small thickness (few monolayers); and free of residues would be extremely beneficial. However, that also means very fragile structures from a mechanical standpoint and an increasing difficulty to obtain large aspect ratio membranes.There are many applications for which a suspended atomically thin membrane (of e.g. graphene) has been proposed to reach the ultimate performance. These include desalination30, direct etching of a metal catalyst31, critical point drying to avoid the drag force from surface tension32 and wet transferring35. The latter is the most popular method to fabricate stand-alone membranes of various aspect ratios. However, the membranes are frequently ruptured because of the surface tension when the rinsing liquid is evaporating and, consequently, the size and yield are limited. Typical sizes of monolayer graphene membranes on a perforated substrate are in the order of tens of micrometers36. In 2014, Lee et al., published a strategy called an inverted floating method (IFM)35. With this strategy, the sample is rinsed without being immersed in liquid, but rather letting the sample float on the surface of an acetone bath. This prevents the liquid entering into the holes underneath the graphene membrane and increases the yield of large membranes, providing suspended monolayer graphene membranes with diameters up to ∼ 500 μm35, but it suffers from large amounts of polymer residue on the graphene surface37. Therefore, development of a large scale, free-standing, and clean graphene membrane is still a big challenge.Different techniques for fabricating graphene membranes have been reported, such as patterning and electroplatingIn this paper, we used IFM followed by thermal annealing in vacuum for fabricating ultra clean monolayer and bilayer chemical vapor deposited (CVD) graphene membranes with diameters up to 750 µm. These membranes are free of cracks, wrinkles and pinholes. Also, the graphene membranes are very clean with almost no PMMA residuals. SEM and optical profilometry images confirm our claim. Furthermore, the resonating properties of the membranes with different diameters are characterized by Laser Doppler Vibrometer (LDV) before and after thermal annealing. The results show that the thermal annealing removes PMMA residuals on the graphene membranes significantly.μm to 1000 μm37. Figure 2. After wet transferring, the PMMA/graphene stack remains well suspended over the holes without rupturing, even in the case of 1 mm holes. We then leave the chip for 24 hours in a clean and dry environment in order to evaporate the water between the graphene and the perforated substrate. During this period, the water surface tension drags the film into contact with the substrate. Unfortunately, this also breaks some of the membranes. Finally, PMMA is carefully removed by exposing the PMMA-covered side of the chip to acetone, but avoiding the solvent to go into the holes (IFM)35. To do this, we use a custom-made stand , CVD monolayer with average grain size of 50 µm (vendor #2) and CVD bilayer with average grain size of 50 µm (vendor #2). The survival yield for each of the three types is shown in Fig. −4 mbar). The effect of this annealing is illustrated in Fig. 45) we can see how the surface becomes much cleaner (from 7 nm to 2.5 nm RMS), except for a large defect that we assume it to be a silica residue from the growth. In addition, we can also see how the membrane after annealing changes its conformality on the hole region, as we find a step of around 50 nm when the hole starts. During this annealing process, unfortunately, some of the membranes break . The main reason for this is the thermal expansion coefficient mismatch between the graphene and the substrate, since we observe a larger percentage of ruptured membranes when we anneal at higher temperatures.In addition to grain boundaries, other defects coming from the growth, such as silica particles, metal residues, etc. can also reduce the yield of the membranes. Alternatively, and together with PMMA residues, they can remain on the membranes after release, as it can be seen in Fig. 45, which is then fitted to a Lorentzian curve to extract both resonance frequency and quality factor46. Figure We then characterize the dynamical behavior of the fabricated membranes, by measuring their thermomechanical noise, using an LDV OFV-551 from Polytec46, to obtain for example a responsivity of 43 nm/V in the case of Fig. 46, with a D is the diameter of the membrane, We first measure the power spectral density (PSD) of the signal in volts, and convert it to amplitude at the center of the membrane by using the method described inThe equivalent surface stress that we obtain from the experimental data before annealing is: Q) extracted from our measurements for the devices before and after thermal annealing, which can be seen in Fig. et al47., we do not see an increase of our quality factor with increasing diameter. This might be because we are mostly probing on another range of diameters (from 10 µm up to 750 µm compared to 2.5µm-25µm in47), or because we have a different main damping mechanism.We can also compare the quality factor . This yields ultra clean, monolayer and bilayer graphene membranes with diameters up to 500 µm and 750 µm, respectively, but only if the grain size is large, not for sample with smaller grains. Dynamic mechanical properties of fabricated devices are characterized via interferometric detection of their thermomechanical noise. The resonance frequencies are inversely proportional to the diameter of the membranes, in accordance with theory. After cleaning, we observe a reduction of the effective stress as well as a reduction of its relative dispersion, showing that it is necessary to clean the resonators before estimating the intrinsic stress. We believe our findings will help in the development of different types of mechanical sensors that benefit of very large aspect ratio to optimize their performance.We demonstrate the transfer of monolayer and bilayer CVD Graphene with small and big grain domains on through-hole chips with diameters ranging from 10 µm to 750 µm by a PMMA-mediated approach. The PMMA layer is removed by IFM followed by thermal annealing for 2 hours at 250 °C in vacuum (10μm thickness was then removed by floating the sample on a copper etchant (Ammonium persulfate 0.1 molar) for 3 hours. To rinse the graphene after copper etching, the copper etchant changed three times with fresh DI water. After the DI water rinsing, PMMA/graphene stack was left on the DI water for 24 hours to remove any impurity and residue of ammonium persulfate and copper oxides. Afterwards, the PMMA/graphene stack was scooped up onto a through-hole SiO2/Si chip. Then, the PMMA was removed using acetone and IFM for 2 min followed by thermal annealing at 250 °C for two hours in vacuum (10−4 mbar).A 150 nm-thick PMMA  was spin-coated on the monolayer and bilayer CVD graphene on copper foil at 1000 rpm for 60 seconds. The PMMA was baked in an oven at 180 °C for 3 minutes. Because the CVD process grew graphene layers on both sides of the copper foil, graphene layers the other side (no PMMA) of the copper foil were removed by oxygen plasma (50 W power for 2 minutes). The copper foil with 50 −5 mbar actively pumped vacuum). Frequency sweeps performed using a lock-in amplifier .Optical microscopy, SEM imaging and optical profilometry  were used to observe and characterize the morphology of the devices during and after device fabrication. Confocal Raman spectroscopy (RENISHAW) was used to verify the presence and quality of the monolayer and bilayer graphene. To measure the resonance frequency and quality factor of the structures we used a LDV (OFV-551) from Polytec GmbH with a laser spot size of the order of 2.5 µm to detect the amplitude of the thermomechanical noise of the structures in vacuum (~10


The n-type charge-transfer doping of graphene was maximized by increasing the density of free amine in the macromolecule through a vacuum annealing process. The graphene adsorbed with the n-type dopants was stacked twice, resulting in a graphene FTCE with a sheet resistance of 38 ohm/sq and optical transmittance of 94.1%. The figure of merit (FoM) of the graphene electrode is as high as 158, which is significantly higher than the minimum standard for commercially available transparent electrodes (FoM = 35) as well as graphene electrodes doped with previously reported chemical doping methods. Furthermore, the n-doped graphene electrodes not only show outstanding flexibility but also maintain the doping effect even in high temperature (500 K) and high vacuum (~10−6 torr) conditions. These results show that the graphene doping proposed in this study is a promising approach for graphene-based next-generation FTCEs.Flexible transparent conducting electrodes (FTCE) are an essential component of next-generation flexible optoelectronic devices. Graphene is expected to be a promising material for the FTCE, because of its high transparency, large charge carrier mobilities, and outstanding chemical and mechanical stability. However, the electrical conductivity of graphene is still not good enough to be used as the electrode of an FTCE, which hinders its practical application. In this study, graphene was heavily  PMMA/Gr was floated on deionized water (DI) to remove residual ammonium persulfate. PMMA/Gr floating on DI was lifted to the various substrates (SiO2 (300 nm)/Si, PEI/SiO2/Si, Quartz, PET and so on). And then PMMA/Gr/substrate was baked on a hot plate at 100 °C for 1 h. PMMA was removed by dipping in acetone. The process of forming the octadecyltrichlorosilane (OTS) self-assembled monolayer (SAM) on SiO2/Si (OTS-SiO2/Si) is detailed in our previous reports ,323+, 40nnealing A. The reth light , the G-pgraphene B. In connnealing . n-type doping by the free amine of PEI, the work function to the vacuum level decreases. The work function of HOPG and undoped graphene measured by Kelvin probe force microscopy (KPFM) were 4.61 and 4.39 eV, respectively  of octadecyltrichlorosilane (OTS), the Fermi level of graphene is close to the Dirac-point [2/Si (OTS-SiO2/Si) substrate was as high as 4008 ± 46 ohm/sq through four-point probe measurement  and the resistance significantly further decreased to 229 ohm/sq. The temperature for vacuum annealing to maximize the doping effect was 200 °C. Since 200 °C is a relatively high temperature to be applied to organic optoelectronic devices such as OLED, vacuum annealing at a low temperature  was also conducted  before annealing, in the case of one-side doping after annealing, the Dirac-point shifted to −310 V, and the carrier density increased to 3.9 × 1012 cm−2. After the dual-side doping, the Dirac-point was shifted up to −768 V, and the carrier density was increased to 2.0 × 1013 cm−2. Before doping, since the Dirac-point of GFETs was 1 V and the carrier density was 3.6 × 1011 cm−2, the PEI doping method that maximized free amine through vacuum annealing proved to be a heavily effective doping method. The change of carrier concentration and resistance due to the vacuum annealing was evaluated by fabricating GFETs . Under hsurement A. When gonducted . As a rent range . Comparen-doped and are thus hardly affected by the applied voltage after annealing  structures, the sheet resistance was reduced to 118 ± 15 ohm/sq at 96.7% transmittance (n-doped graphene (Group I) and p-doped graphene (Group II)  and is evenly formed on the graphene surface . These rmittance B. When gmittance C. This Froup II) C, showinn-doping effect by PEI, the resistance of PEI/Gr/SiO2(300 nm)/Si structure was measured at various temperatures under a vacuum atmosphere (~10−5 torr)  atoms of PEI and graphene. To evaluate the thermal stability of the −5 torr) A. The te2/Si electrode were measured. Solvents were classified and selected into three types: polar protic, polar aprotic and non-polar solvent. Despite being immersed in methoxyethanol, methanol, chloroform and dimethyl sulfoxide (DMSO) with high PEI solubility for 30 min, the overall resistance change was less than 2.1%. This result proved that the charge transfer interaction between graphene and PEI is robust, especially after the vacuum annealing. The change in the doping effect in aqueous solutions of various pH was also investigated by dipping GFETs in various pH solutions (0.1 M) for 30 s. As expected from pKa (9.06) of the amine of PEI [The chemical stability of PEI was also confirmed by dipping the PEI doped graphene in various solutions. First, after dipping in a solvent with high PEI solubility for 30 min, the resistance changes of the PEI/Gr/SiOe of PEI , when th3+). Therefore, the sheet resistance of PEI-doped GFETs was increased because the doping effect was decreased when exposed to air (2SiO3) was formed on the PEI as a passivation layer, and PEI was prevented from being directly exposed to air. As a result, despite being exposed to air for 14 days, the resistance increased by only 25%. In the case where the passivation layer was not formed on the device (PEI/Gr/SiO2/Si), the sheet resistance increased by about 300 times after 14 days, but the sheet resistance could be lowered almost similarly to the resistance measured in vacuum by 150 °C, 2 h annealing. Amine is protonated by moisture or oxygen in the atmosphere to become an ammonium ion  structure was performed. As a result, the sheet resistance increased only 2.7% after 5000 bendings. Based on these results, it was verified that the PEI-doped Gr proposed in this study can be sufficiently used as an FTCE material.n-doping effect of the amine-rich macromolecule on graphene. By maximizing the free amine density through vacuum annealing, the carrier density of graphene was dramatically increased, and the sheet resistance of graphene was significantly decreased to 38 ohm/sq by charge transfer doping. In addition, we were able to fabricate FTCEs with a high figure of merit (FoM ≈ 158) because the dopant layer is very thin and transparent. Besides, the n-type doped graphene using PEI not only showed excellent flexibility and chemical stability, but also maintained the doping effect even in high temperature and high vacuum conditions. These results indicate that maximizing the n-type doping effect on graphene through vacuum annealing is effective in obtaining stable and very conductive graphene FTCEs. Furthermore, our approach can be applied to the surface-doping of other two-dimensional materials without degrading their electrical properties.In conclusion, we demonstrated the strong


Dementia Friendly Nevada (DFNV) aims to develop and promote communities in becoming more respectful, educated, supportive and inclusive of people living with dementia and their care partners. To date, six communities are engaged, representing urban, rural and tribal communities. Each community convened an action group comprised of volunteers from a range of sectors, including people living with dementia as key participants. Each group used a participatory action research process to assess community needs, develop specific goals and actions, and document community change. As part of this process, five of the six communities adopted the Dementia Friends program for community education and awareness raising. This program is licensed nationally by Dementia Friends USA. From June 2018 – September 2019, these communities achieved widespread dissemination of this program, training 68 Dementia Friends Champions , who in turn delivered 55 training sessions to 607 new Dementia Friends  across Nevada. To evaluate the impact of the program, DFNV partnered with the Sanford Center for Aging to conduct a pre-post survey (n = 504) to assess program-related knowledge change as well as commitments to take action. Results showed a statistically-significant (p <.001) increase in participant knowledge, with 17% higher scores at the post-assessment. In addition, given 10 options for specific actions, responses ranged from 12% of participants committing to “start a dementia friendly effort” to 70% committing to “support dementia friendly efforts.” Overall, the early impact of this ongoing effort to educate the public about dementia across Nevada has been very successful.


Investigation of molecular signature, homology, phylogeny, and gene structure suggested that they belonged to lineages 1 (L1) and 3 (L3), and designated Ofcxcl8-L1 and Ofcxcl8-L3. While Ofcxcl8-L1 and Ofcxcl8-L3 revealed quadripartite and tripartite organization, in place of the mammalian ELR (Glu-Leu-Arg) motif, their peptides harbored EMH (Glu-Met-His) and NSH (Asn-Ser-His) motifs, respectively. Transcripts of Ofcxcl8s were constitutively detected by Quantitative Real-Time PCR (qPCR) in 11 tissues examined, however, at different levels. Ofcxcl8-L1 transcript robustly responded to treatments with stimulants, such as flagellin, concanavalin A, lipopolysaccharide, and poly(I:C), and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus, when compared with Ofcxcl8-L3 mRNA. The differences in the putative promoter features may partly explain the differential transcriptional modulation of Ofcxcl8s. Purified recombinant OfCXCL8 (rOfCXCL8) proteins were used in in vitro chemotaxis and proliferation assays. Despite the lack of ELR motif, both rOfCXCL8s exhibited leukocyte chemotactic and proliferative functions, where the potency of rOfCXCL8-L1 was robust and significant compared to that of rOfCXCL8-L3. The results, taken together, are indicative of the crucial importance of Ofcxcl8s in inflammatory responses and immunoregulatory roles in rock bream immunity.CXCL8  is a CXC family chemokine that recruits specific target cells and mediates inflammation and wound healing. This study reports the identification and characterization of two  Homeostatic chemokines are generally involved in lymphocyte trafficking, immune surveillance, and localization of hematopoietic precursors; (2) Inflammatory chemokines are only produced by cells during inflammation to prompt the migration of leukocytes towards an injured or infected site, and also to activate cells to raise an immune response and commence the wound healing process ) or pathogens (bacteria and virus). The biological function of OfCXCL8 homologs was also demonstrated using the purified recombinant proteins by examining their chemotactic and proliferative activities. Collectively, this study provides some insights into molecular aspects of two cxcl8 homologs of teleost origin.Several molecule  and has ivaceous , common s carpio ,14,15, rs mykiss , zebra fio rerio , grass c idellus , ayu Pleltivelis , half-smmilaevis , turbot  maximus , and lars crocea ,23. CXCLs crocea , and gras crocea ,25. Means crocea  and rains crocea . While ta -infections, and three potent pathogens  that devastate the rock bream farming. Transcriptional profiles of Ofcxcl8-L1 and Ofcxcl8-L3 in challenged fish are shown in Ofcxcl8-L1 induction was higher than that of Ofcxcl8-L3 against all the examined challenge conditions. The Ofcxcl8-L1 mRNA was significantly (p < 0.05) and robustly upregulated by LPS , E. tarda  and S. iniae (5.2 and 3.6-fold at 6 and 12 h p.i.) compared to unchallenged control (Ofcxcl8-L1 by poly(I:C)  and RBIV  was only < 5-fold compared to unchallenged control  challenge  . Transcription of Ofcxcl8-L3 was down-regulated across sampling time points, although it was only significant at 3, 12, and 24 h p.t. (p < 0.05).We isolated fresh PBLs and stimulated them with Con A to examine the transcriptional kinetics of e points . Signifitac). The E. coli BL21 (DE3) system was used to in vitro express the MBP-tagged rOfCXCL8 proteins through IPTG-driven induction, and rOfCXCL8 proteins were purified to homogeneity by amylose affinity chromatography. Sample fractions collected during the recombinant expression and purification of rOfCXCL8 were examined by SDS-PAGE under reducing conditions  under a strong promoter  higher than that of both rMBP and rOfCXCL8-L3 at 100 ng/µL. The rOfCXCL8-L3 demonstrated a dose-dependent chemotactic activity, which was not significantly different compared with rMBP control  increase in proliferation at 10 ng/µL and 100 ng/µL compared with respective concentrations of rMBP (450 at 100 ng/µL (p > 0.05), no significant difference in proliferation between leukocytes treated with rOfCXCL8-L3 and rMBP was evident , and cloned. We categorized the two Ofcxcl8 homologs identified in this study under the lineages 1 (L1) and 3 (L3) based on their peculiar molecular features, such as signature motif (ELR-like domain), sequence homology, phylogenic clustering, and exon-intron architecture. Subsequently, two homologs identified in the current study were characterized in terms of (a) exon-intron organization, in a comparative context, with respect to vertebrate cxcl8s, (b) features of aa sequences, (c) characteristics of 5′-flanking regions, (d) basal tissue mRNA expression, (e) response upon in vitro con A treatment, (f) temporal-expression upon injection of PAMP and/or pathogen, and (g) biological activities using recombinant proteins.CXCL8 (IL-8), which is the first and one of the most intensively researched chemokines, represents the prototypical chemokine of the CXC subfamily . The firviatilis , followi species ,16,27,29asciatus . With thasciatus ,61,62,63Ofcxcl8 orthologs featured several properties of known cxcl8 members  that precedes the ELR-like (SLH) motif, where the sixth Leu residue was proposed to be critically important for its function . MSA revEvidence has been clearly established for the existence of three distinct CXCL8 lineages in teleosts ,24,25,70Ofcxcl8s identified in the present study indeed belong to L1 and L3. While OfCXCL8-L1 demonstrated higher sequence homology with orthologs of L1 compared to L2 and L3, OfCXCL8-L3 showed higher homology with orthologs of L3 than L1 and L2. It was interesting to note that OfCXCL8-L1 shared a relatively higher identity with tetrapod sequences than OfCXCL8-L3  is not CXCL8-L3 . The topCXCL8-L3 , where tCXCL8-L3 , as repocxcl8s of different origins  of several teleost-species. The exon/intron arrangement was essentially demonstrated to be the same with quadripartite structure, in which four exons are separated by three introns with widely varying sizes. However, members of L3 (including Ofcxcl8-L3) had a tri-exonic structure that contained three exons split by two introns. This study, therefore, described a novel member of L3 in teleost cxcl8s, which is evolutionarily and genomic structurally distinct from all the previously characterized teleost cxcl8s. The cxcl8-L3 is a newly growing subclass with cxcl8-like sequences in teleost fish [cxcl8-L3 for the first time. Identification of similar orthologs from different teleost species at the genomic level may confirm our findings. Multiple copies of cxcl8 in teleost might have arisen from whole-genome or chromosome duplication events [The above hypothesis was further validated by the inter-species genomic structural comparison conducted with vertebrate  origins . A previost fish ,70, and n events ,25,73. Hcxcl8 are well understood phenomena in mammalian models. The essence of these findings highlighted that the most important characteristic feature of cxcl8 is its dynamic expression under different circumstances [cxcl8 are poorly studied in fish [Ofcxcl8s were obtained by genomic cloning (Ofcxcl8s possessed a TATA box (or a variant), which is located ~25 bp upstream of their TIS, as observed in mammals [cxcl8 [cxcl8 expression [cxcl8 [cxcl8 gene expression is repressed in resting cells through several negative regulators, including NF-κB-repressing factor (NRF) and octamer-1 (Oct-1) [Ofcxcl8s revealed the presence of two NF-κB elements in Ofcxcl8-L1 and an NF-κB1 element in Ofcxcl8-L3 in the vicinity of their TIS. In addition, several C/EBPα, C/EBPβ, Jun:Fos (AP1), and POU2F1 (Oct-1) TFBS were also located in the promoter regions of Ofcxcl8s. These findings, together with data obtained in rainbow trout [cxcl8 in teleosts.Mechanisms of both basal- and induced-gene expression of mstances . However in fish ,75. Tran cloning . As a co mammals . Mutagens [cxcl8 ,77. Synepression ,79. Othen [cxcl8 ,80. Othe (Oct-1) . ScreeniOfcxcl8 transcripts in different tissues of juvenile rock breams. In mammals, although the primary CXCL8 sources are monocytes and macrophages, virtually all the nucleated cells express CXCL8 [Ofcxcl8 transcripts were widely distributed in several tissues of rock bream at different levels  and LPS, which is a structural constituent of Gram-negative strains, had a greater impact on Ofcxcl8-L1 expression compared with Gram-positive bacterium  suggesting that Gram-negative bacteria appear to be the potent stimulant of Ofcxcl8-L1 transcription. This fact corroborates with the previous findings reported in different fish-pathogen models, including mrigal against Aeromonas hydrophila/E. tarda [A. hydrophila [E. tarda [A. hydrophila [Vibrio parahaemolyticus [As a pro-inflammatory chemokine, CXCL8 exerts its function in leukocytes and endothelial cells via their CXCR1 and CXCR2 receptors to promote immune infiltration and angiogenesis. Earlier reports indicated an induced transcriptional response of the bacteria ,21,22,34teleosts ,86. In tallenges A–C. BaseE. tarda , grass cdrophila , turbot drophila  and largOfcxcl8-L1-induction, LPS and bacteria elicited a strong effect compared to that of poly(I:C) and RBIV, indicating that the bacterial induction of Ofcxcl8-L1 was stronger than that of viral induction  resulted in a strong induction in il-8 transcript levels [Lyccxcl8-L3 mRNA expression in large yellow croaker spleen [Ofcxcl8-L3 was transcriptionally non-responsive against the challenges we performed, except for poly(I:C), where it demonstrated a statistically significant but weak response , which varied from the highly-conserved tetrapartite exonic structure of lineage 1 members. Two Ofcxcl8 homologs also demonstrated distinct characteristics in terms of their translated aa sequences, evolutionary aspects, expressional patterns in normal and immune-challenged animals, and biological functions assayed for chemotaxis and proliferation using rOfCXCL8s. Differential involvement of these homologs in pro-inflammatory response was evidenced based on their transcriptional expression and functional properties. Ofcxcl8-L1 was more inducible, and rOfCXCL8-L1 exhibited relatively high chemotactic and proliferative capacity compared to Ofcxcl8-L3 and its recombinant protein, respectively. Collectively, our lines of evidence support the roles of two Ofcxcl8 homologs in the inflammatory responses, and the present study contributes to broadening our current understanding of piscine chemokines.In summary, we have identified, cloned, and characterized two homologs of


Dear Editor,Q-factor, and fast optoelectronic feedback stabilization. Thus, we can use this device to produce mode-locked laser combs with tunable repetition rates, controllable wavelengths, and self-stabilized phase noise down to −120 dBc Hz−1 at 10 kHz. The span of the combs can be further broadened to more than half an octave through convenient supercontinuum amplification. Combining phase-locking techniques and single-atomic-layer optoelectronics, this study provides knowledge for achieving a high-repetition rate optical frequency comb on fibre.Laser frequency combs emitting ultrafast pulses of light, at equidistantly discrete frequencies, are cornerstones of modern information networks. In recent years, the generation of soliton combs in microcavities with ultrahigh-quality factors has established microcombs as out-of-laboratory tools. However, the material and geometry of a laser cavity, which determine comb formation, are difficult to electrically tune. Such dynamic control can further enrich the diversity of comb outputs and help to actively stabilize them. Here we demonstrate electrically controllable laser frequency combs in a heterogeneous graphene-fibre microcavity. By altering the Fermi level of atomically thick graphene, we simultaneously demonstrate the tunable absorption, controllable 2. Stable frequency combs can be achieved via mode locking in rare-earth doped fibre lasers4, generating Kerr solitons in parametric oscillators8, or opto-electrically modulating lithium niobate microresonators with strong second-order nonlinearity10. For many out-of-lab applications, people desire a compact comb device with multiple enhancements, such as an all-in-fibre integration11, low-driven power but high efficiency13, full stabilization15, and diverse comb outputs with fast and convenient tunability16. The rise of graphene has spurred unprecedented advances ranging from material physics, to mechanics, optoelectronics, and biomedicines19. In optics, the symmetrically gapless dispersion of the quasiparticle Dirac fermions of graphene renders its optical permittivity to be defined only by the fine-structure constant20. This unique feature leads to a remarkable tunability in nonlinear absorption and phase shift22, thus enabling diverse optoelectronic modulators25, passively mode-locked lasers29, and photonic sensors32. Via tuning the carrier density electrically, graphene devices also enable distinct lasing tunability either in-fibre cavities or microresonators33. Unlike conventional graphene used in passively mode-locked lasers, we explore a new geometry in which we integrate a graphene p–n junction34 in an erbium/ytterbium fibre-based Fabry–Perot microcavity via the van der Waals assembly technique35. The all-in-fibre configuration enables the generation of high-repetition laser combs with unique electrical tunability and feedback stabilization in situ.The development of laser frequency combs has revolutionized optical communication, photonic sensing, precision spectroscopy, and astronomical observation2O5 and SiO2 using the ion-assisted deposition method. These dielectric Bragg reflectors have a thickness of 4.5 μm, with a high reflectivity of over 98%, enabling the maximum intrinsic Q > 2 × 106, whereas the erbium/ytterbium-based absorption is ≈2%. The group velocity dispersion of the fibre section is anomalous in the C + L band, which is helpful for dissipative soliton formation. Thus, we do not need to chirp the dielectric Bragg reflectors for an additional chromatic dispersion composition. The monocrystalline single-layer graphene is grown by the chemical vapour deposition (CVD) method and connected by Ti/Au electrodes (5 nm/50 nm), forming a current driven p–n junction, which can be directly driven by a source–drain voltage (VSD). The semiconductor channel width of graphene is ~60 μm. Different from the vertically gated graphene field-effect transistors (FETs) in previous studies16, this graphene incorporation without a gating layer simplifies the structural implementation for better in-fibre incorporation. It also decreases intracavity optical scattering loss, which is helpful for preserving the Q-factor and avoiding the risk of heat damage. Device fabrication and characterizations are further detailed in the ‘Methods’ and Supplementary Section Q-factor of the GFPR was 9.6 × 105 in the lasing band (intrinsic Q-factor of 1.5 × 106). Preserving the Q-factor at a value higher than one million helps to generate laser combs at a pump power <1 W. In further applications, one can increase the erbium/ytterbium doping rate to obtain a higher laser power, while also promoting the lasing and mode-locking threshold.Figure VSD = 16 V. By increasing the pump power, the laser finally operates from a continuous wave (cw) state and multimode state to a dissipative soliton mode-locking state36. The laser threshold of the GFPR here is ≈60 mW. When the pump power increases to over 100 mW, the laser operates in an unstable multimode state, and laser lines appear at various resonances. Once the pump power is higher than 450 mW, the high intracavity energy density enables mode locking due to the saturable absorption of graphene. In this evolution, the lasing efficiency of the multimode operation is considerably lower than either the cw operation or the mode-locking operation, determined by fierce mode competition. Here we also illustrate the measured spectra of several states, for example (state i to state v). We discuss the theoretical laser dynamics in Supplementary Section v) in Fig. 2 shape. This value matches well to the spectral bandwidth (≈4.5 THz), considering the sech2-based time–bandwidth product of 0.315.Driven by a 980 nm laser diode (LD) pump, the GFPR generates a laser in the C or L band. Figure p-doped (holes are dominant). Once driven by current, external electrons are injected into the graphene sheet. As a result, the Fermi level of the intracavity graphene is tuned from p-doping to n-doping. Due to Pauli blocking27, graphene shows better optical transparency when the Fermi level is far from the Dirac point. Figure VSD, the nonlinear correlation of the source–drain current (ISD) vs. the VSD indicates that the external VSD modifies the resistance of the heterostructure from 220 to 3350 Ω. Such tuning enables graphene quasi-Fermi level changing, based on the Drude model μ = (Nρ)−1 and the quasi-Fermi energy equation |EF | = ħ|vF|(πN)−1/237. Here, μ is the mobility, ρ is the resistance, N is the carrier density, ħ is Planck’s constant, and vF is the Fermi velocity. When the VSD approaches 12 V, the Fermi level is close to the Dirac point and further increasing the VSD induces n-doping. By tuning the VSD from 12 to 50 V, we can control the |EF| from 0 to 0.45 eV. In addition, the graphene p–n junction is able to control the local temperature via the ohmic heating effect, determined by the surface current ISD2R38. Thus, when VSD increases up to 50 V, we estimate that the local temperature of the graphene approaches 600 K.Figure T(I) = 1 − ΔTexp(−I/Isat) − Tns. Here, T(I) is the transmission intensity, I is the launched-in power density, Isat is the saturation threshold, Tns is the non-saturated transmission, and ΔT is the modulation depth. Determined by the linear Dirac corn of graphene, in which |EF| > ħω/2, the interband photon-electron transition will be dramatically blocked. Hence, when VSD ≈ 10 V , the heterogeneous graphene device has the largest ΔT = 1.4%. Any external doping will decrease the ΔT. When the VSD approaches 47 V, ΔT is only 0.1%. For the laser mode-locking operation, a relatively large ΔT is preferred. Actually, once the VSD is higher than 37 V, it is difficult for the weak modulation depth to support a mode-locking state. A more detailed theoretical analysis of the tunable saturable absorption of graphene is discussed in Supplementary Fig. ΔT changes the total reflectance of the graphene deposited on the distributed Bragg reflector (DBR) facet, RG = R − ΔT; here, R = 98% is the intrinsic reflectance.Figure VSD, the surface current heats the graphene-deposited DBR mirror, thus changing the refractive index nDBR and the grating period ΛDBR. This change causes a shift in the central wavelength of the graphene-deposited DBR mirror and induces an optical reflection imbalance in the spectrum, as determined by the grating model λ = 2nDBRΛDBR39. The temperature-induced ΛDBR shift is at the 8 × 10−4 level, leading to an optical reflection imbalance in the spectrum  and the eighth-order harmonic-locking state with a repetition rate ≈80 GHz , in Fig. VSD , these primary laser lines evolve to varied mode-locking envelopes, as shown in Fig. 41. The experimental setup is shown in Supplementary Fig. 11. By further optimizing the cavity dispersion and combining state-of-the art techniques such as chaos assistance7, it is also possible to achieve a broader comb span. However, in the amplifying and broadening process, noise is also amplified; thus, we need to stabilize the supercontinuum frequency comb.The all-fibre structure can be conveniently connected afterwards to an optic fibre system for further amplification and supercontinuum broadeningp−n junction provides a powerful means for direct laser stabilization via negative feedback. Similar to other microcavities, such as microdisks and microrings43, uncertainty in the spacing of the laser comb lines, or the FSR = c/2neffL, mainly comes from the thermal instability of the effective refractive index neff, where c is the light velocity in vacuum and L is the cavity length. In addition, the intracavity power fluctuation also influences the jitter noise via Kerr nonlinearity44. To compensate for the dynamic noise, a thermal electric cooler (TEC) with a millisecond response is not fast enough for the resonator with a 10 GHz pulse operation. In previous studies, electrical feedback was commonly compensated at the pump14. In the GFPR, we build a feedback loop to control the cavity directly, taking advantage of the fast optoelectronic response of graphene. A modulation bandwidth larger than 200 MHz is shown in Supplementary Fig. In this unique graphene-fibre implementation, the graphene fFSR of the laser, then the beat note of the laser and the RF synthesizer is sent to loop filter, and finally to an electric amplifier. This process is similar to a lock-in amplifier. Afterwards, the amplified voltage is negatively added to the GPFR to compensate for the VSD. Figure −4 dBm2. By mixing with a stable diode laser reference, Fig. A stable RF synthesizer is used for locking the S(f) are shown in Fig. −1 at 1 kHz. At low frequencies (10 Hz to 2 kHz), the phase noise roughly obeys the f−4 dependence on the offset frequency in the vicinity of the carrier. This close-to-carrier behaviour suggests that the phase noise is now dominated by the random-walk frequency (40 dB/decade), rather than the quantum noise phase diffusion. Once locked by the RF synthesizer, the mode-locked laser comb with a 10 GHz repetition demonstrates an S(f) lower than −90 dBc Hz−1 at 1 kHz and even reaches −120 dBc Hz−1 at 10 kHz, which is greatly limited by the instrument. Such noise suppression is comparable to high spectral purity Kerr solions in silica and MgF2 microcavities45. Below 1 kHz, the feedback stabilizes S(f) and demonstrates a f−2.7 dependence on the offset frequency; this is mainly determined by a mixture of flicker frequency noise and white frequency noise from the RF synthesizer. In addition, the thermal noise oscillation at ~80 kHz comes from the measurement system, such as the optical amplifier. Referring to the timing jitter τJ = ∫S(f)/(2πR), wherein R is the repetition rate, we estimate the temporal instability per roundtrip down to 2.5 × 10−15 s. Figure σA(τ) of the ΔfFSR before and after feedback stabilization. Here, τ is the gate time. After down mixing, the beat frequency is counted with a Λ-type frequency counter. In the free running state, σA(τ) increases from 41 to 8724 Hz, approximately meeting the τ1/2 correlation. This result also verifies that the free-running laser comb is limited by technical noise. The optoelectronic feedback improves the laser comb in long-term operation, with a minimum σA(τ) below 0.1 Hz when the gate time is approximately 0.2 s. First, σA(τ) decreases with the trend τ−1/5 and then increases back to the initial value with a slow increasing slope of τ1/4. This in situ feedback scheme of the graphene-fibre resonator is also applicable for other repetition rates.The measured single sideband (SSB) phase noise spectra −1 at a 10 kHz offset, thereby suggesting a timing jitter of less than 2.5 × 10−15 s per roundtrip. The broadened comb envelopes span over half an octave, which is sufficient for 2f–3f self-referencing. Such realization of the dynamic control and stabilization of the microcomb in a heterogeneous graphene-fibre microcavity provides a new platform for the interfacing of single-atomic-layer optoelectronics and ultrafast photonics, and will promote versatile applications for arbitrary waveform generation, fibre communication, signal processing, and spectroscopic metrology.Leveraging the electrical tunability of the heterogeneous graphene device incorporated in a fibre microresonator, we demonstrate its capability to control laser microcomb dynamics in situ. Taking advantage of the tunnelling diode effect, we realize remarkable graphene Dirac fermion tuning from 0 to 0.45 eV. This result leads to a controllable modulation depth in the range of 0.1 to 1.4%. Consequently, mode-locked laser frequency combs with unprecedented dynamic tunability are demonstrated. Moreover, the graphene-integrated microlaser device provides a powerful way to opto-electrically stabilize the comb lines after supercontinuum amplification and the SSB phase noise reaches the instrument-limiting floor of −120 dBc HzVSD via the tunnelling diode effect. Determined by kinetic equations, this alteration dramatically influences the Pauli blocking in graphene. (2) Surface current enables thermal engineering. In the graphene, the surface current in the graphene considerably increases the surrounding temperature, leading to an unbalanced reflection with the DBRs. (3) Tunable intracavity energy manipulates laser operations. Once (PTR)2 > ENEgΔT, stable-mode locking without amplitude fluctuation can be realized. Here, TR is the roundtrip time, P is the laser power, EN and Eg are the saturation energies of the gain media and graphene, respectively, and ΔT is the modulation depth. (4) Active stabilization. The fast optoelectronic response of the graphene device can suppress the noise related to random-walk frequency, flicker frequency, white frequency, flicker phase, and white phase. Details are discussed in Supplementary Section 1) Electrical modulation determines the saturable absorption of graphene. The surface carrier density of graphene relies on the  Electric−1. The insertion loss of the EYDF and standard SMF-28e is <0.12 dB. All fibre sections were carefully cut and capsuled in a ceramic adapter. Before further processing, each fibre end of the cavity was polished to mirror smoothness, forming the F–P resonator. Multilayer dielectric reflective film-based DBRs (SiO2 + Ta2O5) were coated on both sides of the F–P cavity. The reflectance of each DBR was higher than 98%. Monolayer graphene on copper foils was grown by the CVD method. A layer of a poly(methyl methacrylate) (PMMA) protection film was applied by spin coating (3000 r min−1 for 30 s) on the surface of the foil and baked at 150 °C. Then, a free-standing PMMA-supported graphene film was obtained by etching the bottom copper foil with FeCl3 solution and wet transferred onto the surface of the fibre section. Then, the PMMA was dissolved in acetone. A customer-designed soft stencil lithograph made it physically aligned and was transferred on top of the erbium/ytterbium-doped high Q-factor fibre Fabry–Perot resonator. Finally, Ti/Au contact electrodes (5 nm/50 nm) were fabricated through e-beam evaporation. More information about the nano-fabrication and material characterizations are shown in Supplementary Section The erbium/ytterbium-doped single-mode fibre section  offers a 980 nm/1550 nm pump-gain efficiency > 30% m−2). The output power was detected by a high accuracy power metre. For laser generation and control, we used a single-mode 980 nm LD as the optical pump (maximum power of 1.5 W). A high-power 980/1550 wave division multiplexer with a bandwidth of 1520 to 1610 nm divided the pump and laser. The GFPR was fixed on a V-groove fixture with a TEC . The dynamics of the laser combs could be monitored by using an optical spectrum analyser , an oscilloscope, and a 2 ~ 43.5 GHz RF-ESA . The setups are specifically shown in Supplementary Section For optical transmission spectrum measurements, we used a tunable laser  with a stable CW output  scanning from 1520 to 1610 nm. The output power of the tunable laser was fixed at 1 mW to avoid nonlinear excitations. The spectral transmissions were detected by an infrared band PD  and then plotted in a triggered oscilloscope with a 500 MHz bandwidth . For saturable absorption determination, we used a high-power femtosecond laser (mode-locked fibre laser with a 37 MHz repetition rate and 500 fs pulse delay) to provide a sufficiently high peak power  to over 200 nm (3 dB span). Then, we extracted the intrinsic beat frequency (fFSR) and locked it on an RF synthesizer via a loop filter and an electric amplifier. The output voltage of the loop filter was further amplified and then negatively added to the GPFR to compensate for the VSD. In this procedure, we kept the TEC on to obtain better performance. The implementation of this method is shown in Supplementary Section We used erbium doped fibre amplifiers to amplify the comb pulses. Then, a section of dispersion-shifted highly nonlinear fibre with a nonlinear coefficient of 0.04 Wms from 5  dB span SUPPLEMENTARY INFORMATION


The goal of this single case study was to qualitatively investigate the effects of animal-assisted therapy in a patient in a minimally conscious state.via qualitative behavior analysis.We present a 28-year-old female patient in a minimally conscious state following polytrauma after a sports accident leading to cerebral fat embolism causing multiple CNS ischemic lesions. She received eight animal-assisted therapy sessions and eight paralleled control therapy sessions over 4 weeks. We investigated the reactions of the patient during these sessions The patient showed a broader variability and higher quality of behavior during animal-assisted therapy compared to control therapy sessions.The observed behavioral changes showed higher arousal and increased awareness in the presence of an animal. The presented case supports the assumption that animal-assisted therapy can be a beneficial treatment approach for patients in a minimally conscious state. Early rehabilitation is crucial for patients with disorders of consciousness . The tervia neuronal reorganization  and eight control therapy sessions alternately over 4 weeks. Sessions were paralleled and matched as much as possible regarding therapeutic activities, the therapist, weekday and daytime. In this way, each two sessions were comparable with the exception of the presence of an animal in the AAT sessions. Each session included a beginning ritual and a closing ritual that consisted of sanitizing the patient's hands.  concept , see Figvia a qualitative behavior analysis following a humanistic approach defined by Bernhard . The EEG showed slow-wave activity with predominantly delta waves  for the AAT sessions were based on the individual rehabilitation goals and consisted of basic activities selected from a range of occupational therapy assignments. The therapeutic activities were designed to have a connection with the animal present. For example, the task for the patient was to open a box with herbage and feed the herbage to the animal or to cut vegetables and feed the vegetables to the animal. The involved animals were three guinea pigs  and a lop rabbit . We chose to work with these species because they allow patients to observe them within a radius that is observable. These animals show interesting social behavior in groups, have a high interest in humans, and guinea pigs are also very communicative. They can be fed very easily but need mindful movements that they approach a human hand and therefore make it quite easy for the patient to recognize their emotional state. However, it must be noticed that both guinea pigs and rabbits need a special setting to work with patients with disorders of consciousness since they are highly prone to stress. The involved animals should have retreat possibilities at any time and be able to choose to interact with a patient or not to reduce possible stress . To ensuThe following behavior categories were identified in the explorative qualitative video analysis: changes in breathing; tone regulation; verbalization; and movements of the eye, gaze, mouth, chin, head, forehead, nose, and hand/arm. Over all, the patient showed an increased behavior diversity over the course of the 4 weeks, regardless of the intervention type. Additional to this change, there was also a clear difference in the patients' behavioral reactions between the two settings. The variability and the frequency of observed behaviors were higher in the AAT sessions compared to the control sessions. The patient had her eyes open during seven of eight AAT sessions, whereas she had the eyes open only during one of eight control sessions. We observed a clear increase in selective hand and finger movements over the 4 weeks which was more often observed in the AAT sessions. In the beginning of most AAT sessions the tone in the neck, the arms, and the hands and also the breath frequency increased. After a few minutes the patient showed a relaxation in the arms and hands. A tone normalization in the neck was observable nearly in each AAT session. With that, the patient was able to stabilize her head for some minutes by herself. In the control situations the head movements were less controlled and the head had to be held by an assistant most of the time.We observed different persistent behaviors in relation to the context. The patient showed different behavioral reactions toward the different guinea pigs. She showed nose wrinkling and increased movements in both arms and hands toward one guinea pig with a tousled coat, but not toward the other two guinea pigs and the rabbit. Moreover, the patient turned her head away from one therapist persistently but not from others. The same behavioral sign was observed in the direct contact with the more tousled guinea pig. When the patient caressed the other guinea pig with the softer coat, the head turned to the middle and the nose wrinkling stopped. When the patient had herbs in her hands to feed the animals, tone regulation and selective movements in arm and the hand was observable. Behavioral signs as reactions to questions from the therapists were either head movements or nose wrinkling. When the therapist asked if either a spiky ball or a corncob would be suitable for the rabbit, the head turned to the corncob. The patient also showed increased movements with the arms or hands as reactions to questions. At the end of the 4 weeks, she showed attempts of verbalization.The aim of the study was to explore and qualitatively assess the effect of AAT on behavioral signs of a patient in a MCS. Our results show that the patient showed broader variability of different behavioral reactions in the presence of an animal compared to control therapy sessions. Her reactions were also more consistent, shown with higher frequency, and had a higher quality during AAT sessions compared to control therapy sessions.The increase in movements and breath frequency during the AAT sessions in the present case study are interpreted as signs of higher arousal and increased awareness. Moreover, we interpreted some behaviors, such as head and finger movements, as signs of communication because they were seen more than one time over the different sessions and in relation to the context. The patient showed clear stimulus discrimination abilities. She could distinguish the three guinea pigs and we interpret that she had a favorite animal. The head turning in relation to one of the therapists and to one of the guinea pigs might be seen as a disapproval reaction. Also, a repeated EEG two months after the end of the study  from the Swiss National Science Foundation and funding from the Förderverein pro REHAB Basel. Further, this work was supported by the institute of Zurich University of Applied Sciences.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.