We exploit this behaviour to present an in-plane dielectric heterostructure with a spatially dependent bandgap, as an initial step towards the creation of diverse lateral junctions with nanoscale resolution.The ability to control the size of the electronic bandgap is an integral part of solid-state technology. Atomically thin two-dimensional crystals offer a new approach for tuning the energies of the electronic states based on the unusual strength of the Coulomb interaction in these materials and its environmental sensitivity. Here, we show that by engineering the surrounding dielectric environment, one can tune the electronic bandgap and the exciton binding energy in monolayers of WS 2 and WSe2 semiconductors via manipulation of the surrounding dielectric environment rather than by modifications of the materials themselves.Electronic bandgap tuning in semiconductors enables key functionalities in solid-state devices. Here, the authors present a strategy to control the bandgap of atomically thin WS The precise and efficient manipulation of electrons in solid-state devices has driven remarkable progress across fields from information processing and communication technology to sensing and renewable energy. The ability to engineer the electronic bandgap is crucial to these applications2 with M=Mo, W and X=S, Se, Te468911131415The emerging class of atomically thin two-dimensional (2D) materials derived from bulk van der Waals crystals offers an alternative route to bandgap engineering. Within the family of 2D materials, much recent research has focused on the semiconducting transition-metal dichalcogenides (TMDCs)—MXImportantly, the Coulomb interactions between charge carriers in atomically thin TMDCs are remarkably strong18192022232425212723242627282930313436372 and WSe2, we achieve tuning of the electronic quasiparticle bandgap, as well as of the exciton binding energy of the two TMDC monolayers by several 100's of meV. We note that graphene is particularly well-suited to demonstrate and explore the concept of dielectric heterostructures. It combines a high dielectric screening with the possibility of adding an arbitrary number of additional layers as thin as only 3 Å. Furthermore, the TMDC/graphene structures have been heavily studied recently in a variety of contexts with potential applications in optoelectronics and photovoltaics394041In this report, we provide direct experimental demonstration of control of the bandgap and exciton binding energy in 2D materials using Coulomb engineering through the modification of the local dielectric environment. By placing layers of graphene and hexagonal boron nitride above and below monolayers (1L) of WS2 partially covered with bilayer (2L) graphene, is presented in 243n=1, 2, 3, …, analogous to the states of the hydrogen atom. (Throughout the rest of the manuscript we omit the term quasiparticle for clarity of presentation.) The difference between the bandgap Egap and the exciton resonance energies defines the respective exciton binding energies. In particular, the energy Δ12 between the exciton ground state (n=1) and the first excited state (n=2) scales with the ground state exciton binding energy EB. Along with the experimentally determined transition energy E1 of the exciton ground state, we can determine the electronic bandgap via Egap=E1+EB.An optical micrograph of a typical sample, 1L WSR/R=(Rsample−Rsubstrate)/Rsubstrate, of the bare 2L graphene, 1L WS2 and the resulting heterostructure at T=70 K are presented in R/R is predominantly determined by the imaginary part of the dielectric function, which is proportional to the optical absorption452 is dominated by the creation of so-called A excitons at the fundamental optical transition in the material, at the K and K′ points of the hexagonal Brillouin zone. In particular, the ground state (n=1) excitonic resonance occurs at 2.089 eV. The first excited state n=2 appears as a smaller spectral feature at 2.245 eV, with an energy separation between the two states of Δ12=156 meV. In addition, the first derivatives of ΔR/R are presented in n=1 state is scaled by factor 0.03 for better comparison. Here, the energies of the peaks correspond to the points of inflection of the asymmetric derivative features, as indicated by dashed lines for the n=2 states. Finally, the shoulder on the low-energy side of the n=1 peak at 2.045 eV arises from charged excitons, indicating slight residual doping in the WS2 material472 response matches our previous observations on uncapped samples supported on fused silica24Typical linear reflectance contrast spectra, Δ2 capped with graphene, the overall reflectance contrast is offset by the graphene reflectance, similar to findings in TMDC/TMDC heterostructures2 exciton resonances to lower energies, where the n=1 transition and the n=2 states are now located at 2.060 eV and 2.167 eV, respectively . In all cases, a decrease in Δ12 separation was observed with increasing dielectric screening of the environment . The bandgap of WSe2 can be thus tuned by more than 100 meV and the largest shift of almost 300 meV is observed for graphene-encapsulated WS2, the structure with the highest dielectric screening. For comparison, the influence of an arbitrary dielectric environment is presented in 2 encapsulated between two thick layers of varying dielectric constants. As we have shown, the change in the bandgap is roughly the same as the change in the binding energy and thus can be as high as 500 meV .In addition to the graphene-capped WS2 and WS2 covered by a bilayer of graphene. The corresponding path is illustrated schematically in 2 exciton n=1 and n=2 resonances, respectively. Each spectral trace corresponds to a different spatial position x on the sample; the bilayer graphene flake covers the WS2 monolayer between 5 and 12 μm on the x axis. Like the data shown in 2 excitons shift to lower energies in the presence of graphene. The peak energies are extracted from the points of inflection of the derivative, indicated by circles in 2 monolayer not being in close contact with graphene . The thickness of the layers was confirmed by optical contrast spectroscopy. The heterostructures were fabricated using well-established polymer-stamp transfer techniques described in refs 2 based samples and ref. 2 samples  calculationsThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.How to cite this article: Raja, A. et al. Coulomb engineering of the bandgap and excitons in two-dimensional materials. Nat. Commun.8, 15251 doi: 10.1038/ncomms15251 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Notes and Supplementary References


The resolution of conventional terahertz (THz) imaging techniques is limited to about half wavelength, which is not fine enough for applications of biomedical sensing and nondestructive testing. To improve the resolution, a new superlens, constructed by a monolayer graphene sheet combining with a grating voltage gate, are proposed in this paper to achieve deep super-resolution imaging in the THz frequency range. The main idea is based on the Fabry-Perot resonance of graphene edge plasmon waves. By shaping the voltage gate into a radial pattern, magnified images of subwavelength targets can be obtained. With this approach, the finest resolution can achieve up to λ/150. Besides, the superlens can be conveniently tuned to work in a large frequency band ranging from 4.3 THz to 9 THz. The proposal could find potential applications in THz near-field imaging systems. In recent years, terahertz (THz) imaging applications56891319Graphene, emerged as the most promising 2D plasmonic material in the THz to mid-infrared frequency range2427nm is demonstrated, which indicates our proposal could be suitable for biomedical applications.In this paper, a new graphene-based superlens is proposed to break the diffraction limit in the THz frequency range. The superlens is composed by a monolayer graphene sheet and a metallic grating voltage gate. The working mechanism of the superlens relies on Fabry-Perot resonance of graphene edge plasmon waves. The working frequency can be conveniently tuned over a large range by changing the gate voltage. Furthermore, magnified images of subwavelength targets can be obtained if we shape the voltage gate into a radial pattern. A deep resolution of 400 2 substrate. The permittivity of the SiO2 is 3.9. On the back of the substrate, there is a metallic grating voltage gate. Different separations between the graphene and the metallic surface of the grating lead to non-uniform modification of the carrier density, which eventually generates the periodical conductivity along the x direction. This is now a common method used for tuning the conductivity of grapheneσg = σg,r + iσg,i, depends on angular frequency ω, electron relaxation time τ representing the loss mechanism, temperature T, and chemical potential μc. The imaginary part of the conductivity determines the propagation of SPPs waves, the real part of the conductivity represents the losses. When σg,i > 0, a single free-standing layer of graphene effectively behaves as a very thin “noble metal” layer with negative permittivity. Hence, the graphene sheet can surppot a transverse-magnetic (TM) polairized SPPs surface wave. The dispersion of the two dimensional graphene surface plasmon (2DGSP) supported on the sheet can be expressed as32The schematic of the near-field superlens is illustrated in εr1 and εr2 are the relative permittivity of the dielectrics above (here it is the air) and under the graphene sheet, ε0 is the permittivity in vacuum, e is the electron charge, ħ is the Plank’s constant. pq represents the in-plane wave-vector of the plasmon waves.where p, corresponding to the period of the grating. We assume the conductivities of the two adjacent regions (regions 1 and 2) are respectively σg1 and σg2. If the conditions that Im(σg1) > 0 and Im(σg2) < 0 or Im(σg2) ≪ Im(σg1) are fulfilled, Fresnel reflection will occur on the boundary of the two regions. Hence, the surface waves will be confined to a fictitious boundary3336σg1. The width of the ribbons is d, and the duty ratio is 0.5. Below, we will use this equivalent model to present the analysis. Although the microscopic structure of graphene edges modifies the electronic spectrum significantly, the excitation of the edge mode depends only on total charges and total currents. When the separation between the Femi energy and the Dirac point is much larger than the temperature or relaxation energy, the nature of what kind of edge does not affect the calculations reported hereFor the model in λep) and the 2D surface plasmons (λp) differ by a universal numerical factorλep = C0·λp, where C0 is the factor, λp = 2π/Re(qp). The value of C0 increases with the width of the ribbon, but we always have C0 < 1. The physical reason for the smaller wavelength of the edge plasmons compared to the 2D ones is the effective reduction of the Drude weight at the edges, where free carriers exist only on “one side”. The electric field of the edge plasmons decays exponentially away from the edge. For a given ribbon width, high order modes, which are actually the hybrids of 1D edge mode and 2DGSP mode, can be excited at large frequencies. But the 2DGSP mode disappears at low frequencies. Each of the high order modes has a cutoff frequency. Hence, there is always a frequency band for the single fundamental mode (i.e. the 1D edge mode) operation. The narrower the width of the ribbon is, the broader the frequency band will be. In the frequency range we concerned, the condition of single fundamental mode operation is always satisfied by cutting down the width of ribbons to nanometer scale .It has been well demonstrated that the wavelengths of the 1D edge plasmons . These parameters are typical for graphene plasmons observed in THz to infrared frequency range32λep is calculated by 2(DR-DL), DR and DL are respectively the right and the left positions of two adjacent peaks, as illustrated in the inset of d = 0.2 μm, 1 μm and 2 μm are presented in d decreases, the wavelength of the edge mode becomes shorter, which means that the 1D localized mode is more confined to the rims . As we can observe, in the lower frequency range, the gap of the plasmon wavelengths between the edge mode supported on the nanoribbon and the 2DGSP ·m′p) and propagate along each of the nanoribbons, n and m′ are both integers and refers to the n-order harmonic and the m′-th nanoribbon respectively counting from left to right. One of the electric field components on the m-th nanoribbon can be mathematically expressed asFor the graphene nanoribbon array shown in r and Δφ are respectively the reflection coefficient and the phase jump of the reflected surface wave at the boundary, Δφ = tan−1[Im(r)/Re(r)], E0 is related to the coupling strength. As can be observed, regardless of the angle of the oblique incidence (including the evanescent harmonics), the incident plane waves will always be coupled to the graphene edge mode with the same longitudinal wave-vector, i.e. qep. Hence, the phase accumulations of any incident waves are the same at the emergence interface of the graphene nanoribbon array. The scattering waves of subwavelength targets are an integration of all spatial harmonics with corresponding amplitudes and phases. In order to obtain an undistorted image of the targets, the amplitude and the phase at the emergence interface must keep synchronous with the amplitude and phase at the incoming interface. Under this requirement, we can deduce the following equation after considering the boundary condition at both the incoming and the emergence interfacesin which, lg is the length of the nanoribbons. where φ and qep into lg. An analytical method to determine Δφ has been reported in ref. φ is approximately constant during a broadband frequency range. For simplicity here, we can determine Δφ by numerical simulation. When the parameters of the nanoribbons are uc = 0.15 eV and d = 0.2 μm, we get Δφ ≈ 0.54π in the 4 THz-9 THz frequency range. When the working frequency is chosen to be f = 5 THz, we have qep = 46.12k0, in which k0 is the wave-vector in free space. The conductivity of the graphene nanoribbons at 5 THz is σg1 = 0.035 + i0.56 mS. For m = 2, we obtain lg = 0.95 μm.30x-z plane) of two targets are presented. Here, the targets are surface current sources with their directions parallel to z-axis. The x-direction width of the targets is 250 nm. The two targets are separated by 650 nm and located 60 nm away from the incoming interface. The 2D images are detected at the distance of 60 nm away from the emergence interface. As the above theory predicted, the two subwavelength targets are able to be perfectly distinguished in the near-field by the graphene superlens. The x-y plane electric field mapping above the graphene is shown in λ/92 imaging resolution is demonstrated. The near-field 2D images of the two incident targets which are separated 400 nm are illustrated in x-y plane electric field mapping is illustrated in λ/150. Theoretically speaking, this is the ultimate resolution of the designed graphene superlens because the resolution cannot be smaller than the period of the nanoribbon array. To improve the resolution, one can further narrow the width of the nanoribbons.Next, numerical simulation using Comsol is performed to demonstrate the super-resolution imaging of the superlens. In σg1) = 0.56 mS and Im(σg2) = 0.0012 mS. The two targets are separated by 800 nm. As shown in x-y plane electric field mapping in this case is almost the same as y-direction length of the graphene sheet (or nanoribbons) is lg = 1.15 μm, the images of the two targets are disturbed. Hence, we cannot simply regard each of the nanoribbons as a field probe. When lg = 1.53 μm, as shown in the inset of rd order (m = 3) Fabry-Perot resonance is established. The normalized 1D images corresponding to the 2nd, the 3rd and the 4th order Fabry-Perot resonance, plotted along a x-direction line which is 60 nm away from the emergence interface, are also shown in lg = 0.95 μm, 1.53 μm, 2.12 μm, respectively corresponding to m = 2, 3, 4 in Even though all of the above simulations are done on the graphene nanoribbons array, the same results will be obtained if we replace the graphene nanoribbons with a monolayer graphene sheet tuned by a grating voltage gate. At 5 THz, we have Im can be −0.5 mS ~ 0.01 mS. Therefore, the performance of the superlens is not sensitive to σg2. We only need to modulate the value of σg1 for frequency tuning. This is totally different from those hyperbolic-dispersion-based super-resolution imaging mechanisms which need to accurately control σg1 and σg2 simultaneously29Apart from the deep subwavelength resolution, another advantage of our designed graphene superlens is that it can be easily tuned to work over a broadband frequency range. As we have pointed out, what dominates the edge modes propagation on the graphene sheet is qep at different working frequency when lg is fixed. To achieve this, uc must follow a linear relation with the square of f, i.e. uc = α·f2, in which α is a positive constant, known from uc must increase from 0.111 eV to 0.486 eV accordingly, as shown in i0.481 mS to 0.0357 + i1.01 mS. The upper limit of the frequency range is a conservative estimation. If high quality graphene with large carrier density (could be achieved by highly doping) is available, a higher working frequency can be expected.As mentioned above, the principle of realizing super-resolution imaging is to set up the Fabry-Perot resonance. To ensure this, we only need to adjust the gate voltage to get a identical L = [Im(qp)]−1. As shown in μm to 11.66 μm if we tune the frequency from 4.3 THz up to 9 THz. The square dots are the simulated results of the 200 nm nanoribbon. The theoretical and simulated results are in good agreement. The losses decrease when the frequency increases. The minimum propagation length is much larger than the length of the nanoribbons. In nm. The images are detected along a x-direction line which is 60 nm away from the emergence interface. From 4.3 THz to 9 THz, the two peaks corresponding to the two targets can always be distinguished. The image curves at the low (4.4 THz) and the high (8.8 THz) spectral window are also presented in the inset of The lower limit of the frequency range is mainly subjected to the losses. The losses can be characterized by the propagation length of the edge mode, which should refer to the imaginary part of the wave-vector. In the working frequency range, the propagation length can be roughly calculated by r1 and r2 are respectively the inner and the outer radius of the ribbons, θ is the angle of the ribbon. The basic principle is the same as above. But in this case, the plasmon wavelength of edge mode is no longer constant at a given frequency because the width of each ribbon is gradually broadening. Hence, the Fabry-Perot resonance condition now becomesIn practical applications, we always hope magnified images could be obtained so that two objects could be distinguished by conventional imaging system. Hyperlenses which allow magnification of deep subwavelength scale objects have been proposed by using anisotropic materials with hyperbolic dispersionslg = r2 - r1. When the working frequency is 8 THz, the conductivities of the graphene ribbons become σg1 = 0.0357 + i0.898 mS and σg2 = 2.38×10−4 + i5.98×10−3 mS. The magnification factor of image can be calculated by γ = r2/r1. If r1 is fixed, increasing the value of r2 would enlarge the magnification factor. Nevertheless, the value of r2 can only be discrete because the Fabry-Perot resonance condition must be fulfilled. To prove it, we choose r1 = 0.8 μm, θ = 8°. The duty ratio of the gate is 0.5. Two surface sources separated by 800 nm are located 60 nm away from the incoming interface. The simulated electric field mapping at the z = 10 nm plane for the cases of r2 = 3.2 μm and 4.88 μm are respectively shown in z = 200 nm plane is also presented. Because of the evanescent decay of the fields in the z direction, the strongly confined edge modes become 2D “bulk” modes. As shown in in which, The lossless simulations have perfectly demonstrated the mechanism of realizing magnified super-resolution imaging. But in reality, the compromise should be made between the loss and the magnification factor. As we can see in μm). Although in our demonstrations, the length (corresponding to the magnification factor) is not sufficient for compressing the evanescent spatial spectrum of the deep subwavelength targets into propagating spectrum, far-field super resolution imaging could also be predicted if we lower the designed maximum resolution  or mitigate the loss influence. The method of realizing frequency tuning for this superlens is the same as above, therefore, we will not cover it again.Realizing far-field super-resolution imaging requires a relative long graphene ribbon . Even though the demonstrated propagation lengths of the edge plasmon modes are limited, the lengths could be further increased by optimzing the parameters of the graphene and the frequency to reduce loss. We believe it is possible for using the graphene superlens in practical applications. nm. Furthermore, the superlenses can be tuned to work over 4.3 THz ~ 9 THz frequency range. Owing to the strong confinement of the graphene plasmon edge mode, the images are well resolved in both the x and the z directions. It indicates that our proposal may be suitable for 2D super-resolution imaging. The mechanism could be extended to the infrared frequency range. The proposal could find potential applications in THz near-field imaging systems.To conclude, we have introduced a new way to realize deep super-resolution imaging in the THz frequency range by using a monolayer graphene sheet tuned by a grating voltage gate. Magnified images of subwavelength targets also can be obtained by shaping the voltage gate. The finest resolution of the superlenses can reach 400j∥ = σgE∥, where the symbol “∥” means the component is parallel to the surface of the graphene. According to the Kubo formula, the two specific intraband and interband electron transitions contribute to the surface conductivity of a monolayer graphene, which can be calculated from42Numerical simulations are performed by the commercial software, Comsol Multiphysics, which is based on the Finite Element Method (FEM). The direct and iterative solvers are employed. The monolayer graphene sheet or ribbon is characterized by surface currents e is the electron charge, ħ and kB are the Plank’s constant and the Boltzman’s constant, respectively. The chemical potential depends on the carrier density ns which is controlled by the gate voltage. Notice that for relatively small frequencies(ħω/μc ≪ 1), the intraband electron transition dominates the conductivity, hence, In the condition of μc ≫ kBT, a simple semiclassical model for conductivity, which possesses a Drude-like expression, is obtainedin which, The imaging targets in the simulating models are two rectangular surfaces. The width and the length of the two surfaces are 250 nm and 500 nm, respectively. The excitation sources are surface currents added on both of the two rectangular surfaces. The direction of the currents is perpendicular to the graphene. The minimum size of the grid is smaller than 1/6 of the plasmon wavelength.How to cite this article: Tang, H.-H. et al. Tunable Terahertz Deep Subwavelength Imaging Based on a Graphene Monolayer. Sci. Rep.7, 46283; doi: 10.1038/srep46283 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo. F, GF, and A) as well as nearly 600,000 L1 copies rendered immobile by 5′ truncation and the accumulation of internal mutations (Long interspersed element 1 (LINE-1 or L1) is a mobile genetic element active in nearly all mammals . L1 sequutations . The ongutations , and numutations .F, GF, and A subfamily L1 elements, B1 and B2 SINEs, and IAP and ETn long terminal repeat (LTR) elements (mRC-seq) . Using data from mRC-seq reads and PCR validation followed by capillary sequencing, we discerned the complete structures of 17 polymorphic insertions , comprising 11 L1 TF subfamily insertions, two B1 SINEs, and four B2 SINEs. Despite efficient detection of reference IAP and ETn retrotransposons , we identified no polymorphic LTR insertions in our mice. This result may reflect differences in the activity levels of retrotransposon families among inbred mouse strains, revealed by numerous recent studies . Using PCR and capillary sequencing, we validated 11 de novo L1 insertions . All 11 were TF subfamily elements, consistent with previous reports of disease-causing L1 insertions in mice wherein all insertions for which sufficient L1 sequence was present for subfamily distinction were identified as TF elements . Insertion #5 also occurred within an intronic L1mA5 element, and two additional intergenic insertions landed within existing L1 repeats .We regarded insertions detected in one or more offspring but absent from the corresponding parents as potentially de novo , reflecting depletion of mouse L1 3′ termini observed during Illumina sequencing , possibly due to the GC-rich nature of the mouse L1 3′ end sequence , and a previous analysis suggested that TF L1s undergo 5′ truncation less frequently than other L1 elements . Insertions #1 and #2 were identified by mRC-seq in mice SRAB2 and SRAB15, respectively . We did not detect these insertions by PCR genotyping in the somatic tissues of parental mice SRA and SRB; however, insertions #1 and #2 were detected by PCR in both testicles of paternal mouse SRB . Inheritance of each insertion by only 1/20 offspring, coupled with their presence in both testicles of the paternal mouse, suggested germline-restricted mosaicism for insertions #1 and #2 in mouse SRB .Next, we used PCR genotyping to investigate the developmental origin of each de novo L1 insertion . An insertion-specific genomic DNA qPCR assay targeting the 5′ L1-genome junction of insertion #7 revealed its prevalence of ∼11% and ∼4% in the germ cell fraction of the left and right testicle of SRCD14, respectively . As evidenced by our failure to detect L1 insertions #1, #2, and #7 from mesoderm-, ectoderm-, and endoderm-derived tissues of the respective paternal mice, we reasoned that these insertions likely arose in early primordial germ cells (PGCs) during paternal embryonic development, prior to PGC colonization of the genital ridge and the formation of the testes. Thus, we conclude that L1 retrotransposition can occur in early PGCs, resulting in germline-restricted genetic mosaicism and heritable de novo L1 insertions.In an inverse approach to identify germline-restricted mosaic insertions, we performed deep (∼260×) mRC-seq on the germ cell fraction of each testicle of mouse SRCD14. We detected and PCR-validated insertion #7 in both testicles and did not detect it in the somatic tissues of SRCD14 . PCR genotyping revealed bands of varying intensity among the somatic tissues and ovaries of mouse SRE , #6 , and #11  were each identified by mRC-seq in multiple offspring and genotyped as mosaic in the respective maternal tissues. Insertions #8  and #10  were identified by mRC-seq and were confirmed as mosaic in the tissues of mouse SREF17 and mouse F2-145, respectively, but were not transmitted to the limited offspring produced by these animals  . Therefore, we can neither confirm nor rule out the contribution of insertions #8 and #10 to the germ lineage. Taken together and consistent with previous studies . Upon crossing mouse SREF16 to mouse SRCD14, insertion #4 was transmitted to 11/19 offspring . We therefore conclude that mouse SREF16 was heterozygous for insertion #4 and that this insertion arose sufficiently late during germline development of parental mouse SRE or SRF to preclude its detection in bulk gonad tissues, or possibly post-conception in SREF16 at the mature zygote stage.Of the remaining two de novo L1 insertions, insertion #4 likely represented a late germline event. This insertion was identified by mRC-seq in mouse SREF16, one of seven offspring of parental mice SRE and SRF, but was not detected in somatic and germ tissues of SRE or SRF . This rate of transmission suggested either parental heterozygosity or mosaicism with a high degree of germline prevalence. However, we could not detect insertion #9 by nested PCR in the gonads of SRCD11 or SREF20 . The developmental origins of insertion #9 are therefore unclear. We speculate that this event was germline-restricted mosaic in SRCD11 or SREF20, but that the subset of germ cells carrying insertion #9 was depleted by the time the gonads of these animals were harvested, at age 39 and 40 wk , one early embryonic insertion (#5) , and two presumably recent polymorphic L1 TF insertions differentially present/absent among our animals (Poly_L1TF_3 and Poly_ L1TF_4)  carried 3′ transductions, implicating five distinct full-length L1 TF elements present in the C57BL/6J reference genome. Thus, we have identified progenitor elements responsible for de novo heritable L1 insertions.We next sought to identify the progenitor L1 elements responsible for heritable de novo insertions. During transcription of an RC-L1 element, the native L1 polyadenylation signal is occasionally bypassed in favor of a downstream genomic polyadenylation signal, and upon retrotransposition the nascent L1 insertion incorporates a genomic sequence tag, or 3′ transduction, that identifies the progenitor L1 element . Two earSupplemental Fig. S9A; Supplemental Fig. S9B). Given our mouse data indicating each heritable early embryonic L1 insertion was detectable in tissues derived from all three germ layers , we suggest that insertion JH-27 was very likely maternal germline-restricted.Having observed transmission of germline-restricted and somatic/germline mosaic insertions in mouse, consistent with previous studies of human patients , we next2 test with one degree of freedom, two-tailed P-values of 0.49 and 0.67, respectively). Thus, as expected, within a single generation we observe no evidence for positive or negative selection on a de novo L1 insertion. However, future studies tracking the transmission of de novo L1 insertions through many generations may reveal evidence for selection on particular insertions, perhaps dependent on their genomic locations and functional consequences.We uncovered 11 de novo L1 insertions among 85 mouse genomes, providing an estimate of one new insertion per eight mice (11/85 = 0.13 or ∼1/8). This figure is consistent with but more conservative than previous estimations that a new L1 insertion may arise in every two to three mice  and is mWe established for the first time that heritable endogenous L1 retrotransposition events arise in early PGCs, before the PIWI/piRNA retrotransposon defense pathway becomes active in male embryonic gonads , resultiSupplemental Tables 1, 5), suggesting that additional mosaic insertions may have fallen below the detection threshold of mRC-seq. Thus, it is possible that early embryonic retrotransposition frequently generates low-level somatic-restricted mosaicism . Thus, early embryonic insertions present at low prevalence in somatic and germ tissues of a parental mouse nevertheless have the potential to contribute substantially to genetic diversity in the next generation, presumably by their fortuitous presence in a subset of germ cells that ultimately give rise to offspring.Consistent with previous studies , we tracosaicism , as wellosaicism C but wasIn contrast, insertions arising during later germline development  may contribute a broad spectrum of genetic diversity to the gamete pool, yet each insertion individually would have a small likelihood of transmission C. IndeedDe novo heritable L1 retrotransposition is a component of the ongoing evolutionary interplay between retroelements and mammalian genomes, the importance of which is exemplified by recent studies demonstrating the exaptation of LTR retrotransposon sequences and protein products for pluripotency maintenance and embryonic development . FurtherAll animal breeding and handling procedures were carried out in compliance with the guidelines set forth by the University of Queensland Animal Ethics Committee. To establish breeding pedigrees, adult wild-type C57BL/6J mice were ordered from the University of Queensland Biological Resources Facility (UQ-BRF), which in turn sources animals from the Animal Resources Center . The UQ-BRF and ARC provided helpful information regarding the source and breeding history of the animals used in this study. The “P-generation” mice used to initiate the breeding pedigrees in this study were no more than 10 generations removed from the Jackson Laboratory C57BL6/J strain.Supplemental Methods.Genomic DNA from animals and tissues of interest was used to construct Illumina libraries for mRC-seq as described in F GF and A, SINEs B1 and B2, and the LTR elements IAP and ETn . Illumina libraries were pooled to achieve a total mass of 1 µg . A detailed description of mRC-seq hybridization can be found in Supplemental Methods.Hybridization reactions were performed as described in Supplemental Fig. S1B; Supplemental Table 1). We performed mRC-seq on the individual somatic tissues and gonads of mice SRA and SRE and the germ cell fraction of the left and right testicles of SRCD14. We also performed 30× whole-genome sequencing on P-generation animals SRE and SRF, and 22× WGS on their F1 offspring SREF15-SREF21, using pooled somatic tissue genomic DNA . Across all DNA sequencing libraries, we detected 92.8%, 97.8%, and 98.7% of recent reference genome L1 , LTR retrotransposon , and SINE  insertions, respectively .We performed mRC-seq on either pooled genomic DNA from somatic tissues representative of the three embryonic germ layers  or DNA extracted from tail tips and ear punches , generating 2×150-bp read pairs and 2×250-bp read pairs. Some libraries were also sequenced on an Illumina MiSeq platform, generating 2×250-bp and 2×300-bp read pairs. WGS libraries were sequenced on an Illumina HiSeq 2500 platform (Macrogen), generating 2×250-bp read pairs. mRC-seq and WGS data were processed as follows: read pairs were first trimmed from their 5′ and 3′ ends to remove any bases with quality <10, then assembled into contigs using FLASH  (A summary of library pooling and sequencing reads per library is included in ng FLASH  and defang FLASH  (parametETn LTR) , using LETn LTR)  (parametETn LTR) . ClusterETn LTR)  or in alhttp://serialbasics.free.fr/Serial_Cloner.html) and the BLAT tool on the UCSC Genome Browser . Oligonucleotide primers were ordered from Integrated DNA Technologies (IDT).Putative insertions were called as potentially de novo if they appeared in one or more offspring from the same pedigree and were absent from the parental mice. In addition, some insertions were readily detected in offspring and were also detected with 1–2 reads upon deep sequencing of individual tissues of maternal mice SRA and SRE. Such insertions were treated as potentially de novo and chimeric in the maternal mouse. Reads were then manually inspected using SerialCloner . The full details of PCR validation can be found in Supplemental Methods.Empty-filled validation PCRs were carried out using primers specific to the 5′ and 3′ genomic sequence flanking putative insertions. Validation PCRs for 5′ and 3′ junctions were carried out using the appropriate flanking genomic primer paired with a primer internal to the L1 sequence; where necessary, hemi-nested and fully nested PCR reactions were carried out using appropriately designed genomic and L1-specific primers  at a ratio of 3 µL to 1 µg plasmid DNA. G418 selection (400 µg/mL) was initiated at 72 h post-transfection and carried out for 10–12 d  at the Translational Research Institute Flow Cytometry Core. The percentage of EGFP positive cells for each L1 reporter construct was used to normalize the G418-resistant colony counts obtained in the retrotransposition assay . Full deCt values were calculated on the LightCycler software using absolute quantification 2nd derivative max. Details of mosaicism analysis qPCRs can be found in Supplemental Methods.Quantitative PCR using genomic DNA as template was carried out using primers and dual-labeled PrimeTime qPCR probes (5′ 6-FAM-ZEN-3′ Iowa Black FQ) from IDT. Control reactions for DNA input were performed using a predesigned PrimeTime qPCR assay for the single-copy mouse gene RPP25 (Mm.PT.58.21641426.g), with a dual-labeled probe (5′ 6-FAM™-ZEN-3′ Iowa Black FQ). Reactions were run on a Roche LightCycler 480 II with the following cycling conditions: 95°C, 5 min, followed by 45 cycles of 95°C for 10 sec, and 57°C for 1 min, then melt curve (0.11°C per sec from 57°C to 95°C). http://www.ebi.ac.uk/ena/) under project accession number PRJEB10299. Sanger trace files from this study have been submitted to the NCBI Trace Archive (http://www.ncbi.nlm.nih.gov/Traces/home/index.cgi) with TI numbers TI2344112704–TI234412736 and TI234412752.mRC-seq and WGS data from this study have been submitted to the European Nucleotide Archive (ENA; J.A.J. is employed by Roche Sequencing Solutions, Inc., and Roche Sequencing Solutions reagents were used in the study.


Ultra-high molecular weight polyethylene (UHMWPE) is the most common bearing material in total joint arthroplasty due to its unique combination of superior mechanical properties and wear resistance over other polymers. A great deal of research in recent decades has focused on further improving its performances, in order to provide durable implants in young and active patients. From “historical”, gamma-air sterilized polyethylenes, to the so-called first and second generation of highly crosslinked materials, a variety of different formulations have progressively appeared in the market. This paper reviews the structure–properties relationship of these materials, with a particular emphasis on the in vitro and in vivo wear performances, through an analysis of the existing literature. Ultra-high molecular weight polyethylene (UHMWPE) has been used as a bearing material in total joint arthroplasty for more than 50 years now. The idea to replace degraded cartilage with a polymer liner dates back to the late 1950s. At that time, Sir John Charnley chose polytetrafluoroethylene (PTFE), a low friction polymer, as the bearing material to replace the natural acetabulum, articulating against a metallic femoral head, for hip replacement. Due to the unacceptably low wear resistance of PTFE though, the first “low friction arthroplasties” dramatically failed after few years of implantation. In 1962, UHMWPE, a similarly low-friction, but much more wear resistant polymer, replaced PTFE in Charnley’s hip arthroplasty, with remarkably better performances. From then on, arthroplasty has known considerable evolution, but UHMWPE remains the gold standard for artificial hips and now other artificial joints, including the knee and shoulder ..42].Similarly, acetabular cups machined from a polyethylene gamma-irradiated in air at doses ranging from 33 to 1000 kGy, remelted and EtO sterilized, demonstrated an 87% wear reduction with increase in the radiation dose from 33 to 95 kGy, when tested for 5 million cycles in a hip simulator; again, the wear decreased to undetectable levels at a radiation dose higher than 200 kGy . In the Kurtz et al.  summarizThe influence of the type of counterface on the wear behavior of crosslinked polyethylene was also investigated. Bistolfi and coworkers  showed tA previous study  also demRegarding the oxidation stability of the two formulations of 1st generation highly crosslinked polyethylenes, McKellop and coworkers compared the oxidation stability and wear resistance of the same set of samples mentioned above , with orMuratoglu and coworkers  tested tIn another study , the samCollier et al. in an extensive study that compared the physical and mechanical properties of commercial, 1st generation crosslinked polyethylenes from six orthopaedic manufacturers , also coMost of the aforementioned studies attributed the differences in the oxidative stability between remelted and annealed crosslinked polyethylenes to that a thermal treatment above the melting temperature (remelting), leads to a complete melting of the polymer crystallites, hereby allowing termination of the radicals trapped in the crystalline phase and thus brings the amount of residual radicals to undetectable levels ,73. ConvSeveral clinical studies have reported superior in vivo wear performance of highly crosslinked PE, compared to conventional polyethylene in hip replacements ,85,86,87A number of retrieval studies have also reported on the chemical, physical and mechanical properties of these highly crosslinked PE following in vivo aging. According to predictions of in vitro studies, oxidation of the highly crosslinked/annealed formulation was reported by a number of authors ,93,94,95On the other hand, failures of some specific designs of highly crosslinked/remelted have also been described in the literature ,98. Rim As already mentioned, crosslinked/remelted polyethylenes did not contain free radicals at the time of implantation and were expected to be stable to oxidation. Unexpectedly, some recent literature studies reported that measurable levels of oxidation were observed in some retrievals ,103,104.Although the clinical significance of such adverse behaviors of the 1st generation highly crosslinked PE is still under debate, their occurrence stimulated the research of alternative polyethylene formulations to overcome the observed drawbacks.The rationale at the basis of a new generation of highly crosslinked polyethylene was to maintain the superior wear resistance demonstrated by the 1st generation crosslinked PE, while also retaining the mechanical properties and fatigue resistance of the uncrosslinked material and ensuring stable properties, thus oxidative stability, over time.As discussed in the previous paragraph, annealing below the melting temperature preserves most of the original UHMWPE microstructure. Highly crosslinked/annealed PE had been demonstrated to retain mechanical properties and fatigue resistance similar to conventional, gamma-nitrogen sterilized (but unoxidized) material; however, the presence of free radicals in the latter leaves the polyethylene exposed to oxidation . DumbletThis 2nd generation crosslinked PE  was terminally sterilized with gas plasma, so as to avoid the introduction of more free radicals with sterilization. Literature studies reported similar or only slightly reduced mechanical and fatigue properties compared to conventional PE ,107,108 An alternative approach to the 2nd generation highly crosslinked polyethylene involves the addition of an anti-oxidant stabilizer, in order to efficiently inhibit the oxidative degradation, without the need for a thermal treatment of the irradiated polyethylene, so as to preserve the original morphology, mechanical properties and fatigue resistance . AlthougTwo methods are currently used to incorporate vitamin E into UHMWPE ,115. OneSeveral studies have investigated the mechanical properties of both formulations of vitamin E stabilized PE. No significant differences in the static mechanical properties, nor in the fatigue crack propagation resistance have been observed when comparing virgin UHMWPE with blends containing up to 5000 ppm of vitamin E ,135. ConIn addition, Vitamin E-stabilized PE has demonstrated superior oxidation stability under a variety of accelerated aging conditions. For example, blends of UHMWPE with 500, 1000 and 5000 ppm of vitamin E, gamma irradiated to doses up to 100 kGy were found to be more oxidatively stable than virgin, unirradiated UHMWPE, following accelerated aging at 90 °C in air . Oral etThe wear resistance of this 2nd generation highly crosslinked formulation has been extensively tested in several in vitro experiments, under a variety of challenging conditions, and has been compared to conventional and 1st generation highly crosslinked formulations before and after both accelerated and shelf-aging. In a POD wear test, highly crosslinked Vitamin E-doped PE exhibited comparable wear rates to 1st generation highly crosslinked PE irradiated to the same dose , while iBellare et al.  comparedSimilarly, while comparing the wear behavior of standard, unirradiated PE to that of two formulations of crosslinked polyethylene, with and without vitamin E (1000 ppm), both irradiated to 70 kGy, Affatato et al.  found thThese observations suggest that, if vitamin E is necessary to avoid oxidation, then the radiation dose must be optimized in order to obtain enough crosslinking density to preserve the wear resistance of unstabilized, crosslinked PE.Given the concerns mentioned above on the use of 1st generation highly crosslinked PE in knee arthroplasty, the vitamin E crosslinked formulations represented an attractive alternative, in particular in this application. For this reason, many experimental studies investigated the performance of vitamin E stabilized PE for use in the knee with promising results. For example, while comparing the wear rate of two designs (cruciate-retaining and posterior-stabilized) of highly crosslinked UHMWPE doped with vitamin E to that of γ-inert–sterilized direct compression-molded UHMWPE, Haider and coworkers  found thIn another knee simulator experiment, UHMWPE blended with 1000 ppm of vitamin E and radiation sterilized with 30 ± 2 kGy, was artificially aged according to ASTM F2003-2 and tested for 5 million cycle without showing structural failures, as conventional PE did under similar conditions . SimilarTeramura and coworkers  evaluateBladen et al.  demonstrTo investigate the effects of particulate debris in vivo, Bichara et al.  comparedIn addition, it has been shown that vitamin E may also exert some antibacterial activity ,147,148.Literature reports on the clinical outcome of vitamin E stabilized PE are still quite limited in number, but their results are overall promising. One recent study  comparinProspective studies at two to three years follow-up demonstrated no significant differences in femoral head penetration rates between vitamin E doped and virgin highly crosslinked liners ,151,152.However, one study reporting head penetration into vitamin E doped highly crosslinked liners at five years demonstrated less wear compared to that previously reported for 1st generation highly crosslinked PE, at the same interval of five years. Further, the study demonstrated that, after settling of the liners in the early period, no significant head penetration occurred from two- to five-year follow-up and the authors remarked that, overall, the wear observed in the study was well below that at which osteolysis becomes a serious concern .Rowell et al.  analyzedSimilarly, Currier et al.  analyzedUHMWPE remains the most commonly used bearing material in total joint arthroplasty due to its long and relatively successful history. However, UHMWPE is a complex material and its mechanical and tribological behavior is strongly dependent on the morphology and chemical alterations induced by processing conditions, such as sterilization using ionizing radiation or crosslinking. The need for increasing the life expectancy of the implants, in order to meet the demand in younger and active patients, has led to a considerable evolution in the manufacturing of the UHMWPE orthopedic devices over the last few decades. From gamma-air sterilized “historical” polyethylenes, to the first and second generation of highly crosslinked materials, the wear resistance, mechanical properties and overall performances of the UHMWPE biomaterials have greatly improved, as has been extensively reported in the literature. Advances in the sterilization techniques have enabled a substantial reduction of the oxidative degradation experienced by “historical” implants, greatly lowering the incidence of wear-induced osteolysis and early mechanical failures. First generation crosslinked PE has further reduced wear, but at the expenses of fatigue resistance and/or oxidative stability, while 2nd generation crosslinked PE has been introduced to overcome the latter drawback and, overall, it has shown promising, yet early results, despite some issues regarding the oxidative stability of the sequentially irradiated and annealed PE.Nevertheless, the clinical outcome of some of the newest formulations is still largely unexplored. It is therefore of paramount importance to ensure a close, continuous monitoring of the clinical performances of the contemporary UHMWPE biomaterials.


Although chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) seem to be opposite entities from a clinical perspective, common initial pathogenic steps have been suggested in both lung diseases. Emphysema is caused by an elastase/anti-elastase imbalance leading to accelerated elastin degradation. Elastinolysis is however, also accelerated in the IPF patients’ lungs. The amino acids desmosine and isodesmosine (DES) are unique to elastin. During the degradation process, elastases liberate DES from elastin fibers. Blood DES levels consequently reflect the rate of systemic elastinolysis and are increased in COPD. This is the first report describing elevated DES levels in IPF patients. We also demonstrated that the age-related increment of DES concentrations is enhanced in IPF. Our current study suggests that elastinolysis is a shared pathogenic step in both COPD and IPF. Further investigation is required to establish the relevance of accelerated elastin degradation in IPF and to determine whether decelerating this process leads to slower progression of lung fibrosis and better survival for patients with IPF. Although chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) seem to be opposite entities from a clinical perspective, they often coexist in the same patient , and shaet al. recently demonstrated that elastin degradation accelerates during ageing in subjects with or without obstructive lung disease, but that this age-related DES increment is amplified in patients with COPD [The elastin precursor tropoelastin is mainly produced perinatally . Tropoelith COPD . WhetherBy analogy to COPD, we hypothesized that pDES levels would be elevated and that the age-related pDES increment would be enhanced in IPF patients compared to controls with no lung disease.pDES levels were measured by liquid chromatography tandem-mass spectrometry in 154 IPF patients from Maastricht , from Nieuwegein  and from Ann Arbor  as previously described . For eacp<0.0005) compared to controls (337±32 ng/L). No significant differences in pDES levels were found between IPF and COPD patients (p=0.2). FVC data at baseline were available from the Nieuwegein and Ann Arbor cohort; no significant correlation was found between pDES levels and %FVC of predicted at baseline (p=0.3). FVC follow-up data were available from the Nieuwegein cohort; a significant correlation was demonstrated between pDES levels and the change in %FVC of predicted per year (p=0.02).A significant correlation was found between age and pDES (p<0.0005). The equation for the pDES regression line in IPF patients was 76 (95% CI -117–270)+6.76 (95% CI 3.75–9.77)*age (ng/L). Significantly higher pDES levels were found in IPF patients 507±8 ng/L) compared to age-adjusted reference values of non-smoking controls , of smoking controls  and of COPD patients  . Based pPrevious research has mainly focussed on pDES as a biomarker of systemic elastin degradation in COPD . Our prepost hoc study in which AAT augmentation therapy resulted in a decrease of pDES levels in AAT deficient COPD patients [We regard deceleration of elastin degradation as a potential attractive novel therapeutic target in COPD. This intriguing new concept was illustrated by a recently published patients . In IPF,patients . Althougpatients . Future In summary, this is the first study describing elevated pDES levels in IPF patients, which were even higher than in COPD patients. We also demonstrated that the age-related increment of pDES concentrations is enhanced in IPF. Additional studies are needed to establish the relevance of accelerated elastin degradation in IPF and to determine whether decelerating this process translates into slower progression of lung fibrosis and better survival for patients with IPF.


Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi.In  Cryptococcus is now recognised as a major fungal human pathogen responsible for thousands of deaths every year around the world , Cryptococcus gattii exists in several sub-lineages, some of which can infect immunocompetent patients (Cryptococcus lives in the environment (mainly in soil and decaying wood), its biology has been shaped through its interactions with environmental microbiota, as well as seasonal modifications in the physical and chemical soil parameters  and identified two small introns of 52 and 49 nucleotides  was originally reported to be intronless  , becoming a template for dsRNA synthesis and siRNA production  formation in the wake of the RNA polymerase II, thus avoiding the associated accumulation of DNA damage . Bonnet an cells . They fotability . AltogetSummary and future directions - In summary, these studies have revealed introns as key features in the biology of Cryptococcus and, in the maintenance of their genome integrity. They also suggest that their structure have been finely selected throughout evolution. Nevertheless, a number of questions remain to be answered. (1) For instance, the mechanisms regulating intron gain and loss in fungi is still a very active domain of research. A better description of intron structure and position in the different lineages of C. gattii and C. neoformans  Additionally, the role of introns in gene regulation requires further analyses. The current model on Cryptococcus intron retention-dependent regulation of gene expression  Finally, introns have been identified in miscRNAs, but the function, splicing, and evolutionary conservation of this intron subclass have not been studied yet.oformans  will likoformans , a precipression  suggestsentified . In metaentified . In fung


Immunotherapy targeting PD-1/PD-L1 axis showed benefits in cancer. Prognostic significance of tumour infiltrating lymphocytes (TILs) has been determined. We evaluated PD-L1 protein expression in tumour cells and TILs, PD-L1 mRNA level and various histopathologic factors including TILs using 167 formalin-fixed paraffin embedded tissues and 39 fresh tissue of HER2-positive breast cancer. TILs level and PD-L1 expression in tumour cells and TILs were significantly correlated one another. PD-L1 positivity in tumour cells was associated with high histologic grade and high TILs level . High PD-L1 immunoscore in TILs and high total immunoscore (in tumour cells and TILs) of PD-L1 were correlated with high histologic grade , absence of lymphovascular invasion , negative hormone receptor expression  and high TILs level . High PD-L1 mRNA expression was associated with high TILs level . PD-L1 positivity in tumour cells was associated with better disease-free survival in HR−/HER2+ breast cancer (p = 0.039). PD-L1 expression in tumour cells and TILs are significantly associated with TILs level in HER2-positive breast cancer. PD-L1 expression in tumour cells might be positive prognostic factor in HR−/HER2+ breast cancers. Evading immune destruction was added as a component of the hallmarks of cancer by Hanahan and Weinberg in 20112, with emphasis on the tumour microenvironment. There are three mechanisms of immune escape in cancer, including loss of both antigenicity and immunogenicity, and an establishment of an immunosuppressive microenvironment3. To lose immunogenicity, tumour cells use immune checkpoints, which are essential for the maintenance of self-tolerance under normal physiological conditions. Immune checkpoints, including co-stimulatory and inhibitory signals, regulate the level of the T cell response. Inhibitory ligands and receptors, which regulate T cell effector functions, are generally overexpressed in tumour or non-transformed cells in the tumour microenvironment4.Recent progress in the field of immunology supports the theory that the immune system has a role in cancer development through “cancer immunoediting”. This encompasses three processes: elimination, equilibrium and escape13. Remarkably, Schadendorf et al.5 observed a plateau in the survival curves in a meta-analysis of overall survival involving the use of anti-CTLA4 (ipilimumab) in patients with advanced melanoma, which started at approximately three years. This suggests that immunotherapy could be a potentially curative treatment option for advanced malignancies. Despite the evidences surrounding the clinical benefits of immune checkpoint blocking agents, particularly those targeting the PD-1/PD-L1 axis, the value of PD-L1 expression as a predictive biomarker for patient response to anti-PD-1/PD-L1 therapy has not been established. Although the expression of PD-L1 in tumour cells was considered a potential biomarker for PD-1/PD-L1 blocking agents, it has demonstrated effectiveness even in PD-L1 negative patients15. Furthermore, several reports have demonstrated that lymphocyte PD-L1 expression was associated with the response to anti-PD-1/PD-L1 therapy12. Moreover, the prognostic significance of PD-L1 expression has not been conclusively determined.Immune checkpoint blocking agents targeting the cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed death -1 (PD-1)/programmed death-ligand 1 (PD-L1) axis have been developed and possess notable clinical efficacy16. In HER2-positive breast cancers, treatment with trastuzumab appears to boost the immune system response17. A high level of TILs is associated with a better response to trastuzumab, and is a good prognostic factor in HER2-positive breast cancers19. Moreover, there is a consensus regarding the methodology for evaluating histological levels of TILs in breast cancers20.The prognostic impact of tumour infiltrating lymphocytes (TILs) has been reported in various organs, including the breast, and specifically in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancers (TNBCs)et al.21 classified cancers based on TIL and PD-L1 expression and proposed immunotherapeutic strategies according to their classification. As previously mentioned, immune mechanisms are associated with the efficacy of trastuzumab treatment, and several attempts have been made to use immune checkpoint inhibitors in combination with trastuzumab. Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent emtansine. The combination of T-DM1 and anti-CTLA4 or anti-PD-1 antibodies, elicited responses in tumour xenografts that had previously been resistant to T-DM1 monotherapy in a mouse model22. An international phase Ib/II trial  involving trastuzumab in combination with the anti-PD-1 antibody is currently being investigated. In a mouse model, the synergistic effect of anti-PD-1 agent on trastuzumab therapy was demonstrated in HER2-positive breast cancer17. However, the immune checkpoints in HER2-positive breast cancers, particularly PD-L1, are not completely elucidated. Whilst reports indicate the significance of PD-L1 expression in TILs for predicting the response to anti-PD-1/PD-L1 inhibitors, many recent studies have focused on the expression of PD-L1 in tumour cells alone. Despite efforts to establish a combination therapy involving trastuzumab and anti-PD-1/PD-L1 inhibitors, PD-L1 expression in HER2-positive breast cancers has not been fully determined.In addition to the progress made in immunotherapy and cancer immunology, Teng In this study, the expression of PD-L1 in tumour cells and TILs and the relationship between the expression levels of PD-L1 and various clinicopathological parameters, including TILs, were evaluated. The prognostic significance of PD-L1 expression was also assessed.in situ (DCIS), the remaining 65 cases (38.9%) were categorized as having a high level of TLSs around the DCIS. Patients were also grouped by abundance of TLSs around invasive margins, 109 patients (65.3%) and the remaining 58 patients (34.7%) were classified as having a high and low level of TLSs respectively (Supplementary Table A cohort of 167 patients with HER2-positive breast cancers was included in our study. Patient age ranged from 23 to 85 years (mean: 52.59 ± 10.126 years) and the size of the breast tumours ranged from 0.2 to 15.5 cm (mean 3.213 ± 1.773 cm). There were 93 (55.7%) and 74 cases (44.3%) of left and right breast cancers, respectively. Eighty-nine cases (53.3%) were of hormone receptor (HR)+/HER2+ breast cancers and 78 cases (46.7%) were of HR−/HER2+ breast cancers. Sixty-one patients (36.5%) had TIL levels of 10% or less, 74 patients (44.3%) had TIL levels between 20% and 60%, and the remaining 32 patients (19.2%) had TIL levels of more than 60%. One hundred and two cases (61.1%) were categorized as having a low level of tertiary lymphoid structures (TLSs) around the ductal carcinoma 23, 28 cases (16.8%) were type I tumours, 82 cases (49.1%) were type II, 53 cases (31.7%) were type III and four cases (2.4%) were type IV (Supplementary Table Eighty-one patients (48.5%) showed positive immunostaining for PD-L1 in tumour cells according to the Allred scoring system , high TIL level (p < 0.001), and an abundance of TLSs around the invasive component (p = 0.002). High PD-L1 immunoscores for TILs was significantly correlated with high histologic grade (p = 0.001), absence of lymphovascular invasion (p = 0.012), absence of HR expression (p = 0.044), high levels of TILs (p < 0.001) and abundance of TLSs around the invasive component (p = 0.034). High levels of total PD-L1 immunoscore (in tumour cells and TILs) showed significant association with high histologic grade (p < 0.001), absence of lymphovascular invasion (p = 0.007), absence of HR expression (p = 0.001), and high levels of TILs (p < 0.001).The association between PD-L1 protein expression and variable clinicopathological factors is shown in Table p = 0.068), though it was not statistically significant. In correlation analyses, the TIL level, each PD-L1 immunoscore for tumour cells and TILs, the total PD-L1 immunoscore in tumour cells and TILs, and mRNA expression levels of PD-L1 showed a significant positive association with one another (Table p = 0.001) and there was no significant association between mRNA levels of PD-L1 and other clinicopathological factors .Twenty-three cases (59.0%) and the remaining 16 cases (41.0%), were categorized as having low and high expression levels of PD-L1 mRNA respectively based on the mean fold difference value. In the t-test, the fold difference value of the PD-L1 mRNA showed difference between the low and high total immunoscore group . During the follow-up period, there were five cases of local recurrence (2.99%) and 14 cases of distant metastasis (8.38%). Five patients died because of the disease (3.0%).23. The expression of PD-L1 was not significant for the prognosis of total HER2-positive breast cancers and HR+/HER2+ breast cancers  is a ligand of PD-1. PD-1 is a transmembrane receptor and an important negative immune controller, regulating the activation, exhaustion, and tolerance of T cells and the resolution of inflammation. The PD-1 pathway is activated upon engagement of its ligands, PD-L1 or PD-L2, and can modulate T cell activity in several ways. The interaction of PD-1 with its ligand induces epigenetic modification in T cells, which alters transcription factor expression and reduces the phosphorylation of T cell receptor (TCR) signalling molecules. Additionally, the PD-1 signal stimulates the expression of proteins that decrease T cell proliferation and cytokine production, thus decreasing T cell survival and killing capacity4. The adaptive immune resistance mechanism of PD-L1 upregulation is an adaptation to an endogenous tumour-specific immune response. The tumour cells use the normal physiology that occurs during pathogen infection to protect the host from immune-mediated damage. Simply, PD-L1 is induced in tumour cells in response to inflammatory signals such as interferons (IFNs). In a previous study of HER2-positive breast cancers, expression of Myxovirus resistance A (MxA), an excellent marker of IFNs activity, in tumour cells was significantly associated with high TIL levels19. Furthermore, the current study found that TIL level, PD-L1 immunoscore in tumour cells and in TILs, and the total PD-L1 immunoscore in tumour cells and TILs, significantly correlated with each other. Innate immune resistance demonstrates that PD-L1 is driven by constitutive oncogenic signalling pathways in the tumour cells. Constitutive anaplastic lymphoma kinase (ALK) signalling has been reported to induce PD-L1 expression25. Phosphatase and tensin homolog (PTEN) loss or downregulation, which reflects the phosphatidulinositol-4,5-biphosphate 3-kinase/protein kinase B (PI3K/Akt) pathway, was related to PD-L1 expression in TNBCs and glioblastoma27. The current study showed that 53 cases (31.7%) involved type III tumours (positive for PD-L1 with low TIL levels) suggesting tumour intrinsic induction. Tokunaga et al.28 demonstrated that Akt activation was significantly associated with HER2 overexpression in breast cancers. However, the intrinsic induction mechanism of PD-L1 in HER2-positive breast cancer should be determined.Tumour cells use two mechanism for expressing PD-L1: adaptive immune resistance (tumour extrinsic mechanism) and innate immune resistance (tumour intrinsic mechanism)35. This study showed that 81 patients (48.5%) were positive for PD-L1 in tumour cells, whereas 51 cases (30.5%) showed high levels of PD-L1 expression in TILs. Overall, 89 patients  were associated with poor disease-free survival, and type III tumours had intermediate disease-free survival rates in HR−/HER2+ breast cancers, although it was not statistically significant. Better disease-free survival of type III tumours than type II tumours in HR−/HER2+ breast cancers suggests probable positive prognostic role of PD-L1 expression regardless of the status of the tumour microenvironment. Further in vitro studies are required to clarify the prognostic value of PD-L1 in cancer. Type II tumours can be described as tumours with immunological ignorance21. For such tumours, adoptive T cell therapy with the delivery of costimulatory signals, the blocking of inhibitory signals and vaccination could be considered. Adoptive T cell therapy is a form of immunotherapy whereby ex vivo isolation and expansion of antigen-specific T cells is performed and they are transferred back into patients that already showed clinical efficacy in recurrent viral infections37. Furthermore, Bald et al. showed that reversion of immune ignorance with peritumoural injections of the immunostimulant, polyinosinic:polycytidylic acid (poly:IC), induced upregulation of PD-L1 expression in a mouse model of human immune cell-poor melanoma. Additionally, they demonstrated the synergistic effect of anti-PD-1 therapy with immunostimulants38. In the present study, type III tumours showed intermediate outcomes in disease-free survival compared with the remaining tumour types in patients with HR−/HER2+ breast cancers. As mentioned above, intrinsic tumour expression of PD-L1 is associated with constitutive oncogenic signalling. However, these results suggest that blocking anti-PD-1/PD-L1 is unlikely to induce T cell response to the tumour because the tumour microenvironment does not have enough TILs to elicit effective anti-tumour immunity. An approach similar to that used for type II tumours involving the recruitment of TILs into the tumour microenvironment can also be used for this group of patients.Results regarding the prognostic significance of PD-L1 in breast cancers are conflicting. Several reports suggest a good prognostic role of PD-L1 in breast cancer40. Moreover, Lee et al. recently demonstrated that HLA expression was negatively associated with estrogen receptor expression and positively correlated with high TILs level and IFN-associated molecules in breast cancer41. However, the biologic determinants of PD-L1 expression in tumour cells as a positive prognostic factor in HR negative tumours, but not in HR positive tumours, should be further explored.IFNs, inducers of PD-L1, stimulate the expression of human leukocyte antigen (HLA)Studies with large cohorts with long term follow up periods are required to classify all consecutive breast cancers, as each type should have a different treatment strategy. Furthermore, functional studies are required to understand the intrinsic tumour expression of PD-L1 in breast cancer. The importance of defining the molecular pathways that regulate the expression of immune checkpoints cannot be overemphasized, as immune checkpoint intervention will likely lead to clinical benefits.In conclusion, TIL levels and PD-L1 protein expression in tumour cells and in TILs are significantly correlated with one another. High expression of PD-L1 mRNA was significantly associated with high TIL levels. Protein expression of PD-L1 in tumour cells was significantly associated with a better disease-free survival rate in HR−/HER2+ breast cancers.A total of 167 patients with HER2-positive breast cancer were included in our study. Patients were recruited following surgery for primary breast cancer between 2011 and 2013 at Pusan National University Hospital. All included patients were pre-operatively chemo- and radiotherapy naïve. Also, all of them received adjuvant treatment. All patients were treated with 4 cycles of anthracycline and cyclophosphamide (AC) and 1 year of trastuzumab as a standard treatment. Paclitaxel or docetaxel was added between 4 cycles of AC and 1 year of trastuzumab based on the risk stratification encompassing the size of the tumour, lymph node metastasis, Ki-67 proliferation index, histologic grade, age, family history of breast cancer, and general condition. Among them, 14 patients discontinued trastuzumab during treatment, because 4 patients died, 11 patients had recurrence or metastasis and 3 developed cardiac toxicity. Formalin-fixed, paraffin-embedded tissue specimens were available for all patients. Clinicopathological information was collected by reviewing each patient’s electronic medical records and pathology reports. Among these, 39 fresh tissue samples were provided by the Pusan National University Hospital, a member of the National Biobank of Korea, supported by the Ministry of Health, Welfare, and Family Affairs. All samples from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols.20. TILs level was evaluated by visual estimation. In brief, TILs level was calculated as the percentage of stroma within the invasive area covered by mononuclear cells over total intratumoural stromal area. A full evaluation of average TILs in the tumour was performed without focusing solely on hotspots. The TILs level was evaluated as a percentage in 10% increments. If the TIL level was less than 10%, a 1% or 5% criteria was used18. The ducts affected by DCIS that had TLSs surrounding themselves of the total ducts affected by DCIS were assessed, and the amount of TLSs as a percentage of the total circumference of the invasive front was evaluated.Histological evaluation of full face haematoxylin and eosin (H&E) slides was performed. Various histopathological factors, including histological grade, nuclear grade, necrosis, lymphovascular invasion, lymph node metastasis, TIL level, TLSs around the DCIS and invasive component were evaluated. Histological and nuclear grades were determined by the modified Bloom-Richardson classification. TILs level was assessed based on the recommendation by the International TILs Working GroupFor statistical analysis, patients were subdivided into three categories  or two categories (≤60% and >60%) according to the TIL level as appropriate. TLSs level was divided into two subgroups (low and high) based on the mean value.All available H&E stained slides were reviewed to define a representative tumour area. A tissue arraying instrument was used to array each sample with a tissue core of 2 mm in diameter. To reduce tissue loss and the effects of tumour heterogeneity, all samples were arrayed in duplicate.in situ hybridization (SISH) for HER2 was performed in cases of equivocal immunostaining with a score of 2. An automated immunohistochemical staining device  was used for immunohistochemical analysis. The EZ prep was used for deparaffinization with an auto-staining device on 3 µm paraffin sections. For antigen retrieval, cell conditioner 1  or cell conditioner 2  was used, and optimally formulated antibodies targeting ER , PR , and HER-2/neu  were used. An anti-PD-L1 antibody  was applied to the tissue microarray sections. A HER2/CEP17 chromosome dual-probe  was used for SISH.At the time of diagnosis, immunohistochemical staining of the oestrogen receptor (ER), progesterone receptor (PR), and HER2 was performed, and silver 42. Tumours were classified into two subtypes according to HR (ER and PR) expression: HR+/HER2+ and HR−/HER2+.ER and PR positivity was defined as at least 1% of tumour nuclei showing positive staining. Tumours that were positive for ER or PR were classified as the HR-positive group. HER2 positivity was defined as an immunostaining score of 3, or by gene amplification identified by SISH. SISH was performed in cases of equivocal results, such as in the event of an immunohistochemical staining score of 2. The HER2 testing was interpreted according to American Society of Clinical Oncology/Cllege of American Pathologists Guideline 2007, which was used at the time of diagnosis43. TILs expression levels were divided into two categories (low and high) based on the mean value of the PD-L1 immunoscore of the TILs. Additionally, tumours were classified into two categories (low and high) based on the mean value of the total PD-L1 immunoscore of tumour cells and TILs. For survival analysis, tumours were categorized into four groups according to the PD-L1 expression of the tumour cells and the TILs level, which is similar to the criteria of Teng’s classification, which are as follows: type I, positive for PD-L1 and high TIL level; type II, negative for PD-L1 and low TIL level; type III, positive for PD-L1 and low TIL level and type IV, negative for PD-L1 and high TIL level23.PD-L1 expression was evaluated separately for tumour cells and TILs. Membranous stains were assessed for PD-L1 positivity, using human placental tissue as a positive control. The intensity and the percentage of membranous positivity were considered collectively . After blocking non-specific binding sites, the membrane was immunoblotted with the anti-PD-L1 antibody , followed by incubation with the secondary antibody conjugate. The bands were detected using an LAS-3000 Imaging System .® universal PCR Master mix  and TaqMan® Gene Expression Assays . An ABI 7500 Real-Time PCR System  was used for qRT-PCR. Each assay was performed in duplicate. The comparative CT method (ΔΔCT method) was used to determine the relative expression levels of PD-L1 mRNA. Relative expression levels of PD-L1 mRNA were normalized to those of beta-actin, and fresh human placental tissue was used for calibration. Expression levels of PD-L1 mRNA were subdivided into two categories (low and high) according the mean fold difference value.Total mRNA was isolated from 39 cases of HER2 positive breast cancers provided by the National Biobank of Korea, using an RNeasy mini kit  according to the manufacturer’s instructions. Extracted mRNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kits  and amplified using TaqManp-value of less than 0.10 in the univariate analyses. The likelyhood ratio test was done to ensure the proportional hazard assumptions in the Cox regression model. Differences with p-values of less than 0.05 were considered statistically significant.All statistical analyses were carried out using SPSS statistical software program . The t-test, Chi-square test, Fisher’s exact test, linear-by-linear association tests, Spearman rank correlation test, Kaplan-Meier survival analyses, log-rank test and Cox proportional hazards regression model were used as appropriate. For multivariate analysis, a forward stepwise selection method was used with covariates showing Exemption from informed consent after de-identification of information was approved by the Institutional Review Board of Pusan National University Hospital.Supplementary Information


Distributed measurement of humidity is a sought-after capability for various fields of application, especially in the civil engineering and structural health monitoring sectors. This article presents a method for distributed humidity sensing along polymethyl methacrylate (PMMA) polymer optical fibers (POFs) by analyzing wavelength-dependent Rayleigh backscattering and attenuation characteristics at 500 nm and 650 nm wavelengths. Spatially resolved humidity sensing is obtained from backscatter traces of a dual-wavelength optical time domain reflectometer (OTDR). Backscatter dependence, attenuation dependence as well as the fiber length change are characterized as functions of relative humidity. Cross-sensitivity effects are discussed and quantified. The evaluation of the humidity-dependent backscatter effects at the two wavelength measurements allows for distributed and unambiguous measurement of relative humidity. The technique can be readily employed with low-cost standard polymer optical fibers and commercial OTDR devices.  The measurement of humidity or moisture is an important task in various fields of applications, for example in industrial process control, agriculture, domestic applications, air condition control, or the civil engineering and structural health monitoring sectors. Especially for the monitoring of extended structures and water ingress detection, a distributed  measurement is important. This cannot be adequately achieved with conventional humidity point sensors. Common principles for the measurement of humidity are summarized in ,2. ApartThe approach presented in this article has the advantage of true distributed  humidity measurement using intrinsic scattering and absorption effects in polymer optical fibers (POFs). Distributed measurement is achieved by analyzing backscattering and attenuation parameters along the fiber. We previously proposed distributed relative humidity (RH) measurement in standard poly(methyl methacrylate) (PMMA) POFs at 650 nm wavelength ,19: backIn this article, we present the detailed results of Rayleigh backscattered power dependencies and transmission dependencies on RH changes at 500 nm and 650 nm wavelengths. The combination of these two effects and their differing characteristics at different wavelength measurements  are analyzed for unambiguous identification and localization of RH changes and to distinguish other backscatter-dependent effects.υ-OTDR by Luciol Instruments) with a sampling resolution of 2.6 cm and a spatial resolution of about 10 cm. The pulse optical wavelength of the OTDR can be switched between 500 nm and 650 nm, the favorable optical transmission windows for PMMA POF. The photon counting technique provides very high sensitivity and spatial resolution [All measurements have been conducted with standard step-index PMMA POFs (Mitsubishi’s Eska GK-40) with a 980 µm diameter PMMA core, a 10 µm thin fluorinated polymer cladding and a numerical aperture of NA = 0.5. The OTDR technique provides spatially resolved backscatter measurement analogous to the radar technique: short optical pulses are sent into the fiber and backscattered power is sampled as a function of time or distance. The OTDR measurements have been performed with a photon counting OTDR (solution . Howeverl ≈ 21–37 m and l ≈ 58–69 m). The POF sections in the water basins have a saturated water content and act as a reference during the RH cycles presented in Our aim was to investigate all backscatter effects at different optical wavelengths (in this case at 500 nm and 650 nm) and analyze their differing humidity-dependent characteristics for distributed RH sensing. Since the observed response times of the humidity-dependent backscatter changes are relatively slow, the integration time for each backscatter measurement has been chosen to be 1 h. OTDR traces at both wavelengths have been recorded alternatingly at constant temperature and varying RH settings. All OTDR measurements have been conducted with the setup depicted in T = 30 °C.PMMA can take up to about 2% water by mass. Investigations on different PMMA materials show that the rate of water sorption increases with increasing temperature but the equilibrium concentration of water does not depend on the temperature . We alsoIn this section, the measured RH-dependent effects of spectral transmission change, transmission change at the OTDR wavelengths, backscattered power change and detected fiber length change are presented. Each effect is briefly discussed at the end of the respective sub-section. A more general discussion of the POF RH sensor characteristics and the impact of cross-sensitivities is given in fl = 40.5 m long POF. Three transmission measurements have been conducted during saturated RH values of 30%, 60% and 90%. In order to verify the measured OTDR transmission dependence and identify favorable wavelength measurements for future sensor designs, spectral transmission changes have been conducted. υOH and 4υOH + δOH [a/b,OTDRP at the two wavelengths are calculated from the measured transmission spectra (counts) of RH,a(λ)P at RH a relative to RH,b(λ)P at RH b and the actual OTDR source spectra OTDR(λ)P at 500 nm and 650 nm (Pnm500(λ) and Pnm650(λ), respectively) as generalized in this equation:The RH-dependent transmission change results in PMMA POF show increasing transmission loss with increasing RH for wavelengths exceeding about 513 nm with distinct absorption peaks at 612 nm and 665 nm. The cause for the transmission changes are impacts of the OH vibrational absorptions, presumably 5OH + δOH ,31, and P, calculated from measured transmission and emission spectra, are summarized in The transmission changes ΔAs expected from l ≈ 21–37 m and l ≈ 58–69 m and are used as a reference power level for relative backscatter change evaluation.Distributed backscatter change and transmission change is obtained from relative backscattered power evaluation of OTDR measurements. l = 35 m and l = 65 m). Only the backscattered power change can be observed at l = 37–58 m and l > 69 m. Two effects are evident at 650 nm: increased backscattered power level and superimposed transmission loss related to increased water content in the fiber at higher RH settings. As visible in the spectral transmission results in y-axis for direct comparison with the spectral transmission results in All following calculations of backscatter change and transmission change are conducted by comparing OTDR traces relative to a reference OTDR trace at a certain RH setting. The averaged backscattered power level of a fiber section subjected to varying relative humidity is compared to the averaged power level of a saturated fiber section in the water basin. The calculation of backscatter changes at 650 nm has additionally been adjusted by proportionally subtracting the measured optical loss changes over the compared fiber sections. It has to be noted that all relative change values are defined as change of backscattered power as it is detected by the OTDR. That means that transmission change over a given fiber length constitutes optical power loss that is accumulated by passing the fiber section twice: as forward-propagating pulse and as backscattered power. Transmission measurement readings at the end of the fiber would show half of that value for changes over the same fiber length. This definition corresponds to twice the “transmitted intensity” changes and “scatter intensity” changes presented in ,19. FiguThe results show reproducible and reversible characteristics. As indicated in The same backscatter and transmission change evaluation as above has been conducted during gradual RH change cycles between 30%and 90% while maintaining constant RH for 1 day at the reversal values. The resulting backscatter change and transmission change characteristics are shown for slopes of 2 days (a) and 4 days (b) at 500 nm in The results show good reproducibility and reversibility and agree well in magnitude with the results obtained during step-wise humidity changes between 30% and 90% RH in The apparent “hysteresis” is due to the response time of the sensor and is less distinct for lower RH gradients during the 4-day slope. Saturated water content in the fiber shows reproducible results without hysteresis, as presented in −9 cm2s−1 and 9 × 10−9 cm2s−1 for sorption and desorption [Significant response time differences between sorption and desorption are not visible in sorption , respectsorption  reportedsorption . It is asorption . We assume that the Rayleigh backscattered power dependence may be partly caused by the reduction of refractive index differences at the interfaces between the polymer network and the microvoids as well as within the polymer structure itself due to water uptake and the filling of gaseous voids by water molecules. This may result in a reduced inhomogeneity of the refractive index along the fiber and, as a consequence, decreased scattering. It can also be speculated that the humidity-induced swelling of the polymer causes a mechanically forced closing of the microvoids and micro-crazes which in turn leads to an increase of material homogeneity and, therefore, reduced scattering. The exact reasons, however, are unknown. The response time is further discussed in l, normalized to the fiber length that is exposed to RH changes, is obtained from the interpolated shift of the rising slope of the fiber end reflection peak . The measured length changes during step-wise RH changes (a) and gradual changes (b) are shown in The absorption of water into the fiber has also an effect on the optical runtime of the light. Lower RH values result in decreased runtime, meaning a reduced measured fiber length. This is visible in the shift of the fiber end reflection peak see a. The le−6 (%RH)−1) more significant than the opposite impact of the RH-dependent refractive index change of about Δn ≈ −11 × 10−6 (%RH)−1 [n/ΔRH = f(T) and a change of sign (from positive to negative) at temperatures exceeding about 60 °C [n/ΔRH for PMMA may also exhibit a significant temperature dependence.The measurements indicate reduced “fiber length” at lower RH values and a response time comparable to that of the backscattered power change and transmission change effects in  (%RH)−1 ,35. Measut 60 °C . This inn/ΔRH dependence of PMMA, the temperature-dependent refractive index change of PMMA is negative with about −137 × 10−6 K−1 at 20.1 °C [−6 K−1 [−6 K−1 to 92.8 × 10−6 K−1 at 50 °C [−6 K−1 [If the measured fiber length change is intended to be used for humidity measurement, temperature variations of the sensor also have to be considered: besides the unknown Δ [−6 K−1  and alsoat 50 °C . We dete [−6 K−1 . DependiThe aim of this article is to identify the suitability of PMMA POF for distributed humidity measurement in general, and at different wavelengths in particular. The results are very promising. The resolution of the RH changes depends on the signal-to-noise ratio of the OTDR trace, i.e., the fiber distance, the OTDR integration time and the length of the evaluated fiber section. For a saturated condition, a resolution in the order of 1–2% RH could be achieved. The following cross-sensitivities and limitations have to be considered in an application scenario. temperature. We already investigated this effect for PMMA POF [A relevant cross-sensitivity is the fiber’s backscatter dependence on PMMA POF ,38 and lPMMA POF ,24. Tempstrained fiber sections cause increased backscatter levels [It has to be noted that also r levels ,25. Therresponse time of the sensor is slower than that of most other humidity sensors. The field of application is thus limited to slowly varying RH changes or applications where the immediate response to humidity change is not crucial. However, the response time can be improved to a few hours or less by using commercially available standard PMMA fibers with smaller core diameters, for example 500 µm or 250 µm diameters. Response times below one hour may be possible with a 250 µm PMMA fiber. For comparison: a response time of 30 min has been reported for a FBG in a 200 µm PMMA fiber [The MA fiber . FurtherMA fiber  and evenMA fiber . Also, fMA fiber .referencemeasurement or relative to a reference fiber section with a known RH or water content. We used water-saturated fiber sections as a reference. If such a reference condition cannot be provided, only relative changes along the sensor fiber can be measured. This could be viable for applications where water ingress or moisture change has to be detected in an otherwise homogeneous structure. For direct RH measurement, however, one or more reference sections are necessary. Such a reference backscatter power level may also be provided by inscribing constant scattering damage structures into the POF core using focused femtosecond laser pulses [The RH measurement is based on the evaluation of backscattered power changes relative to a r pulses . In thisThe advantage of the approach presented in this article is the distributed measurement capability over an extended sensor length of up to 200 m. The technique has the potential for humidity or moisture measurement not only in gases but also in solid state materials and structures. Due to the distributed measurement along a flexible sensor fiber, continuous and spatially resolved humidity measurement can be conducted. Also humidity changes of fiber sections shorter than the spatial resolution could be detected since power changes are accumulated on the backscatter trace . The combined advantages of distributed and gapless measurement is beneficial in various fields of application.Application examples can be found in the structural health monitoring sector for humidity/moisture monitoring in building foundations, the measurement of moisture content in concrete and screed or moisture ingress detection in sealed structures such as bridges. Other applications that can benefit from distributed RH measurement are seepage line monitoring in dykes and dams or the detection of leaks in landfills, industrial facilities and reservoirs. In most of these applications, the humidity ingress mechanisms are either slow or the required response time to take action is not crucial. Moisture penetration, for example in buildings and foundations, is a very slow process with critical consequences on the long-term structural integrity or public health, for instance due to mold formation. The limited response time is not an issue for these applications. As mentioned above, decreasing the response time by using smaller diameter fibers would further extend the range of applications.The distributed sensing capability is essential if local water ingress or leakage in extended structures is to be detected. These applications can often not be sufficiently serviced with a point sensor approach which may require the installation of hundreds of point sensors and the associated wiring effort. Distributed sensing, on the other hand, allows for precise localization of humidity changes as well as the spatial spreading of the humidity influx over time along a single fiber. Another application example is the distributed measurement of concrete curing and setting processes inside large concrete infrastructures. The information of the spatial humidity distribution inside the structure can be used to optimize the moisture content during curing as well as the construction progress.The increased cost of an OTDR unit can be economical for applications where water ingress or leakage has to be monitored over extended distances and a great number of point sensors can be substituted. OTDR-based sensing could also be cost-efficient if the unit does not have to be installed permanently, for example during a temporary curing monitoring application, or when regular service interval measurements are sufficient. The fiber sensor itself is low-cost and can be installed in any orientation (linear or meandering) to monitor an area, volume or along extended structures. Sensor fibers can also be installed without the initial intent of monitoring the structure at close to zero cost, providing the potential for later activation for distributed humidity measurement. A reduction in cost can be further achieved in large-scale applications since multiple sensors can be multiplexed in a sensor network and silica fibers could be used as lead fibers to extend the attenuation-limited measurement distance of a POF RH sensor network.