Growth mechanism for synthesizing PVD based Graphene using Amorphous Carbon, catalyzed by Copper is investigated in this work. Different experiments with respect to Amorphous Carbon film thickness, annealing time and temperature are performed for the investigation. Copper film stress and its effect on hydrogen diffusion through the film grain boundaries are found to be the key factors for the growth mechanism, and supported by our Finite Element Modeling. Low temperature growth of Graphene is achieved and the proposed growth mechanism is found to remain valid at low temperatures. Currently, the Graphene synthesis method used is Chemical vapor deposition (CVD) technique8910et al.1113Growth of Graphene on Copper using CVD has been demonstrated by Li On the other hand, a simpler and low cost technique to obtain non-transferable Graphene using amorphous carbon as solid source and Nickel or Cobalt thin film as catalyst annealed at elevated temperatures has been reported1516181916As the applications of Copper material is vast and important, such as printed circuit boards, transmission wires and cables, its application in ULSI interconnects, etc, the growth of Graphene on Copper (Cu) can overcome some limitations of Cu materials such as its ease of corrosion, ease of oxidation, limited Electromigration performance as interconnect, and push the electrical and thermal conductivity limits of Cu to higher values.et al.et al.In view of the limitation of the reported methods, the use of amorphous carbon as solid source for Graphene synthesis on Cu was re-explored by Ji Recently, we successfully demonstrated the feasibility of crystallization of amorphous Carbon under sputtered Copper thin film which acts as a catalyst in order to obtain Graphene at the top surface of Cu experimentallyIn this work, various experiments are designed and performed in order to explore the growth mechanism of our Physical Vapor Deposition (PVD) Graphene growth method using amorphous carbon as the carbon-source. The proposed growth mechanism can also explain the claim of inability of Graphene growth on Copper thin film using this method as reported by others162 (300 nm) substrates using Physical Vapor Deposition (PVD) method. The samples are labelled as S1, S1.1 and S1.2 respectively as shown in The experiments in this work involve deposition of amorphous Carbon (a-C) thin film of different thicknesses followed by Copper (Cu) (99.99%) film of 800 nm thickness on Si/SiO2 environment with a flow rate of 50 sccm at a low pressure of 1 torr and different annealing temperatures and annealing times as shown in the annealing process schematic in After samples S1, S1.1 and S1.2 are produced, they are annealed in an Het al.The graphene synthesis method used has been described in detail by Narula Raman scattering spectra are collected using PTT RAMaker Micro Raman/PL/TR-PL Spectrometer with laser excitation wavelength of 473 nm and laser spot-size of about 0.5 μm.In order to understand the underlying mechanism of this graphene growth, experiments and characterization are performed to examine the effect of thickness of a-C, annealing time and annealing temperature for Graphene crystallization on Cu. In order to avoid any changes or modifications in the properties of synthesized Graphene, the experimental analysis is performed completely on the growth substrate. Therefore, all the reported data have been obtained with graphene still being on the Copper thin film.G peak is associated with the doubly degenerate phonon mode at the Brillouin zone center which comes from a normal first order Raman scattering process in graphene and originates from the interplanar sp2-bonded C-C stretching vibrations2D and ID peaks originate from a second-order process, involving two iTO phonons near the K point for the 2D band and one iTO phonon and one defect in the case of the D band which can be often used to evaluate the number of graphene layers and the grain size of graphene respectivelyIt is well known that the I2D/IG peak ratio of sample S1.1 is much higher (close to 1) than that of sample S1  than sample S1, and its ID/IG peak ratio is much less as compared to S1. I2D/IG peak ratio equal or greater than 1 indicates the presence of few layers (i.e. layer number around 3–9) of Graphene while the peak ratio less than 1 indicates multi-layer Graphene (i.e. layer number more than 10)2D/IG peak ratio less than 1, their graphene have multi-layer, with sample S1 having the largest number of layer, and sample S1.1 having the least number of layer, despite that the a-C thickness of S1.1 is not the least. This will be explained later from our proposed growth mechanism.From these information, we can see that in ample S1 , and itsfor S1.2 , its I2D2D/IG and ID/IG vs annealing time. Samples S1.1 and S1.2 are subjected to annealing with different annealing times, and Raman signatures with higher 2D peak are indeed observed. 2D/IG ratio greater than 1 represents the presence of few layers of graphene, a sharp increase in the ratio is expected from zero (where no graphene is present) to a value much greater than 1, followed by a decrease in the ratio towards less than 1 as the number of graphene layer is increasing. Likewise, when the graphene layer is being etched away, a sharp change in the ratio from some values less than 1 to a value much greater than 1 and then down to zero should be expected. However, due to the discrete annealing time used in our experiments, such sharp change in the ratio is difficult to observe, and only dotted lines are shown in As ID/IG ratio. Graphene formation can be found with 20 minutes of annealing time while the ID/IG ratio drops to a lower value. The largest I2D/IG ratio is observed with 30 minutes of annealing time which is a signature of few layers Graphene (since I2D/IG ratio is higher than 1) and an increase in ID/IG ratio can also be observed. This is followed by a drop in I2D/IG and ID/IG ratio with 40 minutes of annealing time and then a small rise in the I2D/IG for 50 minutes of annealing time while the ID/IG ratio also increases at this point.For sample S1.1, no Graphene is present for 10 minutes of annealing time and its surface layer is highly defective as indicated by its I2D/IG ratio during 20 to 30 minutes of annealing time. Beyond 30 minutes of annealing time, its I2D/IG and ID/IG ratios decrease continuously, in contrast to sample S1.1, and approaches zero for 60 minute of annealing time. The defects in sample S1.2 is much less than that in sample S1.1 for annealing time more than 40 minutes.On the other hand, the thin a-C layer in sample S1.2 undergoes early crystallization within 5 mins of annealing to produce few layers of Graphene which continues to form multi layered Graphene with a small increase in IIn the light of the above findings, we can understand the reason of the conclusion made by the reported works on the infeasibility of crystallization using Cu as catalyst because they were either having annealing time less than 10 minutes and/or the thickness of a-C film is too thick where the formation of graphene formation will begin only at longer annealing timeSamples S1.1 and S1.2 are annealed comparatively at lower temperature of 920 °C and 820 °C for 50 mins. The distinct difference in the behavior of the graphene formation with annealing time and temperature for S1.1 and S1.2 is interesting since the difference in the two samples is only the thickness of the a-C layer. This kind of distinct behavior is believed to be due to the thermo-mechanical stress driven crystallization of a-C into Graphene, and we would like to verify it using finite element analysis.Finite Element Analysis (FEA) is performed using ANSYS Workbench R16.2 platform. As thermo-mechanical stressEf and vf are the Young’s modulus and Poisson’s ratio respectively, α is the coefficient of thermal expansion, and ΔT is the range of temperature excursion; the subscripts ‘s’ and ‘f’ refer to substrate and film, respectively, we can see that the stress is independent of the thickness of the a-C layer, and thus the thermo-mechanical stresses in each sample are the same. On the other hand, the thermo-mechanical stress causes the sample to warp, and the resultant Maximum Principal Stress is given by the Stoney’s equation26where (1/R) = (1/R−1/R0), and R0 and R are the radius of curvature of the samples before and after the thermo-mechanical stresses are developed respectively. ‘t’ is for thickness of the layer in the sample with subscripts ‘s’ and ‘f’ refer to substrate and film, respectively. Here Maximum Principal Stress is considered in the Stoney’s equation for a-C as a-C is known to be a brittle materialwhere ΔAs Δ(1/R) is due to the warpage of the sample caused by thermo-mechanical stress which is independent of layer thickness in the sample, the value of Δ(1/R) can be assumed to be a constant for the three samples.According to 2 diffusion in Cu film through its grain boundaries3132Furthermore, thermal strain analysis has also been performed on synthesized Graphene on Cu using ANSYS and is depicted in Based on our FEA simulation results, we revisit and explain our experimental results in the following discussion.2D/IG and ID/IG ratio,  has been confirmed by Ji et al.Sample S1 has the most number of Graphene layers as observed and shown in 2D/IG peak and very high ID/IG in For annealing time below 10 minutes, the Graphene obtained for sample S1.1 is totally defective as indicated by the nearly zero I2 gas has a dual role of carbon activator and Graphene etchant, etching of graphene by the H2 gas occur concurrently, and the number of Graphene layers is reduced. With the availability of the Hydrogen radicals, the exothermic etching reaction (Reaction (3)2D/IG ratio beginning from the 20 minutes of annealing. Due to discretization of annealing times used in our experiment, the exact extent of crystallization and etching of Graphene during 10 to 20 minutes of annealing times are unknown. For 20 minutes of annealing, the quality of Graphene film is improved as compared to that with 10 minutes of annealing and is less defective as reflected by a decreased value of ID/IG value. The production of carbon radicals is also accompanied by production of hydrogen radicals as per the Reaction (1)As annealing time increases to 20 minutes, the crystallization rate increases with time. The increase in crystallization rate is according to Reaction (2)2, primarily at defective sites because these sites facilitates preferential etching of Graphene2D/IG and ID/IG ratios up to 30 minute of annealing time in As the annealing time increases to 30 minutes, the amount of hydrogen radicals increases to sufficient quantity to trigger Reaction (3). Also the heat of absorption due to Reactions (1) and (2) reduces the temperature of the Cu film surface. As a result, the crystallization rate is dominated by the etching rate of Graphene, and thus Graphene is continuously etched by H2D/IG ratio, by the end of 40 minutes of annealing in D/IG is observed because the newly formed layers fill the defective sites created due to etching in the earlier annealing process.When the annealing time goes beyond 30 minutes, the remaining graphene layer is so thin and the heat generated from Reaction (3) plus the increasing amount of carbon radicals as a by-product of Reaction (3) enhance the crystallization Reaction (2) in the forward direction. This facilitates further crystallization, and more number of Graphene layers is formed as indicated by a decrease in I2D/IG and ID/IG ratios in For annealing time beyond 40 minutes, the scenario is similar to what happened during 20 to 30 minutes of annealing time where the hydrogen radicals are also produced with carbon radicals. This increased number of hydrogen radicals and reduction of the Cu film surface temperature because of heat of absorption due to Reactions (1) and (2) triggers Reaction (3) and shifts it in the forward direction, resulting in reduction in the number of Graphene layers. Thus only few layers of Graphene are formed by the end of 50 minutes of annealing. Also the defects increase due to loss of carbon atoms from the Graphene crystal forming defective sites. This is indicated by an increase in ID/IG ratio as can be observed even for annealing time as low as 5 minutes in 2D/IG ratio in D/IG ratio also decreases because the newly formed layers fill up the defective sites, thus reducing the effective defects in the Graphene crystal so formed. The high rate of Reaction (1) produces more H*, and the heat of absorption due to Reactions (1) and (2) reduces the temperature of the Cu film surface. These two factors enhance Reaction (3) in its forward direction and reduces the layer of graphene synthesized when the annealing time is prolonged to 30 minutes. Thus, the number of graphene layer is no longer increasing continuously with the annealing time, and a slow reduction in the number of layer takes place as Reaction (3) starts dominating slowly, and this is indicated by a small increase in I2D/IG while the ID/IG ratio also increases due to etching of graphene as explained before. Thereafter, the growth Reaction (2) becomes active swiftly for the annealing time beyond 30 minutes, and I2D/IG and ID/IG ratio keep on decreasing. This is because the Reaction (3) in the previous step produces carbon radicals as a by-product and Graphene growth Reaction (2) will be shifted to its forward reaction, giving hydrogen radicals lesser chance to etch away the graphene. When the annealing time is long enough (40 minutes and beyond), unlike the situation in sample S1.1, a different mechanism kick in as can be seen from the different rate of change of the I2D/IG ratio as seen in Similar behavior depicted in 2 substrate and on a-C film with SiO2 substrate. Our finite element calculations using One mechanism is stress induced migration38Another mechanism is the sharp increase in the a-C film compressive stress as computed using finite element as shown in 2 gas. The points at which the growth of Graphene layers stops and etching reaction takes over is unknown due to discretization of annealing times in our experiments.As a result of the above two mechanisms, the rate of crystallization of Graphene is much faster than the rate of etching, and the number of layer of the synthesized Graphene increases at a higher rate in S1.2, resulting in multi-layered graphene formation. With such a higher rate of diffusion of carbon radicals and formation of graphene, and the thin layer of a-C in S1.2 at the start, when annealing time go up to 60 minutes and beyond, the remaining a-C is quickly depleting. As a result, the Reaction (2) shifts in the backward direction and Graphene etching reaction becomes dominant and hence all the grown Graphene is etched away by HOn the other hand, the above-mentioned two mechanisms are unlikely to happen in S1.1. First of all, its a-C thickness is 36 nm to start with, and after 40 minutes of annealing, its thickness is still well above 4 nm, and thus the increase in the compressive stress of a-C is slow as seen in 2 and a-C. Consequently, carbon radicals’ supply is reduced but Graphene etching by H2 gas still persists. Thus, only formation of nearly single layer or very few layers Graphene are possible.When the annealing temperature is lowered, the value of the von-Mises stress in Cu film and the area of maximum stress are reduced. 2 through the Cu film will be so slow that Reaction (1)2 gas on top . Thus no formation of graphene layers is observed below a certain temperature, where we term it as threshold temperature. This threshold temperature is lower for S1.2 since it has thinner a-C film and thus the stress in Cu film is higher than S1.1 for a given temperature as shown earlier, and this is consistent with our experimental results. ANSYS simulation for total deformation as shown in When the temperature is further lowered, the diffusion of HMoreover, the growth mechanism discussed for annealing temperature value of 1020 °C stands true for lower temperature regimes as well because the trends in stress variation are similar to the high temperature regimes.  vs 2D (ω2D) modes. Dashed line in purple color with a slope (Δω2D/ΔωG) of 2.2 represents Unstrained Graphene40The thermal strain results presented in D peak intensity, which is also connected with defects, is on a higher side but these defects are advantageous with respect to contact resistance on metals42A worth-noting point observed in our samples is that the IFrom the above investigations, we can see that graphene synthesis on Cu is having a different mechanism as compared to that in the conventional CVD and gaseous carbon-source based graphene synthesis. Based on the results obtained in this work, we propose the plausible mechanism for the Graphene synthesis using amorphous carbon as the solid carbon source in presence of copper catalyst as depicted in Step I- The process begins with native copper oxide reduction by H2 gas at high temperatures ranging from 820–1020 °C, releasing water molecules in the gas phase as by-product and stress induced grain growth44Step II- The high annealing temperature causes thermo-mechanical stress to develop in the films of the sample, and its subsequent warping, rendering high tensile stress in the Cu film.Step III- The high tensile stress due to thermo-mechanical stress in Cu film then enhances hydrogen gas diffusion through the Cu film and reach out to the underlying a-C layer for graphene crystallization.Step IV- H2 gas in the meanwhile reacts with the a-C to form carbon radicals in the presence of Cu catalyst and moves out through the grain boundaries and crystallizes into Graphene. The Graphene formation and etching are simultaneous in the presence of the H2 gas based on discussed reactions. For a given annealing time and annealing temperature, the supply of carbon radical depends on the a-C layer thickness and different stress levels, and hence different quality of Graphene is obtained for different samples.2 gas through copper grain boundaries to interact with a-C underneath and thermo-mechanical stress in copper and a-C films are proposed to be the mechanisms for the Graphene synthesis. Therefore, even though samples S1.1 and S1.2 are similar in configuration, their dependence of the graphene growth rate on the annealing time and temperature are different due to their difference in thickness of a-C, which result in different film stresses in the two samples. The proposed growth mechanisms are found to valid for in the temperature range of 1020 to 820 °C. With this mechanism, the infeasibility of Graphene formation on copper film as reported by previous works can also be explained.Various experiments are successfully conducted in this work in order to explore the growth mechanisms of Graphene synthesis using a-C as the carbon source and Cu as the catalyst. Diffusion of HHow to cite this article: Narula, U. et al. Growth Mechanism for Low Temperature PVD Graphene Synthesis on Copper Using Amorphous Carbon. Sci. Rep.7, 44112; doi: 10.1038/srep44112 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The candidate was derived from the evolutionary-based algorithm and molecular simulations was then profoundly analysed using first-principles density functional theory from the structural, mechanical, phonon, and electronic properties point of view. The proposed polymorph of graphene (rP16-P1m1) is mechanically, dynamically, and thermally stable and can achieve semiconducting with a direct band gap of 0.829 eV.A potentially new, single-atom thick semiconducting 2D- Carbon is probably the most versatile element of the periodic table due to the possibility of creating (C−C) bonds with different atom hybridization sp1, sp, and sp3graphene”, has attracted tremendous interest in this polymorph of carbon [Graphene is a zero-gap semiconductor with linear energy band dispersion close to the Fermi level. The absence of a band gap in the electronic spectrum of graphene can prohibit the direct implementation of this material in electronics and that is why scientists and engineers have started to look for ways to improve this. It was found that mechanical deformations of graphene can give rise to a band gap of up to 1 eV and narrow graphene ribbons may possess the properties of semiconductors, where their band gaps are narrowed as the ribbons become wider [Graphene armchair nanotubes are metallic, whereas zigzag and chiral nanotubes can be also semiconducting with a band gap depending inversely on its diameter [graphene so as to widen the band gap up to 1.2 eV [The Nobel Prize for Physics in 2010, “for groundbreaking experiments regarding the two- dimensional material f carbon  due to if carbon ,5. Graphme wider . Graphendiameter . Patterno 1.2 eV .graphene has accelerated interest in exploring graphene-like materials, see [graphene, graphyne and its family were proposed and analysed theoretically in [Graphyne can be seen as a 6-fold symmetry lattice of benzene rings connected by acetylene (−C≡C−) linkages. The structures proposed therein are, according to linear combination of atomic orbitals (LCAO) calculations, semiconductors with moderate band gaps. More than 30 years later, some of these structures, called graphdiyne and Ene-yne, were synthesized in [Coro-graphene, consisting of 4, 6 and 10 rings, with a direct band gap ∼0.63 eV was found in [Graphenylene, consisting of 4, 6 and 12 rings, with a direct and narrow band gap (0.025 eV) was found in [T-Graphene, 2D carbon allotrope with pza-Undoubtedly the blossoming of studies on als, see ,10,11,12ype, see ,6,13,14.cally in ,16. Grapsized in ,18. Semiposed in  and simifound in . Graphenfound in . T-Graphrings in  is metalrings in  as a plarings in  the samed gap 0.0 eV was ftwin graphene  eV with generalized gradient (Green’s function) approximations was proposed in [In addition to the single-atom thick 2D structures mentioned above, multi-atom structures were also proposed. The tions in . Its banlated in . Even a posed in ,29 but qposed in  because Anisotropic-cyclicgraphene and is organised as outlined below: Anisotropic-cyclicgraphene, The present paper is focused on an in-depth analysis of new, one-atom thick 2D carbon semiconducting material, called graphene-like carbon structures with predefined mechanical properties was shown in [rP8-P1, wherein the designations mean: 2D Pearson symbol, 2D space group, have served here as input for more accurate and reliable first-principles calculations. Consequently, the first that structure with dimensions 6.42 × 6 Å from first-principles calculations found to be metallic, so quite common 2D carbon material, but the second with dimensions 3.922 × 8.472 Å found to be semiconducting, so quite rare and interesting one and for this reason, it was decided to analyse only this one of them.A description and application of the memetic algorithm ,32 and mshown in . The resFirst-principles calculations with the use of density functional theory (DFT) ,39 withiNVT  ensemble; see [The thermal stability of 2D structures is typically examined by performing ab initio molecular dynamics (AIMD) or classical molecular dynamics (MD) with the use of interatomic potentials. Due to the significant calculation cost of AIMD, simulations are limited to only 200 atoms and a few ps  in ble; see ,28. Howerequired , which irequired .All the molecular simulations in this work have been performed by using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS)  and the Anisotropic-cyclicgraphene conventional supercells in the plane of the model, and non-periodic and shrink-wrapped in the normal direction to the model [graphyne.In all computations, periodic boundary conditions were applied to a 2D sheet, consisting of 10 × 10 he model , thus atwas used ,34. Therwas used  to examigraphene.Applying the methodology outlined in Secsion rP8-P1, see oP8-P1m1, see rP16-P1m1 conventional unit cell, see Whereas, the initial unit cell was rectangular, primitive with 8 atoms in unit cell and P1 plane group symmetry: Anisotropic-cyclicgraphene-rP16-P1m1 (C3 + C17 carbon rings), where the basic cell is portrayed in cyclicgraphene, was studied by classical molecular dynamics simulations in [For tions in .s character the bond has, the more tightly held the bond will be and carbon bonds in representative hydrocarbons, sp − sp ≈ 1.37 Å, sp2 − sp2sp ≡ sp ≈ 1.20 Å, see [sp bonds in long carbon chain.Unlike other single-atom thick 2D carbon semiconductors, our structure contains only such odd type of rings. Due to very low symmetry of the proposed structure the bond lengths vary, in the three-membered ring: 1.39–1.41 Å and in the long carbon chain: 1.24–1.39 Å. A general principle from organic chemistry says, that the more 0 Å, see . SimilarAnisotropic-cyclicgrapheneAnisotropic-cyclicgraphene with respect to pristine graphene is graphynes [If we look at the calculated cohesive energy in raphynes ,53 and araphynes , i.e., EAnisotropic-cyclicgraphene than for pristine graphene, which has graphynes it is known that an increase in the number of acetylenic linkages corresponds to degradation of the stiffness [The in-plane elastic constants .9 (N/m) . ElongatC66 > 0)  and is itiffness .Anisotropic-cyclicgraphene is not only mechanically but also dynamically stable.Phonon dispersion curves for investigated structure, plotted along the high symmetry k-points, k-points, s-states minimally contribute to the total DOS and it is clearly evident that the p-states are primarily responsible for the formation of VB and CB near to the Fermi level. The calculated Fermi energy for Anisotropic-cyclicgraphene system equals −4.243 eV with the PBEsol GGA functional and −3.106 eV with the HSE06 hybrid functional.The electronic band structure (EBS) and density of states (DOS), total and partial, for the analysed structure are plotted along the high symmetry Anisotropic-cyclicgraphene does not change and atoms vibrate around equilibrium positions. At T = 900 K topological changes occur and many smaller rings appear. We therefore conclude that the polymorph is thermally stable up to a temperature of 800 K.We see in graphene consisting of C3+C17 carbon anisotropic rings within the framework of DFT from point of view of the structural, mechanical, phonon, and electronic properties was carried out in the paper. All above calculations have been completed using ultra-fine quality settings, the modified PBEsol GGA for solids exchange-correlation functional and additionally for electronic band structure computations the hybrid exchange-correlation functional HSE06. In addition the thermal stability of the proposed Anisotropic-cyclicgraphene was examined by performing a series of classical molecular dynamics simulations.The extensive analysis of potentially new polymorph of graphene (rP16-P1m1) is mechanically and dynamically stable contrary to other C3-cyclicgraphenes.The proposed polymorph of The proposed structure is thermally stable up to a temperature of at least 800 K.Anisotropic-cyclicgraphene with respect to pristine graphene is similar to other graphynes and cyclicgraphenes.The relative energy of Anisotropic-cyclicgraphene can be semiconducting, with a direct band gap with a value of 0.829 eV.The semi-empirical potential AIREBO seems to be surprisingly suitable for carbon structures.The following conclusions can be stated:Some results in this paper are unique and we trust that they will be verified by other works. Naturally, the synthesis of the proposed structure is a separate task and goes beyond the area of this work. The effect of carbon rings anisotropy and chain lengths on the properties of potentially new 2D structures will be the subject of further research.


Nonsmall cell lung cancer accounts for approximately 80% of all lung cancers, and approximately 75% of cases are diagnosed in the middle and late stages of disease. Unfortunately, limited treatment does not improve the prognosis of advanced disease. Monoclonal antibodies targeting programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) represent a new treatment paradigm for nonsmall cell lung cancer. Nevertheless, the immune-related adverse events (irAEs) associated with PD-1 and PD-L1 inhibitors are unique, and early recognition and treatment of these events are essential.A systematic literature search will be performed using the EMBASE, MEDLINE, and Cochrane databases to identify relevant articles published in any language. Randomized clinical trials, case series, and case reports of PD-1 and PD-L1 inhibitors in the treatment of nonsmall cell lung cancer will be included. All meta-analyses will be performed using RevMan software. The quality of the studies will be evaluated using the guidelines listed in the Cochrane Handbook. If the necessary data are available, then subgroup analyses will be performed for high-, median-, and low-dose cohorts. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses statements will be followed until the findings of the systematic review and meta-analysis are reported.This will be the first systematic review and meta-analysis to describe previously reported irAEs related to PD-1 and PD-L1 inhibitors in the treatment of nonsmall cell lung cancer. Based on its pathology, NSCLC can be further subdivided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Compared with small cell lung cancer, the growth of NSCLC cells is slower, and the diffusion occurs relatively late. If diagnosed early, the disease generally has a good prognosis, with surgical resection providing a clinical cure. However, NSCLC is diagnosed as locally advanced or metastatic disease in 70% of patients, leading to an extremely poor 5-year survival rate of 1% to 5% among patients with stage IV NSCLC, a figure that has not changed over the past 25 years.Lung cancer remains a leading cause of cancer-related mortality, Currently, it is well established that tumors suppress antitumor immune responses via numerous mechanisms including the production of inhibitory cytokines, recruitment of immunosuppressive immune cells, and upregulation of coinhibitory receptors known as immune checkpoints. In normal physiological environments, the immune checkpoints contribute to inhibiting immune responses against self-antigens to prevent excessive autoimmunity. However, these immune checkpoints are upregulated in many cancers, resulting in cancer-associated immune suppression and immune evasion. PD-1 binds to PD-L1 and decreases effector cytokine production and cytolytic activity, preventing the elimination of cancer. This discovery prompted the development of different monoclonal antibodies targeting either PD-1 or PD-L1 as new treatments for advanced NSCLC.Although first-line systemic treatment for NSCLC consists of surgery followed by cytotoxic chemotherapy and/or radiation therapy, increasing recognition of the critical roles of the immune system in the development and progression of cancer has spurred the development of numerous targeted therapies with significant potential to improve overall survival (OS) in specific NSCLC populations.,9 PD-L1 and PD-L2 both act on PD-1.,11 However, the main function of T-cell regulation within the tumor microenvironment depends on PD-L1, which is expressed on the same cells that express PD-1. In addition, some cancer cells also express PD-L1, and approximately 60% of NSCLCs are PD-L1–positive. Blockade of the PD-1 pathway is believed to increase the anti-tumor immune function of T-cells, enhance natural killer cell activity, and promote antibody production in PD-1–positive B cells.,9PD-1 is a type I transmembrane protein receptor expressed on the surface of numerous immune cells such as macrophages, dendritic cells, T-cells, B-cells, and natural killer cells.,14,15 Other PD-1/PD-L1 inhibitors are in various stages of clinical development. The induction of a tolerance break against the tumor via PD-1/PD-L1 blockade can lead to immune dysregulation, namely immune-related adverse events (irAEs), which clinically manifest as symptoms including dermatologic, gastrointestinal, hepatic, endocrine, and pulmonary events. In addition to these unique AEs, treatment-related adverse events (trAEs) include fatigue, anorexia, nausea, and diarrhoea.,14,17–21Currently, pembrolizumab and nivolumab, which target PD-1 receptors, have been approved for the first- and second-line treatment of NSCLC, respectively.,23 Our study will assess the incidence and nature of irAEs in the oncologic treatment of NSCLC using available anti-PD-1 or anti-PD-L1 antibodies through a systematic review and meta-analysis of the qualifying literature.As these treatment-related symptoms affect patients’ quality of life, sufficient knowledge, and appropriate management of these events are important.2A systematic review and meta-analysis will be performed to clarify the profile of irAEs associated with PD-1 and PD-L1 inhibitors in the treatment of NSCLCs in a systematized manner and summarize the differences in the adverse effects associated with different inhibitors compared with conventional chemotherapy and radiotherapy, including whether these events are dose-dependent.2.1To describe the profile of irAEs associated with PD-1 and PD-L1 inhibitors in the treatment of NSCLCs in a systematized manner.2.2To compare the irAEs associated with PD-1 and PD-L1 inhibitors with those associated with conventional chemotherapy and radiotherapy.To compare the trAEs associated with PD-1 and PD-L1 inhibitors with those associated with conventional chemotherapy and radiotherapy.To determine whether the severity and frequency of irAEs associated with PD-1 and PD-L1 inhibitors are dose-dependent.To assess whether the profile of irAEs differs among different PD-1 and PD-L1 inhibitors.33.13.1.1The included participants will be adults who were diagnosed with NSCLC and treated with anti-PD-1 or anti-PD-L1 antibodies. Previous oncologic therapy will not preclude study inclusion, but anti-PD-1 or anti-PD-L1 antibodies must be administered separately without combination therapy. Eligible comparison treatments will be chemotherapy, radiation therapy, targeted therapy, immunity therapy, and multiple combination therapies. There will be no restrictions regarding sex, race/ethnicity, education, and economic status.3.1.2This study will include randomized controlled trials (RCTs), case series, and case reports published in peer-reviewed journals without language or time restrictions. Cohort studies, case-control studies, cross-sectional studies, postmarketing vaccine surveillance studies, and other nonrandomized studies will be excluded.3.1.3Review articles, non-RCTs, or RCTs combining anti-PD-1 antibodies or anti-PD-L1 antibodies with other treatment modalities will be excluded.3.2The incidence evaluation will be based on the number of irAEs and their grades . Grades ≥ 3 will be considered high-grade.3.2.1The incidences of irAEs and trAEs associated with PD-1 and PD-L1 inhibitors in the treatment of NSCLC.The incidences of irAEs and trAEs associated with conventional chemotherapy and radiotherapy in the treatment of NSCLC.The incidences of organ-specific adverse events (AEs), especially immune-related pneumonia, and deaths related to AEs associated with PD-1 and PD-L1 inhibitors in the treatment of NSCLCs.The incidences of organ-specific AEs, especially immune-related pneumonia, and deaths related to AEs associated with conventional chemotherapy and radiotherapy in the treatment of NSCLCs.3.2.2Comparison of the incidence of irAEs and trAEs between PD-1 and PD-L1 inhibitors and conventional chemotherapy/radiotherapy.Comparison of the incidence of organ-specific AEs, especially immune-related pneumonia, and deaths related to AEs between PD-1 and PD-L1 inhibitors conventional chemotherapy/radiotherapy.3.3A systematic literature search through December 2016 will be performed using EMBASE, MEDLINE via PubMed, and the Cochrane Central Register of Controlled Trials for relevant articles published in any language.3.4The relevant searching terms will match Medical Subject Heading terms, and the searches will be repeated immediately before the final analyses to identify additional studies for inclusion. An example of the PubMed search strategy is shown in Table 3.53.5.1An electronic parent folder will be created for this study. The folder will contain several subfolders to incorporate all records retrieved using the following criteria: database of retrieval, the full text of the included studies, the results of risk of bias assessments and subgroup analyses, and full systematic review manuscript drafts. The aforementioned task will be the responsibility of the first author (XYS) in consultation with another author (SYC). The parent folder will be backed up on 2 author's mobile hard drives.3.5.2Two authors (XYS and SYC) will screen the search outputs using titles and abstracts to assess whether they meet the inclusion criteria as defined by the protocol and then read the full text of all potentially eligible studies independently for further discrimination of eligible studies. The full text will be obtained through databases or by contacting the corresponding author of the article that will be in the charge of another 2 authors (BF and RR). Discrepancies regarding study inclusion between the 2 authors will be resolved via consensus with the assistance of the senior author.3.5.31.Study characteristics: author, clinical trial information, design, and enrolment size.2.Characteristics of monoclonal antibodies: type and dose.3.Diagnostic methods: Version 2, 3 or 4 of the Common Terminology Criteria for Adverse Events of the National Cancer Institute4.IrAEs/trAEs: type, frequency of any grade, frequency of severe grade, and the median time to onset.5.Frequency of organ-specific AEs, especially immune-related pneumonia, and the incidence of death related to irAEs.Data will be extracted from the included studies by 2 authors (XYS and SYC) independently and recorded on a predesigned extraction list. The following data will be captured in the systematic review and meta-analysis:3.6 and to address selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases. Two authors will independently conduct this quality assessment, with agreement reached via consensus.The Cochrane Collaboration's ‘risk of bias’ tool will be used to assess the risk of bias and evaluate the quality of articles included in the systematic review and meta-analysis,3.7χ2 test and I-squared statistic. Subgroup analyses will be performed to explore potential causes of heterogeneity. The relative risk will be calculated for dichotomous data with 95% confidence intervals for all analyses.All meta-analyses will be performed using RevMan 5.2 software. Statistical heterogeneity between the selected studies will be assessed using the 3.7.1If sufficient data can be obtained, then subgroup analyses will be conducted for high-, median-, and low-dose cohorts. Other variables that will be considered for subgroup analysis include the type (anti-PD-1 vs anti-PD-L1) and brand of antibody drugs.3.7.2Sensitivity analysis will be performed to confirm whether the results are robust and credible by excluding highly biased studies.3.8The preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement will be followed until the findings of the systematic review and meta-analysis are reported. The identification and selection of studies for inclusion will be summarized using flow diagrams. Characteristics of the studies and the incidence of global adverse events associated with anti-PD-1 and anti-PD-L1 therapies will be presented in tables. The quality assessment and summary of other adverse events will be described in the text.4To our knowledge, this will be the first systematic review and meta-analysis reporting all published irAEs related to PD-1 and PD-L1 inhibitors in the treatment of NSCLC. Currently, the study of anti-PD-1 and anti-PD-L1 transitions from basic research to clinical research; however, safety concerns represent the primary limitation of the clinical application stage. Using all RCTs, case series, and case reports included in the pooled analysis, we will determine the incidences of all-grade and high-grade irAEs, which may highlight the high risk of irAEs associated with anti-PD-1 and anti-PD-L1 drugs in the treatment of NSCLC.; thus, security details need to be observed and monitored in the clinical practice. This systematic review will be conducted to clarify essential details associated with anti-PD-1 and anti-PD-L1 drugs in the treatment of NSCLC and the findings of the review may facilitate early prediction, comprehensive observation, and prompt management of irAEs in addition to better patient compliance.PD-1 and PD-L1 inhibitors are demanded because of the poor prognosis of NSCLC despite their potentially high risk of irAEs. Several antibodies have been approved for the second-line and higher treatment of NSCLC, and they are associated with significant improvements in progression-free survival and OS versus conventional chemotherapy We will report the review results according to PRISMA guidelines and search, screen, assess, and extract valuable data from several databases comprehensively and meticulously as previously mentioned. As trAEs may be reported instead of irAEs in studies of anti-PD-1 and anti-PD-L1 drugs in patients with NSCLC, it may be impossible to conduct the review as described in this article. This consideration will be addressed in the discussion of published studies, and the study results will be disseminated in peer-reviewed journals.


We theoretically investigate light absorption by a graphene monolayer that is coated on the outside of dielectric-metal core-shell resonators (DMCSRs). We demonstrate that light absorption of graphene can be greatly enhanced in such multi-layered core-shell architectures as a result of the excitation of the hybridized bonding plasmon resonance supported by the DMCSRs. We also demonstrate that the absorption enhancement in graphene can be easily tuned over a wide range from the visible to the near-infrared, and particularly the enhancement factor can be optimally maximized at any selective wavelength, by simultaneously varying the dielectric core size and the metal shell thickness. Our results suggest that the graphene-wrapped DMCSRs with strong and highly wavelength-tunable absorption enhancement in graphene could be attractive candidates for applications in graphene-based photodetectors and image sensors. Graphene is an excellent light-to-current converter with internal quantum efficiency close to 100%6, however, the relatively low absolute value of light absorption (~2.3%) for one atomic layer graphene7 could severely limit its applications in many optoelectronic devices such as photodetectors8 and tunable modulators9. It has been demonstrated that graphene with appropriate doping could support either propagating12 or localized surface plasmon resonances16, and associated with the excitation of these graphene-plasmon resonances light absorption in graphene could be greatly enhanced. For example, Abajo et al. demonstrated that 100% light absorption could take place in a single patterned sheet of doped graphene13. However, this enhancement mechanism only works in the infrared region and is not applicable for the absorption enhancement in graphene in the visible and near-infrared (vis-NIR) spectral range, because of the absence of graphene-plasmon in the vis-NIR range17. In order to improve the interaction between graphene and vis-NIR light, many approaches have been employed. For example, the integration of graphene with an optical waveguide has been shown to allow the increase of the interaction length through coupling between the evanescent waves and graphene, and thus resulting in greatly enhanced graphene absorption18. Similarly, by placing graphene monolayer in optical micro-cavities such as resonant Fabry-Perot20 and photonic crystal cavities22, which enables light to pass through the graphene film multiple times, absorption enhancement in graphene has been demonstrated as a result of the increased effective interaction length. Recently, based on the ability of plasmonic nanostructures to concentrate light into subwavelength volumes and produce a dramatic enhancement of the local electric field23, the combination of graphene with plasmonic nanostructures has also been theoretically predicted and experimentally demonstrated as an efficient way to enhance the vis-NIR light absorption in graphene33. Although most of these metal-graphene hybrid nanostructures exhibit large absorption enhancement in graphene, the implementation of these nanostructures typically relies on high-cost and time-consuming top-down processes such as electron-beam lithography or focused-ion beam milling32. Therefore, simple metal-graphene hybrid nanostructures that can greatly enhance graphene absorption and be readily prepared either chemically or physically are highly desirable.Graphene, a two-dimensional honeycomb lattice of sp34. Because of the finite thickness of the shell layer, these two primitive plasmons can interact with each other and result in hybridized bonding and antibonding modes35. The resonance wavelengths of these two hybridized plasmon modes can be broadly tuned by varying the relative dimensions of the core and shell35. Over the past few years, the DMCSRs have been routinely synthesized by using physical deposition process37 or wet-chemistry approach38 where the metal shell can be as thin as ~2 nm39, and also have found widespread applications such as thermal-therapy40, low-threshold plasmonic lasing41, fluorescence emission shaping42, and refractive index sensor43. Motivated by these investigations, in this work, we propose the use of DMCSRs to enhance light absorption in graphene monolayer that is wrapped on the outside of the DMCSRs. We demonstrate that excitation of the hybridized bonding plasmon resonances supported by the DMCSRs can lead to a dramatic enhancement of the light absorption in the graphene layer, which can be easily tuned over a wide range from the visible to the near-infrared by varying either the dielectric core size or the thickness of the metal shell layer. Furthermore, we also demonstrate that there exists a particular combination of the core size and metal shell thickness to make the graphene absorption reach the maximum value at any selective wavelength.Dielectric-metal core-shell resonators (DMCSRs), composed of a spherical dielectric core and a thin metal shell layer, are simple nanostructures that support both sphere and cavity plasmons44. In the calculations, the monolayer graphene is assumed to be 0.335-nm-thick17, and its permittivity within the vis-NIR region is described by a Drude-Lorentz model,m = 3, ε∞ = 1.964, Δεj = , ħωgra = 6.02 eV, ħγgra = 4.52 eV, ħΩj =  eV, and ħΓj =  eV45. The metal is assumed to be silver, and its dielectric constant is described by a Drude model,ωAg = 1.38 × 1016 rad/s is the plasma frequency of bulk silver and γAg is the frequency of electron collisions. The efficiencies of absorption in graphene (Qgra) and silver (QAg) components are given by Qgra = Cgra/σgeom and QAg = CAg/σgeom, respectively, where Cgra and CAg are absorption cross section for the graphene and silver, respectively, and σgeom = πr2 is the geometrical cross-section with r being the outer radius of the core-shell nanoparticle. In order to reveal the essence of the underlying physics, we will begin our discussions by analyzing the ideal scenario where silver is assumed to be lossless (γAg = 0 rad/s). In this case, only the graphene layer will absorb light (QAg = 0). Later in this work, we will discuss the effects of metal loss on the graphene absorption.Throughout this paper, the scattering (absorption) of electromagnetic-plane waves from core-shell nanoparticles is analyzed using Mie theorya) of the silver nanosphere. Therefore, the achievable maximum graphene absorption efficiency at any given wavelength and the corresponding optimized core radius can be found by calculating the dependency of the absorption efficiency at this selective wavelength on the radius of the silver core. Figure Qgra) as a function of silver core radius calculated at four different vis-NIR wavelengths of λ = 455 nm, 610 nm, 875 nm, and 1195 nm. As indicated by dashed lines in Fig. a = 54 nm, 81 nm, 120 nm, and 167 nm, the graphene absorption efficiency can achieve the maximum value at the given wavelengths of λ = 455 nm, 610 nm, 875 nm and 1195 nm, respectively, which reveal that the spectral position of the maximum graphene absorption can be tuned by varying the radius of the silver nanosphere. However, it should be noted that the achievable maximum absorption efficiency is very weak in the vis-NIR region and decrease with red-shifting the spectral position of graphene absorption by increasing the core radius  to Qgra ≈ 8.5 [red solid circles], while the silver absorption efficiency correspondingly increases from QAg = 0 [blue dashed line] to QAg ≈ 2.3 [blue solid triangles]. In addition, Fig. n = 1.59 and parameters of , which are optimized for the wavelength of λ = 1195 nm. Compared with the results for the graphene-wrapped DMCSR with a lossless silver shell [dashed lines in Fig. QAg ≈ 4.1 and the achievable maximum graphene absorption efficiency in the presence of metal loss decreases from Qgra ≈ 14.1 to Qgra ≈ 6.8. Although the finite dissipation could lead to the reduction in the achievable maximum graphene absorption efficiency, the graphene-wrapped DMCSR with a lossy silver shell could still be optimized to achieve a relatively large graphene absorption efficiency, for example, Qgra ≈ 6.8 at the wavelength of λ = 1195 nm, which is about 1360 times larger than the achievable maximum graphene absorption efficiency at λ = 1195 nm in a graphene-wrapped solid silver nanosphere [Qgra ≈ 0.005, Fig. 38, which could lead to the increased electric field intensity within the silver shell. Therefore, at the longer wavelength the silver absorption efficiency becomes larger. Consequently, the electric fields distributed outside the silver shell are less enhanced, resulting in a relatively small enhancement of the light absorption in the graphene layer at the longer wavelength. For this reason, the absorption efficiency ratio of the graphene to the silver is found to be relatively large (Qgra/QAg ≈ 3.7) at the short wavelength of λ = 610 nm [Fig. Qgra/QAg ≈ 1.7) at the long wavelength of λ = 1195 nm [Fig. In the practice the metal loss has to be taken into account. Figure  nm Fig.  and rela nm Fig. .Figure 739, followed by electrostatic wrapping of graphene sheets on the surfaces of the as-prepared DMCSRs49. The strong optical absorption in graphene and excellent tunability make the proposed graphene-wrapped DMCSRs attractive candidates for optoelectronic applications. We also hope our strategy could be straightforwardly applied to enhance absorption in other quasi-2D materials.In summary, we demonstrate that the graphene-wrapped dielectric-metal core-shell resonators exhibit large graphene absorption enhancement arising from the excitation of hybridized bonding plasmon resonance supported by the DMCSRs. We show that the core-shell aspect ratio dependence of the hybridized plasmon resonance allows the spectral position of the graphene absorption enhancement to be easily tuned over a wide range from the visible to the near-infrared by varying the relative dimensions of the dielectric core and silver shell. We also demonstrate that the graphene absorption enhancement can be maximized at any given vis-NIR wavelength by simultaneously varying the dielectric core size and the silver shell thickness. Although the graphene absorption can reach the maximum at a given vis-NIR wavelength only for the graphene-wrapped DMCSRs with a particular set of optimized parameters, a flexible choice of the parameters of the DMCSRs is allowed for the achievement of relatively large graphene absorption. We suggest that the proposed graphene-wrapped DMCSRs can be prepared by using physical deposition process or wet-chemistry method to coat dielectric spheres with a metal shell layer44, with the software Matlab. Since the considered spherical nanoparticles are much smaller than the wavelength of the interest, only the dipolar effects in Mie theory is taken into account. Initially, the silver is assumed to be perfect metal without loss. In this case, we can either integrate the dissipation power over the volume of the graphene shell to directly obtain the graphene absorption efficiency (Qgra), or calculate the graphene absorption efficiency based on the energy conservation: Qgra = Qabs = Qext – Qsca (because only the graphene can absorb light). In the final part of the paper, we considered the effect of realistic metal loss on the graphene absorption. Since both graphene and silver can absorb light, the only way to calculate the losses caused by the silver and the graphene shells is to integrate the dissipation power over the volume of the silver shell and the graphene shell, respectively.The plane wave scattering (absorption) by a spherical concentric core-shell nanoparticle is solved analytically using full-wave Mie theory


A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43G298S ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases.TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43 TDP-43 is associated with most cases of FTLD and ALS. Point mutations and elevated TDP-43 levels cause FTLD-TDP coupled with neuronal death. Affected neurons have nuclear depletion and cytoplasmic accumulation of TDP-43. Interestingly, accumulation of TDP-43 in neurons is characteristic of many neurodegenerative diseases in addition to FTLD-TDP and ALS, including Alzheimer’s disease. TDP-43 seems to play roles in the onset and progression of neurodegeneration but mechanistically how it triggers and contributes to disease and cell death is not known.The nucleotide binding protein TDP-43 regulates a multitude of cellular processes including gene transcription, RNA splicing, mRNA stability, localization and translation, and microRNA biogenesis2. It belongs to the family of heterogeneous nuclear ribonucleoproteins (hnRNP) and is ubiquitously expressed. Deletion of the Tardbp (the gene encoding TDP-43) in mice leads to early embryonic lethality between E3.5 and E6.54 indicating an important role during early development. TDP-43 contains two RNA-recognition motifs (RRMs), RRM-1 and RRM-2, and a glycine-rich region at its C-terminus5. The RRM-1 is necessary and sufficient for nucleic acid binding to single stranded RNA at GU-rich sequences6. The C-terminus of TDP-43 is necessary for the formation of hnRNP-rich complexes7 and contains most of the TDP-43 point mutations identified in FTLD and ALS patients. TDP-43 is localized predominantly nuclear in cells and has both a nuclear localization sequence (NLS) and a predicted nuclear export sequence (NES) and seems to be continuously shuttled between the two cellular compartments8.TDP-43 was originally identified as a factor binding to the TAR DNA of human immunodeficiency virus where it is implicated in transcriptional regulation9, which was suggested to drive, at least in part, disease pathogenesis. However, the cause and function of TDP-43 aggregates remains to be shown unequivocally. In mice, robust cytoplasmic TDP-43 aggregation is associated with dramatic neuron loss and features of human pathology, which can be reversed by increased clearance of TDP-4310. Interestingly, mislocalization of TDP-43 to the cytoplasm of mouse neurons is sufficient to induce apoptosis even in the absence of aggregation, suggesting that cytoplasmic TDP-43 aggregates may not be necessary to induce cell death and early mortality in mice13.TDP-43 is one of the main components of cytoplasmic inclusions, which are a characteristic feature of many neurodegenerative disorders. Apoptotic neurons that display cytoplasmic inclusions show a partial loss of TDP-43 in the nucleus14. Cross-linked immunoprecipitation and RNA sequencing (CLIP-Seq) revealed that TDP-43 can bind thousands of RNAs via a UG-rich consensus sequence in the 3′ untranslated regions of target RNAs17. Whereas the RNAs bound by TDP43 in the mouse brain are relatively consistent in the different analyses, TDP-43 targets vary considerably between cell types17.Aberrant TDP-43 causes pleiotropic effects in cells and results in extensive changes in splicing and RNA metabolism18, and other factors of the apoptosis pathway including Bim, Bax and Bcl may be involved in TDP-43-induced cell death19. Components of the proapoptotic pathway are downstream targets of p53 and elevated p53 levels have been detected in affected neurons of ALS patients21. However, the absence of p53 in a transgenic mouse model for ALS (hSOD1G93A) did not rescue apoptosis, suggesting that cell death in these animals occurred in a p53-independent manner23. Although aberrant TDP-43 expression is associated with stress responses24, a causal link between p53 and TDP-43 induced cell death has not been reported. TDP-43 is expressed in the developing and adult brain, therefore, we addressed the role of TDP-43 during development of the telencephalon by gain- and loss-of-function experiments. We thereby hoped to gain insights into TDP-43 functions in the formation and maintenance of the nervous system.Aggregates in diseased neurons contain hyper-phosphorylated and fragmented TDP-43 protein. Interestingly, TDP-43 can be cleaved by caspasesA315T results in p53-mediated apoptosis in neural stem/progenitor cells and immature neurons of the developing mouse telencephalon. In addition, we observed cell death of cortical neurons derived from human iPS cells following TDP-43 expression and found that this neuronal death could also be rescued by p53-inhibition. Expression of the proapoptotic BH3-only genes Bbc3 and Bax was increased in mice and human neural cells as a result of aberrant TDP-43 expression, supporting a role for p53 in the TDP-43 induced cell death. Furthermore, we show that TDP-43 is associated with the mRNA of Cdkn1a and increases Cdkn1a levels, likely explaining the altered neural stem/progenitor cell cycle regulation following TDP-43 and TDP-43A315T expression.Here we show that expression of TDP-43 and its mutant form TDP-43Tardbp is expressed by neural progenitors in the developing central nervous system   Fig. . TDP-43 H3) Fig. .Figure 1in vivo targeting the ventricular zone stem and progenitor cells by in utero electroporation (IUE) with hairpin RNAs targeting its mRNA (shTDP-43). TDP-43 mRNA and protein levels were reduced in neural progenitors by up to ~70% 48 hours after transfection . The percentage of transfected cells that expressed detectable levels of TDP-43 in vivo reduced from 93.9 +/− 4.2% in controls to 3.7 +/− 2.5% in shTDP-43 transfected embryos   TDP-43 ion Fig. . The per07) Fig. . After 4in vivo  was reduced and generation of newborn neurons (Tbr1+) in the cortical plate was blocked by TDP-43 overexpression indicating cell death and impaired neuronal differentiation  increase in wild type TDP-43 expression induced by IUE Fig.  resultedivo Fig. . TDP-43 lon Fig. . The fewlon Fig. . Further25. The mechanisms of mutant TDP-43 induced toxicity remain unclear but are associated with an increase in TDP-43 protein levels26. We examined the effects of mutant TDP-43 expression in neural progenitors in vivo. Mutant human TDP-43A315T   Fig. . These d27. Given that TDP-43 induces cell death, the link between TDP-43 cleavage and caspases, and demonstration of elevated p53 in neurons of ALS patients21, we investigated whether TDP-43 induced apoptosis of neural progenitors cells depends on p53 activity. In contrast to expression in control (p53wt) embryos, IUE-mediated TDP-43 overexpression in p53-deficient embryos (p53−/−) did not disrupt neural progenitors in the ventricular zone or affect their morphology  alleles, to lineage trace the Cre-expressing neural stem/progenitor cells and their progeny, which were either wild type or homozygous for floxed Trp53 alleles. We induced deletion of Trp53 from ventricular zone progenitors in embryos at e11 by Tamoxifen induction28 and expressed TDP-43 by IUE in these embryos at e13.5  underwent apoptosis (caspase-3)  were viable and showed a normal morphology  induced by TDP-43 overexpression  Fig. . Conversogy Fig. . By thision Fig. . This su30, into p53wt mothers rescued TDP-43 overexpressing cells and blocked caspase-3 activation  and basal progenitors (Tbr2+) were comparable to controls  were decreased upon TDP-43 expression, but this was not rescued by inhibition of p53  or TDP-43 overexpressing embryos (18.1 +/− 2.8% versus 23.4 +/− 3.0) ion Fig. . We analp53 Fig. . Further31. TDP-43 binds thousands of RNAs, including its own, regulating many aspects of RNA biogenesis, stability, splicing and transport32. The RNA recognition motif 1 (RRM1) of TDP-43 is necessary and sufficient for RNA binding33. We addressed whether RNA binding is required for TDP-43 induced toxicity in neural progenitors by expressing a mutant form where the RRM1 had been deleted (TDP-43∆RRM1)  and basal progenitors (Tbr2+) were comparable to controls following TDP-43∆RRM1 expression  and Bax as well as cell cycle regulators including Cdkn1a. Consistent with an activation of p53, TDP-43 and TDP-43A315T induced Bbc3 and Bax expression in neural progenitors but not the antiapoptotic gene Bcl2 , we examined the cell cycle defects in more detail. Therefore, we assessed a potential TDP-43 regulation of Trp53 and Cdkn1a by CLIP (cross-linked RNA immunoprecipitation). In accordance with previous findings35, TDP-43 bound and decreased endogenous Tardbp mRNA levels  die in utero  after 48 hours and examined gene expression by qRT-PCR. In agreement with our findings in the mouse telencephalon, overexpression of TDP-43 in human cells significantly increased the expression of TRP53, CDKN1A, BBC3, and BAX but also BCL2 , which enhances expression of ATF3 and CHOP (CCAAT/enhancer-binding protein homologous protein). CHOP inhibits Bcl2 functions at the protein level and induces Bim to enhance cell death through activation of Bax. In agreement, we found that neither TDP-43 nor TDP-43A315T induce Bcl2 expression in mouse cells, whereas Bax is increased. However, it is conceivable that Eif2α inhibition of antiapoptotic BH3-only proteins and the p53-induced proapoptotic gene (Bbc3 and Bax) expression synergize to promote cell death in TDP-43 mutant cells. Hence, TDP-43 seems to activate a bifurcated pathway to control BH3-only protein-mediated apoptosis.Our findings identify activation of proapototic gene expression as a mechanism through which TDP-43 accumulation and mutations can induce cell death in murine neural progenitors A315T in cortical neurons derived from human iPS cells also resulted in an apoptosis phenotype. In concordance with the overexpression in mouse cells, we observed a strong increase in TRP53, CDKN1A, BBC3 and BAX in the human cortical neural cells in vitro. However, BCL2 was also increased in the human cortical neurons in contrast to mouse neural progenitor cells. This opposing result might stem from differences in pathway regulation between species. However, inhibition of p53 by PFT-α rescued the TDP-43 mediated increase in the proapoptotic genes in mouse neural progenitor cells and human cortical neurons.Increase of TDP-43 and TDP-43Cdkn1a mRNA levels. Concomitant, we observed that TDP-43 overexpression induced a block in cell cycle progression in mouse neural progenitors in vivo. Cdkn1a is activated by p53, however, we additionally show an association of TDP-43 to Cdkn1a mRNA, which suggests a regulation of Cdkn1a expression by TDP-43. This is supported by the observation that blocked cell cycle progression could not be rescued by p53 inhibition. Thus, TDP-43 expression not only induces apoptosis but also induces a cell cycle block, probably at G2/M phase transition through cyclin dependent kinase inhibition via Cdkn1a, which in turn could stimulate cell death. Due to the fact that we performed the CLIP experiments with PFA cross-linking, we cannot be sure that TDP-43 binds directly to Cdkn1a mRNA. Confirmation of direct binding of TDP-43 to Cdk1a mRNA will require further analysis. Previously, it has been reported that TDP-43 overexpression induces G2/M arrest in HeLa cells42. It was shown that the effect was partially dependent on p53 but apoptosis could not be rescued by p53-inhibition. These partially contradictory results may be explained by the differences in cells used in the experiments. HeLa cells have an altered cell cycle regulation and might not respond in the fashion as neural progenitors and neurons to inhibition of p53.Besides the TDP-43 mediated increase in p53, we also observed a strong increase in A315T and partially rescued the animals by p53 inhibition. Similar rescue experiments have been preformed in hSOD1G82A transgenic mice. Interestingly, p53 deletion did not rescue the early lethality in these hSOD1G82A animals, which indicates a p53-independent mechanism, possibly involving death receptors34. This is supported by findings that death receptor 6 (DR6) levels are elevated in hSOD1G82A transgenic mice43. In addition, treatment with blocking antibodies against DR6 increased motor neuron survival and, therefore, provided a neuroprotective effect in hSOD1G82A expressing animals. The partial rescue of TDP-43A315T embryos indicates probable involvement of additional factors triggering lethality of these animals. However, this remains to be addressed in the future.Many lines of transgenic mice expressing wild type or mutant forms of TDP-43 have been analyzed in the last years, all of which show early mortality. We employed homozygous mice expressing TDP-43in vivo. Although TDP-43 is indispensable for early development3, it does not seem to have a crucial function in neural progenitor maintenance in the brain at the time points we analyzed. This might be due to the fact that we only achieved a 70% reduction in TDP-43 levels at the protein level. However, we did observe a significant increase in the number of proliferating cells in the in vivo KD experiment. Cdkn1a KD induces proliferation by promoting S-phase entry44. We hypothesize that loss of TDP-43 results in a reduction in Cdkn1a, which in turn activates proliferation, reduces cell cycle exit and delays differentiation.We also investigated how loss of TDP-43 affects cell survival and differentiation of neural stem/progenitor cells. In striking contrast to the overexpression experiments, we did not observe signs of apoptosis upon KD of TDP-43 in vivo as well as in human cortical progenitors and neurons and in TDP-43G298S mutant iPS-derived cortical cultures induces p53-dependent apoptosis. This is associated with up regulation of apoptotic genes including Bbc3 and Bax. We did not observe a direct interaction between TDP-43 and p53 on the protein or mRNA levels. It therefore remains elusive how TDP-43 activates p53 expression but this is likely, at least in part, to be indirect via induction of cellular stress response30. Future work should address this as well as how the multiple pathways downstream of TDP-43 contribute to cell death in disease.In conclusion, we show that TDP-43 accumulation in mouse neural progenitors Trp53tm1Tyj, Trp53tm1Brn, TDP-43A315T, Rosa-CAG::GFP and Hes5::CreERT2 mice have been described elsewhere47. Mice were maintained on a 12-hour day/night cycle with adequate food and water under SPF conditions. All methods were carried out in accordance with guidelines and regulations of Max-Planck Institutional and German Federal regulations and under license numbers H-05/01, 0-06/02, G-09/18, G-09/19, G-08/26  and 2437 and 2438 (Veterinary commission Basel). All experimental protocols were approved by Max-Planck Institutional and German Federal regulations and under license numbers H-05/01, 0-06/02, G-09/18, G-09/19, G-08/26  and 2437 and 2438 (Veterinary commission Basel). The day of vaginal plug was counted as embryonic day 0.5 (e0.5).Trp53tm1Brn/Hes5::CreERT2/Rosa-CAG::GFP mice were given 2 mg Tamoxifen by gavage at e11.5 to induce recombination. Stock solutions of Tamoxifen (Sigma) were prepared at a concentration of 20 mg/ml in corn oil (Sigma). Bromodesoxyuridine  was administered to the adult animals via a single intraperitoneal injection (50 mg/kg body weight). PFT-α (stock solution 20 mM in DMSO) was injected intraperitoneal into pregnant mice (2.2 mg/kg).Trp53tm1Brn mice were used for the in utero expression analyses at 13.5 days after detection of vaginal plug. DNA constructs were injected into the forebrain telencephalic vesicles of the embryonic mice using a microinjector  and pulled Borosilicate glass capillaries (Kwick-FilTM). The capillaries were pulled in a micropipette puller (Sutter Instrument Co.) and sharpened using a capillary sharpener (Bachofer). The capillaries were backend-loaded with 10 μl of the plasmid. Plasmid stocks were prepared under endotoxin free conditions and suspended in sterile phosphate buffered saline (PBS) at a concentration of 3 μg/μl. Fast green contrast dye (10%) was added to the plasmids to visualize the targeted area of the telencephalon. The overexpression or shRNA knockdown vectors were electroporated in a molecular ratio of 3:1 with the transfection reporter vector (pCAGGS::eGFP). The pregnant female mice were administered analgesic  by intraperitoneal injection and anesthetized with 1–2% isoflurane (Baxter) in a constant flow of O2, secured on a heated operating table. Body temperature was monitored continually. The fur was removed from the abdomen using depilation cream. Throughout the operation, the peritoneal cavity was moistened with sterile Hank’s buffered saline solution (HBSS). The uterine horns containing the embryos (e13.5) were manipulated under sterile conditions by hand. A cold light source was used to illuminate the embryos. 1–2 μl of DNA solution were injected into the telencephalic vesicles of each embryo. The embryos were electroporated  with 10 pulses of 40 V and a pulse length of 50 ms at 950 ms intervals. The anode of the electrode was oriented toward the injected side. After injection and electroporation, the uterus was returned to the abdomen, the muscle and the skin sutured and the females allowed to recover under a heating lamp with constant observation. Operated females had free postoperative access to analgesic  in sterile agar. The animals were sacrificed after a defined time and the embryos isolated and prepared for sectioning.Female C57BL/6 J and A315T and human TDP-43∆RRM1 were subcloned into pCAGGS expression vectors with a beta-globin 3-prime untranslated and polyadenylation sequence. pSuper-shRenilla as well as pSuper-shTDP-43 were cloned according to the manufacturer’s instructions (Oligoengine). p3X-FLAG-myc-CMVTM-26 expression construct was obtained from Sigma and p3X-TDP-43-flag was cloned using Not1 and Xba1 restriction sites (Primers: fwd 5′- TTCTGCGGCCGCCACCATGTCTGAATATATTCG-3′ rev 5′-CTTTCTAGACTACATTCCCCAGCCAGAAG-3′).Full-length coding region cDNAs for GFP, Cdkn1a, human TDP-43, human TDP-43in situ RNA hybridization a digoxigenin- (DIG)-labeled antisense RNA probe was generated for mouse Tardbp RNA (amplified from mouse cDNA using primers: fwd 5′-ATTCCTTCCCGTCTGTGCTT-3′ rev 5′-TGCTTAGGTTCAGCATTGGA-3′) using the procedures described previously. Expression was detected by colorimetric reaction using NBT (nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolylphosphate p-toluidine) as reaction substrates and images taken using an Axioplan microscope (Zeiss) with an Axiocam CCD camera (Zeiss).For TM 6000 Real-time PCR machine (Corbett) and analyzed using Rotor-gene 6000 series software 1.7. β-actin and GAPDH mRNA levels were measured as endogenous controls and for quantification.For RNA isolation, cells were lysed directly in Trizol (Invitrogen) reagent. RNA was prepared according to the manufacturer’s instructions. 1 μg of total RNA was used for cDNA synthesis by Oligo-dT priming and BioScript (Bioline). For quantitative RT-PCR the SensiMix SYBR kit (Bioline) was used following the manufacturer’s instructions  The reaction was run in a Rotor-Genehttp://www.bio-protocol.org/e398. In summary, N2A cells were transfected using Lipofectamin2000 (Invitrogen) according to manufacturer’s instructions with p3X-flag-GFP or p3X-flag-TDP-43 and trypsinized after 48 hours. The cells were then fixed in 3% formaldehyde in PBS for 10 minutes and lysed by sonication (10 pulses for 10 seconds). Immunoprecipitation was performed overnight at 4 °C using anti-Flag M2 Affinity Gel (Sigma-Aldrich). After collection by centrifugation at 2000g and washing 3–4 times with lysis buffer the complexes were reverse cross-linked and RNA extracted using Trizol reagent (Invitrogen) according to the manufacturers instructions. Isolated RNA was treated with RNase-free DNaseI (Roche) to remove any genomic DNA contamination. First strand cDNA was generated using BioScript (Bioline) and random hexamer primers followed by real-time PCR using SensiMix SYBR Kit (Bioline).The detailed protocol can be found at p3X-flag-TDP-43 or p3X-flag-GFP as control) and cells were harvested after 24 hours. Cells were lysed and 1/10 of the lysate was used as input control. The remaining lysate was added to pre-blocked beads coupled to anti-flag antibody (ANTI-FLAG® M2 Affinity Gel) or to un-coupled beads (Sepharose-G beads) and incubated at 4 °C. Thereafter the beads with the bound proteins were washed and the samples were incubated for 1 hour at 70 °C to release the bound proteins. The supernatant containing the immunoprecipitated proteins was then analyzed on a SDS-gel. For low molecular weight proteins like TDP-43, 15% separation gels were used according to the manufacturer’s instruction. 20–40 μg proteins with sample loading buffer were denatured and loaded on the gel. Proteins were separated and after blotted onto a PVDF membrane. To detect proteins the membrane was incubated with specific antibodies after blocking with blocking buffer for 1 hour . The membrane was thereafter incubated with a secondary antibody coupled to horse reddish peroxidase and the antibody was detected using Amersham™ ECL™ Western Blotting Detection reagent.For immunoprecipitation, N2A cells were transfected with expression plasmids  solution in 0.1 M phosphate buffer (PB) overnight. Brains were cryoprotected in a 30% sucrose solution in 0.1 M PB for 24 hours, embedded and frozen over dry ice in OCT (TissueTEK). Horizontal sections (20 μm) were collected on Superfrost glass slides (Thermo Scientific), and stored at −20 °C until use. For immunostaining, sections were incubated overnight at 4 °C with the primary antibody diluted in blocking solution of 2% normal donkey serum (Jackson ImmunoResearch), 0.3% Triton X-100 in 0.1 M PB. Sections were washed three times in PBS and incubated at room temperature for 3 hours with the corresponding secondary antibodies in blocking solution. When signal amplification was needed, sections were washed and incubated for 1 hour at room temperature in streptavidin–FITC  and counter-stained with DAPI (1 μg/ml). For BrdU detection, sections were treated with 2 M HCl at 37 °C for 15 minutes prior to primary antibody incubation. HCl-treated sections where then equilibrated in borate buffer  for 10 minutes. For active caspase-3, Tbr1, Tbr2 and TDP-43 detection, antigens were recovered at 80 °C for 30 minutes in Sodium Citrate solution  with 0.05% Tween. Stained sections were embedded in mounting medium containing diazabicyclo-octane  as an anti-fading agent. Antibodies, dilutions and conditions used for immunolabeling are described in the supplementary tables. Sections were analyzed with an Axioscope (Zeiss) or confocal (Zeiss LSM510) fluorescence microscope. Images were acquired using Axiovision or Zeiss LSM 4.2 (Zeiss) and processed with ImageJ 1.33 or Photoshop CS (Adobe) software.48 were differentiated into cerebral cortex neurons for 39 days according to the protocol from Shi et al.37. Patient-derived TDP-43G298S mutant iPS have been characterized and described previously by Alami et al.38. iPS cells were washed, filtered through a 70 µm cell strainer (Miltenyi Biotec) and sorted on a BD FACS Aria III. Live cells were discriminated by forward and side-scatter (for live cells – from the control) and gated for GFP- (non transfected) or single GFP+ populations.Human iPS cells (Nas2 cells)Randomly selected, stained cells were analyzed with fixed photomultiplier settings on a Zeiss LSM510 confocal microscope (Zeiss). n numbers represent the number of animals used, and images from at least 3 sections per animal were quantified. Data are presented as average percentages of co-labeled cells and statistical comparisons were conducted by two-tailed unpaired Student’s tTest. Significance was established at P < 0.05. In all graphs, error bars are standard deviation (SD).Supplementary Figures and legends


Temperature-dependent changes in biomass and community composition depended on the initial composition of phytoplankton communities. Abiotic conditions had a major effect on biomass of phytoplankton communities exposed to different temperature conditions, however, the effect of biotic and abiotic conditions together was even more pronounced. Additionally, phytoplankton community responses to pulse temperature effects depended on the warming history. By disentangling abiotic and biotic effects, our study shows that temperature-dependent effects on phytoplankton communities depend on both, biotic and abiotic constraints.Effects of temperature changes on phytoplankton communities seem to be highly context-specific, but few studies have analyzed whether this context specificity depends on differences in the abiotic conditions or in species composition between studies. We present an experiment that allows disentangling the contribution of abiotic and biotic differences in shaping the response to two aspects of temperature change: permanent increase of mean temperature versus pulse disturbance in form of a heat wave. We used natural communities from six different sites of a floodplain system as well as artificially mixed communities from laboratory cultures and grew both, artificial and natural communities, in water from the six different floodplain lakes (sites). All 12 contexts (2 communities × 6 sites) were first exposed to three different temperature levels  and afterward to temperature pulses (4 °C increase for 7 h dayThe online version of this article (doi:10.1007/s00442-016-3693-3) contains supplementary material, which is available to authorized users. An increasing number of studies focus on how phytoplankton primary productivity and species composition are influenced by water temperature, triggered by an interest to understand how global warming affects ecosystem processes and properties  are strongly interlinked: differences in abiotic conditions might alter temperature effects by direct interactions, e.g., between nutrient availability and temperature effects, as well as by indirect effects mediated by differences in species composition.a concentrations showed positive, none or negative temperature effects  a community is exposed to determine niche availability and thus the type of functional groups that fill these. Whereas functional group composition tends to be deterministic, species composition within these functional groups often arises stochastically from the history of species arrival , we conducted an experiment allowing for the first time to disentangle direct from indirect contingencies: We imposed the same temperature treatments on communities from six different sites (different abiotic conditions and composition) and on the same artificial community inoculated in water from the same six sites (different abiotic conditions only). Thereby, we are able to disentangle the effects only due to conditions from the effects of the presence/absence of certain species, as community composition determines the traits constraining productivity at different temperatures as well as how environmental conditions affect community productivity  Temperature-dependent changes in biomass and community composition depend on the initial composition of a community (composition is determined by the different sites of origin including their disturbance histories).The aim of this study is to analyze potential mechanisms leading to divergent responses to temperature change. The factorial design of the study [3 temperature levels × 6 sites of origin × 2 community types ] is combined with a two-stage application of temperature, pressed disturbance for 14 days and a heat wave (pulse disturbance) of plus 4 °C for 7 h on top of the temperature of the first phase, to test the following hypotheses:(H2) Abiotic conditions have an effect on the response to different temperature conditions of phytoplankton communities. This will be investigated by comparing the effect of changes in temperature on natural and artificial communities under the same experimental conditions.This hypothesis will be investigated by comparing phytoplankton communities from six sites, which differ in composition. However, since the sites also differed in temperature, disturbance history, and chemical parameters, the difference between sites of origin includes abiotic and biotic differences. To separate these, we replaced the natural communities by one artificially assembled community (removing the biotic factor), leaving only the abiotic differences between sites. This allows testing the next hypothesis:(H3) Phytoplankton community responses (composition and biomass) to pulse temperature effects depend on the warming history.These hypotheses will be tested for both the press and the pulse disturbance phase of our experiment, which additionally allows addressing our third hypothesis:We performed laboratory experiments with natural as well as with artificially assembled phytoplankton communities. The natural communities originated from six different sites of the Danube river floodplains near Vienna, Austria: Danube River site 1) and so-called Eberschüttwasser (site 2), Hanselgrund (site 3), Kühwörther Wasser (site 4), Schwarzes Loch (site 5), and Schönauer Traverse (site 6). Previous studies , Cyanophyceae (cyanobacteria), Bacillariophyceae (diatoms) .The respective cultures were obtained from algal culture collections (SAG Göttingen and CCALA Trebon) and pre-cultured prior to the experiment for months at 18 °C in sterile WC medium according to Guillard and Lorenzen  and autoclaved to eliminate organisms. These pre-treated media from the six sampling sites were used to incubate an artificially assembled community consisting of 12 phytoplankton species from three taxonomic groups: Lorenzen .The experiment was divided into two phases: experimental phase I—incubation at permanent increased temperatures for 2 weeks, and experimental phase II—pulse temperature exposure for one additional week. For the first phase of the experiment, three constant temperature levels  were established; natural and artificially assembled communities were grown for 14 days to adapt to these temperatures (press disturbance). These temperatures were chosen based on the temperatures at different sites during initial sampling ranging from 12 to 18 °C ; the position of the culture flasks was changed randomly during the experiment to assure equal light conditions for all treatments and to avoid any position effects. The experiment was conducted with an exchange of 10 % day−1 . The medium used for the daily exchange was pre-filtered and autoclaved water from each of the sites the communities were sampled from  or incubated in , respectively. Light intensity and water temperature were controlled by continuous data logging . Incubation of the natural communities started off at their natural biomass concentrations of the respective site and sampling day. Artificial communities were started with even proportions of the 12 species used and all treatments were incubated with an equal amount of initial biomass (based on chlorophyll a concentrations). Each treatment was replicated three times. At the end of experimental phase I, 50 % of each of the samples was used for analysis and the culture flasks were refilled with fresh medium (as described above) up to the initial sample volume (200 mL). This way, the experimental setup was the same as in a previous laboratory experiment described in Schabhüttl et al. (Cell culture flasks (250 mL) were used as experimental units and were shaken twice a day to keep the algae in suspension. All communities were exposed to a light:dark cycle of 16:8 h at a light intensity of about 100 µmol photons ml et al. .a concentrations were measured fluorometrically  and samples for particulate organic carbon (POC) and particulate organic phosphorus (POP) analysis were filtered onto pre-combusted and acid-washed glass-fiber filters (Whatman GF/C) at the start of the experiment, after experimental phase I (permanent temperature increase), and again after experimental phase II (temperature pulses). POC was measured by infrared spectrometry . Total phosphorus (TP) and particulate organic phosphorus (POP) concentrations were measured by molybdate reaction after sulfuric acid digestion  to temperature between the sites of origin (ANOVA for ‘site’ and ‘temperature’), where a significant interaction between site and temperature indicates differences due to biotic and abiotic conditions. Comparing the effects of abiotic conditions  with the interactive effect of abiotic and biotic conditions  will support these results , where a significant interaction between site and temperature supports H1. Redoing this analysis for the artificial communities tested the importance of the abiotic difference alone (H2): if the interaction between site and temperature remains significant for artificial communities, then abiotic differences between sites suffice to create different temperature effects. If not, then the compositional differences alone drive differential temperature effects. For ANOVAs, we calculated the Eta-Squared measure of effect size  on temperature-(‘temperature’) dependent changes in phytoplankton biomass (H1), the effect of abiotic vs biotic conditions (‘community’) on changes in phytoplankton biomass (H2), and whether the phytoplankton community response to pulsed temperature (‘time’) effects depended on warming history (H3). When analyzing the temperature effect on the natural communities after the pulsed temperature disturbance treatment , a significant time × temperature interaction supports H3 that warming history alters the response to a heat wave (pulsed temperature disturbance). Additionally, the log response ratio was calculated as the log ratio of chlorophyll a concentrations of pulsed disturbance treatments and pressed disturbance treatments [LRR = ln(chlorophyll a pulse/chlorophyll a press)]. For rmANOVAS we calculated the generalized Eta-Squared measure of effect size (Bakeman We used a repeated measures (rm-)ANOVA with log-transformed chlorophyll  Bakeman .To further investigate the effects of temperature, abiotic conditions and pulse temperature disturbance on changes in phytoplankton community composition, we calculated an algal response factor on a taxonomic group level Sarnelle . This reFor natural communities, we calculated Bray–Curtis dissimilarity in phytoplankton community composition (using relative biovolume) between the different sites of origin (‘site’) using the R package “vegan”  was affected by temperature , the biomass of artificial and natural phytoplankton communities increased with higher temperature Fig. . PhytoplFor natural communities, a significant interaction of temperature × site , phytoplankton biomass in all artificial communities was lowest at 12 °C (+4 °C) while no general trend for temperature-dependent differences in biomass existed in natural communities Fig. . Howevera concentrations .While the absolute biomass of the artificial communities was generally higher than the biomass of natural communities , n = 54; pulse disturbance: r2 = 0.34, p < 0.0001, slope = −0.03 (SE = 0.006), n = 54].Although all six natural communities had started off at different taxon and functional group composition, all natural communities developed toward diatom-dominated communities Fig. b was higTemperature-dependent effects on phytoplankton biomass were observed in natural as well as in artificial communities after press and pulse disturbance. Increasing temperature tended to result in a positive effect on phytoplankton biomass in natural , as well as the frequency of medium exchange were exactly the same for the respective natural and artificial communities, the initial community composition remained as the exclusive difference. Thus comparing the results obtained from natural communities with those from the artificial communities, we can conclude that temperature-dependent changes in community composition depend on the initial composition of a community including its disturbance history.Considering only the abiotic differences between sites , we still observed an interaction of temperature and site of origin Fig. ; Table 4Jöhnk et al.  showed tDifferent temperature effects on phytoplankton biomass were observed between natural and artificial communities, as artificial communities showed generally higher absolute biomass while natural communities were able to obtain high biomass after pulsed temperature disturbance Figs. , 2, 3. EWhile the dissimilarity in functional groups between the different sites  was strongly negatively affected by press and pulse disturbance as well as increasing temperature , the compositional dissimilarity between the different sites was high at the beginning of the experiment and remained high after press and pulse disturbance. These data corroborate results from experiments with plant communities, which also showed communities’ functional convergence over time alongside taxonomic divergence  of phytoplankton communities. Abiotic conditions had an effect on biomass of phytoplankton communities exposed to different temperature conditions, however, the effect of biotic and abiotic conditions together was even more pronounced. Phytoplankton community responses to pulse temperature effects depended on the warming history concerning the effects on biomass and community composition: taxon composition diverged while functional group composition converged.Supplementary material 1 (PDF 3123 kb)Below is the link to the electronic supplementary material.