Mesangial cell proliferation is the most basic pathological feature of immunoglobulin A nephropathy (IgAN); however, the specific underlying mechanism and an appropriate therapeutic strategy are yet to be unearthed. This study aimed to investigate the therapeutic effect of triptolide (TP) on IgAN and the mechanism by which TP regulates autophagy and proliferation of mesangial cells through the CARD9/p38 MAPK pathway.We established a TP‐treated IgAN mouse model and produced IgA1‐induced human mesangial cells (HMC) and divided them into control, TP, IgAN, and IgAN+TP groups. The levels of mesangial cell proliferation  and autophagy  were measured, with the autophagy inhibitor 3‐Methyladenine used to explore the relationship between autophagy and proliferation. We observed CARD9 expression in renal biopsies from patients and analyzed its clinical significance. CARD9 siRNA and overexpression plasmids were constructed to investigate the changes in mesangial cell proliferation and autophagy as well as the expression of CARD9 and p‐p38 MAPK/p38 MAPK following TP treatment.Administering TP was safe and effectively alleviated mesangial cell proliferation in IgAN mice. Moreover, TP inhibited IgA1‐induced HMC proliferation by promoting autophagy. The high expression of CARD9 in IgAN patients was positively correlated with the severity of HMC proliferation. CARD9/p38 MAPK was involved in the regulation of HMC autophagy and proliferation, and TP promoted autophagy to inhibit HMC proliferation by downregulating the CARD9/p38 MAPK pathway in IgAN.TP promotes autophagy to inhibit mesangial cell proliferation in IgAN via the CARD9/p38 MAPK pathway. Tripterygium wilfordii hook F., promotes autophagy to inhibit mesangial cell proliferation in IgAN via the CARD9/p38 MAPK pathway.The proliferation of glomerular mesangial cells is the most basic pathological feature for IgA nephropathy (IgAN). Galactose‐deficient IgA1 (Gd‐IgA1) immune complex deposition activates mesangial cells to mediate specific intracellular signal transduction, promotes mesangial cell proliferation, and initiates kidney damage. Autophagy is involved in mesangial cell proliferation. CARD9 is a risk gene for IgAN. The triptolide (TP), purified from  We speculated that TP promotes autophagy via the CARD9/p38 MAPK pathway to inhibit mesangial cell proliferation, thereby improving IgAN.22.1Twenty‐six five‐week‐old female BALB/c mice weighing 20–25 g were obtained from Hunan Slack Jingda Experimental Animal Co. Ltd. . All animal procedures were approved by the Experimental Animal Ethics Committee of the Second Xiangya Hospital of the Central South University. After 7 days of adaptive pre‐feeding, the mice were randomly divided into four groups: control, TP, IgAN, and IgAN+TP. The IgAN mouse model was established by oral mucosal immunization. Gavage with acidified bovine serum albumin (BSA) was performed once every other day (800 mg/kg), and the mixed solution (CCL4: castor oil = 1:5) was subcutaneously injected weekly (0.1 ml) and intraperitoneally injected every 2 weeks (0.06–0.08 ml). Lipopolysaccharide (50 μg) was injected into the tail vein at weeks 6 and 8 week. The IgAN mouse model was successfully established at the end of the 11th week, as confirmed by glomerular IgA immunofluorescence. The TP and IgAN+TP groups received intragastric TP treatment (300 μg/kg/day). The control and IgAN groups were administered the same dose of normal saline. At the end of the 17th week, mice were sacrificed by cervical dislocation. Blood, urine, and kidney tissue samples were collected for the subsequent experiments.According to the manufacturer's standards, the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), serum creatinine (SCr), blood urea nitrogen (BUN), and uric acid (UA) were detected with enzyme‐linked immunosorbent (ELISA) assay kits . The urine protein and creatinine levels of the mice were detected using ELISA kits , and the ratios of urine protein/creatinine were calculated.2.2The human glomerular mesangial cell line (HMC)  was cultured in DMEM/F‐12 medium  with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C and 5% CO2. As previously described, monomeric human IgA1  was heated at 65 °C for 150 min to polymerize into polymer IgA1.2.3The sequence of the CARD9‐siRNA (siCARD9)  was 5′‐CCTCACGCATCACACCTTA‐3′. The CARD9‐overexpression plasmid (Exp‐CARD9)  was designed according to the GenBank reference sequence (NM_052813). Lipofectamine 2000  was used to transfect CARD9‐siRNA or overexpression plasmid into HMCs according to the manufacturer's instructions.2.4All patients were diagnosed based on renal biopsy at the Second Xiangya Hospital of Central South University. All biopsies were obtained from the kidney pathology laboratory of the hospital, and clinical information was obtained from the electronic medical records. All procedures were performed in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Thirty patients with IgAN were enrolled, and mesangial cell proliferation was assessed based on pathological diagnosis reports .2.5Kidney tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin–eosin (H&E) and periodic acid‐Schiff (PAS) staining was performed to observe the proliferation of glomerular mesangial cells. According to the instructions of the immunohistochemistry kit , the expressions of cyclin D1 , PCNA, P62 , and LC3 II  were detected. Quantitative analysis was performed using ImageJ software.2.6Immunofluorescence analysis of mouse IgA was performed as previously described.2.7Total RNA was extracted from cells using TRIzol reagent . The HiScript III RT Supermix (+gDNA wiper)  was used for mRNA reverse transcription. Real‐time PCR was performed on a LightCycler 96 System  using the SYBR Green Master Mix . The primer sequences were CARD9: F‐CAGCCCCTACATCCAGGTA and R‐CAGGGAGAAGATGGTGTTGG. CARD9 and internal reference primers were purchased from Sangon Company (China).2.8Radioimmunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride and phosphatase inhibitors was used to extract cell and tissue proteins. Equal amounts of protein samples were loaded on SDS‐PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% BSA at room temperature for 1 h, followed by incubation overnight at 4°C with the following primary antibodies: cyclin D1 , PCNA, P62 , LC3 II , β‐actin , GAPDH , CARD9 , p‐p38 MAPK , p38 MAPK . Membranes were then incubated with secondary antibodies conjugated to horseradish peroxidase. Chemiluminescence signals were identified using ECL kits  and quantitatively analyzed using ImageJ.2.9A Cell Counting Kit‐8 (CCK‐8) assay  was performed to detect cell viability, and the number of live cells was estimated by measuring the optical density (OD) at 450 nm. Cell cycle distribution was analyzed using flow cytometry . The proliferation index (PI) was calculated as the sum of the percentages of cells in the S and G2/M phases. Cells were fixed with 75% ice‐cold ethanol at 4 °C overnight and added propidium iodide and RNase A  mixed working solution, followed by staining for 30 min at 4 °C in the dark. FlowJo V10 was used to process and analyze the data from the flow cytometer.2.10As mentioned in previous reports, the dynamic process of autophagy was analyzed in cells expressing mRFP‐GFP‐LC3.2.11t‐test was used to determine the statistical difference between the two groups, and one‐way analysis of variance was used to analyze the differences between multiple groups. Bivariate correlation analysis was adopted for the two variables. All experiments were independently repeated at least three times, and statistical significance was set at p < 0.05. All statistical analyses were performed using GraphPad Prism 8.0 and SPSS 26.0 software.Data are presented as the mean ± SD. Two‐tailed Student's 33.1First, we established a TP‐treated IgAN mouse model. IF showed that IgA was significantly deposited in the mesangium of IgAN mice compared to that in control mice Figure . H&E and3.2We also investigated the effects of TP on HMC proliferation and autophagy. IF and WB showed that high expression of PCNA and cyclin D1 in IgA1‐induced HMCs was reduced by TP Figure . The CCKAs shown in Figure 3.3We used the autophagy inhibitor 3‐MA to explore the relationship between autophagy and proliferation under IgA1 and TP conditions Figure . 3‐MA fu3.4p < 0.05, log2 [FC] = 0.7)  from the GEO database to analyze differentially expressed genes in human kidney biopsy tissues. CARD9 was among the top 30 differentially expressed genes Figure . The lev) Figure . Notablyp < 0.05)  Figure . In addi) Figure .3.5Consistent with the sequencing results Figure , the CARIn addition, we transfected a CARD9 overexpression plasmid into IgA1‐induced HMCs. The CCK‐8 assay showed that CARD9 overexpression promoted cell viability Figure . However3.6p38 MAPK is a transcription factor regulated by CARD9. Figure 4In this study, we obtained the following results: (1) Mesangial cell proliferation was increased and autophagy was suppressed in IgAN mice and IgA1‐induced HMCs; (2) TP treatment applied to IgAN mice inhibited proliferation and promoted autophagy in mesangial cells and was safe and effective; (3) TP inhibited IgA1‐induced HMC proliferation by promoting autophagy; (4) high expression of CARD9 in IgAN was positively correlated with the severity of HMC proliferation, with CARD9 knockdown promoting autophagy and inhibiting HMC proliferation; and (5) TP promoted autophagy to inhibit HMC proliferation by downregulating the CARD9/p38 MAPK pathway in IgAN.Tripterygium preparations are widely used for the treatment of chronic kidney disease, rheumatic and autoimmune disease, and tumors because of their immunosuppressive and anti‐inflammatory medicinal value.This study is the first to focus on TP in HMCs for IgAN. Previous studies have reported that tripterygium glycosides reduce Gd‐IgA1 production, proteinuria, and hematuria, inhibit inflammation, and protect renal function in IgAN rats.Glomerular mesangial cell proliferation is the most important pathological feature in the early stages of IgAN; however, its underlying mechanisms have not yet been clarified. Our previous studies suggested that autophagy is involved in HMC proliferation.Our study is the first to investigate the role of CARD9 in kidney disease. CARD9 is an intracellular adaptor protein of the CARD protein family that has been identified for its selective binding to the CARD domain of B‐cell leukaemia‐lymphoma 10 (BCL10) Figure . CARD9 amTOR and class III PI3K complexes act in the early stages of autophagy, but little is known about their regulation in later stages. Henson et al. found that p38 MAPK inhibited autophagy independently of mTOR.In conclusion, this study investigated the therapeutic effects of TP on IgAN and the important role of CARD9 in mesangial cell autophagy and proliferation. TP downregulated CARD9 to suppress p38 MAPK phosphorylation, thereby inhibiting HMC proliferation by promoting autophagy. Moreover, the findings provide new theoretical bases for mesangial cell proliferation and suggests new ideas for future targeted therapy of IgAN.The results presented in this article have not been published previously in whole or part. Conceived and designed the experiments: Lu Zhao, Hong Liu, Chengyuan Tang. Performed the experiments: Lu Zhao, Zhixin Lan, Lili Wan. Analyzed the data: Liang Peng, Xia Tan, Di Liu, Guochun Chen. Wrote the paper: Lu Zhao.The authors declare no conflict of interest.FIGURE S1 The optimal concentration of TP in HMCs. (A) Cell viability was assessed by CCK8 assay. All data presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01 vs. Control. (B) Representative western blot of PCNA, cyclin D1, and P62 in different concentrations of TP‐treated HMCs.FIGURE S2. CARD9 is the differential gene in the dataset GSE116626 (11 control and 37 IgAN samples). (A) Heat map of the top 30 differential genes. (B) The level of CARD9 mRNA in IgAN patients compared to control .FIGURE S3. Immunofluorescence of CARD9 in glomeruli of mice. Bars = 50 μm. All images ×400.FIGURE S4. CARD9 overexpression in IgA1‐induced HMCs. (A) Cell viability was assessed by CCK8 assay. Representative western blots (B) and quantitative analysis (C) of PCNA, cyclin D1, P62, and LC3 II in CARD9‐overexpression HMCs. All data presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01 vs. Control, ###p < 0.001 vs. IgA1 + NC, ns: p > 0.05 vs. IgA1 + NC.Click here for additional data file.


Among various metal ions, L1 showed a highly selective and sensitive turn-on fluorescence response to the presence of Zn2+ ions in methanol. The detection limit for Zn2+ by L1 was calculated to be 1.76 × 10−7 M. The 1 : 1 complexation ratio of the L1–Zn complex was confirmed through Job plot measurements. Complexation studies were performed by FT-IR, NMR and HR-ESI MS measurements and DFT calculations. With the gained insight, it was possible to successfully apply L1 in water sample analysis.A new fluorescent chemosensor based on 8-aminoquinoline L1 bearing a benzimidazole moiety was synthesized, which exists as two predominant tautomers L1A and L1B in diluted DMSO-d The sensor shows a high selectivity and sensitivity toward zinc cations, accompanied by a distinct green fluorescence emission. Thus, soil microbial activities are unfavorably affected which subsequently leads to phytotoxicity and, in general, to lowered crop quality.4 This is particularly problematic in former mining regions such as the Harz mountains  where we are located. Increased zinc concentrations are not only caused by contaminated sites as a result of centuries of mining activity, but also unintentionally by the local metal industry.5Zinc is known to be a common essential element to all living organisms. It has proven to be of vital importance to various biological processes such as enzyme activity or DNA protection.10 electron configuration. Even though various methods are known for its detection, most of them have not proven to be field-operational. Oftentimes, expensive equipment and extensive sample preparations are necessary for obtaining results.6 Therefore, the demand for low cost and easy monitoring systems has been high. Fluorescent chemosensors have proven to be a valuable asset in the sensing and monitoring of heavy and transition metal ions.Zinc is difficult to detect spectroscopically due to its 3d2+-sensing have been designed over the past decades.7,8 Among many trace metal ion sensors, quinoline is a common fluorophore used as a backbone in zinc sensing structures.9,10 The application of various 8-hydroxyquinolates in the fluorometric detection of zinc dates back as far as the 1960s.11 8-Aminoquinoline based Zn2+-chemosensors are also known.10,12,13 However, fluorogenic Zn2+-sensors are not limited to quinoline structures only. Contemporary examples range from systems based on e.g. coumarin,14 fluorescein,15 benzimidazole16,17 or silsesquioxane.18 Further contemporary examples can be found in Table S1 , and fluorescence spectroscopy. DFT calculations have been carried out. Apart from the fact that L1 could successfully distinguish Zn2+ from Cd2+ and Hg2+, its potential use in water sample analysis is shown.In continuation of our interest in metal adducts and metal complexes of heterocycles such as mesomeric betaines and 6 was used as NMR solvent with chemical shifts (δ) being reported in ppm. IR spectra (ATR-IR) were recorded on a BRUKER Alpha T in a range of 400–4000 cm−1. Mass spectra were recorded on a BRUKER Impact II mass spectrometer. UV-vis measurements were performed on a JASCO V-550 spectrophotometer. Fluorescence measurements were performed on a JASCO FP-8500 spectrofluorometer using a prismatic cell to avoid inner-field effects. All measurements were conducted at room temperature. The precursor 2-chloro-N-(quinolin-8-yl)acetamide was synthesized according to known literature procedures.26All chemicals used were purchased and used as received unless noted otherwise. NMR spectra were taken on a BRUKER Avance FT-NMR AVANCE III (600 MHz). DMSO-dN-(quinolin-8-yl)acetamide, 80 mg (0.58 mmol) of potassium carbonate and 104 mg (0.58 mmol) of 5-methoxy-2-mercaptobenzimidazole was dissolved in 5 mL of acetone and refluxed for three hours. Upon completion, monitored by tlc, the reaction mixture was filtered and the solvent was removed in vacuo to afford 191 mg of a light brown solid in 91% yield, mp 180 °C. 1H NMR  of a concentrated solution: δ = 12.56 , 11.20 , 8.80 , 8.65 , 8.35 , 7.64 , 7.59 , 7.54–7.57 , 7.40 , 7.02 , 6.77 , 4.33 , 3.77  ppm. 13C NMR : δ = 167.4  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O), 155.4 , 148.8 , 148.5 , 139.8 , 138.1 , 136.4 , 134.8 , 134.5 , 127.8 , 126.9 , 122.0 , 122.0 , 116.3 , 114.9 , 110.5 , 97.1 , 55.5 , 35.7  ppm. IR (ATR): 3202, 3008, 2947, 2825, 2191, 1660, 1628, 1594, 1522, 1485, 1445, 1426, 1403, 1358, 1340, 1322, 1301, 1264, 1244, 1225, 1202, 1153, 1106, 1087, 1064, 1030, 967, 947, 875, 821, 788, 739, 697, 642, 618, 584, 541, 516, 473, 436 cm−1. HR-ESI-MS: calcd. for C19H16N4O2S [M + Na]+: 387.0892, found 387.0864.A sample of 127 mg (0.58 mmol) of 2-chloro-x-solution  were titrated to the aforementioned solution of L1. After shaking the sample for a couple of seconds, the fluorescence spectra were measured.9 μL of a 10 mM solution of L1 (0.01 mmol in 1 mL of MeOH) were added to 2.991 mL of MeOH to make a final concentration of 30 μM. Afterwards, 30 μL of a 30 mM MCl2O) were titrated gradually to the aforementioned solution of L1. After shaking the sample for a couple of seconds, the UV-vis spectra were taken.9 μL of a 10 mM solution of L1 (0.01 mmol in 1 mL MeOH) were added to 2.991 mL of MeOH to make a final concentration of 30 μM. Afterwards, 0.50–6.00 μL of a 30 mM Zn-solution (0.03 mmol in 1 mL of H2O) were titrated gradually to the aforementioned solution of L1. After shaking the sample for a couple of seconds, the fluorescence spectra were measured.9 μL of a 10 mM solution of L1 (0.01 mmol in 1 mL of MeOH) were added to 2.991 mL of MeOH to make a final concentration of 30 μM. Afterwards, 0.25–6.00 μL of a 30 mM Zn solution  were titrated to the aforementioned solution of L1 followed by the addition of 30 μL of a ZnCl2 solution. After shaking the sample for a couple of seconds, the fluorescence spectra were taken.9 μL of a 10 mM solution of L1 (0.01 mmol in 1 mL of MeOH) were added to 2.991 mL of MeOH to make a final concentration of 30 μM. Afterwards, 30 μL of a 30 mM MCl2. Then, 2.7, 2.4, 2.1, 1.8, 1.5, 1.2, 0.9, 0.6, and 0.3 mL of L1 were transferred to individual vials. Afterwards, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4 and 2.7 mL of the Zn2+ solution were added separately to yield a total volume of 3 mL. After shaking the sample for a couple seconds, the fluorescence spectra were taken.90 μL of a 10 mM solution of L1 were added to 29.91 mL of MeOH to make a final concentration of 30 μM. This procedure was repeated for ZnCl2  were dissolved in DMSO-d6. Afterwards their 1H NMR spectra were measured.Samples of L1 in presence of different equivalents of anhydrous ZnCl2O  samples at different pH values were prepared by addition of dilute NaOH or HCl. After the desired pH value was set, 9 μL of L1 were added to 2.991 mL of pH-adjusted MeOH : H2O to make a 30 μM concentration. Afterwards, 6.0 μL of a 10 mM ZnCl2-solution (0.03 mmol in 1 mL H2O) were added to the aforementioned sample. After shaking the sample for a couple of seconds, the fluorescence spectra were taken.A series of MeOH : H27–29 This DFT package was run on a MS Windows 10 Pro based (Version 21H1) PC system equipped with an AMD Ryzen Threadripper 3970X 32-Core and 128 GB RAM in combination with the appropriate message passing interface MS-MPI 10.0.12498.5. MMFF optimized structures were used as starting geometries for the geometry optimizations with the recently published robust “Swiss army knife” composite method r2SCAN-3c of Grimme and co-workers30 with D4 dispersion correction and geometrical counter poise correction applying the modified triple-zeta basis set def2-mTZVPP. Subsequent frequency calculation of the final structure evidenced the absence of imaginary frequencies and thus the presence of true minima on the potential energy surface. In case of calculations that include a solvent, the Conductor-like Polarizable Continuum Model (CPCM) implemented in ORCA 5 was applied.DFT calculations were performed using ORCA 5 of Neese and co-workers.www.wavefun.com) with the implemented NMR calculation method based upon the hybrid density functional with dispersion correction ωB97X-D by Chai and Head-Gordon31 and the standard basis set 6-31G*. The calculation software was run on the abovementioned MS Windows 10 Pro PC system equipped with the AMD Ryzen Threadripper 3970X 32-core and 128 GB RAM.Additionally, DFT calculated anisotropic NMR shifts of tautomers L1A and L1B were obtained by means of SPARTAN'20 acetamide and the subsequent reaction with 5-methoxy-2-mercaptobenzimidazole in acetone yielded the desired chemosensor L1 in 91% yield  in comparison to L1B (Δδ = 1.6 ppm). The structure elucidation was strongly supported by DFT NMR shift calculations using the ϖB97X-D functional and the 6-31G* standard basis set within the concurrent Spartan'20 software.31 An additional shift prediction33 also promoted the structural assignment  in the diluted DMSO-d6 solution .35 Further addition of Zn2+ past 1 eq. did not cause significant changes regarding the fluorescence intensity.To examine the binding properties of L1, titration experiments were conducted 35Additionally, UV-vis experiments have been conducted to further examine the binding properties. The UV-vis absorbance spectra of L1 (30 μM) in methanol display two distinct absorption bands at 241 and 300 nm, respectively . These b12,21,35 Furthermore, the incremental addition of Zn2+ (0–2 eq.) showed saturation at a L1–Zn-ratio of 1 : 1, as the absorption ratio A360/A300 did not change significantly after 1 eq.  427.0199 and 462.9965 were attributed to [L1 + Zn2+-1]  and [L1 + Zn2+ + Cl−] , respectively. The 1 : 1 complexation was further confirmed by the Benesi–Hildebrand method .8,39The binding constant was calculated to be σ/s.40 Herein, σ represents the standard deviation of blank measurements and s is the slope between the fluorescence intensity and Zn2+ concentration  was calculated by the equation 3n Fig. S3. The sta−7 M, which proved to be much lower than the WHO guideline (76 μM) for Zn2+ ions in drinking water.41 In comparison to other studies, our determined LOD appeared to be lower than reported Zn2+ chemosensors . As seen in 2+, exhibits the strongest fluorescence emission at a pH value of 8. In contrast, under strongly acidic or basic conditions a considerable fluorescence quenching is observed. At low pH values, this might be attributed to possible protonation of nitrogen sites in quinoline or benzimidazole moieties.46 Fluorescence quenching at higher pH values might be due to the deprotonation of NH fragments resulting in a stronger PET towards the fluorophore. Nevertheless, in the pH range from 4 to 10 L1 exhibits a satisfactory fluorescence response with a peak at a pH value of 8, thus demonstrating that the detection of Zn is possible under physiological pH conditions.The pH-dependence of various quinoline chemosensors has been reported.2+ ions  = 0.11 ppm; e.g. Δδ(H-7′) = 0.16 ppm]. The third complexation site can be identified by 13C NMR spectroscopy. Thus, the addition of Zn2+ ions induced a significant shift of the 13C NMR resonance frequencies of the carbonyl carbon atom to higher fields [Δδ(CO) = 0.159 ppm, Fig. S8−1 in the zinc complex  this crucial band is found with excellent congruence at 1598 cm−1  of L1A–Zn . Spectroscopic studies have shown that a 1 : 1 complexation takes place, which upon addition of EDTA showed the possible reversibility of the L1–Zn complex. The prototropic tautomerism exhibited by the benzimidazole moiety was used as proof to successfully identify the binding sites. Furthermore, the capability of L1 to quantify Zn2+ in water samples was shown. Hence, we believe that L1 shows a great potential for use in both biological and environmental applications.To summarize, we have designed and synthesized a new chemosensor L1 based on 8-aminoquinoline bearing a benzimidazole moiety. L1 showed a high selectivity and sensitivity towards ZnThere are no conflicts to declare.RA-011-D1RA05591G-s001


Graphene-based nanomaterials have gained a lot of interest over the last years in flexible electronics due to their exceptional electrical, mechanical, and optoelectronic properties, as well as their potential of surface modification. Their flexibility and processability make them suitable for electronic devices that require bending, folding, and stretching, which cannot be fulfilled by conventional electronics. These nanomaterials can be assembled with various types of organic materials, including polymers, and biomolecules, to generate a variety of nanocomposites with greater stretchability and healability, higher stiffness, electrical conductivity, and exceptional thermal stability for flexible lighting and display technologies. This article summarizes the main characteristics and synthesis methods of graphene, its oxidized form graphene oxide (GO), and reduced GO derivative, as well as their corresponding polymeric composites, and provides a brief overview about some recent examples of these nanocomposites in flexible electronic applications, including electrodes for solar cells and supercapacitors, electronic textiles, and transistors. It is considered as the starting point of other carbon nanomaterials with different dimensionalities, such as 0D fullerenes, 1D CNTs, and 3D graphite, since these can be assembled based on this unit . Ot. Ot45]. Exfoliated graphene have also been deposited on flexible poly (PEN) substrates, and the resulting nanocomposite was used as an anode in OSCs. This anode showed a high transmittance, a small sheet resistance, an efficiency of 4.2% and a high stiffness that were maintained after 150 bending cycles. Flexible OSCs incorporating a low-pressure CVD graphene with a PEDOT:poly(ethylene glycol) (PEDOT:PEG) block copolymer were developed. They had very good conductivity and transparency and were applied as anode and cathode in conventional and inverted cells , reachin−2, corresponding to a specific capacitance of 550 F g−1 when the entire surface area is used , th, th−2, c−1, which preserved around 90% of the original value after a hundred of cycles [−1.A self-standing 3D PANI/rGO foam was prepared via an in situ polymerization method with the aid of a template, to obtain a specific capacitance of 700 F gf cycles . In addif cycles , which s2SO4–polyvinyl alcohol (PVA) electrolyte was prepared, which was converted into a textile for wearable electronics. Binder free composite electrodes with multilayer graphene and PPy nanowires have also been prepared, which showed a maximum capacitance of 160 F g−1 for the highest scan speed. Novel flexible graphene/PPy nanocomposite films were manufactured using a pulsed electropolymerization technique [−1 was attained for a whole buildup time of only 2 min  was used as green reducing agent to obtain rGO/TiO2 films. Third, annealing was performed at different temperatures to obtain films with diverse titania crystalline phases. Lastly, PPy was deposited onto rGO/TiO2 composites. The TiO2 nanoparticles improve nanocomposite wettability, leading to high capacitance and good cycling stability. Moreover, different TiO phases had different behavior. Anatase had higher capacitance while rutile had better stability.Graphene/PPy composite fibers with diameters ranging from 15 to 80 μm have been developed via wet-spinning approach . The fibechnique . A maximly 2 min , about fr stages : first, Phenolic resin-based laser-induced graphene patterns have also been prepared for application in flexible supercapacitors . This tyWearable electronic devices, like e-textiles, are of great interest for use in multifunctional fabrics, portable electronic devices, and wearable displays . To accoA novel e-textile that can be prepared as yarns or fabrics was prepared with rGO and nylon-6 . They weIn a recent study, the production scale dyeing approach was used to prepare a fabric coated with GO, which was converted into rGO with a sustainable reductor to yield extremely conducting textile electrodes. Then, using the layer-by-layer method, these textiles were subsequently coated with conductive polymers to develop breathable, flexible, and washable electrodes . The effGraphene is highly valuable for flexible electronics but simultaneously needs band-gap opening for digital applications. This nanomaterial can be applied as an electrode in field-effect transistors (FETs), which need high transparency combined with elevated conductivity. It can act as a source and drain, as well as a channel layer in any type of FET structure. The idea is to find a great capacitive, easily printable, tough, and well-matched material for gate dielectric. Polymeric electrolytes can be an optimum selection, though their time-consuming response with frequency impedes attaining a good TFT performance. Taking into account compatibility with graphene, GO can be chosen as an insulator, though the development of high-quality GO film is essential. In this regard, organic field-effect transistors (OFETs) are more attractive as they can easily be fabricated by printing method and exhibit a good on/off ratio.Highly conductive and flexible graphene-based textile composites for OFETs have been recently developed. They were arranged by vacuum filtration and wet-transfer of GO onto flexible PET textiles combined with the addition of AgNPs. In particular, the transistor devices were fabricated with a bottom-gate top-contact structure, as depicted in −1. The transistors presented excellent stability in terms of device performance, which was preserved following a thousand of bending cycles and preserved for 1 month out of the glove box [Analogously, flexible fiber-type FET with graphene/Ag hybrid fibers as highly conductive electrodes were developed via wet-spinning and an adapted wet-drawing process. The fibers were then mixed with AgNPs, which showed elevated electrical conductivity, up to 16,000 S cmlove box .Portable and wearable electronics are becoming extremely popular, since devices make life more secure, healthier, and more relaxed. In particular, flexible electronics have gone through important progresses in recent years, due to the miniaturization of technology and the wireless revolution. Henceforth, novel materials need to be explored. Graphene is regarded as one of the most talented materials for the next-generation of flexible electronic applications, due to its brilliant optical, mechanical, and other characteristics. However, most of the graphene-based devices developed up to date are time-consuming and need multi-stage fabrication routes, which are neither scalable nor suitable for industrial production. Moreover, they typically display poor electrical conductivity, washability, and flexibility. In order to solve these issues, they can be combined with polymers to manufacture nanocomposites with enhanced stretchability, superior mechanical strength, conductivity, and stability. Another approach is the combination with other nanoscale fillers like silver nanowires, that also display optimal properties for flexible electronic applications . However


Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research.In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of thein vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of anin vivo model in terms of complexity but are also genetically manipulable and optically transparent.Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches.Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos.Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existingin vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research. Research highlightsScientific benefit(s)•Endothelial cell (EC) proliferation can be visualised at the single cell level in live embryos.3Rs benefit(s)•Zebrafish embryos can be used to partially replace mouse models in early cardiovascular disease research.•This time-lapse zebrafish embryo proliferation assay is a non-terminal refinement ofimmunolabelling and cell labelling approaches, which require tissue fixation.Practical benefit(s)•Only one tank of adult zebrafish are required to produce transgenic embryos for this method (20-40).•Fewer than 30 embryos are required for time-lapse experiments, thus fewer adult pairs are required which reduces breeding stress.•Use of transgenic zebrafish lines allows for proliferating cells to be visualised in real-time without use of embryo fixation, compared to immunolabelling and cell labelling approaches which require embryo fixation.•The zebrafish EC proliferation assay allows simultaneous quantification of proliferation in up to 24 embryos. Assuming four embryos are used as controls, this makes it possible to compare four treatment groups where each group has n = 5 animals. A comparable experiment using 24 mice would require each mouse to be culled, which incurs significant additional cost and effort, and may be difficult to justify, particularly for screening experiments where the potential benefits of the research are unknown.Current applications•Screening for genes involved in EC proliferation in response to blood flow.Potential applications•As a screening assay to identify new drugs/agents that prevent pro-atherogenic changes in endothelial cell proliferation in response to flow.•Screening for genes involved in migration and apoptosis in response to flow by observing cell movement throughout the time-lapse, effects on apoptosis could be screened by counting the number of ECs undergoing apoptosis either by looking for blebbing of EC nuclei, or using a different transgenic.Endothelial cell (EC) proliferation is vital for vascular development and homeostasis, but is also associated with increased vascular permeability which is a key driver of atherosclerosis.In vitro models can be used for high throughput analysis of the genes and pathways that control EC proliferation, but do not model the interaction of different tissues and are therefore reductionist. Conversely, rodents and other mammals have provided physiologically relevant models, such as the ApoE mutant mouse line which is thede facto model of plaque formation in CVD research. Whilst mice are an excellent model for study of genes involved in atherosclerosis , they are unsuitable for screening multiple genes at once as generating mutants is costly, time consuming and may be difficult to justify due to concerns about the effect of mutations on animal welfare and the number of animals that would be required in such a study. Zebrafish embryos survive by oxygen and nutrient diffusion, thus mutations which are embryo lethal in mammals such as abolished development of ECs (cloche) or targeted knock-down of cardiac troponin (sih) are not lethal in zebrafish embryos. These characteristics mean zebrafish embryos are uniquely amenable to study of genes involved in ECs, and EC responses to flow.Like most forms of human disease research, cardiovascular disease (CVD) studies conventionally identify genes for in-depth research by first applying wider screening approaches. The models used in these types of research have several advantages and disadvantages.in vitro models are multiple. One pair of adult zebrafish can produce hundreds of embryos in one day, and with just an incubator and E3 medium these embryos will each develop a complete cardiovascular system in two days,in vitro models, far greater simplicity than comparable three-dimensionalin vitro models, and comparable biological relevance to mammalianin vivo models but with reduced cost and reduced detriment to animal welfare.Whilst zebrafish embryos are not protected prior to 5 days post fertilization (dpf) in the UK, they are still live organisms and therefore present a partial replacement to use of other animals in CVD research. Nonetheless, the advantages of using zebrafish overThe zebrafish embryo is ideal for studying vascular biology, it is small, optically transparent, and allows manipulation of gene expression using morpholino oligonucleotides–in vivo andin vitro models; immunohistochemistry (a technique widely used to study protein expression in mouse endothelium using theen face technique), DNA labelling (used in cell culture and mice) and time-lapse imaging, to determine which method detected the greatest number of proliferating ECs. Indirect approaches for studying proliferation such as cell number counting, viability, and metabolic activity assays were not pursued as their results can be influenced by migration and apoptosis.Zebrafish have been used to model EC apoptosis and migration in response to blood flow.fli1a:nls-mCherry), (fli1a:LifeAct-mClover),fli1:EGFP) lines were used.,fli1a:nls-mCherry expresses a red fluorescent protein linked to a nuclear localisation sequence under the endothelial promoter, resulting in red fluorescence in EC nuclei. Tgfli1a:LifeAct-mClover expresses a f-actin:mClover fusion protein under the endothelial promoter, resulting in visualisation of the EC actin cytoskeleton. Initially, we intended to compare EC proliferation in embryos with and without blood flow, thus for early experiments (whole mount immunostaining) heart contraction was inhibited by silencing troponin T2A by injection oftnnt2a ATG morpholino at 3 ng final dose (sequence 5′-CATGTTTGCTCTGATCTGACACGCA-3′). As EC proliferation was not detected at this experimental stage, flow and no-flow comparison was stopped to minimise our use of embryos and only embryos with blood flow were used for subsequent experiments.Zebrafish care and experimental procedures were carried out under Project Licence 70/8588 issued by the UK Home Office and local ethical committee approval was obtained. Adult zebrafish were kept at a constant temperature of 28 ± 1°C and at pH 7.5 ± 0.5. They were subjected to a light/dark cycle of 14 hours of light and 10 hours of darkness. Adult zebrafish were fed a diet of artemia and dry Zebrafeed™. For mating, pairs of male and female zebrafish were placed into mating tanks and separated by a divider to allow accurate timing of fertilisation. For experiments where this was not required, a box for embryo collection with a mesh insert and marbles on top was placed in the fish tank to harvest progeny. The Nacre lineAdults: For all experiments described, embryos were produced by pair mating eight male and eight female adults of the same transgenic line. This was done to minimize the chance that zero pairs produced embryos, and to minimise confounders by limiting the genetic diversity of the embryos. Adults do not suffer pain or distress from pair mating and were returned to their home tanks immediately after producing embryos, home tanks contain environmental enrichments such as fake jellyfish and fake seaweed. No adults suffered adverse events as a consequence of this work.Tg fli1:EGFP for PCNA, andTg fli1a:nls-mCherry for EdU) then three embryos were randomly selected for imaging. For time-lapse imaging experiments, embryos were screened forTg flia:nls-mCherry andTg fli1a:LifeAct-mClover, then three embryos were randomly selected for imaging. Embryos not selected for imaging were humanely destroyed using bleach.Embryos: No embryos used in this study exceeded 5.2 dpf. For all experiments described, embryo collection stopped after 60 embryos were obtained. Sex of embryos was not determined (as is standard practice in zebrafish research). For PCNA and EdU experiments, embryos were treated then screened for the endothelial marker ((fli1:EGFP) embryos were fixed in 4% paraformaldehyde (PFA) at 30 hpf to allow screening for proliferation in the main trunk vascular beds, then whole mount immunostaining was performed using anti-PCNA antibody  and AlexaFluor-568 mouse anti rabbit secondary antibody . Colocalization of PCNA andfli1-EGFP was used to define proliferating ECs. Embryos not treated with PCNA antibody were imaged during protocol optimization to ensure the specificity of PCNA antibody. Detection of cell proliferation was defined as where a nucleus is both positive for PCNA and forfli1-EGFP.TgThe Click-iT™ EdU Cell Proliferation Kit (Thermo Fisher) was used to label DNA of proliferating cells with AlexaFluor-488. The protocol was modified for zebrafish as follows:fli1a:nls-mCherry) were collected at 30 hpf and incubated in EdU solution  at 28°C for 1 h. Larvae were fixed in 4% PFA overnight then washed and transferred to permeabilization solution (supplied in kit) for 1 h. Permeabilised larvae were then treated according to manufacturer protocol. Proliferating ECs were defined as cells positive forfli1a:nls-mCherry and EdU.Zebrafish larvae with Tg (tnnt2a morphant embryos that lack a heartbeat. The morphant phenotype (no heartbeat) was observed in all (100%) of injected embryos, survival is indistinguishable from wildtype up to the time point tested (30 hpf).Standard light microscopy was used to screen out dead or undeveloped embryos, and to identifyfli1a:nls-mCherry orfli1:EGFP after they were non-terminally anaesthetised using E3 medium. Fluorescence screening was done using a ZEISS Axio Zoom V16 at 28 hpf. Zeiss filter set 63  was used. Camera mode was used and pixel binning set to 5 × 5 to maximise sensitivity to the fluorophore. Screened embryos were washed in E3 media without tricaine, then transferred into fresh E3 prior to further imaging.Fluorescent microscopy was used to identify and select embryos containingFor confocal imaging, embryos were non-terminally anaesthetised using tricaine and mounted in 1–2% agarose over which standard E3 medium was placed. Still images were taken using a Nikon A1 confocal microscope at 1024 × 1024 resolution (0.62 μm per pixel), z-stacks were taken in 8-μm increments. Time lapses were taken using a Zeiss LSM880 in Airyscan mode. Images were captured at 1848 × 1848 resolution (0.47 μm per pixel), z-stacks were taken in 3.43-μm increments. Images were analysed using NIS Elements or ZEN respectively. Composite images were generated by performing maximum intensity projection of the stack data, no other processing was performed. For both microscopes, excitation was done by a 488 nm and 568 nm laser.,fli1a:nls-mCherry nuclei visibly divide over the course of the time lapse. Experimental embryos were then euthanised using bleach.For light sheet microscopy,The number of proliferation events detected using EdU labelling, time-lapse confocal microscopy and PCNA immunolabelling were compared using a two-way ANOVA (n = 3 embryos per each group). Each animal was treated as a single unit of analysis. Analysis was done using GraphPad PRISM (RRID:SCR_002798), an open-source alternative to this is JASP (RRID:SCR_015823).4 and 0.33 mM CaCl2 in dH2O. 4% PFA: paraformaldehyde diluted to 4% with E3 (One month shelf life when stored at 4°C).tnnt2a MO: diluted to a 100 μM stock with dH2O. Stock concentration should be determined using a Nanodrop and diluted to 3 ng/nL for microinjection. PBS: phosphate-buffered saline (ThermoFisher) diluted from 10× stock. PBST: phosphate-buffered saline with Tween-20. PBS made to 0.1% Tween-20. PDT: phosphate-buffered saline with 1% DMSO and 0.1% Triton X-100. TRIS buffer: tris base diluted in dH2O to 1 M stock. 150 mM solution was prepared from stock and pH adjusted to 8.0. Tricaine: 4 g tricaine methanesulfonate in 1 L dH2O pH 7–7.5. Blocking solution: 1% bovine serum albumin in PBS. Goat serum: goat serum stock (frozen) diluted to 10% in blocking solution. Bovine serum albumin (BSA): stock albumin powder should be added to dH20 to make 10% (w/v) BSA solution, this solution should be aliquoted into 1.5-mL Eppendorf tubes and frozen for use as needed. Click-IT EdU AlexaFluor-488 Flow Cytometry Assay Kit – ThermoFisher C10420.E3 medium: 5 mM NaCl, 0.17 mM KCL, 0.33 mM MgSO1)Collect embryos and transfer to a petri dish of E3 media.2)Incubate embryos at 28°C until at least 24 hpf.3)Insert forceps to gently pierce the embryo chorion (Sup. 1 – 1).4)Gently open forceps to tear the chorion (Sup. 1 – 2).5)Open forceps until embryo is free of the chorion (Sup. 1 – 3).1)fli1-EGFP embryos and transfer to a petri dish of E3 media then incubate at 28°C to 30 hpf.Collect2)Dechorionate embryos.3)Fix in 4% PFA overnight at 4°C – now protect samples from light as far as possible until imaging.4)Wash in room temperature PBST  on a rocker for three 10-min washes.5)Wash in room temperature TRIS buffer (150 mM pH 8.0) on a rocker for 5 min.6)Equilibrate embryos in TRIS buffer at 70°C for 15 min.7)Wash in room temperature PBST on a rocker for two 5-min washes.8)Rinse with distilled water on ice.9)Wash in chilled acetone (−20°C) for 15 min.10)Rinse with distilled water for three 5-min washes.11)Block with goat serum (10%), BSA (2%), and PBT  for 4 h at 4°C.12)Incubate with primary antibody overnight at 4°C (Dilute PCNA 1:250 in blocking solution).13)Wash in PBT for four 30-min washes.14)Incubate with secondary antibody (AF-568 anti-rabbit diluted 1:500 in blocking solution).15)Wash in room temperature PBST five times for 5 min.16)Mount as described in ‘Mounting individual zebrafish embryos for confocal imaging’.17)Image using a confocal microscope as described above.1)fli1a:nls-mCherry embryos and incubate at 28°C until 30 hpf.Collect Tg2)Dechorionate embryos.3)Incubate embryos in labelling solution  at 28°C for 1 h.4)Fix in 4% PFA at 4°C overnight.5)Wash with PBST for three 5-min washes, rock samples each time.6)Permeabilise in 1× saponin solution  for 1 h at room temperature while rocking samples.7)Incubate with reaction cocktail  for 1 h in the dark at room temperature whilst rocking.8)Wash five times in PBST for 5 min whilst rocking.9)Mount as described in ‘Mounting individual zebrafish embryos for confocal imaging’.10)Image using a confocal microscope as described above.1)Take treated embryos and mount on glass slides in vectashield (RRID:AB_2336789). Embryos must be laid flat on their side. This occurs naturally for young embryos (~30 hpf). If older embryos are used, the yolk may prevent embryos from lying flat, in this case the embryo yolk and head can be cut off using a scalpel.1)fli1a:nls-mCherry embryos and incubate at 28°C until 26 hpf.Collect Tg2)Dechorionate embryos using forceps.3A)For live imaging using LSM, suspend embryo(s) in melted low-melt agarose (37°C 0.8–1.2%), then use a plunger to draw the embryo(s) into a glass capillary. Wait 2 min for the agarose to set, then suspend the embryos (within the agarose column) into the LSM by gently pushing the plunger.3B)For confocal imaging, add embryo(s) to melted low melt agarose (37°C 0.8–1.2%) then draw up embryo(s) and decant into an Ibidi μ-Dish 35mm (Ibidi cat no – 81156). Use thin-tip forceps to gently push the embryo into a side-mounted position, then wait for agarose to set.4)Image using appropriate microscope ensuring 587 nm excitation is active. Adjust magnification to visualise as much of the trunk as possible whilst retaining nuclear resolution . Z-range should traverse the entire trunk vasculature and Z-interval should be set no lower than 8 μm.5)For euthanasia, discard embryos into bleach. For revival, return embryos to E3.1)Collect embryos and transfer to 28°C E3 media.2)Take a glass slide and place it in a petri dish so it is unable to move further to one side.3)With a Pasteur pipette, draw up embryos then, holding the glass slide petri dish slightly tilted, dispense embryos along the edge of the glass slide (Sup. 2).4)Prepare a glass needle  using a Sutter P1000 glass capillary puller. Suitable settings are heat 475, pull 60, vel 80, time 135, pressure 500. Refer to the user manual for capillary pulling optimization.5)tnnt2a morpholino to a glass needle and calibrate the ejection volume using a graticule .Add injection media containing 3 ng/nL of6)Using a manipulator (World Precision Instruments M3301) and PicoPump (H. Saur PV820), inject 1 nL of morpholino into each embryo until desired number of treated embryos are obtained (some extra embryos should be treated to account for failure to develop and death).We analysed EC proliferation in the trunk vasculature of zebrafish embryos because this vascular bed allows simultaneous imaging of the intersegmental vessels and dorsal aorta . We atteTg fli1a:nls-EGFP) were fixed at 30 hpf then treated with anti-PCNA antibody. Imaging of the embryo trunk detected many PCNA+ cells in the caudal vein plexus (CVP) and none in the ISVs , whereas immunostaining is restricted to detecting markers of proliferation at a single time point. To assess the effectiveness of EdU staining, transgenic embryos expressing endothelial nuclear localised mCherry (Tgfli1a:nls-mCherry) were treated with EdU from 30 to 31 hpf. Embryos were then fixed and treated with fluorescent azide. Between zero and three proliferating ECs were detected in the trunk of treated embryos, with detection occurring in the DA, but not the ISVs (The cell labelling approach works by incorporation of thymidine derivatives such as EdU into newly synthesised DNA. The modified thymidine is labelled with a fluorescent azide thereby enabling the identification of nuclei which have undergone proliferation.he ISVs .fli1a:nls-mCherry) and cytoplasmic LifeAct-mClover (fli1a:LifeAct-mClover) using a light sheet microscope. Using this approach, EC proliferation was quantified by counting the division of EC nuclei in all visible ISVs from 26 to 30 hpf  which has well described toxic effects above the 2% level. 30 hpf .Detection of EC proliferation in the intersegmental vessels was compared for each method. The ISVs are isolated from other tissues, so it was easiest to spot proliferation in this vascular bed. There are also relatively few nuclei in the ISVs, and ISV nuclei are less densely packed than nuclei in the DA/CVP. PCNA did not detect any proliferation events in the ISVs, EdU labelling detected fewer than one proliferation event on average, whereas time-lapse imaging detected an average of nine proliferation events . Two-wayper se, as evidenced by detection of PCNA positive nuclei . This observation is comparable to those observedfli1a:nls-mCherry, fli1a:lifeAct-mClover) enabled direct visualisation of EC nuclei division in proliferation  because of a larger detection window  and7.•Greater breadth of data collected. Time-lapse data could also be analysed for cell migration and apoptosis as desired.Despite this, EdU labelling is unsuitable to analyse the effects of interventions on proliferating ECs because of the limited window of detection and low number of proliferating cells detected. Therefore, we sought a method to capture greater numbers of proliferating EC from zebrafish embryos. We found that light sheet time-lapse microscopy of transgenic embryos  allow real-time imaging of EC proliferation, but lack biological relevance. Whereas establishedin vivo models  are biologically relevant yet cannot be used for real-timein situ monitoring of EC proliferation, and must be culled to isolate vascular tissue for analysis. By using the zebrafish embryo to study EC proliferation, researchers can achieve real-timein situ monitoring of EC proliferation in a biologically relevant model whilst replacing mammals and reducing or eliminating the use of terminal procedures, thus achieving partial replacement and refinement of use of animals in CVD research.This study demonstrates the utility of time-lapse imaging zebrafish embryos to measure EC proliferation. ComparableThis time-lapse approach is more suitable than other methods for quantifying EC proliferation in zebrafish larvae and has applications for studying the mechanisms of proliferation and for screening to detect compounds that modify this process, we are currently using this approach to use zebrafish larvae to screen for genes involved in EC proliferation in response to blood flow. Whilst the manual counting approach used currently is low-throughput, future work could automate detection of proliferation using image macros to enable widespread use of this system for identifying genes involved in atherosclerosishttps://www.doi.org/10.17605/OSF.IO/NV9XE.OSF: Underlying data for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’.The project contains the following underlying data:Raw images of three embryos imaged using the PCNA method, three embryos imaged using the EdU method, and three embryos imaged using the timelapse method.https://www.doi.org/10.17605/OSF.IO/Y2SCW.OSF: Extended data for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’.The project contains the following extended data:Supplementary Figure 1: Dechorionation of zebrafish embryos. 1) Insert forceps into embryo chorion, be careful not to damage the embryo. 2) Gently open forceps to tear the chorion further. 3) Widen the forceps until the embryo has escaped the chorion.Supplementary Figure 2: Preparing zebrafish embryos for microinjection. 1) Place a glass slide in a petri dish and tilt as shown. 2) Draw up embryos in a Pasteur pipette and decant onto the edge of the glass slide as shown. 3) Use the Pasteur pipette to draw up any excess liquid from the petri dish, leaving a row of zebrafish embryos against the glass slide.https://www.doi.org/10.17605/OSF.IO/Y2SCW.OSF: ARRIVE 2.0 checklist for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’.Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The Article by Bowley et al. describes a novel approach to quantify the relatively slow rates of endothelial proliferation in vivo, using zebrafish embryos, rather than in vitro models. The approach has several advantages over using other animal models to examine endothelial proliferation in vivo, including the potential use of zebrafish lines that use fluorescent reports for cell-type differentiation, the ability to examine proliferation pre-post heart beat, to determine any effects of flow and easy genetic manipulation through genome editing or knockdown with morpholinos. As such, this model provides a flexible platform to examine drug or gene regulation of  proliferation. The text and methodology is clearly described and in sufficient detail to allow other scientists to replicate the experiments described here. As such it is a useful step forward to quantify in vivo proliferation while minimising the use of protected species. The platform is sufficiently flexible to be used to advance the field on this topic.Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Endothelial biomechanics and endothelial dysfunctionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. determine the feasible method to quantify endothelial cell (EC) proliferation which regulates vascular formation and homeostasis in zebrafish embryos to investigate development via a comparison between three different comparative analyses: antibody, DNA labeling, and time-lapse imaging. Although rodent models are predominantly used to explore the mechanism of EC proliferation in cardiovascular disease or development, the authors pointed out there is a limitation - time-consuming, costly, and detrimental to animal welfare. The authors concluded that time-lapse imaging is accurate, precise, scalable, simple, and effective than the other two methods: antibody (PNCA) or DNA labeling (EdU) in EC proliferation study in the zebrafish embryo model. This method paper is necessary to approach EC proliferation in zebrafish and is well designed to compare three different methods, however, there are major and minor points that need to be improved.The authors are focusing on the quantification of EC proliferation using three different methods, however, vascular endothelial growth factor (VEGF) is the most common marker for EC proliferation. Please address why the authors chose PCNA for detecting proliferating cell nuclear antigens and EdU for measuring DNA proliferation instead of VEGF, and it is necessary to be discussed.EdU labeling is a well-established method to quantify S-phase cells. The authors performed EdU labeling in zebrafish embryos for 1 hour and showed that it did not label as many cells as they observed via tracking nuclear division over 6 hours . This does not translate to EdU labelling being unable to provide accurate measurements. Instead, it simply prompts a longer period of EdU incubation. The authors argue that the high concentration of DMSO used in the labeling solution is toxic to embryos for long-term incubation, however, there is no necessity of using DMSO during EdU incubation as EdU is soluble in aqueous buffers. The authors should perform extra experiments on this.On the other hand, there are large amounts of EdU+ cells (green) colocalization with ECs (red) in Figure 3. The authors should also test their hypothesis at different regions of the vascular network, not just ISV.In Figure 2, to see the clear effects of tnnt2 morphant (inhibition of heart contraction) in EC proliferation, the control group (control morphant) should be presented. The authors also need to provide negative control images with only secondary antibodies to validate the staining, as virtually every cell in the image was red, meaning PCNA+. However, PCNA staining should give fewer labeled cells than EdU staining and time-lapse tracking, according to the authors. It can also be that the PCNA antibody is not working well. The authors need to address this.In addition, to rely on the comparison, Figures 2 and 3 should be offered the same enlargement.In Figure 5, two-way ANOVA is an advisable statistical method among the groups.The graphical abstract  is arduous to comprehend because of a lack of a rationale for the limitation of immunostaining and EdU labeling. The authors should offer the reference or evidence to elaborate the experimental design.In this method article, BowleyIs the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?PartlyAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors’ clearly set out the context for their study; developing a non-invasive experimental system to accurately capture spatial and temporal information on EC proliferation in the vasculature would be important step forward. Three techniques are explored for achieving these aims. Two  do not appear to work for the intended purpose but it is not immediately clear from the data shown why. More ‘negative’ data would be very helpful in setting out the limitations of these techniques so that others can appreciate why these approaches ‘failed’. One technique, time-lapse imaging of nuclear division (where labelling driven by a EC-specific promoter) does appear to have some future applicability, although the technique, at present, is limited to manual counting and and very low numbers of experiments are reported in support of this approach. There are a number of issues with the current version of the manuscript that require attention:Abstract The abstract refers specifically to n=24 experimental units, where in the example given, n=4 comprise controls and then four groups of n=5 ‘treated’ groups. These numbers seem rather arbitrary and are not referred to in the paper.Introduction / General It would be helpful to include more detail on how the ZF embryo CV system recapitulates key features of mammalian circulatory system. Please provide clarity (and perhaps illustrative detail) on how ZFe’s “complete cardiovascular system” has a “simplicity comparable with that of two dimensional models in vitro” and “comparable relevance to mammalian in vivo models”. This might help uptake by those researchers typically using mammalian models of vascular plasticity. Blebbing of nuclei is not a unique marker of apoptosis. Autophagy and other activation of modes of cellular degradation are characterised by blebbing. The authors imply that apoptosis is the sole mechanism of cell death in ZF embryos. Is this correct? No data on cell death is reported in the manuscript so the references to apoptosis don’t seem to have much relevance in the context of the paper.MethodsHow are male and female zebrafish distinguished?Does the Nacre line have identical vascularization as wild-type?You state that “both transgenic lines use Nacre as background” but the study uses three TG lines.How leaky, or not, is the EC-specific promoter. Can you be sure that expression is only in EC lineages? This is a key issue since your method relies on typing ‘EC-specific’ expression.A reference to the inhibition of contractility by tnnt2a morpholino should be given.It is presumed that that TG-EGFP was used for PCNA and TG-mCherry was used for EdU to avoid the spectral overlap of fluorophores in each instance? This should be stated.More details on how the commercial EdU labelling kit results in the generation of a detectable 488nm signal should be included. What is the concentration of saponin? What is the range of EdU signal developed (i.e. time-dependent accumulation)? How homogeneous is this across cells/FACS populations?Brightfield microscopy is included as the means for discriminating dead or undeveloped embryos are identified. How? No brightfield imaging is included in the paper.Please define Light Sheet microscopy as LSM  laser scanning microscopy.The statement that “587nm excitation is active”. Please include the excitation source and what is meant by “active”.The methods include non-terminal anaesthesia using E3 medium (“Fluorescent microscopy”) or E3 medium and tricaine (“Light-sheet microscopy”). Please clarify. What is the final concentration of tricaine used in anaesthesia?Statistics. Please check that two-way ANOVA is an appropriate statistical test for data where n=3 .degrees C?).Microinjection - it is appreciated that the settings are specific for the Sutter puller but it would be helpful to include units as applicable  but its not immediately clear what itFigure 3 should be presented at the same magnification/zoom as Figure 2. Figure 3 should also be better integrated with your conclusion that EdU labelling was ruled out of contention as a technique for quantifying EC proliferation. Do you have any images showing  the effect of 15% DMSO on sample morphology beyond 1 hour? It is also somewhat misleading to state, as you do in Figure 7, that EdU labelling is limited by low detection of EC proliferation. The problem seems to be that the reagent is toxic which prevents longer-term imaging. It needs to made more clear that EdU is of limited use for mapping cellular proliferation occurring on a time-scale incompatible with reagent toxicity.Figure 4. Your findings suggest that to capture sufficient EC proliferation, an experimental window of up to 12 hours is required. Figure 4 only shows a plus-15min window. Some idea of how time-lapse capture of EC proliferation changes with time would be very helpful. Also, the arrowheads appear squashed and point away from the area you mean to depict.Figure 5 shows the number of proliferation events but this is not that useful. It would be much better if you showed this the number of as the proportion or percentage of mCherry cells scrutinized. ‘n’ is very low here and statistical analysis is not convincing .DiscussionIt is not possible to conclude, from the data shown, that nuclei-imaging is “accurate” . This will need much more data and deeper modes of analysis.As you note, “computer assisted quantification” would augment your approach, but this phrase is vague. What would you want to focus on, and what would need to be taken into account / eliminated etc. More discussion on how you see this approach being potentially worked-up into a medium- to high-throughput screening platform would be helpful.OtherZebrafish welfare and use section. The second-to-last sentence in the first paragraph needs splitting into two sentences. It would be helpful to define what constitutes an “adverse event” here.PCNA and EDU should be described in full at first mention. Has PCNA ever been used as a marker of proliferation during zebrafish CV development? (You state kidney but what about CV…….)The phrase “humanely destroyed using bleach” is inappropriate. It might be better worded as “embryos were destroyed using bleach”. Ditto “euthanised” on pages 5 and 8.The Supplemental Figures do not add to the article and should be omitted. Videos of the actual procedures would be much better.Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?NoAre the conclusions about the method and its performance adequately supported by the findings presented in the article?PartlyIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?PartlyAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?PartlyReviewer Expertise:Network behaviour of cardiovascular cell types / cell imaging / imaging technologies / signal decoding. Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Such results indicate graphene is an exceptional nanomaterial for future MIR optical interconnects.The mid-infrared  waveband is of great interest for integrated photonics in many applications such as on-chip spectroscopic chemical sensing, and optical communication. Thermo-optic switches are essential to large-scale integrated photonic circuits at MIR wavebands. However, current technologies require a thick cladding layer, high driving voltages or may introduce high losses in MIR wavelengths, limiting the performance. This paper has demonstrated thermo-optic (TO) switches operating at 2 μm by integrating graphene onto silicon-on-insulator (SOI) structures. The remarkable thermal and optical properties of graphene make it an excellent heater material platform. The lower loss of graphene at MIR wavelength can reduce the required cladding thickness for the thermo-optics phase shifter from micrometers to tens of nanometers, resulting in a lower driving voltage and power consumption. The modulation efficiency of the microring resonator (MRR) switch was 0.11 nm/mW. The power consumption for 8-dB extinction ratio was 5.18 mW (0.8 V modulation voltage), and the rise/fall time was 3.72/3.96 μs. Furthermore, we demonstrated a 2 × 2 Mach-Zehnder interferometer (MZI) TO switch with a high extinction ratio of more than 27 dB and a switching rise/fall time of 4.92/4.97 μs. A comprehensive analysis of the device performance affected by the device structure and the graphene Fermi level was also performed. The theoretical figure of merit (2.644 mW Mid-infrared  is a prAmong the on-chip photonic components, optical switches  (electroπP) of 0.39 mW. However, the low thermal conductivity of polymer leads to a long response time of 92.4 μs, and polymers also become opaque in the mid-infrared waveband. The figure-of-merit (FOM) of a TO device combines π-phase-shift power consumption and response time τ in the form of 1/(π·τP), which could be used to evaluate the performance of devices based on different materials and devices platforms [πP of 9 mW, respectively [Two-dimensional materials ,42 own rlatforms . Recentllatforms ,53. To flatforms  or photolatforms ,56. Siliectively . Taking ectively . Graphenectively . ChalcogπP power consumption of 14.42 mW, and comparable FOM of 0.0175 mW−1μs−1 to previously reported silicon TO switches with graphene heaters. Our MZI device showed a high modulation depth of 27.8 dB. Lastly, a complete numerical analysis of the device’s performance was demonstrated. The design strategy of the graphene heater and the performance limits was given. Optimal structural parameters and graphene with a Fermi-level of 0.43 eV for the MRR-based TO switch could achieve an ultrahigh FOM of 2.644 mW−1μs−1. In this context, we demonstrated the first silicon TO switch with graphene heaters operating at 2-μm mid-infrared waveband, which is also considered a new communication window for the next-generation optical communication  to meet 2 box layer by multi-project-wafer (MPW) involved processes. First, the waveguide structures were patterned by deep ultra-violet photolithography and etched by inductive coupling plasma (ICP) process to a ridge depth of 150 nm and width of 600 nm. Next, a layer of 1-µm-thick SiO2 cladding was deposited by plasma-enhanced chemical vapor deposition (PECVD). Then the silica cladding was polished by chemical mechanical polishing (CMP) to a thickness of about 300 nm (150 nm higher than the waveguides). The titanium/gold  contact electrodes were patterned by electron beam lithography (EBL) and deposited using electron beam evaporation. The contact distances were 2.6/7 μm for the MRR/MZI-based switches, preventing the excessive absorption from metal contacts. Then the chemical-vapor-deposition (CVD) graphene [The schematic illustration of the MZI device is shown in graphene  was trangraphene . The loaThe measurement setup is shown in The MRR-type switches play an essential role in silicon photonics. The MRR can behave as a spectral filter, which can be used for applications such as wavelength division multiplexing and sensing. The position and the shape of the dips are very sensitive to structural parameters. In most cases, the spectrum of the fabricated device could not be predicted precisely because of the fabrication error. Therefore, wavelength tuning is necessary for wavelength-relevant applications, and thermally modulating the refractive index of the waveguide in the microring resonator is a straightforward and effective approach. The switching function of an MRR works in the same principle .We fabricated MRR-based switches with a ring radius of 40 μm and a graphene length of 100 μm. The microscope image of the MRR-based TO switch is displayed in η of the switch was calculated to be 0.11 nm/mW by fitting the curve in πP of 14.42 mW, where πP of a cavity-type switch is defined as the power consumption to detune the resonator by a phase shift with a product of π and the full width at half maximum (FWHM) of the resonance peak [3 and the FWHM was 0.505 nm. By decreasing the propagation loss from the waveguide (1.62 dB/cm) [πP power consumption can be further suppressed. This could be improved by optimizing the fabrication process to reduce the scattering loss of the waveguide and doping graphene to ensure less absorption. The sheet resistance of the heater is calculated to be about 125 Ω from the current-voltage (I-V) curve shown in FOM of 0.0175 mW−1μs−1, which is the highest performance among the reported graphene-assisted silicon TO devices operating at the 2 μm waveband.The resonance wavelengths of measured transmission spectra were red-shifted, as depicted in nce peak . It shou2 dB/cm)  and absoThe MZI-type 2 × 2 optical switch is also one of the indispensable components in photonic integrated circuits, which is conventionally constructed by two 3-dB splitters/couplers using multimode interference (MMIs) and connecting waveguides as the two MZI arms. By tuning the optical phase difference of the light between two arms of the MZI, the optical intensity distribution of the output ports can be modulated, realizing optical switching for applications including cross-connection, optical routing, and information processing.We fabricated balanced 2 × 2 MZI TO switches with 100-μm-long graphene on both arms of the MZI to balance the loss difference between the two arms. The microscope view of the switch is shown in the inset of πP of 57.75 mW. Such high-power consumption was due to an extensively long contact distance which will be explained later.The light from two arms of the MZI with variable phase delay was combined by a 2 × 2 MMI, resulting in different power distribution at the output ports. FOM of the switch is calculated to be 0.003 mW−1μs−1.The dynamic response of the switch was also characterized and demonstrated in FOM comparable with TO switches using doped-silicon heaters. Although the doped-silicon TO modulators show better performance at present, our theoretical prediction implies that graphene is a more appropriate heater material at MIR waveband, which will be explained in the following session.To investigate the theoretical performance limits of graphene-based TO-switches, optical modeling of the waveguide modes and finite element method thermal simulations of the graphene phase shifter were carried out using Lumerical MODE solution and the COMSOL Multiphysics package, respectively. 0λ is the free-space wavelength, Δeffn represents the variation of effective refractive index, and gn is the modal group index. Based on the perturbation theory, the effective refractive index tuning ∆effn should be given by a surface integral about temperature, electric field, and refractive index [dn/dT is the material thermo-optic coefficient. The TO coefficients are approximately 1.76 × 10−4 K−1 for silicon [−5 K−1 for silicon dioxide [The thermally induced refractive index change causes the wavelength tuning of TO switches. The wavelength shift corresponding to the refractive index could be expressed by :(1)Δλ=λ0ve index :(2)Δneff silicon  and 1.3  dioxide  at 2 μm,FOM benefiting from the resonance effect. The critical factors for the graphene-on-SOI phase shifter are the cladding thickness (clh), contact distance (cd), and the resistance of the heating region. The measured resistance of the graphene-based MRR resonators is 125 Ω. It contained resistance (gR) from the graphene heater and contact resistance (Rc) between metal and graphene. Considering the sheet resistance of graphene is 1307 Ω/sq, cR would be 4550 Ω·μm. Thus, the divided voltage dU applied on the graphene heater in the simulation is equal to gU∙Rt + R/(Rc), where U is applied voltage across the electrodes. For a given length of graphene heater, the contact resistance would keep the same while distance between the two electrodes varied. clh) and contact distance (cd) on the temperature of the waveguide core. Although the increase of the contact distance cd would entail an increased divided voltage dU on the graphene heater, the increase of total resistance and thermal mass would lead to a decrease in temperature change ΔwgT in the waveguide with the increasing contact distance, shown in clh becomes thicker, the graphene heater will keep further away from the waveguide core. The ΔwgT will also decrease. For an MRR type graphene-based TO switch with a radius r of 40 μm and a heater length hl of 100 μm, the overall wavelength shifting could be expressed as follows:In this section, we focus on the analysis of MRR type TO switch since it could gain much higher λ profile, which is illustrated in η of switches with variable design parameters could be calculated byP represents the change of electric power. The electric power can be calculated by 2/RtU, where tR is equal to g + RcR.Based on Equations (2) and (3), we can convert the thermal profile to the wavelength shift Δclh and cd are presented in clh and cd are also desired for faster operation. The simulated response time is 3.735 μs which is very close to the experimental results of 3.96 μs.The response time of the thermo-optic switch is significant for high-speed light intensity modulation and optical routing. Finite element method (FEM) simulation could also be applied to study the dynamic heating progress. The temporal evolution of the temperature of the waveguide core was simulated under a square-wave driving bias with a period of 30 μs, and the time-domain response progress of devices with different FE) of graphene was 0.25 eV. Based on this Fermi-level, we studied the performance matrix of MRR type TO switches with different design parameters, as depicted in clh with cd being 2.6 μm and 7 μm, respectively, and the propagation loss from the metal contact could be neglected if the distance between two electrodes is larger than 2.5 μm. Thicker cladding is preferred for less propagation loss, but it will sacrifice the heating effect. Thus, if we want to design an optimal heater structure with graphene, a compromise should be made between the heater loss and heating efficiency. Here, we can notice that TO switches could achieve a higher heating efficiency and faster response if the distance between the graphene and the waveguide and the distance between the two electrodes are close enough. However, the overall performance of TO switches will also be limited by the absorption from graphene heaters and the metal electrodes. Although metal absorption is much larger than graphene, graphene is still a lossy material if its Fermi level is low. Q factor and the FWHM of the resonance peaks and consequently the power consumption. The power consumption for π = FWHM×Pπ/η and the relation between the total Q factor and the FWHM is expressed byrλ is the resonance wavelength. To obtain the best performance, the ring resonator should work in the critical coupling condition where the intrinsic Q factor equals the external Q factor:Q factor can be obtained byα is the loss of the ring, including the absorption of the graphene gα and the waveguide loss wgα (1.62 dB/cm). The gn is the group index of the fundamental TE mode. The loss of the resonator is expressed by:For the ring resonator, the loss directly impacts the FWHM of resonator covered by graphene with the same Fermi level (0.25 eV) and length as our device’s with different clh and cd can be obtained and depicted in gn is 3.68 and rλ is 2022.74 nm. Thicker cladding leads to a narrower FWHM, implying that a smaller wavelength shift is necessary to achieve effective modulation. Based on the FWHM, combined with the efficiency η, power consumption πP was calculated and shown in FOM, which is equal to 1/(π·τP), could be obtained, and they are plotted in Therefore, based on Equations (5)–(8) , and theFWHM of the resonance peak and the FOM of MRR type TO switches. Fortunately, the loss of the graphene decreases significantly with the doping level of graphene (FE), as shown in FE = 0.43 eV  could be achieved with a 371-nm cladding thickness and 2.25-μm contact distance.As discussed before, the loss induced by graphene could significantly influence the hievable ). Thus, FOM in −1μs−1 is already an ultrahigh value. Hence, graphene TO devices would allow much larger fabrication-error tolerance to achieve high performance than other technologies. Relatively high FOM can be obtained with thin cladding, and the current device design can achieve an extremely high FOM of 2.177 mW−1μs−1.As mentioned, the heavily p-doped graphene heater owns obviously smaller absorption loss, and therefore the minimum FOM of a switch with a metal heater could also be evaluated. The red curve in FOM of a 50-nm-thick gold heater MRR TO switch with the same cladding thickness and contact distance is only 0.005 mW−1μs−1, which is three orders of magnitude less than that with graphene heaters. As known, the absorption coefficient for the metal increases with the wavelength. Thus, metal heater should be placed much further away from the waveguide core in the mid-infrared range. On the other hand, graphene could become transparent in the mid-infrared wavelength range through tuning its Fermi level. Thus, the FOM of graphene-based TO switches could be much higher than those based on metal heaters. Graphene will become an exceptional nanomaterial for future MIR photonic circuits.Using these models, the FOM can be as high as 2.644 mW−1μs−1, which outclasses reported works. The doping engineering can make graphene an almost transparent heater in MIR waveband, which can be directly placed on the waveguide. Such unparalleled performance can hardly be achieved by resorting to metal heaters or doped silicon heaters. Our work proves that graphene heater owns exceptional advantages compared to other kinds of heaters, and graphene is a superb photonic material for future photonic integrated circuits working at MIR waveband.We demonstrated MRR-based and MZI-based TO switches operating at 2 μm waveband for the first time, where single-layer graphene was adopted as the heater. For the MRR device, the modulation efficiency was 0.11 nm/mW, corresponding to minimum power consumption of about 5 mW. The 10–90% rise time/90–10% fall time is 3.7/3.96 μs. Such dynamic response is close to the fastest silicon TO devices ever reported. As for the MZI-based device, an insertion loss of 2.0 dB and an outstanding extinction ratio of 27.08 dB with broadband operation wavelength range was achieved. The switching time was also characterized to be 4.92/4.97 μs respectively. The insertion loss could be minimized by the chemical doping of graphene, and the relatively high power consumption can be largely suppressed by a more appropriate design of contact distance. The numerical analysis showed that the performance of the graphene-assisted TO switch can be improved significantly by appropriately designing the structure of the heater and doping the graphene. The