Furthermore, we stack graphene with CVD hexagonal boron nitride and MoS2 layers to heterostructures, and fabricate encapsulated field-effect graphene devices, with high carrier mobilities of up to Integrating two-dimensional (2D) materials into semiconductor manufacturing lines is essential to exploit their material properties in a wide range of application areas. However, current approaches are not compatible with high-volume manufacturing on wafer level. Here, we report a generic methodology for large-area integration of 2D materials by adhesive wafer bonding. Our approach avoids manual handling and uses equipment, processes, and materials that are readily available in large-scale semiconductor manufacturing lines. We demonstrate the transfer of CVD graphene from copper foils (100-mm diameter) and molybdenum disulfide (MoS The existing integration approaches for 2D materials often degrade material properties and are not compatible with industrial processing. Here, the authors devise an adhesive wafer bonding strategy to transfer and stack monolayers, suitable for back end of the line integration of 2D materials. Transition metal dichalcogenides are often semiconducting and promise advancements for high- and low-power transistors9, infrared photodetectors10, and emerging device concepts such as memristors13 and single-photon emitters for optical quantum communication16. The scaling limit of transistors is set by short-channel effects that arise from intrinsic semiconductor properties. These effects degrade the off-state leakage current and are the limiting factor for silicon transistors with sub-10 nm gate length. On this scale, molybdenum disulfide (MoS2) transistors are expected to achieve more than two orders of magnitude lower leakage current than silicon transistors17. Furthermore, the carrier mobility in MoS2 transistors degrades less with decreasing channel thickness than in silicon transistors19. Hence, 2D semiconductors may be the ultimate channel material for ultra-scaled MOSFETs20. Vertical stacking of 2D materials forms van der Waals heterostructures, a novel class of materials with new material properties that result from synergetic effects between the stacked layers at their interfaces26. Integrating 2D materials into conventional semiconductor fabrication lines is critically important to exploit their material properties in commercial devices while benefitting from established silicon-based infrastructure with low-cost and high-volume device manufacturing on large20 substrates27. Yet, there are several bottlenecks that have prevented this last decisive step so far. The synthesis of 2D materials by chemical vapor deposition (CVD) is scalable to large areas, and the best contemporary processes provide close to intrinsic material quality31. However, the high process temperatures required to obtain high material quality rules out direct growth on preprocessed silicon electronics substrates, and hence mandates a material transfer from dedicated growth substrates to the device substrate. Commonly used wet transfer approaches rely on an intermediate polymeric carrier, typically poly(methyl methacrylate) (PMMA)34 or polycarbonate36, which mechanically supports the 2D material during its removal from the growth substrate and the transfer to the target substrate by scooping from the surface of a liquid. This process may be implemented at various stages of the device fabrication and allows the placement of the 2D material directly on a target substrate of choice, including complementary metal oxide semiconductor electronic wafers. For graphene, wet transfers degrade the material properties by causing defects, wrinkles, and strain in the transferred layer37. In addition, residuals of the polymeric carrier layer remain on the surface of the 2D material and degrade its electronic transport properties39. Attempts to minimize these adverse effects of wet transfer include bubble delamination40, advanced cleaning procedures41, and replacing poly(methyl methacrylate) with paraffin as carrier polymer42. Nevertheless, they do not eliminate the noted issues entirely. Dry transfer approaches using rollers, laminators, and hot presses enable the reuse of metallic growth substrates and avoid submersion of the target substrate in liquids, which is beneficial, for example, for the integration of 2D materials with suspended devices50. However, dry-transferred layers often suffer from microcracks, wrinkles, reduction in charge carrier mobility, and contamination by residuals from the sacrificial carrier layers. On the micrometer scale, cleaning techniques can mechanically manipulate the location of encapsulated contaminants and restore intrinsic material properties51. Delamination of single-crystal CVD graphene from copper foil by strong van der Waals forces with a stamp of exfoliated hexagonal boron nitride (hBN) yields almost intrinsic graphene properties, but is limited in size to areas of a few hundred micrometers30. Most importantly, neither of the reported transfer methods is compatible with industry routines for large-scale manufacturing while still preserving the high quality of the 2D material as on the growth substrate, which is required for many applications52. Here, we present a versatile approach for the transfer and stacking of 2D materials to heterostructures by adhesive bonding with bisbenzocyclobutene (BCB) and commercial wafer bonding equipment. BCB is a hydroxyl-free dielectric material that was developed for the semiconductor industry and is being used as an interlayer dielectric, for heterogeneous system integration, and as an organic gate dielectric layer since it has low fixed-charge and trap densities57. Our approach avoids manual handling of the 2D material and does not require sacrificial carrier layers that may contaminate the surface of the 2D material. The proposed method only utilizes equipment, processes and materials that are readily available in large-scale semiconductor production lines, which are crucial advantages for the integration into the semiconductor ecosystem59. We demonstrated the transfer of monolayer graphene from copper foils to 100-mm-diameter silicon wafers, the transfer and stacking of multilayer hBN, and monolayer graphene to form graphene/hBN heterostructures, and the stacking of two graphene layers to form double-layer graphene. We also fabricated field-effect graphene devices to demonstrate the utility of our methodology for wafer-level device manufacturing with conventional semiconductor processes. Furthermore, we demonstrated the transfer of multilayer MoS2, as a representative for transition metal dichalcogenides, from a centimeter-sized silicon chip to a 100-mm-diameter silicon wafer. All transferred layers and heterostructures were of high quality, that is, they featured uniform coverage and little strain in the transferred 2D materials. These findings indicate that our proposed integration approach preserves similar mechanical properties of the 2D materials as present on the growth substrate while minimizing degradation of the transferred layers through the introduction of wrinkles or excessive strain.The astonishing properties of two-dimensional (2D) materials have aroused tremendous interest in the semiconductor industry. These new types of atomically thin materials promise to continue the trend of shrinking transistors and better sensors. For graphene, an atomically thin layer of carbon atoms, the ultrahigh charge carrier mobility, and its strong light absorption enabled the demonstration of high-frequency analog electronicsOur method for transferring 2D materials from their growth substrate to a target wafer comprises four consecutive steps Fig. : first, 3 solution and rinsing in deionized water, we placed the monolayer graphene on a copper foil (100 mm diameter) on top of the target wafer and performed a second bonding process in a vacuum atmosphere. Finally, etching in FeCl3 uncovered the transferred layers. Due to the difference in the size of the growth substrates, the graphene/hBN heterostructure in the center of the wafer is surrounded by a monolayer of graphene on BCB. Note that in the heterostructure, neither the graphene nor the top surface of the hBN was in contact with polymers or adhesives at any time. Moreover, the second transfer was performed in a vacuum and at elevated temperature, which reduces the amount of water and gas molecules that may get trapped between the layers. Residuals of copper and the copper etchant are still potential contaminants at the interlayer interface, which potentially can affect the properties of the heterostructure60. Next, we integrated Ti/Au electrodes and patterned the 2D material heterostructure to vdP devices, which allow electrical material characterization on the target wafer , carrier density (n), and mobility of majority charge carrier (μ) of the transferred graphene layer, both for graphene resting on BCB and for graphene resting on hBN , whereas the n and μ showed differences for graphene resting on BCB and on hBN, respectively. The carrier density in the graphene on BCB was lower than for graphene on hBN  is mainly responsible for the discrepancy in material characteristics. For our materials, the microscopic surface roughness of hBN on Cu foil is ~50% higher than for graphene on Cu foil (root mean square (RMS): 264 and 176 nm, respectively, see Supplementary Fig. 67. In addition, large topography of the transferred materials may potentially cause problems for very high-resolution lithography. Using growth substrates with lower surface topography may mitigate this issue70.To demonstrate the viability of our methodology for wafer-level transfer of 2D materials and formation of large-area van der Waals heterostructures, we integrated graphene/hBN heterostructures . We characterized the transferred layers using van der Pauw (vdP) devices, noncontact terahertz (THz) near-field spectroscopy, and Raman spectroscopy , which records the amplitude and phase information of a THz signal traveling through the sample. Scanning across the sample surface yields spatially resolved images of the graphene sheet resistance on wafer scale. For our measurements, we set the spatial resolution to 500 µm by adjusting the distance of the near-field detector to the sample and the pitch between acquisition sites. The spatial resolution is significantly larger than the intrinsic length scale that is probed by the THz-TDS. This transport length is estimated by the distance lD, which a charge carrier transverses during one cycle of the alternating THz field73. For our sample and setup, we estimate 63. Rahimi et al.61 reported a higher mean value of We used terahertz time-domain spectroscopy (THz-TDS) to further characterize the electrical properties and homogeneity of the wafer-level transferred graphene and large-area graphene/hBN heterostructure. The THz-TDS measurements are recorded in transmission mode using a photoconductive near-field detectorfer Fig. . The sheωG) and 2D (ω2D) peaks and the full-width at half-maximum of the 2D peak (Γ2D)76. In particular, stress variations are of interest since they limit the charge carrier mobility in high-quality graphene, a crucial parameter for high-performance graphene devices67. In Fig. ωG and ω2D for the acquired Raman spectra of the transferred graphene placed on BCB (triangles) and on hBN (filled circles), respectively. The location of the acquisition sites on the wafer is marked in Fig. 74. Hole doping of graphene shifts ωG and ω2D with a relative slope of 0.7 (dashed line), while strain results in a relative shift with a slope of 2.2 (solid line)75. For both substrate materials on our sample, the peak locations indicate slightly tensile strain and predominant hole doping. For graphene on BCB, the charge carrier doping is more pronounced than for graphene on hBN, while the peak locations are less scattered and more confined within a smaller area. Our observation of hole doping and reduced doping level of the graphene when placed on hBN compared to placement on polymeric substrate materials agrees with literature77. However, multilayer hBN grown by CVD does not have the same strong effect as exfoliated hBN, which restores almost intrinsic conditions77. The color scale of the data points in Fig. Γ2D and averages to We performed Raman measurements to extract detailed information about the local graphene quality, including the relationship between doping and nanometer-scale strain variations, by analyzing the positions of the Raman G  in Fig. Rsh in the center clearly distinguishes 1- and 2-layer graphene on the target wafer and confirms the presence of double-layer graphene. We attribute the difference in the 1-layer graphene Rsh between these experiments and the dataset presented in Fig. ωG and ω2D  (coloration of data points) is similar in both regions and averages to We performed two consecutive transfers of monolayer (1-layer) graphene from their copper growth substrates to a 100-mm-diameter silicon wafer to demonstrate that our transfer methodology is suitable for high-yield formation of double-layer 2-layer) graphene on large areas . First, we transferred CVD graphene from a quarter of a 100-mm copper foil to the center of the target wafer with BCB. For the second transfer, we used a full 100-mm copper foil with CVD graphene. To provide a reference for THz spectroscopy, we removed the graphene on parts of the growth substrate before transfer , where the mobility is zero at the Dirac point due to a zero of the transconductance. At this point, the charge carriers convert from electrons to holes or vice versa. In our measurements, the mobility is not exactly zero because of the limited number of data points. The maximum field-effect mobility of holes from 16 devices averages to 2O3 deposition, multiple lithographic layers, metal depositions, and etching processes. Consequently, these results suggest that our transfer methodology is compatible with large-scale manufacturing of graphene devices using conventional semiconductor process technologies.We fabricated and evaluated top-gated field-effect devices on 100-mm-diameter silicon wafers using four photolithography layers and conventional processing technology Fig. , left sh2 from a SiO2/Si growth substrate to a 100-mm-diameter silicon wafer (see “Methods” for parameters of film growth and transfer). First, we bonded the centimeter-sized MoS2/SiO2/Si substrate to a BCB-coated 100-mm-diameter target wafer. Next, we submerged the bonded stack in a potassium hydroxide (KOH) solution, which permeated into the MoS2/SiO2 interface and detached the MoS2 from its growth substrate. This process took tens of seconds, after which the MoS2 remained transferred on top of the BCB layer of the target wafer. The surface roughness of the transferred MoS2  remained low compared to the original surface roughness of the MoS2 on the growth substrate . Typical Raman spectra prior transfer  and 407.2 cm−1 (A1g), that is, without detectable shifts in the position of the E12g mode within the accuracy of our Raman setup. Since the E12g mode is highly sensitive to uniaxial strain84, we conclude that the strain in the transferred MoS2 film was the same as present on the growth substrate and was not significantly affected by our transfer methodology. In contrast, the A1g mode is sensitive to changes in the substrate material, which could cause the observed shift of 1.8 cm−186. To further investigate the quality of the transferred MoS2 film, we compare the average of 25 normalized photoluminescence (PL) intensity spectra of pristine and transferred MoS2  to BCB (after transfer)86. The additional features of the PL intensity between 1.96 and 1.97 eV are Raman modes of the underlying BCB, and hence do not belong to the excitonic transitions in the MoS2 (see Supplementary information for the Raman spectrum of BCB). We formed MoS2/graphene heterostructures by stacking a centimeter-sized layer of MoS2 onto a sheet of graphene in two consecutive transfers (see “Methods”). The map of the graphene sheet resistance extracted from THz spectroscopy indicated the presence of graphene in both the MoS2/graphene heterostructure and its surroundings  averaged to 12g mode of MoS2 exhibited a minor shift from 1g mode remained unchanged . Taken together, the Raman and PL measurements confirm that the layer quality of MoS2 is preserved during the transfer and the stacking to heterostructures. Hence, our methodology is potentially suitable for large-area transfer of MoS2 films and their heterostructures.Our proposed transfer methodology is generic and applicable to a broad spectrum of 2D materials available on various types of growth substrates. We demonstrated this versatility by transferring a multilayer of CVD MoSfer Fig.  exhibiteoS2 Fig. . Before 2. Raman and PL spectroscopy indicated little strain in both transferred graphene and MoS2. We fabricated top-gated field-effect graphene devices  was spin-coated at 5000 r.p.m. on the 100-mm target wafer. A softbake on a hot plate at 100 °C for 4 min removed solvents and solidified the adhesive layer. Next, the target wafer was brought in proximity to a 100-mm sheet of monolayer CVD graphene on copper foil (Graphenea Inc.) with the graphene facing the BCB layer. This stack was bonded in a commercial wafer bonder  at 190 °C for 20 min using a bond force of 3 kN (bond pressure: 0.95 bar) in a nitrogen atmosphere. Etching the copper foil in FeCl3 and subsequent rinsing in deionized water for 30 min uncovered the hBN, transferred to the target wafer. To form a graphene/hBN heterostructure, a 100-mm sheet of CVD graphene on copper foil was placed on top of the target wafer and bonded at 190 °C for 20 min . Etching of the copper foil in FeCl3 and rinsing in deionized water for 30 min uncovered the graphene, which formed a graphene/hBN heterostructure with the previously transferred hBN.A 100-mm wafer was spin-coated with a 2.5-µm-thick layer of BCB . A 2.5 cm × 2.5 cm sheet of multilayer hBN (thickness: 2–5 nm) synthesized by CVD on copper foil (2D Semiconductors Inc.) was placed on top of the target wafer (hBN facing the BCB layer) and bonded at 190 °C for 20 min at a bond force of 250 N (bond pressure: 4 bar) in a nitrogen atmosphere. Etching of the copper foil in FeCl3 solution and rinsing in deionized water for 30 min uncovered the graphene, transferred to the target wafer. To form 2-layer graphene, a 100-mm sheet of CVD graphene on copper foil was placed on top of the target wafer and bonded at 190 °C for 20 min . Etching of the copper foil in FeCl3 and rinsing in deionized water for 30 min uncovered the graphene, which formed 2-layer graphene with the previously transferred layer. Note, the bond force was adapted to account for the difference in size of the growth substrates. To form reference areas for THz spectroscopy, the 100-mm copper foil was cut into two pieces and graphene was partially removed from one piece by etching in O2 plasma prior transfer. During transfer, both parts were placed side by side, covering the entire target wafer.A 2.5-µm-thick adhesive layer of BCB  was spin-coated at 5000 r.p.m. on the 100-mm target wafer. Softbake on a hot plate at 100 °C for 4 min removed solvents and solidified the adhesive layer. The target wafer was brought in proximity to a quarter of a 100 mm sheet of monolayer CVD graphene on copper foil (Graphenea Inc.) with the graphene facing the BCB layer. This stack was bonded in a commercial wafer bonder (Suss-SB8) at 190 °C for 20 min using a bond force of 600 N (bond pressure: 3.1 bar) in a nitrogen atmosphere. Etching of the copper foil in FeCl2) (see above). Thermal evaporation of 20 nm Ti and 200 nm Au through a shadow mask formed electrodes (300 µm × 300 µm) on top of the transferred graphene layer. Ablation by a pulsed femtosecond laser electrically insulated individual vdP devices with an edge length of ~3 mm. These noncontact processes prevent potential material degradation by lithographic layers in contact with the graphene. For device characterization, the wafer was resting on an electromagnet , which was placed inside a probe station (Cascade Microtech Inc.). Four-point probe measurements of the voltage across the device edge (device current: 100 µA), and the Hall voltage  yielded the sheet resistance, carrier density, and mobility of the transferred graphene. Electrical measurements were performed under ambient conditions at room temperature, using a parameter analyzer (Keithley SCS4200).A graphene/hBN heterostructure was formed on an oxidized 100-mm silicon wafer (100 nm SiO2O3 was deposited by atomic layer deposition and top-gate electrodes were fabricated by e-beam evaporation of 10/150 nm Ti/Al (3rd photolithography layer). To access the graphene metal contacts under Al2O3, reactive ion etching with SF6 and O2 was used to open vias through the Al2O3 (4th photolithography layer). All resist layers were removed by immersion in acetone . In this process sequence, the first and second lithographic layers were in direct contact with the graphene, which may have degraded the graphene quality. However, encapsulation of the graphene before fabricating the contacts eliminates this potential source of degradation89. Electrical measurements were performed under ambient conditions at room temperature. The sheet resistance (Rsh) was measured by 4-probe vdP method with top gating. A current (I12) was forced between electrodes 1 and 2  was measured. The sheet resistance (Rsh) was calculated by90μ) was then derived byVG and CG are the gate voltage and the capacitance of the dielectric per unit area (k = 7 for Al2O3), respectively.Graphene-based top-gated field-effect devices were fabricated on a 100-mm silicon wafer. First, large-area graphene (10 cm × 10 cm) was transferred by adhesive wafer bonding (see above). Contacts to graphene were fabricated by sputter deposition of 25 nm Ni and e-beam evaporation of 135 nm Al, followed by a lift-off process in acetone  (1st photolithography layer). The graphene sheet was then patterned by etching in oxygen plasma (2nd photolithography layer)). As a top-gate dielectric, 40 nm Ald 2 Fig. , while t91. The microscope was connected to a 600 mm ultrahigh-throughput spectrometer (UHTS 600) using a photonic fiber. An 1800 g mm−1 grating was used for the dispersion and an EMCCD camera for the detection of the scattered light. To compensate the topography of the substrate, a ×50 objective with a NA of 0.5 was chosen for the measurements. Note that we have used a high-resolution 1800 g mm−1 grating in combination with the 600 mm spectrometer to obtain more intense signals with a pixel resolution of 0.1 cm−1 @ 2800 relative cm−1. For each measurement site, the spectra were acquired in areas of 25 µm × 25 µm with 25 single spectra per line and 25 lines per site (integration time: 3 s). Peak positions were extracted by a Lorentzian fit. To ensure the validity of the extracted values, only data with a peak intensity exceeding a threshold of ten counts and sufficient separation to the noise level were considered.Raman measurements were conducted using a confocal Raman microscope , equipped with a 532-nm laser, which was coupled to the microscope using a single-mode optical fiber. A power meter in the optical path determined the laser power (0.6 mW), which ensured a high reproducibility of the experiments. The laser power was chosen to stay in the noninvasive regime4 Ω·cm, to avoid inadvertent absorption of the THz signal by the target wafer. For the measurements, we used a THz pump/probe setup equipped with a femtosecond fiber laser (generating pulses of 100 fs duration and 780 nm center wavelength) to pump a terahertz emitter and to gate a photoconductive near-field micro-probe detector (TeraSpike TD-800-X-HR-WT). By measuring the THz transmission through the sample, we extracted the local sheet resistance of the graphene72. Lateral scanning of the sample with the tip of the near-field detector held at close distance yielded a spatially resolved map of the graphene sheet resistance with a pixel size of a few hundred micrometer. See Supplementary information for a description of the setup and the estimation of the pixel size.We used THz-TDS in transmission mode to measure the sheet resistance of the transferred graphene on the target wafer. The samples were prepared on high-resistivity wafers with a resistivity of >102 films were grown in a CVD furnace with a 40 mm-diameter quartz tube as the reactor, similar to that described by Luo et al. 92. In short, the SiO2/Si growth substrates were located 12–15 cm downstream from the center of the heated tube (500 mm). MoS2 powder  was loaded into a quartz boat and placed in the center of the quartz tube of the furnace. The furnace was purged by forming gas (Ar 95%/H2 5%). For the growth, the furnace temperature was ramped up to 950 °C within 30 min, which was then maintained for 60 min. After the growth, the quartz tube was cooled down within 4 h. The growth yielded quasi-continuous films of MoS2 multilayers, with micrometer-sized domains.The MoS2, grown on SiO2/Si chips (10 mm × 7 mm), was placed on top of the BCB layer (MoS2 facing the target wafer) and surrounded by silicon dummy chips of the same height to ensure a uniform force distribution during the bonding process. This stack was bonded at 190 °C for 20 min using a bond force of 400 N (bond pressure: 2 bar) in a nitrogen atmosphere, which attached the MoS2 layer on its growth substrate to the target wafer. Etching in O2/SF6 plasma cleaned the edges of the growth substrate from potential BCB residuals. Submersion of the bonded stack in KOH solution detached the growth substrate from the MoS2 layer by permeation of KOH into the MoS2/substrate interface within tens of seconds. The MoS2 layer remained transferred on the target wafer.First, a 2.5-µm-thick adhesive layer of BCB  was spin-coated at 5000 r.p.m. on the 100-mm target wafer. A softbake on a hot plate at 100 °C for 4 min removed solvents and solidified the adhesive layer. Next, a multilayer of MoS4 Ω·cm) was spin-coated with a 2.5 µm-thick BCB layer . A quarter of a 100-mm sheet of monolayer CVD graphene on copper foil (Graphenea Inc.) was bonded to the target wafer with the graphene facing the BCB layer . Etching of the copper foil and rinsing in deionized water for 30 min uncovered the transferred graphene on top of the BCB on the target wafer. Next, a multilayer of MoS2 on a SiO2/Si chip (10 mm × 10 mm) was placed on top of the transferred graphene (MoS2 facing the graphene) and surrounded by silicon dummy chips to ensure a uniform force distribution over the wafer during the following bonding process. Bonding at 190 °C for 30 min attached the MoS2/SiO2/Si chip to the target wafer . Etching in O2/SF6 plasma cleaned the edges of the chip while a resist mask protected the remaining surface of the target wafer. Submersion of the bonded stack in acetone stripped the resist mask and detached the growth substrate, leaving the MoS2 film transferred on top of the graphene, forming a MoS2/graphene heterostructure.First, the silicon target wafer , which achieves a spectral resolution of about 1.2 cm−1. For PL measurements, the excitation power was 0.2 mW (600 g mm−1 grating).Raman and PL measurements were carried out in a confocal Raman system  in ambient conditions. For all measurements, an excitation laser with a wavelength of 532 nm and a 100× objective was used. For Raman measurements, the excitation power was 1 mW (1800 g mmSupplementary InformationPeer Review File


On the purpose of detecting communities, many algorithms have been proposed for the disjointed community sets. The major challenge of detecting communities from the real-world problems is to determine the overlapped communities. The overlapped vertices belong to some communities, so it is difficult to be detected using the modularity maximization approach. The major problem is that the overlapping structure barely be found by maximizing the fuzzy modularity function. In this paper, we firstly introduce a node weight allocation problem to formulate the overlapping property in the community detection. We propose an extension of modularity, which is a better measure for overlapping communities based on reweighting nodes, to design the proposed algorithm. We use the genetic algorithm for solving the node weight allocation problem and detecting the overlapping communities. To fit the properties of various instances, we introduce three refinement strategies to increase the solution quality. In the experiments, the proposed method is applied on both synthetic and real networks, and the results show that the proposed solution can detect the nontrivial valuable overlapping nodes which might be ignored by other algorithms. Determining the group with some particular properties helps the analysts to capture the common properties from the members in the community. Many applications could be considered based on the community detection. For example, the precise information delivery, e.g., Google AdWords  increaseMany results focus on the disjoin community sets that each node belongs to exactly one community ,3. HowevThe modularity function proposed by Newman and Girvan  is the fResolution limits Fortunato et al. introduced that small communities cannot be detected in large networks [networks ,18. Sincnetworks ,20, specnetworks ,22,23, snetworks  and mathnetworks .Overlapping community Some nodes may belong to several communities, so simply assigning the nodes to one community is difficult. Thus, the straightforward solution is to modify the modularity for allowing the nodes belonging to multiple communities at the same time [B and C. Thus, we get three communities, and they are {{A and B in ame time ,28,29,30On the other hand, besides the computation complexity, the modularity maximization has two problems in detecting communities:In this paper, we focus on the overlapping community detection, and propose the node weight allocation problem denoted by ms (GAs) ,3,8. Thems (GAs) . Shang ems (GAs) ,5,6,31. ms (GAs) . To emphms (GAs) . Shakya Even if some approaches provide the solutions with high modularity, the partitions may not reflect the properties of the real-world networks in some situations. We found that the solution quality could be refined by considering following issues: ignoring overlapping nodes, merging clusters, and reweighting nodes. Therefore, we consider the modularity to design the solution searcher of the approach In the simulation, we consider the synthetic network and popular networks that include Zachary Karate Club Network, Books about American Politics, and American College Football to evaluate the solution quality calculated by This paper is organized as follows. The overlapping communities and the problem definition are introduced and formulated in n is the number of nodes, and k is a given number of communities. Each entry c. The constraint of the relationship between U with optimal result The community detection of a given network involves two processes. The first one is to find out the network structure and the other one is to determine the numbers of communities. Here we introduce the works proposed by Nepusz et al.  to explaction is:DG(U)=∑i,k, Nepusz et al. iteratively increase the value of k from 2, and then choose the value of k with the highest fuzzy modularity value calculated by Equation (To determine an appropriate number of communities Equation .(3)QovNk, and a null model weight The overlapping community detection problem is considered as a node weight allocation problem, denoted by We consider By solving the Equation  is the sEquation , which mB in G4415 while A in G415, and they are not overlapping nodes.Although Griechisch et al.  apply thThe major difference between Equations  and 5)  5) is thComputing the partition with maximum modularity has been proved as the NP-complete problem . Even ifP with c. The initial population is generated randomly, and each row of M must satisfy the problem constraints. Given a maximum number of iterations The iterative process of GA as shown in Algorithm 1 includes three major processes: crossover, mutation, and selection. Before invoking the iterative process, the initial population Crossover: we randomly select two chromosomes P, and a random column. The offspring is generated by the selected column of Mutation: the mutation process is launched in 80% probability after finishing the crossover. Once the mutation is invoked, one Selection: we consider the modularity to be the objective function, and finding the partition with maximum modularity is the purpose of GA. We use Algorithm 1: Genetic algorithm for allocating node weightTo keep the heavily overlapping nodes, a threshold Equation .GA provides an elite solution from the population, but this solution may not be suitable for all instances. In the pre-analysis phase, we observed three situations derived by Ignore slight overlapping nodes The overlapping degree of each Equation  to deterEquation . When λ<Merge clusters Some small communities should be merged by other large community. If the overlapping ratio of any two communities is larger than a given merge threshold Reweight node values To calculate the weight distribution of each overlapping node, directly converting c. Moreover, if the neighbors of c are more than the average number of nodes in c, c is more important than others for c and Equation  results We consider a synthetic network and three real networks including Zachary Karate Club network, Books about American Politics, and American College Football to evaluate the performance of G210 as our synthetic network which has 210 nodes and four pre-defined communities A, B, C and D. Each of them has 60 nodes and 10 shared by any two continuous communities, i.e., A and B share nodes B and C share nodes We consider ng parts .Nodes are grouped by the detected community id. For the overlapping nodes, only the smallest id is counted.c, all nodes are sorted in descending order of For each Since the fuzzy modularity is a special case of the Although the maximum number of communities is six, only four communities were detected while the other two were empty communities. Since the number of communities could be captured by modularity , it is uZachary karate club network  is a popWe first evaluate the communities captured by Q. It implies that the overlapping structure is easier to be detected as assigning higher weight to the overlapping nodes.Considering that a node has only one edge connecting to an overlapping node, e.g., Here we consider an extreme case that all nodes are overlapped, i.e., Even if we derive the result with maximized value of ignore step. The result with and without the ignore step are 0.427114 and 0.427117, respectively. We consider the network with To emphasize the importance of the communities, we propose a reweight strategy to assign various weights. The result with reweight strategy is identical to that shown in We examine the network with exactly two communities to verify the property illustrated in In the above simulations, munities , and theChen et al. captured two overlapping communities , and theFrom the above observation, the communities are split more precisely by liberal, neutral, or conservative. Each category has 43, 13, and 49 nodes respectively. In this simulation, we consider This network is built from the transaction data from amazon.com . The netW, X, Y, and Z. Most nodes belong to two large communities W and X, which are mainly consisted of conservative and liberal books respectively. Most neutral books belong to two small communities. This result is similar to that obtained by Newman [neutral nodes, that is neutral nodes. The result implies that neutral books are often co-purchased with different books.y Newman . Table 6W and X into two parts respectively denoted by mization . TherefoX. Besides, the overlapped part is even denser as shown in Moreover, we also detect an extra community The overlapping ratios of  and Sun Belt (Label 10). There are total seven overlapping nodes in these two conferences. From Independents has five teams, and only one game was held. This is the major reason that makes this conference undetectable. However, the teams often play with other teams in varied conferences, and this phenomenon results in the overlapping property. For example, G with four edges. Sun Belt is in the similar situation. In this example, the heavily overlapping nodes could be detected by our method.The conference The Dolphin Network is a common benchmark for evaluating the overlapping communities. Some results consider the Dolphin Network to evaluate the community quality ,43. We cThe distribution of Nicosia et al. found four communities in Dolphin network . The oveQ=0.385. The sepleury in , but theleury in . In summGiven a network, the modularity is used for measuring the partition quality while the fuzzy clustering recognizes the overlapping communities. Combining above concepts together to be the fuzzy modularity is an appropriate method to formulate the structure of the given network with overlapping communities. Maximizing the modularity outputs the partition with well network structure, but computing the partition with maximum modularity requires huge computation cost. Therefore, the heuristic algorithms are outstanding in seeking high quality solution from a large search space, and we can find some research results of using heuristic algorithms for finding the partitions with maximum modularity. However, there are some special cases that we have to deal with. We find out three common situations from the partitions derived from the GA with modularity maximization and propose three solution refinement strategies to ignore overlapping nodes, merge clusters, and reweight nodes to separate the network to be closer the real-world behaviors. Moreover, we modify the fitness function of the GA to consider the null model for measuring the distance between the derived partition and the random graph. Thus, the simulation results show that the proposed In our simulations, we got an interesting result as shown in The proposed algorithm invokes GA to compute the preliminary partitions and then adopts proposed refinement strategies to correct the partitions by the secondary processes. The refinement strategies could be considered as the local search to improve the partition quality in each iteration. However, it is a tradeoff between the computation cost and the partition quality. Once the refinement strategies are modified from the external processes to the internal processes in GA, the computation cost will be increased. Moreover, the given networks may not always consist of the target properties that could be improved by the refinement strategies. Therefore, the refinement strategies could be designed as local search approaches, but the trigger of launching the local search approaches should be analyzed in the future.The overlapping nodes could be detected and provided appropriate allocation by


Staphylococcus aureus (MRSA) is a pathogen commonly found in bone and joint infections, including septic arthritis. S. aureus virulence and the frailty of affected patients can cause several complications; a prompt and specific antibiotic treatment can positively affect the outcome of patients. We carried out an in-depth genomic characterization by Illumina whole genome sequencing and bioinformatics of two biofilm-producing M1 and M2 ST398 MSSA causing septic knee arthritis not-responding to antimicrobial therapy. The strains were characterized for antibiotic resistance, biofilm and adhesive properties as well as genomics, single nucleotide polymorphism phylogeny, resistomics and virulomics. Our results showed that M1 and M2 MSSA were ST398-t1451-agrI-Cap5, susceptible to cefoxitin and resistant to erythromycin and clindamycin, traits consistent with the lack of the SCCmec-locus and the presence of the sole blaZ and ermT. Furthermore, M1 and M2 were biofilm-producing and largely potentially adhesive strains, as indicated by the adhesion gene profile. Our data characterized a new human-adapted ST398 MSSA lineage, representing a “fusion” between the human-animal independent ST398 and the Livestock Associated (LA) ST398 lineages, forming biofilm and genomically predicted high adhesive, characterized by different genomic adaptation conferring a great ability to adhere to the host’s extracellular matrix causing septic knee arthritis.Methicillin-susceptible (MSSA) and methicillin-resistant  Staphylococcus aureus can cause severe and chronic septic arthritis associated with a high rate of relapse [Invasive  relapse , in part relapse . Biofilm relapse ,4.Bacterial communities in biofilms alter some of their phenotypic  and genotypic features (gene expression and protein production profiling) . A biofierm(T), and a livestock-associated (LA) CC398 SA clade with the SCCmec locus (methicillin-resistance), the tetracycline-resistant gene tetM and carrying the ϕ2, ϕ6, or ϕAvb phages [S. aureus (MSSA) isolated before and during different antibiotic treatments came to our attention. The strains were related to a devastating infection sustained by MSSA.In recent decades, the clonal complex 398 (CC398) methicillin-resistant SA (MRSA) spread in animals and humans worldwide . Human ib phages ,11,12. Mb phages ,14,15. IThe present work is the full characterization of the two MSSA strains, immediately confirmed as biofilm producers of the ST398 lineage.A 25-year-old athletic man, admitted to a private clinic with a diagnosis of contusive right knee trauma with complete rupture of the proximal middle third anterior cruciate ligament, underwent surgical ligament reconstruction with autologous patellar tendon, medial meniscectomy after chemoprophylaxis with 2 g of cefazolin.S. aureus.Then, 11 days after surgery, the patient presented with intense pain associated with fever and evidence of edema and erythema of the medial face of the thigh, knee, and right leg. Arthrocentesis revealed corpuscular fluid positive for MSSA (M1 strain); consequently, the patient was treated with teicoplanin 800 mg and rifampicin 600 mg. Despite the antimicrobial therapy, the fever and inflammation persisted, hence the switch to 850 mg daptomycin + 750 mg levofloxacin + 4.5 g × 4 days tazobactam-piperacillin and a second surgery after 4 days—revealing the presence of fibrinoid tissue incorporating the neoligament, the screws of both the tibial and femoral graft and the meniscal root button—with all new cultures positive for Next, 5 days after the second surgery, the persistence of inflammation, pain and fever, together with repeated isolation of MSSA from joint drainages (M2 strain)—even though in the presence of negative blood cultures—determined a new change in the antimicrobial therapy: first to daptomycin 850 mg and linezolid 600 mg × 2 days, and then to linezolid suspension. Nevertheless, the patient remained febrile and with persistent knee inflammation. Finally, 25 days after the second surgery, the new arthrocentesis revealed negative cultures, determining a new change in therapy, with discontinuation of daptomycin and initiation of dalbavancin infusions. The patient repeatedly underwent arthroscopic arthrolysis with biopsy samples negative for cultures.A timeline of M1 and M2 MSSA isolation in relation to the surgeries and treatment regimens is shown in M1 and M2 MSSA antibiotypes were determined using minimum inhibitory concentration (MIC) determinations versus a panel of anti-Gram-positive antimicrobials, i.e., cefoxitin, vancomycin, teicoplanin, daptomycin, gentamicin, ciprofloxacin, cotrimoxazole, tetracycline, mupirocin, linezolid, dalbavancin, erythromycin, clindamycin, and rifampicin, according to the European Committee on Antimicrobial Susceptibility Testing guidelines version 10.0 (2020).Biofilm and slime production were evaluated as previously published ,17. BrieS. aureus was cultured on blood agar plates  as well as on homemade sheep blood agar plates prepared with Columbia blood agar powder . The bacteria were incubated at 35 °C in 5% CO2 (v/v) for 24 h and then underwent serial passages. The haemolytic properties were then investigated. Positive control strains and S. aureus ATCC25923, having a complete hemolytic phenotype and therefore to be considered as a positive control strain, were also used for comparative analysis.Haemolytic ability was investigated as previously published . BrieflyWhole genome sequencing (WGS) was performed with an Illumina Mi-Seq sequencing system using paired-end (PE) read libraries prepared by Nextera XT DNA Library Prep Kit  following the manufacturer’s protocol and whose quality was evaluated as previously published . Raw reahttp://cgview.ca/).The de novo genome assembly was performed using SPAdes software (v3.12.0), producing a contig file for each sample. Post-assembly controls and metrics were generated using Quast (v.4.6.3). The CG viewer server was used to generate circular maps of the M1/M2 genomes (http://RAST.nmpdr.org).The assembled contigs were processed by Rapid Annotations using Subsystems Technology (RAST) for microbial genome annotation (The genomic reads were deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) Genome database under study accession n° SAMN16688054 and SAMN16688055 (BioProject: PRJNA675099).S. aureus Reference Genomes (RefGen) deposited in GenBank. The LA ST398 MRSA S0385 (acc. n° AM990992.1) and human animal-independent ST398 MSSA NM01 (acc. n° CP003045.1) were selected as Reference Genomes for SNP mapping in agreement with the phylogenetic results.SNP calls were carried out from the PE library raw reads as previously published . The CSIThe wgSNP effects of both mappings were evaluated by SnpEff (v.4.3T). High (HI), Low (LI), Moderate (MI), and ModiFier Impacting (MFI) effects were assigned according to the criteria previously published and in use in the tool. High impact: the variant is assumed to have disruptive impact in the protein, probably causing protein truncation and loss of function or triggering nonsense mediated decay. Low impact: the variant is assumed to be mostly harmless or unlikely to change the protein behavior. Moderate impact: the variant is a nondisruptive variant that might change protein effectiveness. Modifier impact: the variant is a usually noncoding variant or a variant affecting noncoding genes where predictions are difficult or there is no evidence of impact .Whole Genome Sequencing raw data were analyzed to investigate the genomic epidemiology with ResFinder (v.3.2) and Point Finder (v.3.1.0) services for the detection of acquired antimicrobial resistance (AMR) genes and to detect the known nsSNPs related to AMR genes . Multi LThe core genome SNP (cgSNP) detection shared by all strains was computationally carried out by Nullarbor on the LA ST398 MRSA S0385 (acc. n° AM990992.1) and human animal-independent ST398 MSSA NM01 (acc. n° CP003045.1) Reference Genomes.M1 and M2 MSSA strains showed a nearly complete susceptibility profile, being susceptible to cefoxitin, vancomycin, teicoplanin, daptomycin, gentamicin, ciprofloxacin, cotrimoxazole, tetracycline, mupirocin, linezolid, dalbavancin, and rifampicin; resistant only to erythromycin and clindamycin .490 0.4). Both strains were also not haemolytic on blood agar plates  matrix formation in Congo Red Agar  and biofr plates .The de novo assembly of the M1 and M2 genomes yielded 57/92 contigs with a total length of 2,700,365/2,704,459 bp and a G+C content of 32.84/32.83%. The largest contig size was 412,322 bp for M1 and 405,071 bp for M2; the N50/N75 contig size was 190,663/80,862 for the first and 110,557/44,428 for the second one, with 98% genome coverage for both.A circular graphic representation of the M1 and M2 MSSA genomes is provided in n = 229/231), “Carbohydrates” (n = 172), “Cofactors, Vitamins, Prosthetic Groups, Pigments” (n = 99/101), “Cell Wall and Capsule” (n = 37/38), “DNA metabolism” (n = 57/59), “Fatty Acids, Lipids, and Isoprenoids” (n = 50/54), “Nucleosides and Nucleotides” (n = 80), “Protein Metabolism” (n = 149/151), “Stress Response” (n = 32), and “Virulence, Disease and Defense” (n = 59/120) (n = 23) and “Adhesion” (n = 23/84) in the “Virulence, Disease and Defense” category; “Osmotic stress” (n = 12) and “Oxidative stress” (n = 13) in the “Stress Response” category; “Protein biosynthesis” (n = 125/126) in the “Protein Metabolism” category  . Severalcategory .agrI-Cap5. CSI wgSNP phylogeny distance analysis found the closest genomic similarity with the human animal-independent ST398 MSSA NM01 RefGen and other investigated human ST398 MSSA and MRSA genomes, followed by the LA ST398 MRSA RefGen as shown in the phylogenetic tree  the plasmid rep gene 13; (iii) resistomes including blaZ and ermT; and unique traits that is no-shared features with the two reference ST398 strains: (i) the presence of a single copy of the MGE ISSau8; (ii) the presence of an intact copy of the Staphylococcus phage StauST398-4, Immune Evasion Cluster (IEC) β-converting Sa3 prophage [Comparative genomics of M1 and M2 ST398 MSSA—versus LA ST398 MRSA S0385 and human animal-independent ST398 MSSA NM01—highlighted characterizing human traits: (i) the lack of SCCprophage   located in the chromosome integrated plasmid pUR3912, conferring β-lactam, macrolides/lincosamides and streptogramin B (MLSb) resistance. No mutations were found in rpoB (rifampin resistance) and fusA (fusidic acid resistance), and no known mutations were detected in 23S rDNA  toxin coding genes, namely the hemolysin hlgABC, hlb, hld, and hly/hla; (iii) exoenzyme coding genes, i.e., the zinc metalloproteinase aureolysin aur, the cysteine protease sspABC, the lipase lip, and the sortase strB; (iv) host immune evasion coding genes, i.e., spa (protein A belongs to the MSCRAMMs family) and sbi, both binding the IgG Fc portion interfering with opsonophagocytosis, staphylococcal complement inhibitor scn included in the IEC and chp (the immuno-modulator for the chemotaxis inhibitory protein of Staphylococcus aureus CHIPS), both contained in the conserved 3′ end of β-hemolysin (hlb)-converting bacteriophages βC-φs (β-converting Sa3 prophage), Cap5 biosynthesis genes (the capsular polysaccharide synthesis enzyme implicated in the antiphagocytosis), adsA (adenosine synthase) and geh (activation of innate immune cells); v) essABC, esaAB and esxA (type VII secretion system) (M1 and M2 ST398 MSSA virulomes were shaped by a mosaic of different virulence factors as follows: (i) adherence virulence determinants, namely the cell surface binding elastin  system) .clfA, clfB, fnbA, fnbB, coa, sdrE and sdrC gene sequence as the human animal-independent ST398 MSSA NM01 RefGenomes, whilst the wild type (WT) collagen binding adhesin cna and mutated sdrD as the LA ST398 MRSA S0385  SA S0385 .S. aureus)—in MSCRAMM family adhesin coding genes, i.e., clumping factor clfA/B , fibronectin binding coding gene fnbB , superantigenlike exotoxin coding ssl11 (HI SNPs), extracellular adherence protein coding eap  (HI nsSNPs), serine-aspartate repeat protein sdrD (MI nsSNPs frameshifting to the mutated gene sequence as in LA ST398 MRSA S0385), collagen adhesin gene cna (MI nsSNPs frameshifting to the WT gene as in LA ST398 MRSA), Enoyl-acp reductase FabI, and staphylocoagulase coding gene coa  as shown in dhaM) (frameshifting to the WT gene) and lactose/galactose ; (ii) phosphate (pstS); (iii) amino acid biosynthesis-arginine (argJ), tyrosine (tyrA) (frameshifting to the WT gene) and L-lysine (dapE); (iv) ammonium (nrgA); (v) oligopeptide transport (oppF); (vi) peptodoglycan turnover, i.e., N-acetylmuramoyl-L-alanine amidase LytH and autolysin (amiC gene); (vii) DNA (SAPIG0346 sirtuin2 family protein); (viii) SAPIG1607 (competence protein ComGC)  shared by M1 and M2 were found—only on the LA ST398 MRSA S0385 mapping (Ancestor protype genome of ST398 n ComGC) . MI effeThe strain-specific HI and MI nsSNPs mapped on the LA ST398 MRSA S0385 RefGen are shown in S. aureus has recently emerged worldwide and is a frequent source of human infections, being involved in serious infectious diseases such as bloodstream infections (BSI) [ST398 ns (BSI) ,32, prosns (BSI) ,34 and ins (BSI) , whereasns (BSI) . ST398 Mns (BSI) .S. aureus can switch to different lifestyles moving along a “continuum of cell modalities”—from planktonic, to sessile (biofilm), to quasidormant lifestyles (SCVs)—the modes of growth that are key to the ability to establish chronic, persistent and relapsing infections [S. aureus a high affinity for the synovia, allowing this microorganism to be one of the most common agents in joint infections.fections  under difections . An arrafections ,40,41, cagr-I type and the capsular type 5 as LA indicators [mec cassette, a lower average core genome variation rate versus the human animal-independent ST398 MSSA NM01 RefGen, the IEC β-converting Sa3 ST398-4 prophage, the plasmid replication gene rep13, resistome including only blaZ and erm(T), and the adhesion coding gene sequences as in the human animal-independent ST398 MSSA NM01 markers. The resistome characterized by the presence of the β-lactamase blaZ (β-lactam Resistance), and the 23S rRNA methyltransferase erm(T) (MLSb Resistance) associated with the tetM lack is consistent with β-lactams and MLSb use in human therapy and tetracycline in animal feeds. These genomic traits strongly support the similarity between M1 and M2 ST398 MSSA and the human animal-independent ST398 MSSA genomic background.Our investigations characterized two ST398 MSSA isolates, being M2 a genetic evolution of M1 MSSA emerging under antimicrobial treatments, with a sticky behavior due to their in vitro slime and biofilm production. The severity of the infection, as described in the clinical section, did not correspond to the antimicrobial resistance profile but was instead related to the biofilm production ability and to the presence of several genes related to these sticky properties. The comparison of core genome SNPomes showed the close phylogenetic relationship between M1 and M2 ST398 MSSA compared to the human animal-independent ST398 MSSA NM01. Comparative genomics and genomic epidemiology evidenced that M1 and M2 represent a new human-adapted ST398 MSSA lineage having predominantly human-animal independent ST398 traits and few features associated to LA ST398. In details, they have the spa-type t1541, the dicators , and thePathogenomics indicated that the M1 and M2 ST398 MSSA virulomes were prevalently shaped by a mosaic of “human-adapted like” virulence factors constituted by a large repertoire of wild type MSCRAMM surface adhesins, biofilm associated PIA, MHC-II analogous protein MAP, and the serine-aspartate repeat containing SdrCDE, ClfA/B and FnbA/B . These aS. aureus can persist intracellularly after internalization in cultured osteoblasts [Moreover, eoblasts  and swithlgABC, hlb, hld, and hly/hla, similarly to the human-animal independent ST398 MSSA, even though they were not haemolytic strains in agar plates likely for a dysregulation in hla expression, and showed the lack of enterotoxins and phage-encoded toxins [5 biosynthesis genes involved in antiphagocytosis. In septic arthritis, S. aureus can evade the clearance by the host immune system using different staphylococcal defense mechanisms. First, protein A binds the Fc portion of immunoglobulin G and presents the Fab fragment of the antibody to the external environment; as a consequence, the Fc segment cannot bind the complement or signal polymorphonuclear leukocytes interfering with staphylococcal opsonization and phagocytosis [Regarding toxin coding genes, M1 and M2 ST398 MSSA had a very poor toxin gene content made up of hemolysin coding genes d toxins . A previd toxins . Superand toxins . Concernocytosis . Capsulaocytosis ,47,48,49ocytosis .S. aureus, conferring to this microorganism a great adaptability to be a joint infection pathogen.In conclusion, we characterized a new human-adapted ST398 MSSA lineage, biofilm producing and virtually highly adhesive to the host-derived matrix, characterized to be a “fusion” between a prevalence of human-animal independent genomic traits and few LA ST398 genomic features. Our data confirm the high genomic adaptive plasticity of


A number of international safe communities are displaying increasing interest in the United Nations Sustainable Development Goals (UNSDG). The International Safe Communities Certifying Centre is seeking to establish formal relations with the World Health Organization (WHO).A formal review of the UNSDG and WHO General Program of Work 13 – 2019 to 2023 (WHO GPW13) alignment with the international Safe Communities indicators.UNSDGs were formally reviewed by 4 senior ISCCC certifiers and ranked in terms alignment with International Safe Communities Indicators.While injury imposes a considerable cost burden on communities, safety is a critical precondition necessary for the attainment of the productive physical, physical and social environments that communities require to achieve their full potential. Safety provides a dynamic leverage point for communities to achieve their aspiration for sustainable development. There is very strong alignment between the UNSDGs and the International Safe Community Indicators The holistic understanding of injury prevention and safety promotion adopted by the safe communities movement supports the WHO vision to “Promote health, keep the world safe and serve the vulnerable” articulated in its 13th General Program of Work (GPW): 2019 to 2023. Based on the analysis the ISCCC board has identified 7 domains of injury prevention and safety promotion practice that are of high strategic importance8: • Disaster preparedness • Prevention of Road Traffic Injuries • Prevention of Violence against Women • Prevention Violence against Children • Suicide prevention • Falls prevention • Prevention of workplace injury. All new designation applications submitted after the 1st of July 2019 must provide statistical information regarding the incidence of these issues and to describe how they are addressed in their community. The International Safe Community movement embraces a whole of system  definition of injury prevention and safety promotion that aligns easily with the United Nations Sustainable Development Goals (UNSDG) and the WHO General Program of Work 13, 2019 to 2023 (WHO GPW13).United Nations SDGs, WHO General Program of Work, ISCCC


Growth-associated protein-43 (GAP-43) and brain acid-soluble protein 1 (BASP1) regulate actin dynamics and presynaptic vesicle cycling at axon terminals, thereby facilitating axonal growth, regeneration, and plasticity. These functions highly depend on changes in GAP-43 and BASP1 expression levels and post-translational modifications such as phosphorylation. Interestingly, examinations of GAP-43 and BASP1 in neurodegenerative diseases reveal alterations in their expression and phosphorylation profiles. This review provides an overview of the structural properties, regulations, and functions of GAP-43 and BASP1, highlighting their involvement in neural injury response and regeneration. By discussing GAP-43 and BASP1 in the context of neurodegenerative diseases, we also explore the therapeutic potential of modulating their activities to compensate for neuron loss in neurodegenerative diseases. Axons integrate external cues to grow toward and arborize their terminals onto their correct targets. Such axonal behavior is critical for establishing proper connections, regenerating injured nerves, and retaining anatomical plasticity in adult brains . A group–/–) or BASP1 (BASP1–/–) leads to high neonatal lethality, resulting in 5–10% survival to adulthood  exhibit a selective impairment in contextual memory  and peripheral nervous systems (PNS), different external and internal factors lead to disparate outcomes of injury response. In the CNS, glial scar  and inhiin vitro and synaptogenesis in vivo   and braiGrowth-associated protein-43 and BASP1 are acidic proteins with isoelectric points of 4.4–4.6 . Their mGrowth-associated protein-43 and BASP1 have peptide sequences rich in proline, glutamate, serine, and threonine (PEST) . While P2+ and to dissociate at high Ca2+ (2+ elevation to sharpen the downstream response (2+ (Growth-associated protein-43 and BASP1 have regions termed the effector domain (ED) that are enriched in basic and hydrophobic residues, bind calmodulin (CaM), and are phosphorylated by protein kinase C (PKC) . The basigh Ca2+ . Hence, response . Unlike response . BASP1 lresponse . These aresponse . BASP1 wresponse  and in tonse  was the most potent driver of oligomerization P2-induced oligomers may be important for this process. Oligomerization of GAP-43 and BASP1 results in α-helix formation within their EDs while preserving the overall structural disorder (in vitro and in presynaptic membranes (Growth-associated protein-43 and BASP1 form oligomers in the presence of anionic phospholipids or sodium dodecyl sulfate (SDS) . Among arization . Interesskeleton . The roldisorder . A signidisorder . In suppembranes . In agreembranes .In developing and regenerating neurons, the levels of GAP-43 and BASP1 mRNAs and proteins are upregulated . Studieso, a heterotrimeric GTP-binding protein enriched in growth cones  (Growth-associated protein-43 and BASP1 EDs undergo Caectively . GAP-43 in (CaN) . Phosphoin (CaN) . More imin (CaN)  but alsoin (CaN) . Throughin (CaN) , axon ouin (CaN) , neurotrin (CaN) , and synin (CaN) . The modin (CaN) , Serine-in (CaN) , and Serin (CaN) . Additioin (CaN) . The phoin (CaN) .Regulation of the actin cytoskeleton is important for axon guidance and growth , endocyt2 P2 . In the 2 (2 . Concentproteins . These pproteins . The funproteins .d = 161 nM) compared to the unphosphorylated form (Kd = 1.2 uM) (Growth-associated protein-43 binds and directly regulates actin filaments in a PKC-dependent manner . Phospho 1.2 uM) . In acco 1.2 uM) . Moreove 1.2 uM) . On the  1.2 uM) . CappingGrowth-associated protein-43 has also been proposed to modulate actin dynamics through the functional association with the neural cell adhesion molecule-180 (NCAM-180) and the cytoskeletal protein spectrin . NCAMs eGrowth-associated protein-43 and BASP1 are intrinsically disordered proteins . The funPost-transcriptional regulation allows a rapid adjustment of the location and level of protein expression . This sp2+ signaling through PKC and CaM  and amyloid-β peptides  is the most common neurodegenerative disorder causing dementia . AD is nin vitro scratch lesion model using α-syn mutations causing autosomal-dominant forms of PD, the study found reduced neurite regeneration and subsequent loss of dopaminergic neurons accompanied by a reduction of striatal expression of GAP-43 (Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder . PD is nf GAP-43 . In one f GAP-43 . While tf GAP-43 . The appf GAP-43 . This mof GAP-43 . Treatmef GAP-43 . This cof GAP-43 . This opin vitro regeneration. Low GAP-43 levels in cerebrospinal fluid were associated with a poorer response to treatment of primary progressive multiple sclerosis using fingolimod or alemtuzumab (Growth-associated protein-43 and BASP1 are essential for developing axons to grow toward their correct targets and form synaptic connections during neuronal development and after neural injury. These axonal functions highly depend on their expression levels and phosphorylation status, which when modulated appropriately, can stimulate mature neurons to re- enter a growth state. This transition, which can protect and enable surviving neurons to re-establish functional connections, has been explored as a therapeutic avenue for neurodegenerative diseases. Specifically, the effects of delivering agents that upregulate GAP-43, such as BDNF, have been tested in animal models of PD . BDNF trmtuzumab  suggestimtuzumab . Despitemtuzumab . Such chmtuzumab . At sub-mtuzumab  and othemtuzumab  need to DC contributed by writing and revising the entire review, as well as generating figures. AS contributed to writing the section “Involvement in Neural Injury Response” and building The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zea mays L. seedlings were watered with different concentrations (0–100 mg/L) of graphene to explore the effects and molecular mechanism of graphene on the growth and development of Z. mays L. Upon evaluating root growth indices, 50 mg/L graphene remarkably increased total root length, root volume, and the number of root tips and forks of maize seedlings compared to those of the control group. We observed that the contents of nitrogen and potassium in rhizosphere soil increased following the 50 mg/L graphene treatment. Thereafter, we compared the transcriptome changes in Z. mays roots in response to the 50 mg/L graphene treatment. Transcriptional factor regulation, plant hormone signal transduction, nitrogen and potassium metabolism, as well as secondary metabolism in maize roots subjected to graphene treatment, exhibited significantly upregulated expression, all of which could be related to mechanisms underlying the response to graphene. Based on qPCR validations, we proposed several candidate genes that might have been affected with the graphene treatment of maize roots. The transcriptional profiles presented here provide a foundation for deciphering the mechanism underlying graphene and maize root interaction.Graphene reportedly exerts positive effects on plant root growth and development, although the corresponding molecular response mechanism remains to be elucidated. Maize seeds were randomly divided into a control and experimental group, and the roots of  Nanomaterials have been widely used in the fields of electronics, machinery, energy, and biomedicine –4. ReseaMyriad studies have evaluated the positive effects of graphene on plant growth and development. For a few species, including tomato, spinach, and wheat, graphene treatment reportedly promotes seed germination. In a study conducted using tomato, graphene-treated seeds showed accelerated germination compared to control (CK) seeds, possibly due to graphene penetrating the seed husks and consequently facilitating water uptake . LikewisResearch in several species  have shown that graphene positively affects roots generally by promoting growth and/or by increasing the number of lateral roots , 12–15. Zea mays L. seedlings.To date, multiple studies have focused on the physiological and/or phenotypic response to graphene exposure, with few studies characterizing the molecular response to graphene. The goal of the present study was to explore the effects of graphene on gene expression and to characterize the molecular response to graphene exposure regarding root growth and development in Graphene was obtained and generated in our lab. The characteristics of graphene were analyzed by using ultraviolet-visible absorption spectrogram and Raman spectroscopy . The Raman spectra were obtained using Renishaw inVia™ Qontor with a 532-nm excitation laser. The morphology of graphene was examined using scanning electron microscopy  and transmission electron microscopy .Similar-sized maize seeds were divided into five groups (30 seeds in each group), germinated in potting soil in a growth chamber, and the resulting seedlings were maintained in a controlled environment at 28°C (daytime) and 20°C (night-time), with a 16-h light/8-h dark photoperiod. Graphene was diluted to five different concentrations with deionized water  and neutralized to pH 6.3–6.5 with an aqueous solution of sodium hydroxide (0.1 M). Each solution was used as a treatment for one group of maize seeds/seedlings. Each graphene treatment was applied to the group via irrigation once per week, beginning with seed planting. After germination, seedlings were watered weekly with 1 L of the working solution containing the respective concentration of graphene. After 30 days (d) of exposure to graphene, the maize roots were thoroughly washed with deionized water, dried with absorbent paper to remove surface water, flash-frozen in liquid nitrogen, and stored at -80°C until RNA extraction.Maize seedlings from each treatment group were used for root architecture analysis. Similar to the procedures followed for samples collected for RNA-seq, maize roots were washed and harvested after 30 d of graphene exposure. Roots were scanned using the Epson Perfection V850 Pro  at 600 dpi, and the scanned images were subsequently analyzed by WinRHIZO  , which iTotal RNA of maize roots from the CK and 50 mg/L graphene-treated group was extracted using the RNAprep pure plant kit  according to the manufacturer’s instructions. A total of 1 μg purified mRNA was used for cDNA library construction using the NEBNext UltraTM RNA Library Prep Kit for Illumina , following the manufacturer’s instructions. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was conducted using divalent cations under elevated temperature conditions using the NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3′ ends of DNA fragments, the NEBNext Adaptors containing hairpin loop structures were ligated to proceed with hybridization. The AMPure XP bead system  was used to select cDNA fragments of ~300 bp. PCR amplification of the library was performed using Universal PCR primers and an Index (X) Primer with the Phusion High-Fidelity DNA polymerase. Finally, PCR products were purified (AMPure XP system) and library quality was assessed using the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed using the cBot Cluster Generation System and the TruSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the prepared libraries were sequenced using the Illumina NovaSeq platform and 150-bp paired-end reads were generated. Three biological replicates were performed for both the CK and graphene treatment groups.http://hannonlab.cshl.edu/fastx_toolkit/) to remove adapter-containing reads and/or those of a low quality (including poly-N). These clean reads were then mapped to the Z. mays (assembly B73 RefGen_v4) reference genome [https://bigd.big.ac.cn/) under accession number CRA002623.Raw data (raw reads) were initially processed using the FASTX-Toolkit , using the following formula:FPKM = {cDNA Fragments\over {Mapped Fragments (Millions) *Transcript Length(kb)}}.p-values were adjusted using the Benjamini-Hochberg approach [p-value < 0.01 and two-fold or greater expression change identified by DESeq2 [Differential expression analysis of the two groups was performed using the DESeq2 . DESeq2 approach  for conty DESeq2  were cony DESeq2  was usedftp://ftp.ncbi.nih.gov/blast/db/), nt , Pfam , COG , Swiss-Prot , KO , and GO .DEGs were assigned functions based on the following databases: nr  enrichment of the DEGs was analyzed using the R package goseq, which uses a Wallenius non-central hypergeometric distribution  that canqRT-PCR analyses using the RNAprep pure plant kit  according to the manufacturer’s instructions. The resulting RNAs were treated with DNase I before synthesizing cDNA with oligo (dT) primers and M-MLV Reverse Transcriptase (Invitrogen); these products were diluted 5-fold before use. For quantitative real-time PCR (qRT-PCR), the Primer5 software was used to design gene-specific forward and reverse primers (Z. mays GAPDH (ZmGAPDH) gene was used as an internal reference [Total RNA from the CK and 50 mg/L graphene-treated group was extracted for  primers . Analyseeference , and theeference . RelativThe soil nutrient analyzer TPY-6PC developed by Zhejiang Tuopu Yunnong Technology, Co, Ltd, was used to determine nitrogen and potassium contents of the seedling rhizosphere soil according to the manufacturer’s instructions. The rhizosphere soil was sampled with a 5-point sampling method according to the manufacturer’s instructions. Soil samples were thoroughly mixed and dried for 24 h and passed through a 1-mm sieve before measurement.t-test and differences were regarded as significant at p < 0.05. All statistical analyses were performed using SPSS 21 .Each treatment was conducted in triplicate, and the results are presented as mean ± standard deviation (SD). The data were analyzed using a one-way analysis of variance (ANOVA). The significance of differences between mean values was determined using the least significant difference (LSD) test at a 0.05 probability level. Pairwise comparisons were made using the Student's -1, which is generated by the stretching and movement of sp2 hybridized atoms in carbon rings or long chains, representing the ordered sp2 bond structure. Peak D appeared near 1348 cm-1, which is indicative of a sp3 hybridized structure, representing defects and amorphous structures at the edges of the graphene. A wide 2D peak appeared near 2707 cm-1, indicating that the number of graphene layers prepared was within 10 layers. High-resolution scanning electron microscopy  and used untreated seedlings as the corresponding CKs. All samples were used for transcriptome sequencing with three biological replicates . In totanome B73 , the numZ. mays roots exposed to 50 mg/L graphene and CK treatments exhibited two levels of gene expression  for all genes and generated a heatmap plot showing changes in gene expression . A two-fold change and a pression . We idenpression .To investigate possible biological functions that determined the different responses of the maize plants to 50 mg/L graphene treatment, we used GOseq  to perfoDEGs were referenced against the COG database  to classDEGs were also subjected to KEGG pathway analysis to further inform their functional categorization. Z. mays roots in response to the graphene treatment mostly belonged to the MYB and MYB-like, WRKY, NAC, and bHLH families, suggesting that these transcription factor genes might respond specifically to graphene in Z. mays roots.We found that the GO term “transcription factor activity, protein binding” was significantly enriched in maize roots subjected to graphene treatment . TranscrZ. mays roots to graphene treatment, there were three upregulated and one downregulated ethylene-responsive (ERF) transcription factor (TF) genes  genes , eight uF) genes , five upF) genes , ten uprF) genes , three uF) genes , three uF) genes , and twoF) genes .Z. mays.As mentioned above, the total root length, root volume, and the number of root tips and root forks  of maizePlant hormones, including auxins, cytokinin (CKN), gibberellin (GA), abscisic acid (ABA), ethylene, brassinosteroid (BR), jasmonate (JA), salicylic acid (SA), and strigolactone (SL) play critical roles in plant processes, including growth, development, and adaptation to external changing environments –57. We iThe CKN signaling pathway plays an important role in plant growth regulation. Four genes associated with the CKN signaling pathway exhibited significant differential expression in response to graphene treatment . The exp4+ content in the seedling rhizosphere soil, which was significantly increased by up to 1.64 times in response to the 50 mg/L graphene treatment  were also upregulated based on RNA-seq  and qRT-reatment . These rTo validate the RNA-seq results, 20 DEGs were screened by qRT-PCR, including fourteen TF, three nitrogen metabolism, and three potassium metabolism genes. We analyzed the expression of these genes using quantitative real-time PCR (qRT-PCR) and compared the results with the RNA-seq data . These tRecently, research on carbon nanomaterials has focused on applications in agriculture and forestry , 58–60. Z. mays subjected to graphene treatment. After exposure to graphene, the number of upregulated DEGs was higher than the number of downregulated DEGs. Functional characterization of DEGs show enrichment in the categories of transporter activity, TF activity, metabolisms and plant hormone signal transduction, response to stimulus, and detoxification.Using RNA-seq, we successfully identified 962 DEGs in the roots of Arabidopsis, while Wang et al. [Catalpa Scop. MYB and MYB-like TF families are also important, as they are involved in controlling trichome development and root hair formation [Previous research suggests that plant responses to graphene include altered expression of TF genes, as seen by the several WRKY genes induced by graphene in the roots of treated maize. Importantly, WRKY TFs often act as activators or repressors to modulate important plant processes . For exag et al.  reportedormation –47. Addiormation . Studiesormation –43, and ormation  and enhaormation . In our Z. mays. Thereafter, we systematically characterized the influence of graphene on multiple metabolic pathways of Z. mays roots, including producing alterations in the expression of phytohormones, TF, and transporter genes. These genes are potential candidates that respond to graphene treatment by promoting root growth and development. These results provide a theoretical foundation for subsequent research detailing the molecular mechanisms underlying the interaction between graphene and maize roots.In this study, we demonstrated that low-concentration graphene treatment (50 mg/L) promotes root growth and development in S1 Fig(TIF)Click here for additional data file.S2 Fig(A) Pearson correlation coefficient (PCC) of analysis of all genes between the six samples. (B) Principal component analysis of all samples. Red and light blue colors represent the samples of CK and those exposed to 50 mg/L graphene, respectively. (C) Volcano plot of differentially expressed genes. (D) The number of upregulated and downregulated genes.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Complex skin wounds have always been a significant health and economic problem worldwide due to their elusive and sometimes poor or non-healing conditions. If not well-treated, such wounds may lead to amputation, infections, cancer, or even death. Thus, there is a need to efficiently generate multifunctional skin grafts that address a wide range of skin conditions, including non-healing wounds, and enable the regeneration of new skin tissue. Here, we propose studying pristine graphene and two of its oxygen-functionalized derivatives—high and low-oxygen graphene films—as potential substrates for skin cell proliferation and differentiation. Using BJ cells (human foreskin-derived fibroblasts) to represent basic skin cells, we show that the changes in surface properties of pristine graphene due to oxygen functionalization do not seem to statistically impact the normal proliferation and maturation of skin cells. Our results indicate that the pristine and oxidized graphenes presented relatively low cytotoxicity to BJ fibroblasts and, in fact, support their growth and bioactivity. Therefore, these graphene films could potentially be integrated into more complex skin regenerative systems to support skin regeneration. Because graphene’s surface can be relatively easily functionalized with various chemical groups, this finding presents a major opportunity for the development of various composite materials that can act as active components in regenerative applications such as skin regeneration. Thesegraphene ,4. Becaugraphene ,6, energgraphene ,8, bio-sgraphene ,10, and graphene ,12.In the biomedical field, graphene and its derivatives (two or more layers) have been intensively studied, with focus given to properties such as size, shape, and surface chemical functionalization with different functional groups, as well as its integration into various composite systems. Graphene has been used as a cell scaffold for new tissue formation , as wellin vivo and its inherent long-term impact on organs and organisms. The most significant challenge is to accurately detect graphene and distinguish between its carbon structure and the other carbon-rich environments [in vivo. Furthermore, a combination of Raman and photoacoustic/photothermal spectroscopies has been used to quantify down to single cell level the uptake of graphene into various cell systems [Therefore, it is crucial to study and understand the toxicity effects induced by graphene in each pertinent system associated with a biomedical application. Given graphene’s carbon-based structure, it is extremely challenging to thoroughly understand and quantify graphene’s biological distribution ronments . Spectro systems .Different biological systems respond differently to the same graphene material. For example, it was found that red blood cells (non-adherent cells) and skin fibroblasts (adherent cells) responded differently when exposed to aggregated graphene sheets (GS) and dispersed graphene oxide (GO) . Liao etComplex skin wounds are major health and economic problem, as indicated by their elevated prevalence worldwide and corresponding financial cost for treatment ,25. In tTissue engineering offers the opportunity for biomaterials such as graphene and graphene-based materials to directly address prominent issues, such as the ones mentioned above, that hinder normal wound healing and new tissue formation. These biomaterials are chosen for their unique and useful intrinsic physicochemical properties, as well as for their ability to maintain those properties when combined with other materials and bioactive molecules to make them multifunctional in tissue engineering ,29. In sOur study’s aim was to assess and understand how graphene structures with various levels of oxygen functionalization may be used as substrates for skin growth and proliferation. We used pristine graphene (PG) and two of its oxygen-functionalized derivatives, a low and a high oxygen content graphene , as primary substrates for growth and proliferation of BJ fibroblast cells, a human foreskin-derived fibroblast cell line. Our results clearly indicate that an increase in oxygen content from 2.5% in pristine graphene to 8.5% and 25% in low and high oxygen graphene, respectively, drastically altered the material’s surface properties by decreasing the surface roughness and increasing the surface energy. However, these changes in surface properties did not seem to have a short-term effect on the viability and proliferative ability of BJ fibroblasts. Therefore, our findings could serve as the foundation for more in-depth work with these specific graphene materials for regenerative applications.1–1.2-nm-thick PG  was used as received.The low- and high-oxygen graphene samples were prepared according to previously reported methods ,21,36.Dispersions of each graphene  were made at a concentration of 0.5 mg/mL and then bath-sonicated for 1 hour, followed by tip sonication for 1 hour (5sec On/Off pulse) using a Sonics VibraCell Model VCX-130 equipped with a 6-mm microtip probe. 20 mL of the dispersion were then sprayed over culture-treated 24-well plates or 15-mm-diameter glass coverslips using an air-brush technique.XPS  was used to identify the elemental composition of the samples, following the procedures reported ,36. BrieRq, was determined and averaged over the 8 different locations, after each image had been smoothed to remove any artifacts.Surface morphology of the three graphene films was examined using AFM (Bruker Dimension Icon). For each sample, AFM scans were carried out on a 25-x-25-μm area using tapping mode at 8 different locations. NanoScope Analysis (ver. 1.8) software was used to determine the surface roughness. The root mean square surface roughness, EasyDrop  was used to measure water contact angle by sessile drop method. ~10-μl distilled water droplets were dispensed slowly on each sample using an automated syringe controlled by a computer. A CCD camera was used to digitally capture images of water droplets at 60 frames per second, with 780 x 580 pixels. For each sample, contact angle was measured at 3 different places, and the results were averaged.2 and 100% humidity. The cell medium was changed every 2–3 days, and regular cell passage was performed every 3–5 days depending on cell confluency inside the flask.BJ cells  are skin cells derived from human foreskin. The BJ cells are commonly utilized to study skin cells ,37. Expa5 cells were seeded on each of the graphene films sprayed in 24-well cell culture-treated plate and in 1 ml of the serum-supplemented EMEM cell medium. Cells were allowed to grow for 2 days, then trypsinized (trypsin-EDTA (0.25%), Thermo Fisher Scientific catalog # 25200056), stained with Trypan blue dye , and counted using a hemocytometer. The number of live cells from each treatment was recorded.The cell viability assay based on Trypan blue staining works to distinguish live (light) from dead cells (blue). Because the plasma membrane of dead cells disintegrates and is, thus, permeable, it uptakes the blue dye, whereas the intact plasma membrane of live cells is impermeable. In this experiment, roughly 2.5×10Apoptosis in cells grown on graphene films was detected using Annexin V and propidium iodide (PI); where viable cells are negative for both Annexin V and PI, early apoptotic cells are positive for Annexin V, late apoptotic cells are positive for both, and necrotic cells are only positive for PI. Annexin V  binds to the intracellular receptors of the plasma membrane, and PI  binds to cellular DNA following damage to the nuclear membrane. After the Trypan blue exclusion assay, cells were washed with the binding buffer . Next, they were stained with Annexin V and incubated for 30 minutes at 4ºC, away from the light. Cells were washed several times to remove excess and unbound Annexin V, then stained with PI. Data was gathered with an LRSFortessa  at the Flow Cytometry Core of the University of Arkansas for Medical Sciences . The flow cytometry data was analyzed using FlowJo software .Cells were seeded on 15-mm-diameter glass coverslips coated with graphene solutions, then incubated in 1-ml cell culture medium. After 2 days of cell growth, the cell culture medium was removed, and the cells were washed several times with 1x PBS, pH 7.4 , to remove any traces of cell culture medium. The cells were then fixed in 3% glutaraldehyde  in 0.2-M cacodylate buffer, pH 7.2 , for 24 hours at 4ºC. The samples were washed twice for 10 minutes each time in 0.2-M cacodylate buffer then rinsed twice in distilled water for 10 minutes each time. Next, they were post-fixed in 4% osmium tetroxide solution  for 24 hours at 4ºC. As before, they were washed twice in cacodylate buffer followed by rinsing in distilled water. The samples were then subjected to a dehydration process that involved 10-minute serial incubation in graded ethanol solution at increasing concentrations of ethanol: 30, 50, 70, 80, 95, and 100% (twice for 100%). Excess ethanol was removed by a critical point dryer. Finally, the samples were mounted with double-sided carbon tape onto aluminum cylindrical stubs and coated with ~3-nm-thick carbon layers to enhance conductivity. They were imaged by a JEOL JSM-7000F SEM with a field emission gun  at 5 kV.™, Cat# H3570) was conducted. Finally, the samples were washed to remove excess Hoechst solution and mounted on microscope slides for imaging.After 2 days of growth on graphene film-coated glass coverslips, cells were fixed in 4% paraformaldehyde solution  for 15 minutes at room temperature. Later, they were washed twice in PBS followed by a 30-minute incubation in 2% bovine serum albumin solution . Then, they were incubated in CD44 antibody conjugated with APC  and in TE-7 antibody  conjugated with Alexa Fluor 488 . Excess, unbound antibody solution was washed away with PBS, then nuclei staining in Hoechst solution 33342  antibody conjugated with Alexa fluor 488 by following the same procedure described above. Images were collected using ZEN Black software connected to a ZEISS LSM 880 confocal microscope with Airyscan . ZEN Blue software was used to quantify immunofluorescence intensity.The data was analyzed by GraphPad Prism 8.0.2. software. Statistical analysis was performed using ordinary one-way ANOVA to compare variance of the mean across different populations and with Tukey’s multiple comparison test to compare variance of the mean between each population with the control population. We also used two-way ANOVA with Dunnett’s multiple comparison test where one-way ANOVA could not be applied ,39. P va3/sp2 atom. Due to oxygen functionalization, the C1s photoemission peak broadened towards higher binding energy, with the HOG sample displaying distinct peaks. The carbon atoms bound to oxygen, resulting in new functional groups on the HOG: C-OH, C = O, and -COOH at 286, 287, and 288.5 eV, respectively. However, PG and LOG structures showed little evidence of these C-O functional groups. Instead, they exhibited strong π-π shake-up peaks at 291 eV due to the many π-bonds present in sp2-hybridized systems [Our XPS analysis of the graphene samples corroborates what was reported in our previous study , which r systems .Representational AFM images of the three graphene films’ surface topography are shown in We assessed whether the different levels of oxygen in each of the graphene films impacted cell viability. To do so, we grew BJ fibroblasts on the graphene films for two days and then stained the cells with Trypan blue. Compared to untreated BJ cells and the positive controls (BJ cells treated with 400 μM dexamethasone (Dex.) and 5 μg/mL doxorubicin (Dox.)), HOG showed the highest viability followed by PG, then LOG . NotablyNext, we verified the results of our viability assay by staining for cellular apoptosis. Since BJ cells appeared to be viable with little to no apoptosis on graphene films, we assessed whether these cells maintained their fibroblast-like-shape as well. Using SEM, we confirmed that BJ cells did maintain their normal fibroblastic-like-shape despite going through the rigorous dehydration procedure of SEM biological sample preparation . To furtNext, we examined whether BJ fibroblasts could produce collagen (COL-1), a marker of maturation, on graphene films. In this assay, we included HFF-1 fibroblasts as a positive control for COL-1 and vimentin and human keratinocytes (HaCaT) as a negative control for both markers. As expected, both BJ and HFF-1 showed a positive staining for COL-1 and vimentin on HOG, but HaCaT showed no expression of either . CompareIn this study, we aimed to evaluate whether functionalizing pristine graphene with oxygen to create low and high oxygen graphene affects its use as a potential substrate for skin regeneration. We did so by assessing BJ fibroblast (a human-derived foreskin cell line) viability, morphology, and proliferation on those three graphene films. Incorporation of functional groups such as oxygen in biomaterials changes their behavior in biological systems. Importantly, these functional groups can alter a biomaterial surface’s adhesive properties towards cells . Since nFirst, using XPS, we verified the variation in oxygen content among the three graphene samples, with HOG having the highest oxygen content (~25%) compared to LOG (8.5%) and PG (2.5%) . Second,BJ fibroblasts were seeded and allowed to proliferate on these three graphene films for two days, and various assays were conducted to evaluate their growth and proliferation. First, we assessed their viability using a Trypan blue exclusion assay. From this assay, we saw an increase in cell viability on HOG in comparison to the untreated BJ cells. However, the difference in cell viability across the three graphene films was not statistically significant. Second, we analyzed graphene’s cytotoxicity using an Annexin V/PI apoptosis assay by flow cytometry. Our results showed that graphene causes very low toxicity, with HOG being the least toxic (~5%), followed by PG (<10%) and LOG (~12.5%), all of which were minimal compared to the roughly 30% toxicity of the positive control (BJ cells treated with 400 μM Dexamethasone + 5 μg/mL Doxorubicin). There was no statistical difference between the cell toxicity caused by HOG, PG, or LOG. Combined, graphene induced cell toxicity on average ~9%, significantly lower than the positive control (30%) (p <0.0001). Based on these results, HOG was used as the representative graphene substrate in the follow-up immunofluorescence studies.Third, we assessed the graphene films’ biocompatibility with BJ fibroblasts by analyzing cell morphology and expression of two surface markers, TE-7 and CD44, after two days of incubation on HOG. From this assay, we quantified the expression of both cell surface markers on HOG and compared it to their expression in the absence of graphene (no matrix). Positive distinction of fibroblasts from other cells is challenging, in part due to a lack of specific fibroblast markers . In someUsing SEM, we showed that BJ fibroblasts were able to maintain their normal spindle-like shape  ; this waTaken together, our data indicates that although CD44, a cell adhesion and migration protein, was highly expressed in BJ fibroblasts on HOG, COL-1 expression was decreased. Instead, COL-1 was more abundant in BJ fibroblasts expressing less CD44. Several studies have shown that there is a correlation between CD44 expression and extracellular matrix protein expressions, including COL-1, in fibroblasts from different tissues. However, in each of those studies, it appears that the combination of several other factors such as hyaluronanan (a significant ECM component that promotes cell-matrix interaction through CD44 receptor) and TGF-β (a potent growth factor that triggers excess deposition of collagen) in addition to CD44 expression may have played a role in increasing production of the extracellular matrix proteins, specifically COL-1 –52. TherOur results are in agreement with previous reports that showed pristine graphene to be a biocompatible substrate for adherent cells such as fibroblasts and osteoblasts ,53. FurtWe evaluated the possibility of using graphene as an active material to support cellular (fibroblast) proliferation. We investigated graphene materials with variable levels of oxygen, as induced by acid treatment, and the results showed that the oxidized graphene films had an excellent ability to support BJ fibroblast proliferation with low toxicity. Our work, therefore, clearly indicates that oxidized graphene has high potential to play a beneficial role in artificial extracellular matrices for skin regeneration.Based on our data, we conclude that graphene and its derivatives have strong potential to benefit multiple areas of regenerative medicine, including skin regeneration. Graphitic flakes can be integrated in continuous films that support cellular proliferation and differentiation. In order to develop this graphene application, special attention needs to be focused on understanding the complex interactions between the substrates and the various cell lines, down to molecular level. It is also interesting to study the impact that graphene has on the mechanisms that control ECM-specific protein deposition and how these processes are influenced by the substrate’s properties. Another major consideration should be given to the chemistry of the graphene surface.in vitro and in vivo mechanistic studies to further elucidate the complex interactions between graphene and various cell lines and tissues. Furthermore, the strong foundation laid by this study indicates the need for follow-on in vivo work to explore the potential benefits of graphene as a foundational component in skin regeneration platforms.In summary, we have shown that altering the amount of oxygen on the surface of graphitic nanomaterials does not seem to impact their support of cellular proliferation and functions. The next step should be in-depth S1 FigPristine graphene (PG) (black line), low oxygen graphene (LOG) (red line), and high oxygen graphene (HOG) (blue line). The z-potential of the graphene samples with various levels of oxygen functionalizatrion has been studied and published in our earlier studies ,57.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Figa) Differences in expression of adhesion protein CD44 by TE-7-positive BJ fibroblasts on HOG compared to control (no matrix) (p <0.0001). b) HFF-1 and MDA on HOG are positive and negative controls for TE-7, respectively.(TIF)Click here for additional data file.S4 Figa) Differences in expression of extracellular matrix protein COL-1 by vimentin-positive BJ fibroblasts on HOG compared to control (no matrix) (p <0.0001). b) HFF-1 and HaCaT are positive and negative control for both COL-1 and vimentin, respectively.(TIF)Click here for additional data file.