Lamellar-structured graphene aerogels with vertically aligned and closely stacked high-quality graphene lamellae are fabricated.−1 K−1 at 2.30 vol% of graphene content.The superior thermally conductive capacity of the aerogel endows epoxy with a high through-plane thermal conductivity of 20.0 W mThe nacre-like structure endows the epoxy composite with enhanced fracture toughness.−1 K−1, 100 times of that of epoxy, with a record-high specific thermal conductivity enhancement of 4310%. Furthermore, the lamellar-structured graphene aerogel endows epoxy with a high fracture toughness, ~ 1.71 times of that of epoxy. Although thermally conductive graphene sheets are efficient in enhancing in-plane thermal conductivities of polymers, the resulting nanocomposites usually exhibit low through-plane thermal conductivities, limiting their application as thermal interface materials. Herein, lamellar-structured polyamic acid salt/graphene oxide (PAAS/GO) hybrid aerogels are constructed by bidirectional freezing of PAAS/GO suspension followed by lyophilization. Subsequently, PAAS monomers are polymerized to polyimide (PI), while GO is converted to thermally reduced graphene oxide (RGO) during thermal annealing at 300 °C. Final graphitization at 2800 °C converts PI to graphitized carbon with the inductive effect of RGO, and simultaneously, RGO is thermally reduced and healed to high-quality graphene. Consequently, lamellar-structured graphene aerogels with superior through-plane thermal conduction capacity are fabricated for the first time, and its superior through-plane thermal conduction capacity results from its vertically aligned and closely stacked high-quality graphene lamellae. After vacuum-assisted impregnation with epoxy, the resultant epoxy composite with 2.30 vol% of graphene exhibits an outstanding through-plane thermal conductivity of as high as 20.0 W mThe online version of this article (10.1007/s40820-020-00548-5) contains supplementary material, which is available to authorized users. Therefore, the improvements in thermal conductivity of polymer materials, especially their through-plane thermal conductivity, are highly crucial for their application as thermal interface materials (TIMs) between heaters and heat sinks [−1 K−1) and mechanical properties [−1 K−1 even at the graphene loading of ~ 10 wt% [−1 K−1, higher graphene loading is required, which would seriously deteriorate ductility and toughness of polymers [Although polymers have a wide range of applications in electronic devices, including power electronics, electric motors, and generators due to their lightweight, corrosion resistance, and ease of processing –4, theirat sinks . To achiat sinks , 7, boroat sinks –10, carbat sinks , graphitat sinks , 13, andat sinks –17, are operties . Howeveroperties . The the~ 10 wt% , 21. To polymers .−1 K−1 at a low graphene content of 1.23 vol% [−1 K−1 with its TCE of 8100% and its specific TCE of 1300% [To improve the enhancement efficiency in thermal conductivity, Ruoff et al. fabricated a three-dimensional (3D) graphene foam using a chemical vapor deposition (CVD) method as continuous thermal conduction paths for phase change materials, and its erythritol composite exhibited a high thermal conductivity of 3.44 W mof 1300% .−1 K−1) at low graphene contents .Obviously, because of the effective decreases in both the contact thermal resistance between graphene sheets and the interface thermal resistance between graphene and polymer matrix, preconstruction of a continuous graphene conduction network becomes an efficient approach for improving thermal conductivities of polymer/graphene composites –30. Howe−1 K−1 because of the significantly decreased contact thermal resistance [−1 K−1 [For 3D graphene aerogels used for thermally conductive polymer composites, their skeleton walls can be approximately regarded as ultrathin graphene-based films. The quality of graphene sheets and their dense compaction are crucial for heat conduction along the continuous skeletons of the aerogel. In reality, many graphene aerogels derive from GO sheets, and high temperature annealing is adopted to remove the residual oxygen-containing groups of GO sheets and heal their lattice defects to reduce the phonon scattering –33. But,sistance . Besidessistance . Near-pe [−1 K−1 .−1 K−1 at a low graphene loading of ~ 2.30 vol%, as well as a high TCE of ~ 9915% and a record-high specific TCE of ~ 4310%. Furthermore, the lamellar structure of the LSGA endows its thermally conductive nacre-like epoxy composite with high fracture toughness.Inspired by the ultrahigh thermal conductivity of densely stacked high-quality graphene films, herein, we fabricate lamellar-structured graphene aerogels (LSGAs) with continuous and highly thermally conductive paths by bidirectional freezing of a suspension of polyamic acid salt (PAAS) and graphene oxide (GO), followed by lyophilization, imidization, and graphitization treatments. By regulating polyamic acid (PAA)/GO mass ratios, an optimal lamellar architecture is achieved during the bidirectional freezing process, where PAAS and GO components are expelled by bidirectionally grown ice crystals to form the numerous vertically aligned lamellae. By subsequent imidization treatments at 300 °C, the PAAS monomers are polymerized to polyimide (PI) macromolecules, while the GO is thermally reduced to RGO by partially removing its residual oxygen-containing groups. Finally, graphitization at 2800 °C is adopted to graphitize PI macromolecules to be graphitized carbon with the help of the induced orientation effect of RGO and simultaneously upgrade RGO to be high-quality graphene with negligible lattice defects and large crystal sizes. The resultant lamellar-structured aerogel possesses superior through-plane thermal conduction capacity because of its vertically aligned and closely stacked high-quality graphene lamellae. Besides, the conventional graphene aerogels are proud of their low apparent density , 38, whi1,2-Bisethane as the reactive diluent, methyl hexahydrophthalic anhydride as the curing agent, and 2,4,6-tris(dimethylaminomethyl)phenol as the curing accelerator were purchased from Adamas Reagents (China). Bisphenol-A epoxy resin was supplied by Jiafa Chemicals (China). Sulfuric acid (98%), hydrochloric acid (37%), hydrogen peroxide (30%), and potassium permanganate (99.5%) were supplied by Beijing Chemical Reagents (China). Pyromellitic dianhydride , 4,4′-diaminodiphenyl ether , N,N-dimethylacetamide (DMAc), triethylamine (TEA), and sodium nitrate were purchased from Aladdin (China). Natural graphite flakes (300 meshes) were purchased from Huatai Lubricant & Sealing (China). All chemicals and reagents were used as received without further purification.Graphite oxide was synthesized by oxidizing graphite flakes with a modified Hummers method . GO shee3 or 30 × 50 × 20 mm3) that placed on a copper bridge, and then, one end of the copper bridge was inserted into liquid nitrogen to form a bidirectional temperature gradient. The frozen suspension was freeze-dried in a freeze-dryer  for 72 h. The resultant PAAS/GO hybrid aerogel was designated as PxGy, where x:y is the PAA/GO mass ratio. For comparison, neat PAAS aerogel and GO aerogel were also prepared using the same methodology. Detailed ingredients for the preparation of PAAS/GO hybrid aerogels are provided in Table S1.PAAS was prepared by dissolving PAA and TEA in deionized water under magnetic stirring for 2 h with a mass ratio of 1:0.48. After a GO suspension was added into the PAAS solution, the obtained PAAS/GO suspension was sonicated for 15 min and magnetically stirred for 2 h. The initial PAA/GO mass ratio varied from 9:1 to 3:7 to obtain different PAAS/GO suspensions, but the total concentration of PAAS/GO suspensions is kept at ~ 4 wt%. PAAS/GO hybrid aerogels were prepared by bidirectional freezing and lyophilization  was prepared by conventional freezing the PAAS/GO suspension in liquid nitrogen followed by freeze-drying, imidization, and graphitization. A unidirectionally orientated graphene aerogel (UP6G4-2800) was also fabricated by unidirectional freezing the PAAS/GO suspension followed by freeze-drying, imidization, and graphitization.The graphene/epoxy composites (GEs) were fabricated by vacuum-assisted impregnation followed by thermal curing. Epoxy resin, the reactive diluent, the curing agent, and the curing accelerator were mixed uniformly with the mass ratio of 8:2:9.48:0.0576, and then, the LSGAs were immersed into the mixture under vacuum for 12 h, followed by curing at 80 °C for 4 h and post-curing at 120 °C for 2 h. The resultant composites were designated as GEx, where x varies from 1 to 5, representing the initial mass ratio of PAA to GO from 9:1 to 5:5. To increase the graphene content of the resultant composites, the LSGA (P6G4-2800) was compressed perpendicular to the lamellar surface before epoxy resin was thermally cured. The obtained composites were designated as GE4-y, where y is the compression extents, which can be 30%, 50%, and 70%. As a reference, the IP6G4-2800/epoxy composites and UP6G4-2800/epoxy composites were also prepared using the similar infiltration and curing processes and designated as IGE4 and UGE4, respectively.2 was automatically scanned with a XY stage using a step size of ~ 1 μm, and Raman spectra were recorded at every points. The crystal sizes of LSGAs were calculated with the empirical formula: La(nm) = (2.4 × 10−10)λ4(ID/IG)−1 [ID/IG is the integrated intensity ratio of D band to G band. Thermal conductivities were calculated by k = α × ρ×Cp, where α is the thermal diffusivity, Cp is the specific heat capacity, and ρ is the density. Thermal diffusivities of GEs were measured on a Netzsch LFA467 light flash apparatus from 30 to 80 °C. Specific heat capacities of GEs from 30 to 80 °C were obtained using a TA Q20 differential scanning calorimeter (DSC) at a scanning rate of 10 °C min−1. Densities of GEs were measured by an Mettler Toledo electronic balance with a density determination kit 33,360. Thermogravimetric analysis (TGA) curves were obtained with a TA Q50 thermogravimetric analyzer at a heating rate of ~ 10 °C min−1 in a nitrogen atmosphere. The volume fraction was calculated by: vol% = wt% × (ρcom/ρ*), where wt% is the mass fraction, ρcom is the density of a composite, and ρ* is the true density of graphene (2.25 g cm−3). The heat transfer performances of GEs were recorded by a FLIR E40 infrared camera. High-resolution transmission electron microscopy (HRTEM) images were obtained using a JEM-2100Plus microscope at an operation voltage of ~ 200 kV. Three-point bending tests (span 7.75 mm) were performed on a SUNS UTM4103 tester with a loading rate of 0.05 mm min−1. The loading direction is perpendicular to the lamellar surface of the LSGA. For single-edge notched beam specimens, the epoxy and GEs were cut and polished to beams with dimension of 12 × 2.5 × 2.5 mm3, and the beams were then notched by a diamond blade. The notch was sharpened by a razor blade perpendicular to the lamellar direction. The notch was about half of the thickness of specimens. The initial fracture toughness (KIC) was calculated by Eq.  was calculated by Eq.  is calculated by Eq. . X-ray diffraction (XRD) patterns were recorded using a Rigaku D/Max 2500 X-ray diffractometer at a generator voltage of 40 kV. A Renishaw inVia Raman microscope at an excitation wavelength of 514 nm was used to obtain Raman mapping images. An area of 40 × 40 μmID/IG)−1 , where λFigure −3) in the absence of PAAS presents a completely disordered structure . This is not only because the oxygen-containing groups on the GO sheets are willing to be adsorbed to the surface of the ice crystal, and hence, causing a curved ice crystal during freezing process, the high viscosity of GO suspension at such a high concentration also hinders the unidirectional growth of ice crystal [As expected, the PAAS/GO hybrid aerogels show lamellar and porous structures, and the spacing between adjacent lamellae varies in the range of 20-40 μm Fig. b–f. It i crystal . Fortuna crystal . Consequ−3 as the GO content increases from 10 to 50 wt%  of D band (~ 1350 cm−1) and G band (~ 1580 cm−1) [ID/IG ratios. The blue, green, and red colors correspond to their ID/IG values of 0, ~ 0.2, and ~ 0.4, respectively. Compared to PAA-2800 with an average ID/IG of ~ 0.189 , the graphitization extent of P9G1-2800 is greatly improved with a low average ID/IG of ~ 0.087, although it still presents large green and glaucous areas. Interestingly, as the increase in the GO dosage, the blue area becomes larger, while the green area decreases gradually. The average ID/IG decreases to ~ 0.028 for P5G5-2800, very close to that of GO-2800 (~ 0.026) . Meanwhile, as shown in Fig. ID/IG manifests the stepwise healing of sp2 domains from ~ 87.4 nm of PAA-2800 to ~ 188.9 nm of P9G1-2800, and then to ~ 583.4 nm of P5G5-2800 [The graphene quality in LSGAs is further evaluated with Raman mappings by integrated intensity ratios  . As shown in Fig. Km (W m−1 K−1) and K (W m−1 K−1) are thermal conductivities of epoxy and LSGA/epoxy composites, respectively, and V (vol%) is the volume fraction of fillers. As shown in Fig. −1 K−1 with a relatively high specific TCE of ~ 4750% at the graphene content of ~ 1.23 wt%, because GE4 owns highly efficient thermal conduction paths as well as relatively high filler content.By a vacuum-assisted filtration of LSGAs with epoxy monomer and curing agents followed by thermal curing, the resultant LSGA/epoxy composites still show a nacre-like structure . Due to the anisotropic lamellar structure of LSGAs, their composites exhibit different thermal conductivities in three directions, and the thermal conductivity in Z-direction is higher than those along the other two directions  . The thr−1) Fig. a. Moreov−1 K−1, while that of GE4-70% increases to ~ 20.0 W m−1 K−1, because the lamellar sheets become more compact, while the nacre-like structure of the composites is well retained  [−1 K−1, while that of GE4-70% in Y-direction is only ~ 1.22 W m−1 K−1, which is ascribed to the high interface thermal resistances between graphene and polymer matrix in the conduction path of the Y-direction.To further improve thermal conductivity of the epoxy composites while maintaining their mechanical properties, the graphene content can be increased by slowly compressing the LSGAs perpendicular to the lamellar direction before the epoxy resin is thermally cured. As shown in Fig. . S5d-f) . Note th−1 K−1. As shown in Fig. −1 K−1, much higher than those reported in the literature at similar graphene contents [The influence of temperature on thermal conductivity of the thermosetting epoxy composites is also evaluated Fig. e. When tcontents , 58–61. contents , 59, 62.3 are placed on the same hot stage at 75 °C, and an infrared camera is adopted to record in situ the side temperature variation of the samples  results calculated on the basis of the force–displacement curves. KIC of the IGE4 is ~ 0.64 MPa m1/2, slightly higher than that of epoxy (~ 0.62 MPa m1/2). Fortunately, the lamellar-structured GE4 exhibits an enhanced fracture toughness of ~ 0.88 MPa m1/2, ~ 1.38-fold that of the IGE4 at the same graphene content. Consistent with the force–displacement curves, the KIC decreases gradually to ~ 0.70 MPa m1/2 when the compression extent increases to 70%. As reported previously, when the graphene content is beyond a certain value, the fracture toughness of its epoxy composite would decrease [−1 K−1 as well as a high fracture toughness at a relatively low graphene content. As shown in Fig. KJ) of GE4 gradually increases to ~ 2.06 MPa m1/2 within the American Society for Testing and Materials (ASTM) limit (E1820-13) [J of GE4-70% is lower than that of GE4, it still reaches ~ 1.06 MPa m1/2, which is ~ 1.71-fold that of epoxy.Apart from the high through-plane thermal conductivity, the nacre-like structure also endows the composites with high fracture toughness. The typical force–displacement curves Fig. a show th1820-13) , which iCrack propagation is used to explain the high fracture toughness of GE4-70% Fig. d–g. The −1 K−1 at a low graphene content of ~ 2.30 vol%, with a high TCE of ~ 9915% and a record-high specific TCE of ~ 4310%. In addition, the lamellar-structured graphene aerogel also endows epoxy with an enhanced fracture toughness. Our nacre-like graphene/epoxy composite with high through-plane thermal conductivity and fracture toughness demonstrates an insightful avenue for fabrication of high-performance thermal interface materials.Lamellar-structured PAAS/GO aerogels are fabricated by bidirectional freezing of PAAS/GO suspensions followed by lyophilization and converted to PI/RGO aerogels by thermal treatment at 300 °C, during which PAAS monomers are polymerized to PI macromolecules, while GO is thermally reduced to RGO. The final graphitization at 2800 °C is crucial for obtaining the lamellar-structured high-quality graphene aerogels, during which PI is carbonized and even graphitized to thermally conductive carbon with the inductive effect of RGO, while RGO is simultaneously upgraded to high-quality graphene by thermally removing its residual oxygen-containing groups and healing its lattice defects. By adjusting the initial mass ratio of PAA and GO, an optimal LSGA with superior thermally conductive capacity is obtained because of its continuous network, densely stacked graphene lamellae, and large graphene sizes. Thanks to the excellent compressibility, the lamellar-structured graphene aerogel infiltrated with epoxy monomer and curing agent could be compressed perpendicular to the lamellar direction to adjust the graphene content in the resultant graphene/epoxy composite. The nacre-like anisotropic composite exhibits different thermal conductivities along three directions, and its through-plane thermal conductivity can be as high as ~ 20.0 W mD/IG values of LSGAs; and comparison of thermal conductivities and specific TCE of composites with those reported. (DOC 18656 kb)SEM and AFM images of GO; SEM images of PAAS/GO hybrid aerogels and GO aerogel; SEM images of P6G4-2800; Raman mapping images of PAA-2800 and GO-2800; SEM images of composites; comparison of thermal conductivities of GE4; comparison of thermal conductivities of composites; SEM images and Raman mappings of IP6G4-2800 and UP6G4-2800; XRD patterns of P6G4-2800, IP6G4-2800, and UP6G4-2800; TGA curves of epoxy and its composites; fracture surfaces of epoxy and GE4-70%; detailed ingredients of PAAS/GO hybrid aerogels; filler contents, through-plane thermal conductivities of composites, and average ISupplementary material 2 (MP4 127 kb)Below is the link to the electronic supplementary material.


Staphylococcus aureus (MSSA-PENS). To date, there is relatively little known about the current situation and molecular characteristics of MSSA-PENS in China. In this study, we carried out a laboratory-based multi-region retrospective study to investigate the genomic epidemiology and characterisation of MSSA-PENS isolated from invasive bloodstream infections (BSIs) across 17 provinces. The prevalence of MSSA-PENS isolates increased significantly over the 6-year period, with the proportion increasing from 3.51% in 2014–8.80% in 2019, an average relative increase of 22.14% per year , suggesting that China is experiencing a resurgence of MSSA-PENS. Phylogenetic analysis showed a higher strain diversity occurred; the most frequent clonal complexes (CCs) identified were CC188 (17.14%), CC398 (15.71%) and CC5 (15.71%). Over half of MSSA-PENS strains were pan-susceptible, with erythromycin the most frequent resistance observed. Moreover, 25 isolates were identified as immune evasion cluster negative, including CC15, CC188 and CC1, and 6 strains encoded the Panton-Valentine leucocidin gene. Importantly, virulence assays showed that MSSA-PENS exhibited a level of virulence comparable to that of penicillin-resistant MSSA (MSSA-PENR), indicating that more-sensitive strains should not be mistaken for lacking aggressiveness in vivo. Furthermore, 11 of these isolates were confirmed as blaZ positive but phenotype sensitive, with different amino acid changes in blaZ. Our data support the recommendation to clinicians regarding the usage of penicillin in invasive BSIs caused by MSSA-PENS, which might create a novel opportunity for better antimicrobial stewardship in the future.Many countries have reported increasing rates of penicillin-susceptible methicillin-sensitive  Staphylococcus aureus is an opportunistic pathogen with high capacity to adapt to human hosts and healthcare settings. With the rise of hospital-based medicine, S. aureus quickly became a major cause of health-care-associated infections especially bloodstream infections (BSIs)  × width [W]) for 4 days.Virulence assays were performed as previously described. StrainsA total of 1952 MSSA strains isolated from blood were examined in this study. As shown in S detected among all MSSA isolates from blood cultures identified in the 55 Chinese hospitals was 7.17% (140/1952). Of note, as shown in S increased significantly among the MSSA isolates over the 6-year period, with the proportion increasing from 3.51% in 2014–8.80% in 2019, an average relative increase of 22.14% per year .The proportion of MSSA-PENS isolates from BSIs in patients at 55 hospitals across China, we first undertook a whole-genome phylogenetic analysis. As shown in S isolates, 26 STs were identified, and seven isolates were defined as new singletons. The 26 identified ST types belong to 16 clonal complexes (CCs), with CC188 (17.14%), CC5 (15.71%), CC398 (15.71%), CC8 (9.29%) and CC1 (7.14%) the most frequently detected CCs, comprising >65.0% of all MSSA-PENS isolates. Of note, a higher strain diversity was observed yearly from 2014 to 2019 . We observed that >40% of MSSA-PENS isolates belonged to sporadically occurring clones, such as ST707, ST2315, ST1821 and ST2990.To accurately determine the population structure of MSSA-PENblaZ were observed in this study. Thus, we defined MSSA-PENS harbouring blaZ as MSSA-PENS-blaZpositive(n = 11) and MSSA-PENS that did not harbour blaZ as MSSA-PENS-blaZnegative(n = 129). Among the 11 MSSA-PENS-blaZpositive isolates, most belonged to CC188 , whereas the remaining isolates were CC398 (n = 2), CC7 (n = 1) and CC8 (n = 1). Furthermore, most MSSA-PENS-blaZpositive strains (6/11) were isolated from Zhejiang Province.Of the isolates, 11 strains displaying penicillin sensitivity but harbouring spa type, constituting 15.71% of all strains. All isolates of the t189 type belonged to CC188. MSSA-PENS strains of the second most common spa type, t571, accounted for 10.0% of the isolated strains, 63.64% of which were CC398. Notably, t571 is the spa-type most associated with CC398-MSSA BSIs in Europe  gene and absence of erm[T] gene), as this strain was sensitive to tetracycline and lacked the tet(M) gene. In addition, two CC398 isolates harbouring the PVL gene were found in this study. We identified the two PVL-positive CC398 strains as ST398/t034-carrying/TCY-S/ERY-R/IEC type B and ST398/t1451-carrying/TCY-S/ERY-R/IEC type C, respectively. It is important to emphasize that the human-adapted CC398 MSSA was identified as a predecessor of community-acquired MRSA CC398, which evolved by acquiring SCCmec cassettes imparting methicillin resistance [S strains in other studies [In this study, almost all CC398 isolates belonged to sistance . CC5 is sistance . In addi studies ,41.S-blaZnegative isolates (58.14%). Erythromycin exhibited the poorest antibacterial activity (resistance rate of 32.86%), with most of the erythromycin-resistant isolates belonging to CC5, CC398 and CC59, and the resistance was encoded by the ermB, ermC and ermT genes, as predicted by WGS. Notably, although most of the MSSA-PENS isolates identified in this study were blaZnegative , WGS also confirmed that 11 isolates were blaZpositive with different amino acid changes. As a first-generation cephalosporin, cefazolin is the priority antibiotic that is routinely available to treat invasive MSSA infections [blaZ gene reported early [blaZ. Similarly, we also found a single-nucleotide deletion within the blaR1 gene (c.466delA) in seven isolates that introduced a premature stop codon and suppressed blaR1 expression. Previous study has investigated the effect of different blaZ types and polymorphisms at specific amino acid positions of BlaZ on the CzIE [blaZ and blaR1 genes, respectively. This finding indicates that single-nucleotide deletions might play a significant role in penicillin susceptibility of MSSA. However, the role of nonsynonymous amino acid substitutions in penicillin susceptibility should be investigated further in the future.Pan-susceptibility was identified in over a half of the MSSA-PENfections . Cefazolfections , which hfections . Interesthe CzIE . In addiin vivo. For this purpose, we compared the virulence of MSSA-PENS and MSSA-PENR isolates. Interestingly, MSSA-PENS isolates did not exhibit attenuated virulence. Due to its antimicrobial sensitivity, infections caused by MSSA-PENS isolates are easier to treat than those caused by more-resistant strains. However, based on our study, we propose that the danger posed by MSSA-PENS isolates should not be underestimated, as they can cause infections as severe as those caused by MSSA-PENR isolates.We also investigated whether higher antibiotic sensitivity was associated with less aggressiveness S. aureus infections using broad-spectrum antimicrobials might have led to penicillin becoming a “forgotten” antimicrobial in clinical treatment, and the heavy reliance upon vancomycin has also provided opportunities for the emergence of these sensitive isolates. Alternatively, the emergence of novel successful penicillin-susceptible S. aureus clones may provide another possible explanation for the increasing rate of MSSA-PENS. Continuous investigations should be performed to clarify whether some successful penicillin-susceptible S. aureus clones have emerged, which may be responsible for the increasing rates of MSSA-PENS. Taken together, our integrated epidemiologic analysis and characterisation of the MSSA-PENS isolates indicated that penicillin susceptibility is worthy of research attention and that more-sensitive strains should not be mistakenly considered to lack aggressiveness in vivo. Considering the progressive increase in MSSA in BSIs, our data support the recommendation to clinicians regarding the usage of penicillin in invasive BSIs caused by MSSA-PENS isolates, which might create a novel opportunity for better antimicrobial stewardship in the future.In conclusion, like other developed countries such as Sweden, the USA and Spain, China has also experienced a resurgence of penicillin susceptibility among MSSA strains. In this study, we confirmed increased penicillin susceptibility among MSSA isolates obtained from patients with invasive BSIs. Although variable, in some provinces, more than 10% the MSSA isolates were susceptible to this conventional agent. To date, the reasons for the resurgence of penicillin susceptibility remain unknown. Long-term empiric therapy of Click here for additional data file.


We present detailed Raman studies of graphene deposited on gallium nitride nanowires with different variations in height. Our results indicate that different density and height of nanowires impact graphene properties such as roughness, strain, and carrier concentration as well as density and type of induced defects. Tracing the manifestation of those interactions is important for the application of novel heterostructures. A detailed analysis of Raman spectra of graphene deposited on different nanowire substrates shows that bigger differences in nanowires height increase graphene strain, while a higher number of nanowires in contact with graphene locally reduces the strain. Moreover, the value of graphene carrier concentration is found to be correlated with the density of nanowires in contact with graphene. The lowest concentration of defects is observed for graphene deposited on nanowires with the lowest density. The contact between graphene and densely arranged nanowires leads to a large density of vacancies. On the other hand, grain boundaries are the main type of defects in graphene on rarely distributed nanowires. Our results also show modification of graphene carrier concentration and strain by different types of defects present in graphene. Therefore, the nanowire substrate is promising not only for strain and carrier concentration engineering but also for defect engineering. The combination of excellent electrical and mechanical properties with interesting physical phenomena occurring in two-dimensional structures makes graphene an interesting experimental material to study –3. ImporA novel approach of graphene-based nanostructures are van der Waals heterostructures in which graphene is transferred onto another material with a different morphology and electronic properties . HoweverSystems containing graphene on nanowires have been used in solar cells to increase their efficiency. In particular, it has been shown that the application of nanowires in solar cells decreases light reflection by scattering of light in between nanowires –8. NanowOne of the most common experimental techniques for studying properties of graphene is Raman spectroscopy . Non-invIn this paper, we present detailed statistical studies of Raman spectra of graphene deposited on gallium nitride nanowires (GaN NWs) with different variations in height. The electric field induced in GaN predicted by theoretical calculations could reach 5 MV/cm . This is−2. However, they formed clusters containing several merged NWs. In the second sample (N100) the height of the NWs varied by approx.100 nm, from 300–400 nm, and the density of NWs was approx. 400 NWs·µm−2. In the third sample (N500) the average density of NWs was similar to that in the first sample – approx. 120 NWs·µm−2. However, in this sample, two distinct groups of NWs were observed – approx. 80% of them were 1 μm in height while approx. 20% reached 1.5 μm.Monolayer graphene was grown by chemical vapour deposition (CVD) on a copper foil with methane gas as the precursor . Next, gThe samples were studied by scanning electron microscopy (SEM) using a SU8230 Hitachi microscope equipped with an in-lens secondary electron detector at 5 kV electron beam voltage. The Raman spectra were collected by using a T64000 Horiba Jobin-Yvon spectrometer with a Nd:YAG laser operating at 532 nm wavelength as the excitation source, and with an objective with a magnification of 100× that allowed to obtain a spatial resolution of approx. 300 nm. The laser power was reduced to 3 mW in order to reduce the heating effect. The micro-Raman maps were collected with 100 nm steps with a few square micrometres of mapping area for each sample. The spectra were calibrated by using a reference sample of high-quality silicon.The morphology of graphene deposited on NWs with different variations in height is presented in The large cracks visible in the graphene layer are caused by the transfer process. Graphene on NWs with equal height is smooth . Small w−1 and 2D band at 2680 cm−1) and both defect bands (D band at 1345 cm−1 and D’ band at 1620 cm−1) are observed . The dependence of graphene strain on the 2D band energy shift is described by 2D is the Grüneisen parameter, Δε is a value of strain, and the value of 2D band energy for unstrained graphene  was reported to be 2677.6 cm−1 [where γ7.6 cm−1 . Positiv7.6 cm−1 ,36–39. T7.6 cm−1 –41. Ther7.6 cm−1 ,42.−1) is observed for the N500 sample, while the highest standard deviation of 2D band energy (2.7 cm−1) is observed for the N100 sample  and the lowest for the N500 sample (1.7). Surprisingly, the value of R2DG suggests that the carrier concentration in the N500 sample is the highest of all the investigated samples, which disagrees with the conclusions obtained from the analysis of the G and 2D band energy values and FWHM.The intensity ratio of 2D and G graphene bands in monolayer graphene has been reported to be negatively correlated with carrier concentration . A higheR2DG on the strain and carrier concentration was performed by the analysis of 2D and G band FWHM. As discussed before, 2D band FWHM is positively correlated with the graphene strain, while G band FWHM is negatively correlated with the carrier concentration [R2DG ratio and 2D band FWHM is observed  and described by The G band is generated by the scattering on iTO or iLO phonons near the Γ point of the Brillouin zone. For the presence of D band, the resonant scattering on the iTO phonon near the K point of the Brillouin zone and the defect are necessary. Consequently, the intensity of the G band is proportional to the laser spot area while the intensity of the D band depends on the number of defects in the excited area. Therefore, the density of defects l is the excitation light wavelength. In order to visualize the distribution of defect density on the graphene surface, we performed spatial and statistical analysis of the intensity ratios of the G and D bands  is correlated with the low density of defects. However, a similar number of defects in the N0 and N100 samples remains unclear. Although the density of defects is similar for N0 and N100 samples, a different distribution of the RGD ratio, which reflects defect density on the surface, is observed  depends on defect density and parameters describing the perturbation introduced by the defects in the crystal lattice. These perturbation parameters depend on the type of defect and are different for the D and D’ bands. Thus, the intensity ratio between D and D’ bands characterize the type of defects in graphene [RDD’ ratio equals to 3.5 is characteristic of grain boundaries, five is characteristic of multiple vacancies, seven corresponds to single vacancies, while 13 is observed for sp3 hybridisation defects [RGD ratio, a strong modulation of RDD’ by the NW substrate is observed on the 2D maps  while the grain boundaries are the main defects in the N500 sample . At least 98% of all types of defects in the N500 sample are grain boundaries, which is a higher value than the obtained for vacancy contribution in N0 and N100 samples . The standard deviation of the RDD’ ratio for the main maximum in the N500 sample is equal to 0.6, which is lower than that for N0 and N100 samples . Therefore, the interaction between graphene and rarely distributed NWs is more homogenous than that with densely arranged NWs. The largest number of different types of defects  are observed in the N100 sample, which confirms that graphene interacts with densely distributed NWs in a variety of ways.The intensity of both defect bands D and D’  in these two samples is unclear. From the discussion above, the dependence between the density of NWs supporting graphene and the types of defects is nontrivial. Our results suggest that in graphene deposited on rarely arranged NWs, the grain boundaries are the most dominant type of defects. Densely arranged nanowire substrates introduce vacancies in the graphene deposited on them. Furthermore, the presence of a large number of vacancies in the N0 and N100 samples together with gating by the nanowire substrate could be responsible for increasing the carrier concentration, which is also confirmed by other studies [As discussed before, the analysis of the 2D band energy and  studies ,48. In tTherefore, high differences in NW height and low density of supporting points decrease the observed density of defects and highlight grain boundary defects omnipresent in the graphene layers. The contact with NWs of lower differences in height and higher density of supporting points creates more vacancies and increases their density on the surface. Moreover, graphene strain and carrier concentration can be locally modified by the different density of defects and their types. Thus, further studies on the influence of NWs supporting graphene and graphene strain, carrier concentration, and defects performed with higher resolution are essential.RGD and RDD’ ratios showed that the density of defects in graphene was affected by the nanowire substrate. Our results suggest that NWs supporting graphene with low differences in height introduce vacancies in the graphene. Increasing distances between NWs decreased the density of defects and exposed a larger number of grain boundaries omnipresent in any graphene layer. Furthermore, the vacancies could locally increase the graphene carrier concentration and tensile strain in the N0 and N100 samples together with the nanowire substrate. Thus, the density of NWs supporting graphene substrate and their differences in height impacted graphene carrier concentration and strain. It is, therefore, possible to consider the use of NW substrates for defect engineering in graphene and probably in other 2D materials.We transferred graphene onto GaN NWs with 0, 100, and 500 nm variations in height and studied their properties by SEM and Raman spectroscopy. Graphene on NWs with variations in height was rippled and pierced by the highest NWs. A detailed analysis of the Raman spectra showed that differences in NW height as well as NW density strongly impact graphene strain and carrier concentration. The highest strain value was observed for the sample with the highest differences in height . Unexpectedly, the strain in the graphene on NWs with equal height had a tensile character while the strain in the graphene on NWs with non-equal height had a compressive character. Analyses of G band energy and G band FWHM showed a positive correlation between the density of NWs under graphene and the value of its carrier concentration. In contradiction to previous reports, we found that the intensity ratio between 2D and G bands is correlated with the graphene strain rather than with its carrier concentration. Furthermore, analyses of


Industrial preparation of graphene has been a research hotspot in recent years. Finding an economical and practical carbon source and reducing the cost of production and instrument is significant in industrial graphene production. Coal is a common carbon source. Efficient improvement and utilization in the cleaning of coal has recently been a popular research area. In this study, we developed a set of graphene preparation methods based on Anhui Huainan’s low-rank gas coal (HNGC). Using self-built experimental equipment, benzene precursor was prepared from HNGC and used as carbon source to realize graphene growth. The quality of the graphene was characterized by a high-resolution microscope and Raman spectrometer. This study provides a new idea and method for the preparation of low-rank coal-based graphene. Coal mining destroys groundwater resources and exacerbates water shortage in water-deficient areas. Coal mining leads to the emission of waste gas , which harms the atmospheric environment. In addition, there are few inorganic substances in coal, mainly water and minerals, which reduce the quality and utilization value of coal. Therefore, the development of converting coal into clean and efficient materials enhances the utilization value of coal and provides new ideas for tapping the potential industrial value in materials. Among them, the preparation of graphene from coal is a popular research area in this field in recent years [Graphene, as a new type of two-dimensional material, has attracted wide attention from the scientific research community because of its unique crystal structure and electronic properties, which make it have unique electrical, mechanical and thermal properties. Graphene has a good application prospect in various physical and engineering fields. Therefore, obtaining cheap and controllable technology for the industrial preparation of graphene has been widely pursued by the scientific research community. In this area, obtaining a clean and pure carbon source from cheap and common matter to realize graphene is one of the most popular research areas in recent years. Coal is a kind of carbon-containing organic matter with polycyclic aromatic hydrocarbon as the main structural unit, which exists widely in nature. As a big coal country, China is rich in coal resources. Anhui, as a big coal province, has huge coal reserves, and Huainan gas coal is the main coal resource. However, the conversion efficiency of coal is low because the coal is still limited in practical applications of burning. Nowadays, coal, as the main fossil energy in China, will inevitably pollute the environment during processing and utilization. When coal is burned, most sulfur is oxidized to sulfur dioxide , gasification and liquefaction of coal ,8. The uGenerally, from the basic principle of chemical vapor deposition (CVD) growth of graphene, polycrystalline copper foil is applied as an economical and efficient catalyst to reduce the decomposition barrier of carbon precursors, and the most common carbon precursor is gaseous methane ,12,13,14However, methane is a flammable and explosive gas produced during the coal mining process, which makes the current process complicated and increases the cost of fabrication. In recent years, in order to realize the large-scale industrial production of graphene, more and more new precursor carbon sources have appeared. It is found that graphene can be prepared via some solid and liquid precursor molecules ,16,17. TOur study will explore the technology of producing graphene from gas coal to graphene based on the technical route process: from gas coal to coal tar to benzene. That is,Pyrolyzing gas coal in high temperature and oxygen-free environment to produce coal tar;Separating crude benzene and other fractions by distillation;Refining industrial-grade benzene as carbon source to grow graphene.In view of the mature industrial foundation of the preparation of refined benzene, we focused on the CVD growth of monolayer graphene with benzene as carbon source. For the graphene samples obtained, we used an optical microscope, a scanning electron microscope and Raman spectroscopy to characterize the graphene. In addition, we examined the number of layers and the morphology features of graphene grown under different growth conditions, such as benzene flow rates and growth temperature, in order to explore the possibility of the low-temperature preparation of the graphene in the future.Coal tar is pyrolyzed from collected gas coal at high temperature under oxygen-free conditions, and then crude benzene is obtained by distillation, and refined benzene with high concentration is obtained by refining. The method of producing graphene with refined benzene as carbon source is as follows: introduce 50 sccm hydrogen gas and keep the tube furnace at 1060 °C for three hours. The main purpose of heating for 3 h with added hydrogen is to remove the oxide on the surface of copper foil, and to make the surface smooth by the reconstruction of atoms within the annealing process. After that, reduce the temperature to 500 °C and reduce the flow rate of hydrogen to 30 sccm , meanwhile introduce benzene for 40 min. The flow rate of benzene can be controlled by adjusting the trimming valve in order to avoid the influence and waste caused by volatilization of benzene when the substrate is heated up. Generally, after reaching the growth temperature, the needle valve should be adjusted properly to make benzene fill in the furnace tube at a required rate continuously, which makes the pressure in the tube maintain at the required vacuum condition. After finished the sequence, the ball valve and trimming valve are required to be closed, then the temperature of the tube is cooled down to room temperature naturally. After that, graphene is transferred to silicon oxide by standard wet transfer process.We found that the growth condition of preparing monolayer and multilayer graphene from benzene at high temperature generally has a large range. Since benzene is liquid, it is hard to control the content of benzene with a traditional flow controller process, so the amount of benzene used is supposed to be accurately adjusted by using a trimming valve. The content of benzene can be controlled and monitored by a vacuum gauge. We control the carbon flux in the growth process by different benzene partial pressures in order to study the graphene quality at different benzene partial pressure. We found that at the partial pressure of benzene of 1.1 Pa, the optical microscopic results show the nearly completely monolayer graphene with a regular and uniform surface. From the image of optical microscopy a, the hiIn order to examine the details of the graphene quality, we used the scanning electron microscope (SEM) to show the detailed morphology and the material quality of graphene, as shown in In order to realize the possibility of growing graphene at a low temperature in future, we examined the number of layers and the morphologic features of graphene grown with different growth conditions, such as benzene flow rates and growth temperature. In our experiment, while the partial pressure of hydrogen with a flow rate of 30 sccm was 55.6 Pa, the partial pressure of benzene ranged from 17.3 Pa to 1.1 Pa, and the growth temperature ranged from 1060 °C to 500 °C. When the partial pressure of benzene is 17.3 Pa but the growth temperature is still kept at 1060 °C, it is observed under an optical microscope a that thWhile in order to decrease the amount of benzene, when the partial pressure of benzene is reduced to 4.4 Pa, the quality of the material is obviously decreased and there are some bi-layer/multi-layer graphene structures with the obvious brightness at the edge because of the large thickness from the SEM image e althougThe way to grow graphene at high temperature is to break the benzene ring into carbon atoms and obtain a large area of graphene through the catalytic reaction at copper substrate. However, the benzene ring can directly remove hydrogen atoms and then re-bonds each other to generate monolayer graphene at a low temperature. Following this process, industrial production costs can be greatly reduced. We also prepared graphene at a temperature of 500 °C and the partial pressure of benzene of 1.1 Pa. Under the optical microscope, it was observed that the prepared graphene was very regular g but theIn this study, the coal samples analyzed were from the No. 13-1 coal seam of the Upper Shihezi Formation in Huainan coalfield, northern China. In order to ensure the freshness of the samples, groove sampling was carried out in the underground working face. In the laboratory, vitrified coal strips (removing gangue) were obtained by a manual separation method and they were ground and sieved until the particle size reached 200 mesh. The ground powder sample was then dried and stored in a sealed bag for testing.daf, %), hydrogen , nitrogen , and sulfur  were the average of two parallel samples, and the oxygen  content was obtained by difference subtraction. According to the GB/T212-2008 standard, the moisture , ash  and volatile matter  of HNGC samples were determined. The experimental results are presented in The ultimate analysis of Huainan gas coal (HNGC) samples was carried out on a Vario EL element analyzer, German EA Company . The contents of carbon , D peak (1350 cm−1) and 2D peak (2680 cm−1) are caused by defects or disorders that can be used to preliminarily determine the number of graphene layers, the stacking order between layers and the defect and impurity density, respectively. The light transmittance of the optical microscope and the interference of the substrate can be used to directly characterize the graphene layers transferred to the silicon oxide substrate. In addition, the overall roughness and uniformity of graphene can be preliminarily characterized. The scanning electron microscope can be used to characterize the surface morphology details of graphene [The method of obtaining high-temperature dry distillation coal tar by high-temperature pyrolysis of coal annealing 200 g HNGC to 1000 °C by using a high-temperature pyrolysis program analyzer. The instrument for the preparing of graphene using the CVD method from coal consists of three parts: a tubular furnace of HF-Kejing with a vacuum gauge monitoring the pressure inside, connected to a liquid/gas flow rate control system and a vacuum pump unit  by chemical vapor deposition with different benzene flow rates; the relatively uniform monolayer graphene structure was also obtained by low temperature CVD. Our results show that in future industrial applications, replacing the traditional gas carbon source of methane gas from the Huainan coal with the liquid benzene can produce high-quality monolayer graphene with an economic preparation cost, and has the characteristics of high efficiency and good safety.Our results also found that the layers and structure of graphene made with CVD process are highly related to the flow rate of benzene. We are working on quantifying the relationship and trying to control the structure and layers. We believe, in future, this method can be used for convenient, flexible and economic growth control of monolayer/multi-layer graphene, which can lay a foundation for the exploration and development of new functions of coal.


DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations  reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene  Glued is the largest component of dynactin [Dynactin, a megadalton-sized multimeric complex, acts as an essential cofactor of the microtubule-based motor cytoplasmic dynein-1  in many eukaryotic cells . Dynactidynactin ,3,4. DCTdynactin ,6. The odynactin ,9,10.Interactions of dynein and dynactin, including those of DCTN1, with various partner proteins have been identified through the efforts of many investigators. DCTN1 directly interacts with microtubules in a dynein-independent manner and accumulates at microtubule plus-ends ,12. DCTNDynein also binds to Huntingtin (HTT), the causative protein for Huntington’s disease (HD), while DCTN1 binds to HTT-associated protein 1 (HAP1) ,23,24. TTARDBP gene, is a ubiquitously expressed RNA-binding protein; it is predominantly nuclear but undergoes nucleocytoplasmic shuttling. Physiologically, TDP-43 coordinates multiple aspects of RNA metabolism, for example, regulating gene transcription and RNA splicing in the nucleus, and RNA transport and protein translation in the cytoplasm and axoplasm [Transactive response DNA-binding protein 43 (TDP-43), encoded by the axoplasm ,29. TDP-axoplasm . Evidencaxoplasm ,32. PrLDaxoplasm . PrLD/IDaxoplasm . Recent ALS10) and sporadic ALS; expression of mutant TDP-43 induces neural apoptosis [In 2006, almost simultaneously, two research groups reported TDP-43 to be a major ubiquitinated protein component of insoluble cytoplasmic aggregates in degenerating neurons found in patients with frontotemporal dementia (FTD) and a fatal motor neuron disease, amyotrophic lateral sclerosis (ALS) ,36. Thespoptosis ,39. The poptosis ,32,39. Tpoptosis ,41. HoweDCTN1, closely apposed mutations in its CAP-Gly domain cause two distinct neurodegenerative diseases  have been identified so far -derived neurons, thereby recapitulating several cellular phenotypes found in the brain neurons of Perry disease patients.The first goal of this study was to characterize the biochemical relationship between DCTN1 and TDP-43. To investigate the molecular basis of Perry disease, we have recently been searching for DCTN1-interacting proteins involved in neurodegeneration. However, no novel, promising candidates for such DCTN1 interactors were found. We thus refocused on the findings that, in our immunohistochemical studies, both TDP-43 proteinopathy and dynactin aggregates were detected in all of the Perry disease post-mortem brains examined ,48,50. Rrevealed . These fTo examine whether DCTN1 interacted with TDP-43, we performed coimmunoprecipitation between endogenous Dctn1 and Tdp-43 proteins in murine brains. We prepared whole brain extracts from embryonic day (E) 16.5 mice. An anti-DCTN1 antibody was used to immunoprecipitate Dctn1 from the brain cell lysates, and an anti-TDP-43 antibody was used to detect Tdp-43 in the immunoprecipitates through Western blotting. Results of our coimmunoprecipitation experiment revealed that the endogenous Dctn1 and Tdp-43 interacted with each other in vivo C.To confirm the potential interactions between DCTN1 and TDP-43, we next performed coimmunoprecipitation between tagged DCTN1 and TDP-43 proteins in cultured cells. cDNAs encoding the full-length human DCTN1 C-terminally tagged with 2× myc epitope (DCTN1-myc) or with monomeric green fluorescent protein (DCTN1-mGFP) and/or full-length human TDP-43 N-terminally tagged with mCherry (monomeric red fluorescent protein (mRFP) derivative; mCherry-TDP-43) were transfected into monkey fibroblast COS-7 cells. DCTN1-myc or DCTN1-mGFP were immunoprecipitated using an anti-myc or anti-GFP antibody, respectively, from the cell lysates D,E. In bWe sought to map the TDP-43-interacting domains of DCTN1. A series of truncated mutants of DCTN1-myc were made and subjected to coimmunoprecipitation assays. Aiming at high-precision domain dissection, bioinformatics analyses were performed on the amino acid sequence of DCTN1 ) using online resources, including SMART-EMBL Heidelberg , MulticoBased on these results, we divided the DCTN1-coding sequence to create expression plasmids encoding seven truncated mutant forms of DCTN1-myc, as illustrated in These truncated mutant DCTN1-myc and mCherry-tagged full-length TDP-43 were cotransfected into COS-7 cells, and their interactions were examined by coimmunoprecipitation. TDP-43 was detected in the anti-myc immunoprecipitates from cells expressing full-length DCTN1 or several mutant forms; the CAP-Gly-basic supradomain-containing fragment (D∆2), dynactin domain (D∆4), CC2-containing region (D∆5), and extreme C-terminal region (D∆6) of DCTN1 interacted with TDP-43  or the PrLD-containing C-terminal fragment  and the TDP-43-CTF were sufficient for interactions between DCTN1 and TDP-43 E. AltogeG71A and DCTN1G59S mutants with TDP-43 was significantly higher than those of wild-type DCTN1 . On the other hand, the interactions of DCTN1F52L with TDP-43 were not significantly different from those of wild-type DCTN1 , which was consistent with clinical observations that symptoms of patients with the DCTN1 p.F52L mutation are less severe than with the other mutation types [Two different groups of missense mutations within the DCTN1 CAP-Gly domain are known to cause Perry disease, accompanying TDP-43 proteinopathy, or HMN7B, with no detectable TDP-43 proteinopathy ,48,50. Won types . Taken tWe next examined interactions between wild-type DCTN1 and a defective NLS-carrying mutant TDP-43 (TDP-43-∆NLS). As previously reported , contrarE. coli and purified using glutathione Sepharose beads (We examined whether DCTN1 bound to TDP-43 in vitro. The DCTN1-coding sequence was divided into two parts: to express the NTF composed of the CAP-Gly-basic and CC1 domains (D∆8), or the CTF composed of the dynactin and CC2 domains and extreme C-terminal region (D∆9) , less severe levels of DCTN1 protein aggregates were found in cells coexpressing DCTN1G59S and TDP-43, as compared with the cells coexpressing DCTN1G71A and TDP-43  prevented the cytoplasmic-nuclear transport of TDP-43, and thereby caused dramatic mislocalization and aggregation of TDP-43, revealing that DCTN1 physiologically regulated TDP-43 localization and aggregation, possibly via multivalent interactions between DCTN1 and TDP-43. The results of expression of truncated DCTN1 (D∆4–6) suggest that the disruption of regulated multivalent DCTN1-TDP-43 interactions drive aberrant aggregation of the two proteins.G71A or D∆4 with TDP-43, significant levels of TDP-43 cytoplasmic mislocalization were induced  [Many researchers, including the current authors, have speculated that a direct relationship between DCTN1 aggregation and TDP-43 aggregation was unlikely, and that TDP-43 proteinopathy might be a secondary event that emerges long after dynactin aggregation, mainly for two reasons. First, colocalizations of dynactin components, DCTN1, DCTN2/p50, and DCTN4/p62, with TDP-43 were detected in only a small number of protein inclusions, according to autopsy studies ,50 and cd below) .G71A or D∆4–6) and wild-type TDP-43, the incidence of aggregation of DCTN1 alone, without TDP-43 aggregation, was unexpectedly low. However, coaggregation (or coexistence within the same cells) of DCTN1 and TDP-43, or less frequently, aggregation of TDP-43 alone, was detected in around 79%–85% of the cells  became localized predominantly in the nucleus D. On theytoplasm . We therDrosophila RNA-binding protein Egalitarian (Egl), although Egl is not thought to be evolutionally conserved [mRNA localization underlies precise spatiotemporal control of protein translation in polarized cells, such as neurons, and especially in axons; the dysfunction of this machinery has been implicated in various neurodegenerative diseases ,86. The onserved . It remaonserved ,6. We foonserved ,11. ThusFurthermore, as well as interacting with dynein, DCTN1 interacts with the anterograde microtubule motor kinesins, to coordinate bidirectional transport ,90. DCTNElectron microscopic studies reveal that the N-terminal region of DCTN1, composed of the CAP-Gly-basic, CC1, dynactin, and CC2 domains form a flexible extension called the projecting arm, while its C-terminal region is buried in the shoulder of the dynactin complex ,91. We hAccording to our bioinformatics analysis, five putative IDRs were found in DCTN1. IDRs have been implicated in protein phase separation-driven aggregation . InteresTaken together, the present study showed that DCTN1 can form aggregates of DCTN1 and TDP-43, preferentially via the dynactin domain and C-terminal region, which act as TDP-43-binding sites. Our results also implicate biophysical mechanisms by which aggregates of DCTN1 and TDP-43 are formed in Perry-diseased neurons. It remains to be clarified whether non-aggregated forms of mutant DCTN1 triggers TDP-43 aggregation, and whether mutant DCTN1 aggregation occurs earlier than TDP-43 aggregation, or simultaneously. It also remains to be understood whether coaggregation proceeds at the surface or in an intermingled manner or both; interestingly, as mentioned above, we frequently observed these two modes of coaggregation, colocalization and surface-to-surface contacts of DCTN1 aggregates with TDP-43 aggregates, even in the same cells. In addition, our data indicated that the CAP-Gly-basic domain, dynactin domain, and the C-terminal region of DCTN1 act as distinct functional modules for controlling TDP-43 cytoplasmic-nuclear transport and aggregation.Taken together with the previous studies, our results support the two currently well-discussed aspects of Perry disease-linked mutant DCTN1: loss of function (reduced microtubule-binding and impairment of dynactin recruitment at microtubule plus-ends) and gain of function (induction of TDP-43 aggregation), both of which compromise the cytoplasmic-nuclear transport of TDP-43.Recent studies have revealed that mislocalization of TDP-43 from the nucleus to the cytoplasm and the cytoplasmic aggregation of TDP-43 impairs the machineries for nuclear protein import and RNA export ,63. MoleConsistently, dynein heavy and light chains were found in TDP-43 aggregates purified from transfected neuroblastoma cells . As discG71A, D∆4, or D∆6, nuclear clearance of TDP-43 was observed less frequently (less than 1%) in U2OS cell models coexpressing TDP-43 and mutant DCTN1 (unpublished observations); but significantly (25%–30%) in iPSC-derived neurons coexpressing TDP-43 and mutant DCTN1  at Fukuoka University.cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene  (SC110869). The coding region of DCTN1 was amplified by polymerase chain reaction (PCR) and subcloned into pcDNA3.1/CT-GFP-TOPO plasmid . Following confirmation by DNA sequencing, DCTN1 point mutants  were generated using a QuikChange II XL site-directed mutagenesis kit . The cDNAs encoding wild-type/full-length DCTN1, point mutants or truncated mutants were reamplified by PCR using KOD Plus (Ver. 2) polymerase , and subcloned into SacII/AgeI sites of mEGFP/pRK5 vector , to make DCTN1-mEGFP expression plasmids, or 2× myc/pRK5  to make DCTN1-myc expression plasmids.To clone cDNA encoding human TDP-43, double-stranded Human Liver QUICK-Clone cDNA  was used for reverse transcription-PCR, according to the manufacturer’s instructions. Full-length cDNAs were cloned into XhoI/BamHI sites of mCherry-C1 plasmid  was replaced with the mCherry-coding sequence). mCherry-TDP-43-∆NLS was generated using a QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies), based on the same design as previously described . The codThe nucleotide sequences of DCTN1, TDP-43, their missense mutants and truncated mutants used in this study were confirmed by DNA sequencing .Antibodies against DCTN1 , TDP-43 , and β-tubulin ; Sigma-Aldrich, St. Louis, MO, USA; TUB2.1) were used for immunoprecipitation and/or Western blotting. Antibodies against GFP , RFP , c-Myc , Myc-tag , and horseradish peroxidase (HRP)-conjugated anti-RFP antibody  were also used for the detection of tagged DCTN1 and TDP-43 proteins.HRP-conjugated secondary antibodies used for Western blotting in this study were: goat antimouse IgG , AffiniPure donkey antigoat IgG , Amersham ECL donkey antirabbit IgG , and mouse antirabbit IgG (conformation-specific) mAb .Cercopithecus aethiops) fibroblast COS-7 cells and human osteosarcoma U2OS cells were obtained from ATCC. Cells were maintained in Dulbecco’s modified Eagle’s Medium  supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). All cells were cultured in a 5% CO2 incubator at 37 °C.African green monkey , without any antibiotic treatment, ~24 h before transfection. Transient transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, cells were transfected in Opti-MEM I reduced serum medium  by adding a mixture of 6 μL of Lipofectamine 3000 reagent in Opti-MEM and combinations of plasmids plus 5 μL of P3000 reagent in Opti-MEM I [4Cl for 10 min at room temperature to reduce autofluorescence, and incubated with Hoechst 33,342 (Molecular Probes/Thermo Fisher Scientific) for nuclear counterstaining. Samples were mounted in Permafluor (Thermo Fisher Scientific), and observed under a confocal microscope.For transfection with plasmids, cells were seeded at 2.0 × 10ti-MEM I . The folNEUROG2-inducible human iPSC (201B7) lines, where NEUROG2 is overexpressed upon treatment with doxycycline, were maintained using a feeder-free culture system  [5 cells/well) were replated onto poly-D-lysine/laminin-coated coverslips  in a 24-well plate or μ-dishes , and directly differentiated into neurons by treatment with 1 μg/mL doxycycline for 7 days and then by stimulation with 20 ng/mL brain-derived neurotrophic factor , 20 ng/mL Glial cell line-derived neurotrophic factor , 1mM dibutyryl cyclic AMP , and 200 nM ascorbic acid  in Neurobasal medium  supplemented with B-27  for 3 days. After a total of 10 days of neuronal differentiation, iPSC-derived neurons were cotransfected with the DCTN1-mGFP  and mCherry-TDP-43 expression plasmids using Lipofectamine 3000, following the same protocol as above. The cells were fixed 48 h after transfection by adding 4% PFA + 10% sucrose in PBS, stained with Hoechst 33,342 and observed under a confocal microscope. In the current experiments, the survival rates of neurons coexpressing D∆5 or D∆6 and TDP-43 were too low for observation, possibly due to their cell death-inducing effects in differentiated neurons. Thus, analyses on these cells were excluded from our present study.; AK02N) ,70. The For coimmunoprecipitation between endogenous Dctn1 and Tdp-43 proteins, cell extracts were prepared from embryonic day 16.5 (E16.5) mouse whole-brain isolated from pregnant ICR mice . The buffer used contained 50 mM Tris-HCl (pH7.4), 150 mM sodium chloride, 1 mM EDTA, 0.5% NP-40, and 0.5% sodium deoxycholate; supplemented with 5 mM sodium orthovanadate, 10 mM sodium fluoride, and cOmplete Mini . The brain cell lysates were preabsorbed with Protein G-conjugated Sepharose (Cytiva), and then incubated with a goat anti- DCTN1 antibody at 4 °C for 17 h. The immunoprecipitates were recovered using SureBeads Protein G magnetic beads  for 2 h. Beads were collected by using DynaMag-2 (Thermo Fisher Scientific) and washed five times with the above buffer. Recovered proteins were eluted by adding 1 × Laemmli buffer to the beads and boiled at 95 °C for 5 min. The proteins were detected by Western blotting with the antibodies indicated in the For coimmunoprecipitation of tagged proteins, COS-7 cells were transfected with different combinations of plasmids encoding the wild-type/full-length, truncated mutant or point mutant protein of DCTN1 or TDP-43. Forty-eight hours after transfection, cell lysates were prepared using the same buffer as above except without sodium deoxycholate, and immunoprecipitated with anti-c-Myc, anti-GFP or anti-RFP antibodies and then with SureBeads Protein G magnetic beads.E.coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies) at 15.5 °C for 16 h upon induction with 100 mM IPTG [To detect the in vitro binding between DCTN1 and TDP-43, recombinant GST-fused full-length TDP-43 proteins were expressed in  mM IPTG . After bImmunoprecipitates, pull-down samples, and cell lysates were resolved on an SDS-PAGE gel  together with Precision plus protein dual color standards (Bio-Rad), transferred onto Immobilon-P PVDF membranes (Millipore), and probed with the primary antibody as indicated and HRP-conjugated secondary antibodies as previously described . ImmunorImages of single optical sections and/or z-stacks of fixed cells were acquired using a laser-scanning confocal LSM710 microscope or super-resolution LSM880Airy microscope  . Quantification of images was performed using ZEN 3.0 SR (black) (Carl Zeiss) and MetaMorph . Images presented in The cells to be quantified were randomly selected on each coverslip. In each experiment shown in 2 atmosphere. Approximately 48–72 h after transfection, cells were live-imaged in CO2-dependent FluoroBrite DMEM media (Thermo Fisher Scientific), supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine. Super-resolution images were acquired at intervals between approximately 4 and 6 s, in a single focal plane.Live-cell microscopy was performed using the LSM880Airy microscope (in super-resolution mode) with a 63×/NA 1.40 oil objective lens (Carl Zeiss) and an Incubation System S  at 37 °C and in a 5% COt-test or two-tailed Mann–Whitney test ; p < 0.05 was considered statistically significant.No statistical methods were used to predetermine sample size. Sample sizes were chosen based on previous experience to obtain reproducibility. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. No data points were excluded, and all data collected from each individual experiment were used for analysis. Statistical analysis was performed using the two-tailed paired


Idiopathic pulmonary fibrosis (IPF) is a chronic respiratory disease with high incidence, morbidity, and mortality rates. Jinshui Huanxian formula (JHF) is an empirical formula that targets the pathogenesis of lung-kidney qi deficiency and phlegm-blood stasis in pulmonary fibrosis (PF). The purpose of this study was to explore JHF's potential pharmacological mechanisms in IPF therapy using network intersection analysis. JHF's primary active components and corresponding target genes were predicted using various databases. Two sets of IPF disease genes were obtained from the DisGeNET and GEO databases and two sets of IPF drug targets were collected. The disease and drug target genes were analyzed. The JHF target genes that intersected with IPF's differentially expressed genes were identified to predict JHF's targets of action in IPF. The functions and pathways of predicted targets acting on IPF were analyzed using the DAVID and KEGG pathway databases. Finally, the resulting drug target mechanisms were validated in a rat model of PF. The initial analyses identified 494 active compounds and 1,304 corresponding targets for JHF. The intersection analysis revealed four common genes for the JHF targets, IPF disease, and anti-IPF drugs in the KEGG database. Furthermore, these genes were targeted by several JHF compounds. Seventy-two JHF targets were closely related to IPF, which suggests that they are therapeutically relevant. Target screening revealed that they regulate IPF through 18 pathways. The targets' molecular functions included regulation of oxidoreductase activity, kinase regulator activity, phosphotransferase activity, and transmembrane receptor protein kinase activity. In vivo experiments showed that JHF alleviated the degree of PF, including decreases in collagen deposition and epithelial-mesenchymal transition. This study systematically explored JHF's mechanisms to identify the specific target pathways involved in IPF. The generated pharmacological network, paired with in vivo validation, elucidates the potential roles and mechanisms of JHF in IPF therapy. Idiopathic pulmonary fibrosis (IPF) is a chronic respiratory disease, which is characterized by progressive fibrosis of lung parenchyma, resulting in function and respiratory failure. It is the most common pulmonary interstitial disease with an estimated incidence of 2.8–9.3 cases per 100,000 person per year in Europe and North America [Drugs commonly used to treat IPF include glucocorticoids, azathioprine, cyclosporin, warfarin, N-acetylcysteine, and acid suppressants. However, these drugs may induce adverse events (AEs), such as myelotoxicity associated with cytotoxic drugs or diffuse alveolar hemorrhage . PirfeniRadix Rehmanniae. This blend nourishes yin and dissipates phlegm, promotes blood circulation and regulates qi, and primarily treats the lung-spleen deficiency syndrome and phlegm-blood stasis in the later stage of IPF. In clinical practice, JHF has shown significant improvements in clinical symptoms, inducing delayed down disease development and improving the quality of life [Traditional Chinese medicine (TCM), a natural chemical library, is a main component of medical practice. The characteristics of Chinese herbal compound include being multicomponent, multitarget, and having complex mechanisms of action. They can enhance body functions and reduce drug toxicity through the synergistic actions of their main active ingredients. Jinshui Huanxian formula (JHF) is an empirical formula for the pathogenesis of lung-kidney qi deficiency and phlegm-blood stasis in PF. JHF's monarch herbs are ginseng and  of life .Network pharmacology integrates system-level network analysis and pharmacology to gain insight into the complex mechanisms of herbal formulas used to treat complex diseases , 12. In In order to explore JHF's pharmacological mechanisms associated with IPF, we used JHF-based network pharmacology to study the relationship among TCM, Chinese medicinal ingredients, target genes, and differentially expressed genes in IPF. First, through extensive data mining, we collected information on two groups of IPF-related disease genes, two groups of anti-IPF drugs, and their therapeutic targets. Second, we gathered information on JHF's bioactive compounds and identified candidate target genes using public databases. Potential vital targets and their related pathways involved in JHF-mediated effects were identified by analyzing the collected datasets. Then, based on IPF's differentially expressed genes, a network analysis was conducted to discover JHF's therapeutic targets that might target IPF's differentially expressed genes, their biological functions, and the important pathways involved in this response. Finally, JHF's predicted targets and functions were verified using in vivo experiments.The project's workflow is illustrated in http://tcmspw.com/tcmsp.php) is a unique Chinese herbal medicine system that contains information about absorption, distribution, metabolism, and excretion (ADME) characteristics of compounds [http://119.3.41.228:8000/tcmid/search/) includes comprehensive formulas, herbs, herbal ingredients, and drug and disease information. This database helps researchers in the traditional medicine fields to discover potential new drugs and the mechanisms of drug interactions.The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)  [Homo sapiens” species setting. The STITCH database uses compounds that are structurally similar to JHF's chemical components to identify targets   using th targets . The thrhttp://www.disgenet.org/, ver. 6.0) [https://www.ncbi.nlm.nih.gov/geo/) [Information on IPF-associated genes was collected from the DisGeNET (er. 6.0)  and GEO ov/geo/)  databaseov/geo/)  ran micrhttps://www.genome.jp/kegg/pathway.html) and DrugBank [https://www.drugbank.ca/, ver. 5.1.4) databases. Searching the KEGG pathway database revealed four IPF-associated drugs with 14 target proteins .The constructed compound-target and target-pathway networks were visualized using Cytoscape . The HINT database is a curated compilation of high-quality protein-protein interactions from eight interactome resources  [To screen for JHF targets that may regulate differentially expressed genes in IPF, IPF-associated differentially expressed genes were first mapped to the High-quality INTeractomes database  . The tarhttps://david.ncifcrf.gov/home.jsp, ver. 6.8) to calculate the Gene Ontology enrichment and the KEGG pathways.To clarify the functions and pathways associated with the predicted JHF targets, we used the functional annotation clustering tool in the Database for Annotation, Visualization, and Integrated Discovery  were obtained from the Experimental Animal Center of Henan Province . The rats were housed under standard temperature (26 ± 2°C), humidity (50 ± 10%), and light intensity (12 h light/dark cycle) conditions and were allowed free access to standard laboratory food and water. All animal experiments were performed in accordance with international ethical guidelines and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Experimental Animal Care and Ethics Committee of the First Affiliated Hospital, Henan University of Traditional Chinese Medicine.The PF rat model was established using previously published methods . The PF μm sections. The sections were stained with hematoxylin-eosin solution  and Masson's Trichrome Stain Kit  to determine the collagen distribution. Digital images were captured using light microscopy.The formaldehyde-fixed lung tissues were paraffin-embedded and cut into 4 α-SMA , E-cadherin , N-cadherin , and GAPDH  at 4°C overnight. The membranes were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse antibody  for 2 h and visualized using a Bio-Rad ChemiDocTM MP System  with Super ECL Plus reagent .Lung tissues were homogenized and lysed with RIPA containing PMSF  for 30 min and centrifuged at 12,000 g for 10 min at 4°C. Lysates were mixed with SDS loading buffer and boiled at 100°C for 5 min. The proteins were separated using SDS-PAGE gel and transferred to PVDF membranes . The membranes were sealed with 5% nonfat milk and incubated with the following primary antibodies: P value  < 0.05.All data are expressed as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVAs followed by post hoc analysis with Tukey's tests. Results were considered significant at Ginseng Radix et Rhizoma, 162 compounds), Maidong , Dihuang , Gualou , Zhebeimu , Mudanpi , Yinyanghuo , Baiguo , Baitouweng , Yiyiren , Chenpi , and Gouqizi . Fifty-four of the compounds revealed by the TCMSP and TCMID were duplicates, resulting in 494 unique compounds.JHF contained the following 12 herb materials with a total of 548 compounds: Renshen , C-C motif chemokine (CCL2), interleukin-6 (IL6), and interleukin-10 (IL10). This suggests their important role in IPF treatment. These predictions indicated that TNF was targeted by 13 JHF compounds , CCL2 was targeted by six JHF compounds , IL6 was targeted by eight JHF compounds , and IL10 was targeted by three JHF compounds  (enosine) . The fouβ (TGF-β) signaling pathway, cytokine-cytokine receptor interaction, and cellular senescence. We used ClueGO, a Cytoscape plugin [We used Cytoscape software to construct KEGG's drug-target-pathway network for the anti-IPF drugs . PFD, a e plugin , to analP < 0.01)) (< 0.01)) . Disease< 0.01)) .β1 and SMAD3, that participated in 18 pathways, including the ErbB signaling pathway, thyroid hormone signaling pathway, and TGF-β signaling pathway  signaling is known to play an important role in PF, including their effects on mitochondrial function. Increased transformation of prohormone thyroxine to active 3,5,3-triiodothyronine improved alveolar epithelial cell (AEC) metabolism in IPF, with thyroid hormone playing an important role in mitochondrial biogenesis and bioenergy, thereby regulating AEC apoptosis . CompareMultiple chemokines and cytokines induce fibroblast migration and induce the phenotype switch to myofibroblasts, thereby contributing to the occurrence of IPF. Chemokines and cytokines are expressed and released in alveolar macrophages, which mediate the inflammatory response and fibrotic process. During the middle stage of PF, immune cells secrete chemokines and proinflammatory factors and accelerate macrophage migration and secretion, thereby accelerating the immune response . Single-ErbB signaling is enriched in the plasma proteome of IPF patients . TyrosinNeurotrophin signaling is performed via the binding of NT ligands to their homologous high-affinity receptors, i.e., neurotrophic tyrosine kinase receptors. NT ligands and their homologous receptors have been detected in mice and adult human lung tissues –48. Mounβ1-induced fibrogenesis in pulmonary fibroblasts by activating the Hippo pathway, resulting in the promotion of nuclear translocation and increasing YAP1 inactivation and degradation in the cytoplasm [The Hippo signaling pathway plays a key role in many important pathological processes, such as organ growth control, cell proliferation, apoptosis, tissue regeneration, and tumor suppression , 52. Yesytoplasm .β is an important EMT inducer and is the strongest inducer of extracellular matrix deposition. TGF-β can stimulate fibroblasts to synthesize extracellular matrix components and induce matrix metalloproteinase expression [β1 can stimulate VEGF-A expression in human fetal pulmonary fibroblasts through the Smad3 signaling pathway [In addition, TGF-pression , includipression , 57. VEGpression . In the pression , 59. Som pathway . These rSome “hub” signal molecules show multiple overlapping pathway routes that form complex functional modules . This coThe current study systematically analyzed JHF's pharmacological mechanisms underlying IPF treatment to provide guidance for clinical practice. The study investigated the underlying mechanisms of a common TCM at the levels of the target, pathway, network, and biomedical efficacy; obtained information regarding JHF's therapeutic targets that might affect IPF's differentially expressed genes; and elucidated the biological functions of these targets and their important pathways. In addition, this work lays a foundation for identifying therapeutic treatments for complex diseases like IPF.


CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca2+ levels (caused by CaV channel opening), Ca2+-bound CaM binding to CaV is essential for promoting rapid Ca2+-dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca2+-induced channel opening . In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca2+-free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca2+ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca2+ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca2+ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca2+-bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca2+-bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.L-type voltage-gated Ca Thus, CaM acts as both an accelerator and a brake to control CaV channel opening i = 100 nM vs DappK = 100 nM), which may have functional implications (2+ binding to either CaM or CaBP1) are needed to test whether Ca2+ binding to the C-lobe of CaM (or CaBP1) is essential for the increased CaV channel open probability caused by CaM (or CaBP1) under basal conditions.CaV channels are oppositely regulated by CaM and CaBP1: CaM binding to CaV is essential for channel CDI, whereas CaBP1 binding prevents CDI and promotes CDF. A careful analysis of available Caications . Future


Immobilization of uricase on MBs can decouple the functionalization steps from the sensor surface, which allows the sensor to be reusable. Our microsensor platform exhibits a relatively lower detection limit (1 μM), high sensitivity (5.6 mV/decade), a linear range (from 1 μM to 1000 μM), and excellent linearity (R2 = 0.9945). In addition, interference assay and repeatability tests were conducted, and the result suggests that our method is highly stable and not affected by common interfering substances (glucose and urea). The integration of this high-performance and compact biosensor device can create a point-of-care diagnosis system with reduced cost, test time, and reagent volume.In this study, we developed a low-cost, reusable, and highly sensitive analytical platform for the detection of the human metabolite uric acid (UA). This novel analysis platform combines the graphene chemoresistor detection technique with a magnetic bead (MB) system. The heterojunction (single-layer graphene and HfO The detection and quantification of biomarkers are essential for medical diagnostics, environmental monitoring, and bioresearch. Uric acid is a metabolite in whole blood and is crucial in the diagnosis of many clinical diseases , such asGraphene-based nanomaterials are used as transducers of biosensors, which are involved in converting the interactions between the receptor and the target molecules into detectable measurements ,15. The In the past decades, the combination of MBs with conventional biological assay techniques has achieved great success due to the excellent properties of MBs, which include scalable and reusable capabilities, a high specific surface area, less toxicity, powerful magnetism, and high loading of the sensitive matrix . In the Here, we first proposed combining MBs and graphene chemoresistors as a microsensor platform for uric acid detection. This method overcomes the aforementioned drawbacks of the traditional sensor method. First, the proposed uric acid sensor is portable, reusable, and simple and has a low-cost fabrication process. Based on our calculation, the cost was approximately 10 USD. As it is reusable, the cost of each single test could be much lower. Second, the receptor functionalization step is achieved on the MB surface, breaking the main limitation of performance degradation caused by repeated modification of the graphene channel surface. Test results showed that the analysis platform exhibits excellent performance for the detection of uric acid. Miniaturization of the micro-biosensing platform endows itself with a promising prospect of application in a portable real-time instrument for point-of-care diagnostics.− as the pH increases [− make graphene p-doped; n-doped and p-doped graphene is able to modulate the channel conductance by doping holes or electrons, which is consistent with other graphene studies [The mechanism of our graphene-based biosensor detection of uric acid is shown in ncreases . Therefo studies ,24,25.3) and hydrochloric acid (HCl), which corrode copper foil, were purchased from Aladdin Reagent Co, Ltd. . Deionized water used in this work was purified using Milli-Q water purification. Buffer formulation ingredients, sodium sulfate (Na2SO4), sulfuric acid (H2SO4), sodium dihydrogen phosphate (NaH2PO4), sodium hydrogen phosphate (Na2HPO4), sodium bicarbonate (NaHCO3), and sodium carbonate (Na2CO3) included, were bought from Sinopharm Chemical Reagent Co, Ltd. . Dynabeads M-280 Tosylactivated (MBs), bovine serum albumin (BSA), and uricase, which was used to modify MBs, were purchased from Thermo Fisher Scientific. Phosphate-buffered saline (PBS) needed for this assay was purchased from Aladdin Reagent Co, Ltd. .Iron chloride hexahydrate  of the graphene chemoresistor. A Raman spectrometer (RamTracerTM-200 Raman Spectrometer) was performed to obtain Raman spectra to characterize the quality, layer number, and uniformity of graphene.An H-600 Hitachi scanning electron microscope  was used to characterize the scanning electron micrographs of the graphene morphology. Samples were imaged under the high-vacuum mode at magnifications of 800×. A Keysight 34465A digital multimeter was used to measure the source-drain voltage (4) was introduced into the chamber with a flow rate of 35 sccm for 10 min to initiate the growth of graphene; at the same time, 35 sccm of hydrogen gas was added to the chamber .The single-layer graphene films in our experiment were grown on copper foils by low-pressure CVD (LPCVD). First, copper substrates were pretreated by an annealing process, and the surface morphology of the copper substrate significantly improved. Then, copper foils were pre-cleaned in acetic acid at 35 °C for 10 min to remove the surface oxide . Subsequ2 (20 nm) was deposited by atomic layer deposition (ALD).I) of 1 mA at different pH values of the solutions . The pH change of the solutions was regulated by adjusting the proportion of buffer compositions. Respectively, neutral buffers of different pH values (from pH 6.0 to pH 8.0) were prepared by mixing 0.2 mol L−1 of NaH2PO4 and 0.2 mol L−1 of Na2HPO4, while acidic buffers of different pH values (from pH 1.7 to pH 4.1) were prepared by mixing 0.01 mol L−1 of Na2SO4 and 0.01 mol L−1 of H2SO4, and alkaline buffers of different pH values (from pH 9.17 to pH 10.83) were prepared by mixing 0.1 mol L−1 of Na2CO3 and 0.1 mol L−1 of NaHCO3.To study the effect of pH on the performance of the graphene chemoresistor, acquisition of electrical signals was performed through a Keysight 34465A digital multimeter at a constant source and drain current .−1, 1590 cm−1, and 2700 cm−1, respectively. The related assignments of spectral peaks to chemical bonds can be found in our previously published works [I2D/IG ratio was about 2, and more than 90% of the graphene film was determined as a single layer. In addition, the relatively weak D peak and the large 2D peak indicated the high quality and low defect density of graphene.Scanning electron microscope (SEM) analysis was used to determine the morphology of graphene. SEM photomicrographs made at 800× magnification of graphene on a glass substrate are shown in ed works ,29,30. BtotalV). Measurements of the source-to-drain voltage as a function of time demonstrated stepwise increase along with the discrete change of the buffer’s pH. The results are shown in −1 for the neutral solution, 81 mVpH−1 for the acidic solution, and 70 mVpH-1 for the alkaline solution. The standard deviations of the test were 2.29% (for pH = 6–8), 4.52% (for pH = 1.7–4.1), and 2.69% (for pH = 9.17–10.83).We evaluated the performance of our biosensor in acidic, neutral, and alkaline solutions . To conclude, our biosensor exhibits good corresponding linearity and sensitivity performance in buffer solution, which suggests that our graphene chemoresistor is a microscale pH sensor. The testing results above also prove the feasibility using the biosensor to detect highly efficient reactions of uric acid and uricase.Uricase is immobilized on beads to specifically catalyze the oxidation of UA. Uricase hydrolyzes free uric acid according to Equation (1) below :(1)Uric totalV) of the graphene sensor decreased to varying degrees with the addition of different concentrations of uric acid. 2 = 0.995) was obtained when the UA concentration was changed from 0 μΜ to 1 mM, as shown in As shown in Selectivity is the ability of a biosensor to generate a positive result only from interactions. p < 0.01, N = 3). The above testing results demonstrated similar behaviors with voltage-time plots, which is similar to what was shown in The graphene biosensor was then subjected to a repeatability test. Compared with the traditional methods for UA detection, our system demonstrated better performance in terms of the detection range and limit of detection. The results are summarized in 2 was deposited on the surface of high-quality graphene to improve the pH response of the graphene sensor. After verifying the feasibility of the graphene biosensor to detect the change in pH in acidic and alkaline environments, it was combined with magnetic beads for the detection of uric acid. The low-cost and scalable analysis platform exhibited excellent performance for detecting uric acid, including a lower detection limit (1 μM), good sensitivity (5.6/dec), and excellent linearity (0.995). The sensor showed good real-time detection performance, good stability, simple operation, and specificity for uric acid. Furthermore, this proposed method can potentially be applied to biological sample detection in whole blood and serum in the future after optimization. All of these features suggest that our platform has the potential to be a competitive method in the field of healthcare monitoring and clinical diagnosis.In summary, by atomic layer deposition, 20 nm of HfO