This article is about managing flood disasters affecting the built environment in the rural communities of Zimbabwe. Using Tsholotsho district in Matabeleland North province as a case study, the authors argue that flooding has adversely impacted the built environment through destroying infrastructure. The principal objectives of this study were to establish the impact of flood disasters on the built environment, to demarcate factors that perpetuate communities’ vulnerabilities to flooding and to delineate challenges that negate the management of flood disasters in the built environment. This qualitative study was based on a purposive sample of 40 participants. Data were collected through semi-structured interviews and observation methods. The findings were that floods can damage human shelter, roads, bridges and dams. Locating homesteads near rivers and dams, using poor-quality construction materials, and lack of flood warning were found to perpetuate vulnerability to flooding. Poverty and costs of rebuilding infrastructure, lack of cooperation between the communities and duty-bearers, and failure to use indigenous knowledge were found to be impeding the management of flood disasters. The study concluded that flood disasters can wipe out community development gains accumulated over many years. Further, community vulnerability to flooding in the built environment is socially constructed. The study posits that addressing the root causes, reducing flood vulnerability and avoiding risk creation are viable options to development in the built environment. Lastly, reconstruction following flood disasters is arduous and gruelling, and not an easy exercise. Flood disasters have a propensity to impact the built environment and cause huge losses through destroying property, including buildings and infrastructure Cigler . EvidencThis study focuses on managing flood disasters on the built environment in Tsholotsho district, south-west of Zimbabwe. The principal objectives of the study are threefold, namely, to establish the impact of flood disasters on the built environment, to demarcate factors that perpetuate vulnerability to flooding and to delineate the challenges that negate the management of flood disasters impacting the built environment.The management of flood disasters in Tsholotsho district in the Matabeleland North province, south-west of Zimbabwe, has not been effective enough to save the built environment. Of late, the district has been inundated by destructive flooding events. The floods have severely impacted the built environment, damaging human shelter among other forms of infrastructure. Communities most affected have been those located along the Gwayi, Zombani and Manzamnyama rivers, and communities settled near Gariya and Bhudani dams. The Government of Zimbabwe has at times tried to restore the damaged shelter and infrastructure through some reconstruction processes. However, following subsequent severe flooding, the built environment has always experienced the same impact of being destroyed by the forceful floods. As such, it is imperative that the government comes up with new robust measures that can save the built environment. Such measures have a potential to benefit the communities through minimising the impact of future flooding events. If no meaningful measures are taken by the duty-bearers to deal with the impact of flooding on the built environment in the district, the communities will continue to suffer huge losses. Such losses may even be permanent, considering that many communities in rural districts are poor, with little or no means to recover from disaster impact Cigler .This section of the article reviews the literature relevant to the study. The literature is interrogated and discussed in a manner that agrees with the study’s objectives. The first objective focuses on the impact of flood disasters on the built environment.Globally, flood disasters of various magnitudes have continued to severely impact the built environment. According to Lindell , disasteCertain factors may perpetuate communities’ vulnerability to flooding, leading to disaster occurrence in the built environment. This study chose the three common aspects of flood vulnerability, namely, use of poor building materials and communities’ failure to adhere to building codes, sociological aspects of communities and cultural beliefs. These factors were chosen because they were believed to be the major drivers contributing to flood disasters in modern societies. Mudavanhu  posits tPrevious studies on disaster risk reduction have shown that structures constructed using poor, non-durable construction materials are vulnerable to flood hazards. Therefore, players in the construction industry are encouraged to carefully choose building materials when constructing structures. Substandard building practices are understood to aggravate flood disaster impact on the built environment. Deficiencies, which manifest in the failure to enforce building codes, if not addressed, may prevail even in the post-flood disaster reconstruction period. Vulnerability to flooding in the built environment can, therefore, be reduced through the building of resilient structures, through regulating social behaviours, and through the provision of adequate early warnings, among other strategies Pelling . ApproprSatterthwaite et al.  note thaSocial setting of communities is another factor that can influence flood disasters within built environments. Sociological aspects and configurations of communities can worsen some people’s vulnerabilities and fuel the intensity of flooding events The findings in Three major factors were found to have contributed to and perpetuated flood vulnerability, and flood disasters in the built environment of Tsholotsho district. According to the interviewees, such factors include location of homesteads near rivers, dams and other water basins – which was most mentioned (25.0% – 62.5%), followed by use of non-durable or poor-quality construction materials (10.0%– 25.0%) and lack of timely flood warning (5.0% – 12.5%). These factors are discussed in detail in the subsection ‘Homesteads located near rivers, dams and other water basins’.Homesteads located near rivers and dams proved to be the major factor perpetuating flood vulnerability in the district. Of the interviewed flood victims, 62.5% were of the view that had their settlements been sited away from the rivers and dams, they would have been free from flood risk. This can be interpreted to mean that the communities settled in flood-prone areas were aware of the dangers facing them, but just ignored them. As such, disasters can impact even those communities with enough disaster knowledge and awareness if no proper measures are taken. Through the researchers’ observations, it was also noted that some villagers in Wards 5 and 6 were located very close to the Zombani and Gwayi rivers respectively, while those in Ward 8 were situated close to Gariya and Bhudani dams. One villager narrated how during the rainy season, water from the Bhudani dam would overflow into their homesteads:‘During rainy seasons, water overflows to our homesteads when Bhudani dam is full. This situation has resulted in the community losing some property in the form of huts, barns and school buildings in addition to the destroyed dam wall’. The above findings concur with a study conducted by Parker, Little and Heuser , who fouApart from building their homesteads in flood-prone areas and near rivers and dams, communities were observed to have used non-durable materials for building their structures. Some respondents (10.0% – 25.0%) thought their flood vulnerability was worsened by weak materials used in the construction of shelter (huts and houses), roads, bridges and dam walls. Dwelling structures, according to the respondents and complemented by observations, were constructed from pole, mud and thatch. This type of building material was observed to be inferior and prone to flood damage. The continued use of such material in Tsholotsho district suggests that the district might continue to be a breeding place for flood disasters. Poor workmanship in the construction of bridges was observed by the researchers.One such bridge which was observed to have been damaged by the floods was a small bridge crossing Zombani River in Siphepha area. The bridge was completely washed away by flood waters, rendering it dangerous and unusable. Crossing places along rivers need to be constructed using durable material that does not endanger the lives of the rural communities. The findings are in line with results from Sharma and Joshi , who fouLack of flood awareness within the communities was found to be another factor contributing to flood vulnerability in Tsholotsho district. Some respondents (5.0% – 12.5%) highlighted that most people lacked sufficient information on impending flood hazards, resulting in being caught unawares. The respondents were of the opinion that lack of flood awareness contributed to their ignorance about flood hazards, hence their increased vulnerability to flooding. Their argument was that communities with enough flood awareness would rarely be caught unaware during periods of severe flooding. Respondents felt that flood awareness programmes should be undertaken within the communities, and that these programmes would make known flood hazards existing within their vulnerability contexts. One respondent had this to say:‘We want the responsible authorities, especially the District Civil Protection Unit, to come down here to the communities and provide flood awareness programmes. People living in this part of the district do not have enough information concerning their vulnerabilities to flooding, hence our continued suffering’. However, the study further established from the respondents that awareness campaigns had been provided by the Tsholotsho District Civil Protection Unit (DCPU), although they have been few. From the respondents’ narrations, provision of timely flood awareness by the DCPU may enhance their capacity to prepare for flooding events in the district. As such, this study interprets that the DCPU and its partners are not effective enough to serve the communities well. Disaster knowledge is an empowerment tool against flooding, and communities should possess such knowledge in advance and in abundance (Fabiyi & Oloukoi Three major issues were found to negate the management of flood disasters in the built environment of Tsholotsho district. As highlighted by 20 out of 40 (50.0%) respondents, poverty and cost of rebuilding infrastructure, followed by lack of cooperation between communities and duty-bearers (15.0% – 37.5%), and failure to properly use indigenous knowledge (5.0% – 12.5%) were found to be the main challenges affecting the rebuilding processes.The majority of people in places affected by the floods in Tsholotsho district, as highlighted by the respondents, are peasant farmers, most of whom are languishing in poverty. This study views poverty as being based on the evaluation of the economic or income status of individual members of the community People expect the responsible governments to help them to recover from disasters. Therefore, the government as the main duty-bearer should not impose humanitarian aid on the communities without proper consultation. These results support findings from previous research found in the extant literature. For example, Cannon, Twigg and Rodwell (2003:10) showed that communities and organisations that have cooperated successfully were in a position to assist each other and other groups during disaster events. Lack of cooperation between the communities and the duty-bearers can result in a situation where flood-impacted communities find themselves in worse conditions.inkanku (a bird associated with forecasting rains), cloud patterns and changes in certain indigenous trees. The respondents believed such indigenous knowledge helps them to predict rains with a potential for flooding. From the researchers’ interpretive point of view, the use of indigenous knowledge by the residents is symbolic of the norms, culture and beliefs prevailing in many African communities. It also shows the value that African communities attach to their cultural beliefs and how their culture shapes their daily lives. These findings are consistent with the results of a study by Domfeh (Members of the community living with flood risk were found to command indigenous knowledge for dealing with floods. According to the respondents, they were able to use home-grown knowledge to forecast rainy seasons with a potential to cause flooding. This guided them in taking appropriate action in preparation for flooding. The respondents narrated how they studied and interpreted the behaviour of certain animals and birds to forecast the magnitude of rains. They studied the behaviour of y Domfeh  on indigAlthough the knowledge generated from the local communities is of utmost relevance, the respondents felt that their knowledge was underrated by the DCPU and other players. The respondents thought that local communities should be allowed to contribute their knowledge in managing flood disasters. The authors interpret this as low-rating of indigenous knowledge compared to Western knowledge. Again, disaster risk reduction practitioners see no potential value of indigenous knowledge as an effective tool for flood disaster management. However, the respondents were of the opinion that their indigenous knowledge should be integrated with modern knowledge, so that both spheres of knowledge become more effective. Their argument was that as the practitioners fall short of using indigenous knowledge that communities possess, infusing the two types would be a positive step.Drawing from the findings and conclusions, the authors recommend duty-bearers to assist flood-impacted communities with the means of restoring their built environments. Duty-bearers should assist communities with material for rebuilding of flood-resistant structures. In addition, regular flood awareness is to be undertaken to alert the communities in flood-prone areas. Lastly, duty-bearers should consider integrating indigenous knowledge of the communities with modern knowledge in disaster issues.This article concludes that flood disasters can impact the built environment through damaging infrastructure. Therefore, flood disasters have a propensity to wipe out community development gains accumulated over many years. The article further concludes that perpetuation of flood vulnerability in the built environment is an avoidable social construct created through people’s interaction with their environment. This study views the build-back-better concept as viable option to development in the aftermath of flood damage. However, challenges exist that may impede the management of flood disasters affecting the built environment. Finally, the study concludes that reconstruction following flood disasters is difficult and arduous.


Drug‐resistant bacterial infection is a global threat to public health due to the high mobility of the population. Novel therapy methods have been intensively studied for the eradication of antibiotic‐resistant bacteria, including photothermal treatment, which has established outstanding bacterial killing efficiencies under laser radiation, and superhydrophobic surfaces have exhibited excellent antifouling properties. However, an effective, scalable, and affordable bactericidal coating for eliminating drug‐resistant bacteria is lacking. Herein, a novel graphene coating using one‐step laser‐induced graphene and simultaneous laser‐induced forward transfer is introduced. The graphene coating shows high photothermal conversion and superhydrophobic performance, and these synergistic effects can make the bacteria number decrease with over 99.99% proportions under one sun illumination. The superhydrophobic properties can also reduce 99.87% of bacteria compared to the control sample when the solar energy is not available. This additive and scalable method can quickly coat functional graphene onto various substrates, with bacterial applications in many areas, such as water pipeline robots. A synergistic antibacterial graphene coating is developed for water pipeline robotics and has superhydrophobic and photothermal effects. Solar energy is more abundant and can be used for larger area bacteria eradiation compared to NIR laser. Photothermal sterilization was also proven using Prussian blue nanocages under solar irradiation.E. coli. This additive manufacturing method is scalable and affordable, and can be used in real‐world applications, such as antibacterial coatings for water pipeline robots.Recently, we developed a novel and scalable method for the fabrication of large area graphene materials with superwetting and photothermal properties. Ultra‐high specific capacity supercapacitors have been fabricated using this laser induced forward transfer (LIFT) strategy.2FigureThe LIFT deposition process is schematically illustrated as shown in FigureThe surface energies of graphene are estimated using static contact angle measurement, in comparison with polydimethylsiloxane (PDMS) and glass substrates. The pristine PDMS substrate displayed hydrophobic behavior with a contact angle at 117°, as shown in Figure−2. The temperature increased up to 55 °C after the initial 2 min and stabilized around 60 °C after 10 min, as shown in the red curve in Figure The photothermal effect of graphene compared to PDMS and glass substrates was explored, as shown in E. coli colonies within agar plates were grown on pristine PDMS, pristine glass substrate, and graphene coated glass to examine the antibacteria effects, as illustrated in FigureIn combination with the outstanding superwetting and photothermal properties of such graphene, the synergistic antibacteria performance was examined. The E. coli colonies formed on the agar plates were examined with optical photos and electron microscopy. The optical images of the E. coli colonies on various substrates without solar treatment were characterized using 107 dilution folds. Large numbers of E. coli colonies were observed in both the pristine PDMS . As shown in the field emission SEM, the dense biofilms of E. coli were clearly observed on the PDMS  medium containing 50 mg L−1 kanamycin overnight at 37 °C before use. Then, these E. coli cells were diluted by PBS to prepare the suspensions with optical density at 600 nm (OD600) reaching 0.6. Then, 200 µL E. coli suspensions were used to be incubated on the surface of gelatinous LB agar plates (diameter = 10 cm) containing 1.5 wt% agar at 37 °C for 12 h. Ideally, E. coli cells can totally occupy all the surface. Samples (PDMS/Glass/Graphene) were then incubated with the E. coli‐occupied surfaces at 37 °C for 12 h in the dark whilst one more glass sample was set in each agar plate for the thickness control of E. coli cells. All the samples, except the thickness control, were subjected to 10 min solar treatment on white paper.The E. coli cells were detached from the samples and resuspended into PBS. After appropriately dilution by PBS, these E. coli suspensions were placed on gelatinous LB agar plates (diameter = 10 cm) containing 1.5 wt% agar at 37 °C for 18 h. Consequently, each living cell can develop into a distinct colony after incubation and the number of living E. coli cells was estimated by the visible colony numbers on the agar plates. All the experiments were performed independently twice, while the colony counting was operated in triplicate.For SEM imaging, the above samples were soaked in liquid nitrogen prior to the following treatment. For colony counting assay, all the samples including the thickness control, were transferred to centrifuge tubes filled with 10 mL PBS. After 10 min shaking and 1 min sonication at 37 °C, the The authors declare no conflict of interest.SupplementaryClick here for additional data file.


In this study, a novel biobased poly(ethylene brassylate)-poly(furfuryl glycidyl ether) copolymer (PEBF) copolymer was synthesized and applied as a structure-directing template to incorporate graphene and 1,1′-bismaleimide (BMI) to fabricate a series of self-healing organic/inorganic hybrid materials. This ternary material system provided different types of diene/dienophile pairs from the furan/maleimide, graphene/furan, and graphene/maleimide combinations to build a crosslinked network via multiple Diels–Alder (DA) reactions and synergistically co-assembled graphene sheets into the polymeric matrix with a uniform dispersibility. The PEBF/graphene/BMI hybrid system possessed an efficient self-repairability for healing structural defects and an electromagnetic interference shielding ability in the Ku-band frequency range. We believe that the development of the biobased self-healing hybrid system provides a promising direction for the creation of a new class of materials with the advantages of environmental friendliness as well as durability, and shows potential for use in advanced electromagnetic applications. During the past decade, the development of self-healing substances has become an important research field since these smart materials bear resemblance to the living organisms which possess an intrinsic capability to repair their architectural damage via an automatic process without additional intervention. So far, several approaches have been developed to efficiently provide the self-healing property to fabricated materials, such as microcapsules ,2,3, micAmong these reversible self-healing methodologies, a dynamic-covalent bond technology based on Diels–Alder chemistry has received a great deal of attention since the deformation of the crosslinked molecular structure can be easily triggered via a heating process, resulting in a liquification of the polymer material to rebuild the crack area. In most cases, DA type self-healing materials were prepared by combining furan and maleimide functional groups serving respectively as diene and dienophile moieties to form a thermally reversible crosslinked network through a [4 + 2] cycloaddition reaction ,30,31,32Recently, increasing attention is given to sustainable chemistry with the goal to reduce dependence on fossil feedstock and develop alternative polymers from renewable biomass sources ,47,48. I3/g and size distribution of 5.6 to ~18 μm was supplied by Taiwan Graphene Co., Ltd. . Ethylene brassylate  and furfuryl glycidyl ether  were purchased from Sigma-Aldrich  and Acros , respectively. These two monomers were used as received. Furfuryl alcohol , 1,5,7-triazabicyclo[4.4.0]dec-5-ene  and 1,1′-bismaleimide  were purchased and used as received. Additionally, all the solvents for this study were purchased from Sigma-Aldrich and used without any purification.Graphene with a specific surface area ≥15 m−1) was prepared by dissolving the copolymer in THF solvent, followed by mixing with graphene in different weight fractions of graphene to PEBF of 1 wt %, 2 wt % and 5 wt %. The mixtures were then subjected to a bath sonication for 12 h. Afterward, BMI was added to the PEBF/graphene solutions based on a molar ratio of furan group to maleimide group of 1:1. After mixing for 24 h, the solutions were dried in ambient condition for 48 h. The obtained bulk samples were then placed in a vacuum oven and heated to 180 °C. After heating at 180 °C for 2 h, the hybrid samples were subsequently cooled to 60 °C and then isothermally annealed at 60 °C for 24 h. In addition to the PEBF/graphene/BMI hybrid samples, a self-healing PEBF/BMI sample without graphene was also prepared by following the similar route of the PEBF/graphene/BMI hybrids.First, a PEBF solution  with deuterated chloroform as the solvent. To analyze the molecular weight characteristics of the PEBF copolymer, size exclusion chromatography (SEC) analysis was conducted using a Viscotek TDA 305 system  equipped with a refractive-index detector. THF solvent was used as the mobile phase with a flow rate of 1.0 mL/min, and the measurement was operated at a temperature of 35 °C. Raman analyses were performed by using a Dongwoo Ramboss 500i Micro-Raman/PL spectroscope . For the Raman measurements, thin film samples were prepared by depositing hybrid solutions onto silicon wafer substrates, followed by drying in air for 12 h. The morphologies of the samples were investigated by using a Hitachi H-7650 transmission electron microscopy  operating at 75 kV. ATR–FTIR spectra were recorded on a Jasco FT/IR-4600 spectrometer , and flat bulk samples were used for the measurements. Wide-angle X-ray scattering (WAXS) measurements were carried out on beamline 13A1 of the National Synchrotron Radiation Research Center (NSRRC). Bulk samples with 1 mm thickness were used for the WAXS measurements and the incident X-ray was configured with a wavelength of 1.033 Å. TGA measurements were performed on a Hitachi STA7200 instrument  to analyze the thermal stability of the hybrid system. The sample was heated from 50 to 600 °C at 10 °C/min under nitrogen atmosphere. The thermal properties of the hybrid system were also characterized using a TA Q20 differential scanning calorimeter . The measurements were conducted by heating the sample from 50 to 200 °C at 10 °C/min. A Nikon ECLIPSE LV100N POL microscope  with a Linkam THMS600 hot stage  was used to image the self-healing behavior of the hybrid system. Additionally, scanning electron microscopy (SEM) micrographs were captured by using a Hitachi TM4000 Plus instrument  operating at 20 kV accelerating voltage to monitor the morphologies of the hybrid samples. The EMI shielding performance of the hybrid system was analyzed by the waveguide method using a vector network analyzer  in Ku-band frequency range (12.4~18 GHz). A rectangular bulk sample with dimensions of 16 mm × 8 mm × 3.5 mm was placed between the waveguide holders, and the S parameters  were measured to calculate the EMI shielding effectiveness.For identifying the molecular structure of the synthesized polymer, −1 and a molecular weight distribution of 1.51, indicating the successful synthesis of PEBF. 1H-NMR measurement was further performed to characterize the PEBF sample. As shown in 1H-NMR spectrum, the EB to FGE ratio in the PEBF copolymer was estimated to be 77% by calculating the integrated area ratio between the proton signal of furan ring and methylene protons (δ = 2.3 ppm) neighboring the carbonyl group of EB.The schematic illustration for synthesizing the PEBF copolymer system is shown in −1 and a tangential band (G band) at 1582 cm−1. The relative intensity ratio between the D band peak to the G band peak (ID/IG) corresponded to the bonding transition of graphene from C–C (sp2) to C–C (sp3), indicating the degree of structural defects on the graphene sample. In contrast to the Raman spectrum of the pristine graphene which exhibited a ID/IG value of 0.12, the Raman profile of the PEBF/graphene sample revealed a higher value (ID/IG = 0.19). This sudden change indicated the increase of graphene defects resulted from sp2 to sp3 hybridized carbons, providing the evidence for the occurrence of DA reaction in the PEBF/graphene hybrid. This structural integrity of PEBF attached to the surface of graphene was further confirmed by comparing the solubility of the PEBF/graphene sample and pristine graphene in solvent medium. For the investigations, the PEBF/graphene and pristine graphene were separately added to THF to prepare 0.05 wt % solutions, followed by a sonication for 30 min to disperse these materials in the liquid phase. The prepared solutions were then stored in an ambient environment. The dispersion conditions of these solutions were examined as a function of storage time, as shown in On the basis of the sample preparation method described above, the self-healing PEBF/graphene/BMI hybrid system was fabricated by first preparing a PEBF/graphene hybrid solution, followed by adding BMI into the solution to produce a ternary material system. Recently, it has been found that carbon materials such as carbon nanotubes, fullerene, and graphene can serve as either a diene material in the [4 + 2] DA cycloaddition with various dienophiles or a dienophile material to react with diene compounds to form DA adducts. Therefore, for the PEBF/graphene hybrid, the furan groups of PEBF could be expected to behave as active diene sites to readily interact with graphene (dienophile) via a DA cycloaddition reaction, resulting in grafting PEBF onto the surface of graphene. For the modified graphene sample, the defect condition and ordered/disordered structures could reveal significant differences from those of the non-modified graphene. Thus, Raman analyses were subsequently performed to study the structural features to confirm the DA reaction between PEBF and graphene. As shown in D/IG value of 0.17 higher than that of the pristine graphene sample, showing the evidence for the successful DA reaction. More importantly, the furan groups of PEBF (diene) and maleimide groups of BMI (dienophile) are highly reactive for DA reaction. Therefore, the diene/dienophile pairs from the furan/maleimide, furan/graphene, and maleimide/graphene combinations could efficiently induce multiple DA reactions, resulting in a DA crosslinked network, wherein graphene sheets were uniformly dispersed in the polymeric matrix. In the current study, different amounts of graphene sheets were added to fabricate a series of hybrid samples. The sample designations and corresponding compositional characteristics of the hybrid system are listed in −1, which correspond to the C=C stretching vibration of BMI and C=C stretching of DA adduct, respectively. The occurrence of these two signals clearly indicated the formation of DA adducts within the PEBF/graphene/BMI hybrid system. Additionally, the pristine PEBF sample exhibited a shoulder peak at 750 cm−1 associated with the signal of furfuryl moiety of the copolymer sample. However, the FTIR spectra of DA-PEBF-5 did not exhibit this shoulder peak, which again proved the formation of DA adducts with the rupture of the original furan rings in PEBF [The Raman analysis data and suspension condition of the PEBF/graphene hybrid indicated the successful incorporation of furan functional groups onto graphene surfaces through DA reaction. Furthermore, BMI was added into the PEBF/graphene hybrid to fabricate a PEBF/graphene/BMI ternary system. For the ternary material system, graphene could also serve as a functionalization agent which provided diene sites to react with maleimide groups of BMI (dienophile). Thus formed DA adduct could be also identified by using Raman analysis. For the BMI/graphene hybrid, its Raman spectrum  exhibite in PEBF .To further understand the dispersion of graphene sheets in the hybrid system, SEM measurements for monitoring the fracture surfaces of the nanocomposites were subsequently carried out. For the DA-PEBF-0 sample, its SEM micrograph a showed We subsequently studied the thermal stability of the PEBF/graphene/BMI hybrids. The TGA thermograms of the composite samples with different amounts of graphene sheets can be seen in To further verify the thermal reversibility of the PEBF/graphene/BMI hybrid system, a sol–gel experiment was used. This investigation was taken with a solid/liquid mixture prepared by mixing DA-PEBF-2 with DMSO solvent (60 wt % in DMSO). Shown in Due to the thermally reversible feature of DA mechanism, the PEBF/graphene/BMI hybrid system was expected to have self-healing ability for repairing structural damages. For evaluating the self-healing performance of this system, a rectangular bulk sample with a cut of 1 mm depth on its surface was first prepared, followed by monitoring the morphological change of the cracked sample under different heating temperatures by using an optical microscopy. These measurements were started from room temperature with a heating step of 10 °C. At each temperature, the measured sample was isothermally heated for 10 min to cause the surface morphology of the sample to reach an equilibrium state. Shown in T is the total EMI shielding effectiveness, SEA is the absorption of electromagnetic wave, SER is the reflection from the material surface and SEM is the multiple reflections occurred at the inhomogeneous interfaces within the material [The EMI shielding performance of the hybrid system was further quantified as the relative intensity ratio between the incident electromagnetic wave to the transmitted wave. The total attenuation of the incident electromagnetic wave can be expressed as the following equation:material ,53. Notematerial . Thus, tT, SEA, and SER in Ku-band are further summarized in T of 14.4 dB. Additionally, the SEA and SER of DA-PEBF-1 are measured to be 0.9 and 13.5 dB. From these results, we could observe that the reflection efficiency mainly provided a major contribution to the attenuation of the incident electromagnetic microwave rather than the absorption efficiency. The reflection of electromagnetic waves could be attributed to the impedance mismatch between air and the hybrid system with uniformly dispersed conductive graphene sheets. As for incorporating 2 wt % graphene into the hybrid (DA-PEBF-2), we could observe that the DA-PEBF-2 sample exhibited a SEA of 1.5 dB, a SER of 14.2 dB, and gave a SET of 15.7 dB higher than that of the DA-PEBF-1 sample. Furthermore, an improvement in the EMI shielding performance was achieved in the DA-PEBF-5 sample, showing a SEA of 4.0 dB, a SER of 12.6 dB, and a SET of 16.6 dB. As a comparison, it could be clearly observed that the SEA value gradually rose with the increasing graphene fraction while the level of SER only showed a tiny variation. The changing trend of EMI shielding performance of the PEBF/graphene/BMI system could be attributed mainly to the different microstructures of the hybrid samples. Based on the SEM results discussed above, there was a structural change from a uniform dispersion of graphene sheets to a continuous graphene distribution as the graphene content was increased from 1 to 5 wt %. The gradually contacted graphene sheets could form interconnected networks in the polymeric matrix and provided the ability to restrict the incident electromagnetic microwaves from escaping from the compartments and caused the waves to be multireflected and absorbed [The variation in total EMI SE in Ku-band (12.4~18 GHz) as a function of different amounts of graphene for the PEBF/graphene/BMI hybrid samples is presented in absorbed , leadingT to at least 94% of the initial value  were cut to form a large crack with a width of 1 mm and depth of 2 mm. From the experimental results, it could be observed that the self-repairability for healing the structural defect simultaneously resulted in a promising restoration of SEshown in c, demonsIn summary, we have successfully synthesized a novel biobased PEBF copolymer and fabricated a PEBF/graphene/BMI hybrid material system by incorporating graphene sheets and BMI into the polymeric matrix. Beside the reactive furan-maleimide combination, graphene could also serve as an effective DA crosslinking agent to build the DA crosslinked network. For this hybrid system, the DA adducts induced from multiple diene/dienophile combinations and their thermal reversibility were systematically analyzed by Raman, TEM, FTIR, SEM, WAXS, TGA, DSC, and sol–gel measurements. This organic/inorganic hybrid system provided multiple functions including self-repairability as well as EM wave shielding ability. Additionally, the use of biobased monomer ethylene brassylate further showed the benefits of economics and environmental friendliness. Therefore, this research provides a new way for developing advanced biobased materials with self-repairing and EMI shielding functionalities, which could be applied as self-healing protection layers for EMI shielding applications.


Over the past decade, major discoveries in retrotransposon biology have depicted the neural genome as a dynamic structure during life. In particular, the retrotransposon LINE-1 (L1) has been shown to be transcribed and mobilized in the brain. Retrotransposition in the developing brain, as well as during adult neurogenesis, provides a milieu in which neural diversity can arise. Dysregulation of retrotransposon activity may also contribute to neurological disease. Here, we review recent reports of retrotransposon activity in the brain, and discuss the temporal nature of retrotransposition and its regulation in neural cells in response to stimuli. We also put forward hypotheses regarding the significance of retrotransposons for brain development and neurological function, and consider the potential implications of this phenomenon for neuropsychiatric and neurodegenerative conditions. Neural cell diversity underpins this complexity, and has classically been defined in terms of the morphological differences between cell types, their diverse connectivity patterns, physiological and functional properties and the expression of various transcription factors (TFs), cell surface and secreted molecules ,2. At thSomatic mosaicism is defined as the existence of two or more cells with different genotypes within one individual. Depending on its developmental timing, mosaicism can encompass mutations that are heritable (germline mosaicism), non-heritable (somatic mosaicism) or a combination of these two outcomes ,11–14. R2.in cis, L1 can mobilize in trans non-autonomous short interspersed element (SINE) retrotransposons. These include 7SL-derived Alu elements, composite SVA (SINE–variable number tandem repeat–Alu) elements, and cellular mRNAs, resulting in processed pseudogenes [Alu copies are approximately 300 bp in size and represent the most abundant transposable element by copy number (approx. 11% by sequence) [Alu, and comprise only about 0.2% of the genome sequence [a).Retrotransposons are the main class of transposable elements found in most mammalian genomes. Based on the presence of long terminal repeats (LTR) in their structure, they are classified into LTR and non-LTR retrotransposons. The LTR group consists of ERVs (endogenous retroviruses), which comprise approximately 8% of the human genome and represent remnant retrovirus sequences incorporated into the host germline after ancient viral infections ,20. The udogenes –25. L1 cudogenes . Alu copequence) ,26,27. Ssequence ,28–30  L1s [in vitro to date belong to the L1-Ta and L1-preTa families [Alu subfamilies, such as AluYa5 and AluYb8 [Owing to truncation, deletion, internal rearrangement and other mutations, the vast majority of human retrotransposon sequences are incapable of further mobilization and are considered molecular fossils . Computaive) L1s . All of families –42. Simid AluYb8 , and SVAd AluYb8 ,24,29 had AluYb8 . However3.cis-preference [b) [All retrotransposons mobilize via a replication mechanism involving an RNA intermediate . During eference , meaningeference ,49 or eneference  makes a eference   ,50,51.Retrotransposition does not always result in the faithful duplication of an element, particularly in the case of L1. Owing to errors during reverse transcription, only approximately 40% of L1 copies are identical to their source, or donor, L1 . Also, 5As a result of 5' truncations, internal inversions or deletions  and poin4.Although nearly 70 years have passed since Barbara McClintock's seminal studies of transposable elements in maize ,62, ongoEGFP gene is interrupted by an intron in the same transcriptional orientation as the L1. Thus, only cells with successful L1 retrotransposition events can express GFP because the EGFP intron is removed from the RNA intermediate, followed by reverse transcription and integration into a genomic location. When the L1-EGFP system is employed in neuronal cultures, GFP co-localizes with neuronal markers Map2 and β-tubulin, whereas very little to no GFP co-localizes with astrocyte or oligodendrocyte markers [Over the past decade, three main tools have facilitated the discovery and study of L1 retrotransposition in the brain. Firstly, an engineered L1 reporter construct, previously developed to recapitulate L1 retrotransposition in cultured cells ,70, has  markers . Neurons markers –74. Alth markers , the deset al. [Secondly, to estimate the number of L1 copies across different tissues and brain areas, Coufal et al.  developeet al. ,74,76. Tet al. , this aset al. . New quaet al. ,79.Thirdly, high-throughput DNA sequencing strategies have been developed to detect, quantify and resolve L1 insertions. Whole-genome sequencing (WGS) or targeted sequencing of retrotransposon–genome junctions has been applied to bulk brain, pooled neurons and whole-genome amplified (WGA) material from single neuronal nuclei from post-mortem human brain –7,10,80.Providing a valuable orthogonal approach to single-cell genomic analysis, a recent study used somatic cell nuclear transfer to reprogramme postmitotic olfactory bulb neurons into enucleated oocytes and cloned the neuronal genome to produce enough DNA for WGS without the need for WGA. WGS analysis of the resulting clones revealed several de novo L1 insertions . These s5.a). Neural diversity arises during early embryogenesis through the spatial and temporal patterning of neuroblasts and neuronal progenitor cells. A complex network of gene expression determines a particular cell type-specific progenitor domain and fate [b).Retrotransposition in the brain could occur at any stage of life, from early brain development to brain maintenance and decline during adulthood, and also influence neural diversity and survival a. Neuraland fate . Expressand fate . Althougand fate ,87. An eand fate . Whether5.1.et al. [in vitro, in rodent primary hippocampus-derived NPC cultures, and in vivo, in transgenic mice. L1 expression in transgenic mice was observed as early as embryonic day (E) 12.5, when the first waves of immature differentiated neurons appear in the brain [et al. [Over a decade ago, Muotri et al.  presentehe brain . These d [et al.  in a stu [et al. , the cel [et al. –92. The Another explanation for L1 activity in proliferating NPCs may be a more relaxed chromatin state. A relaxed chromatin state is correlated with pluripotency and proliferation, and differentiated cells have more condensed chromatin than progenitor cells . Open chFinally, TFs can stimulate L1 to retrotranspose during neuronal differentiation. Sex determining region Y-box 2 (SOX2), which is expressed in stem cells and neural progenitors, and is essential in maintaining a proliferative state, can bind to the 5' UTR of L1 and repress promoter activity . During 5.2.et al. [et al. [et al. compared the retrotransposition of a hybrid adenoviral L1-EGFP vector in dividing versus non-dividing neural cells by infecting NPC cultures with the L1 reporter and concomitantly adding 5'-bromo-2'-deoxyuridine (BrdU), which labels dividing cells. Immunostainings of differentiated NPC cultures infected with L1-EGFP at 31 days post-NPC differentiation showed the presence of neurons expressing only EGFP but not the cycling marker, BrdU. When comparing the number of integrated L1-EGFP copies in cultures infected with the L1 reporter at day 0 (multi-potent NPC) versus day 31 of differentiation (in mature neurons), a sixfold increase in L1-EGFP copies was observed by qPCR against the EGFP splice junction in mature neurons, even when correcting for differences in proliferation rate [in vitro assay to compare dividing versus non-dividing neural cells. Going forward, it will be interesting to address this question in vivo, perhaps by using an inducible knock-in reporter model. Furthermore, it would be very interesting to explore in detail the mechanisms through which retrotransposons escape epigenetic silencing and mobilize in postmitotic neurons.If we accept that endogenous L1 mobilization can occur during adult neurogenesis, can it also occur in postmitotic neurons? This is a fundamental question because the lifespan of mature neurons can be as long as a human lifespan, and therefore, any potential for somatic retrotransposition in this context may lead to the largest absolute accumulation of new L1 insertions found in the brain. Crucially, Kubo et al.  showed i [et al.  confirmeion rate . This wa6.It is not established whether retrotransposition in the neural genome is part of normal brain function. It is however plausible that, if retrotransposons did contribute to normal brain function, it would be via altered splicing and DNA methylation of genes, with these being routes to perturb the transcriptional output of cells. For example, pseudogene transcripts can carry microRNA (miRNA) recognition sites and facilitate degradation of their target miRNAs, acting as competitive miRNA targets to the miRNA targets of their parent genes . Such ps7.PDHX gene encoding pyruvate dehydrogenase, which results in pyruvate dehydrogenase deficiency, a neurodegenerative disorder [Retrotransposon insertions can significantly alter protein-coding and regulatory regions of the genome, and thereby affect gene expression and other cellular outputs ,99–103. disorder . Genomicdisorder . Recombidisorder . Transcrdisorder . An antidisorder ,114–116,Glyrβ) gene which leads to aberrant splicing of the pre-mRNA by exon skipping, thus resulting in reduced intact subunit β expression in the brain [Reln) gene, which also leads to exon skipping and a frame shift, in this case generating a 220 bp deletion of the Reln mRNA, and leading to inefficient secretion of Reln truncated protein [Reln mutant mice recapitulate the phenotype seen in patients with lissencephaly caused by other Reln mutations [New L1 insertions can lead to aberrant transcriptional splicing –121. Twohe brain ,119. As  protein ,122,123.utations . Hence, 8.8.1.in vitro when tagged with a fluorescent or selectable marker [et al. [Retrotransposition depends on a mobile element's intrinsic ability to ‘jump’, and its host cell's capacity to defeat this mobilization. Despite their significant presence in the genome, almost all human L1 copies are 5' truncated, or contain internal mutations and rearrangements, and are not able to mobilize. A few L1 copies are full length, remain transcriptionally active and have intact ORFs and therefore have the capacity to act as source, or donor, elements in a cycle of retrotransposition. An even smaller subset  retrotranspose efficiently e marker ,70 and ae marker ,42, althe marker . If a ce [et al.  recently [et al.  identifi8.2.As retrotransposition events can cause such large effects on gene expression, cell function and ultimately organism fitness, several mechanisms exist to limit and regulate L1 mobilization in germ and neural cells. These mechanisms have been reviewed comprehensively ,127. We Several epigenetic factors are known to repress L1. For example, methyl-CpG-binding protein 2 (MeCP2) is present ubiquitously in the body and in higher abundance in mature neurons. MeCP2 binds 5-methyl-cytosine residues in CpG dinucleotides and interacts with histone deacetylase protein (HDAC) and transcriptional SIN3A corepressor complexes to repress transcription –134. MeC. As new L1 insertions carry TF binding sites and potentially attract epigenetic suppression [In addition to epigenetic regulation, retrotransposon activity can be regulated by specific TFs, such as yin yang 1 (YY1), runt-related transcription factor 3 (RUNX3), SOX2 and tumour protein p53 (p53). YY1 is a zinc finger protein expressed ubiquitously in the brain. It is critical for regional patterning of the brain and neuronal differentiation, as it regulates genes including Otx2 and Engrailed 2 –141. YY1pression , the intpression ,154, andpression . Host fapression , or DNA pression . Hence, 8.3.et al. [et al. [in vivo.Numerous studies have proposed that environmental factors may trigger hyperactivation of L1 and other retrotransposons. However, most of these studies are preliminary and the experiments have been largely performed on cell lines. For instance, heavy metals such as mercury, nickel and cadmium seem to increase L1 retrotransposition in HeLa and neuroblastoma cells –159, andet al.  describe [et al.  reported [et al. ,163,165.9.Alu data. The implication of HERVs in neurological disease, such as multiple sclerosis, schizophrenia and bipolar disorder, has been recently reviewed [Retrotransposition in the early embryo and committed germline can result in heritable mutations that cause genetic disease ,63. Severeviewed ,209,210.reviewed ,212. Pro9.1.MeCP2 gene [MeCP2 mutant rat NSC cultures and transgenic mice [MeCP2 mutation.A prominent example of a neurodevelopmental disorder that has been linked to retrotransposon activity is Rett syndrome (RTT). RTT is a progressive, severe neurodevelopmental disease associated with a mutation in the CP2 gene . MeCP2 gCP2 gene ,135. MicCP2 gene . Increasnic mice . Elevatenic mice . Howeveret al. [Psychiatric diseases, such as autism, schizophrenia and bipolar disorder, have also been linked with retrotransposon activity ,190–192.et al.  sought tet al. . WGS peret al. . The autet al. [et al. [Increased retrotransposon copy number and mRNA levels have been reported in schizophrenia and depression ,192. Bunet al.  revealedet al. . A consiet al. . WGS peret al. . Howeveret al. . Very re [et al.  proposed [et al. . As a qPAlu epigenetic regulation has been reported in post-traumatic stress disorder (PTSD), an anxiety condition characterized by persistent re-experiences of a past traumatic event or events [Alu in serum when compared with healthy individuals [Even if elevated somatic retrotransposition ultimately is not found to occur in a given neurological condition, a molecular signature of L1 regulatory disruption may indicate a wider landscape of epigenome abnormality, and prove useful as a diagnostic tool. For example, altered L1 and r events . PTSD paividuals . Non-PTSividuals . These f9.2.ATM gene encoding a serine/threonine kinase [ATM mutation in cultured human NPCs and ATM knockout in L1-EGFP mice significantly increased L1-EGFP retrotransposition, measured by the number of GFP-positive cells, without affecting L1 promoter activity or endogenous ORF1p levels, and with no change in cell division rates or survival [ATM mutant cells [in vivo, is required to corroborate these results, and identify the role of ATM in controlling L1 integration.Neurodegenerative conditions, such as Parkinson's disease, Huntington's disease and Alzheimer's disease, represent a group of nervous system disorders characterized by the selective death of neuronal subsets in particular brain regions . Experime kinase , which le kinase . ATM rese kinase . ATM mutsurvival . Notablynt cells , potentiDrosophila brains, recapitulated these results from patients and mouse models, demonstrating that TDP-43 dysfunction results in de-repression of retrotransposons [gypsy ERV transcript levels as previously reported to be elevated in ageing flies [gypsy ameliorated this phenotype, suggesting that gypsy may be involved in cell death, potentially by mediating DNA damage [As many neurodegenerative conditions are associated with ageing, retrotransposition in neurodegenerative conditions may, in general, reflect conditions encountered in senescent cells. Accumulation of oxidative DNA damage and unrepaired DNA, as well as changes in methylation patterns commonly associated with ageing, might lead to reduced silencing of retrotransposition in the brain. As an example of this, TAR DNA-binding protein 43 (TDP-43) is a nucleic acid binding protein involved in transcriptional repression and RNA metabolism during stress response. Mutant TDP-43 is usually associated with neurodegenerative disorders, but it can also be present in healthy elderly people . Accumulnsposons . Interesng flies . Human TA damage .et al. [Alu within and adjacent to mitochondrial genes [Alu retrotransposon insertions in a number of translocase of outer mitochondrial membrane genes [Alu insertions in two genes involved in mitochondria stabilization and function led to disorders with a neurodegenerative component [Alu activity can preferentially insert into and induce mitochondrial gene disruption.Many neurodegenerative disorders are strongly linked with mitochondrial dysfunction. In intriguing recent work, Larsen et al.  put forwal genes . Previoune genes ,230,231.omponent ,194. Mor10.in vivo, and the regulatory mechanisms involved in allowing L1 retrotransposition in mature neurons require further investigation. Environmental stress, as well as genetic dysfunction in L1 silencing factors, can result in high retrotransposition levels, and possibly lead to neurological disease. This also requires extensive investigation, as we lack even basic understanding of the pathways on which stress factors or dysfunction in silencing factors can act to impact retrotransposition in neural cells. Our understanding of retrotransposition in the brain is still very much in its infancy.It is now well established that retrotransposon-driven mosaicism can occur in the mammalian brain. Dynamism in TF activity, and that of other regulatory mechanisms described to act during embryonic and adult neurogenesis, provides a temporal and spatial niche amenable to retrotransposition. Additionally, retrotransposition may not be limited to NPCs; it may also take place in postmitotic neurons. Further proof is required to be certain that this can occur Further advances in single-cell genomics  to unde


ATG9A is essential during autophagosome biogenesis; however, the function of this multispanning membrane protein is not well defined. Judith et al. report that ATG9A vesicles deliver PI4KIIIβ to the autophagosome nucleation site to produce PI4P and initiate autophagosome formation. ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid–starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes.  Macroautophagy, referred here as autophagy, is a dynamic, highly conserved, lysosomal-mediated degradative process necessary for eukaryote development, survival, and homeostasis. Autophagy can selectively eliminate damaged organelles, protein aggregates, and viral and bacterial pathogens. Autophagosome formation involves a cytoplasmic protein machinery acting on a membrane source to nucleate and form a phagophore, which will close to become a double-membrane autophagosome, which is then degraded after fusion with endolysosomes. The molecular machinery driving autophagy is composed of ATG proteins . While dATG9 is the only multispanning ATG membrane protein essential for autophagy. In yeast, Atg9 vesicles are implicated in the delivery of membrane components to the initiation site or the preautophagosomal compartment . LikewisUnder nutrient-rich conditions, ATG9A is mainly located in the perinuclear region, colocalizing with medial and TGN markers of the Golgi complex, and partially with early and recycling endosomes . During ATG9A vesicles are highly mobile, and their trafficking is controlled by nutrient-regulated signaling: in fed and starved cells, ATG9A trafficking from the Golgi complex is controlled by the ULK1/2 complex . The ULKIn amino acid starvation, ATG9A localizes to tubular-vesicular structures adjacent to nascent phagophores, called the ATG9 compartment . Also foThe relationship of the ATG9 compartment and the highly mobile vesicles seen in both yeast and mammalian cells to the phagophore is not known. Although several properties of ATG9A are similar to those reported in yeast, in mammalian cells, ATG9A undergoes a complex trafficking pathway through the endocytic and secretory pathways . Our worUsing an antibody to endogenous ATG9A, we immunoisolated ATG9A-positive membranes and determined their composition by proteomics. We found Arfaptins (ARFIP1 and ARFIP2) to be components of ATG9A-positive membranes. ARFIPs are Bin/Amphiphysin/Rvs (BAR) domain–containing proteins, proposed to be required for sensing and generation of membrane curvature . The ARFDespite their similar domain structure and subcellular localization, there are notable differences between ARFIP1 and 2. ARFIP1, shown to be required for secretory granule biogenesis , is reguIn addition, we show that the phosphatidylinositol 4 kinases, PI4KIIα and PI4KIIIβ, are components of ATG9A-positive membranes. As PI4KΙΙα acts at autophagosome–lysosome fusion , we studTo identify the protein composition of ATG9A vesicles, nutrient-rich and amino acid–starved HEK293A cells were mechanically lysed, and ATG9A-positive membranes were immunoisolated using a hamster monoclonal antibody raised against human ATG9A or, as a control for specificity, hamster IgM . Stable BIF-1, ARFIP1, and ARFIP2 are BAR domain–containing proteins. Whereas BIF-1 is required for trafficking of ATG9A , 2016, lConsistent with previous reports , ARFIP1 Next, we determined the effect of ARFIP2 depletion during autophagy. Analysis of the localization of ARFIP2 relative to ATG9A revealed an association in the perinuclear region in FM . In ES, Thus, as ARFIP2 is present on ATG9A vesicles, we asked if it behaved like ATG9A and is found associated with forming autophagosomes. We performed live-cell imaging of cells under amino acid starvation stably expressing GFP-ATG13 (a member of the ULK1/2 complex) and mRFP-ATG9A, and transiently expressed iRFP-ARFIP2  cells , quantifARFIP2 contains an AH adjacent to the BAR domain . The AH As our data suggest that ARFIP2 modulates ATG9A trafficking, we assessed whether depletion of ARFIP2 would modify the composition of the ATG9A vesicles upon amino acid starvation. Using immunoisolation-SILAC coupled with liquid chromatography/tandem mass spectrometry, we compared immunoisolated ATG9A-positive membranes from CTRL and CrARFIP2 KO cells in ES . Among tThese data and the fact that ARFIP2 localized at the Golgi apparatus led us to investigate the two major PI4-kinases present on the Golgi apparatus: PI4KIIα and PI4KIIIβ . We confLegionella pneumophila, which labels PI4P . A target sequence in the fifth exon of human ARFIP2 was selected . The appropriate oligonucleotide was cloned into Bbs1 site of pSpCas9(BB)-2A-GFP plasmid obtained from the laboratory of F. Zhang  according to the cloning protocol provided by the laboratory. Following single-cell sorting on GFP-positive cells, colonies were screened for ARFIP2 deficiency by immunoblot.To induce autophagy, cells were washed three times with PBS and incubated in ES for 2 h. Where indicated, cells were treated with 100 nM bafilomycin A1  for 2 h or VPS34 IN1  for 1 h.The antibodies used in this work are as follows. Mouse antibodies: anti-WIPI2 , anti-GMSecondary antibodies for immunofluorescence (from Life Technologies unless otherwise specified) were anti-rabbit IgG Alexa Fluor 488, 555, and 647; anti-mouse IgG Alexa Fluor 488, 555, and 647; and anti-hamster Cy3 (Jackson ImmunoResearch). HRP-conjugated secondary antibodies used for Western blotting were from GE Healthcare.GFP-ARFIP2 was a gift from K. Nakayama  was used for transient transfection of cells according to the manufacturer’s instructions. DNA plasmids were used at 1 µg/ml. For RNAi, cells were transfected with the relevant siRNA oligonucleotide using oligofectamine , followed by a second transfection using Lipofectamine 2000 the following day, both according to the manufacturer’s instructions. Cells were harvested 72 h after transfection. The final concentration of siRNA oligonucleotides was 100 nM. siRNAs used (Dharmacon): D-001220-01 , D-020256-01 (ARFIP1), D-012820-01 (ARFIP2-01), D-012820-02 (ARFIP2-02), D-014294-02 (ATG9A), and D-006777-02 (PI4KIIIβ).Cells were lysed in ice-cold TNTE buffer  containing EDTA-free Complete Protease Inhibitor cocktail  and phosphatase inhibitor cocktail . Lysates were cleared by centrifugation for 15 min at 13,000 rpm, heated at 65°C for 15 min, and resolved on NuPAGE BIS-Tris 4–12% gels (Life Technologies) followed by transfer onto a PVDF membrane (Millipore). Following incubation with primary and secondary antibodies, the Western blots were developed by enhanced chemiluminescence  on Hyperfilm MP  or by chemiluminescence detection using Amersham Imager 680 . Densitometry was performed by using ImageJ software .Cells were lysed using IP buffer  and 1× EDTA-free complete protease inhibitor and phosphatase inhibitor cocktail, and the clarified lysates used for immunoprecipitation were incubated with either rabbit anti-ATG9A  or rabbig at 4°C. Pellets were resuspended using ice-cold isotonic buffer A  supplemented with EDTA-free complete protease inhibitor cocktail and phosphatase inhibitor cocktail and incubated for 15 min on ice. The resuspended pellet was then passed through a 27G needle 15 to 20 times for homogenization before clarification by centrifugation at 3,000 g at 4°C. Supernatants were used for incubation overnight at 4°C with hamster anti-ATG9A or hamster IgM CTRL coupled with protein A Dynabeads . The ATG9A-positive membranes on the beads were washed three times with isotonic buffer supplemented with 150 mM NaCl. The ATG9A-positive membranes on the beads were then eluted by competition with ATG9A peptide (HPEPVPEEGSEDELPPQVHK). The eluted ATG9A-positive membranes were then pelleted by centrifugation at 47,000 rpm for 1 h at 4°C. The pellet membranes were resuspended in 2× Laemmli sample buffer before being resolved by SDS-PAGE and Western blotting. For confocal microscopy, mRFP-ATG9A–positive membranes were immunoisolated following the protocol described above and then fixed with 2% PFA after the washing step.For immunoisolation of ATG9A-positive membranes, HEK293 cells were treated with FM or ES for 2 h. Cells were then washed in ice-cold PBS and harvested by centrifugation at 200 For immunoisolation of GFP-P4MX2–, GFP-ATG13–, GFP-DFCP1–, and GFP-LC3B–positive membranes, mouse anti-GFP antibody or mouse anti-FLAG M2 (CTRL) coupled to protein A Dynabeads was used. After the overnight incubation of the supernatants with the antibody coupled to protein A Dynabeads, GFP-positive membranes were washed with the isotonic buffer supplemented with 75–150 mM NaCl and eluted in 2× Laemmli sample buffer before being resolved by SDS-PAGE and Western blotting.14N, 12C) or heavy  lysine and arginine . ATG9-positive membranes were isolated from SILAC HEK293 cells either from nutrient-rich condition or amino acid starvation, using a hamster monoclonal antibody specific to the C-terminal domain of ATG9 conjugated to magnetic beads. Eluted proteins were subjected to SDS-PAGE until the running front had migrated 2–3 cm into the gel. Proteins were in-gel digested using trypsin, and peptides were analyzed using a Q Exactive mass spectrometer coupled to an Ultimate3000 HPLC equipped with an EASY-Spray source (Thermo Fisher Scientific). Raw data were processed using MaxQuant v1.3.0.5, with SILAC selected as the quantification algorithm. The proteingroup.txt output table was imported into Perseus software for further processing, statistical analysis, and visualization. Data were analyzed to screen for proteins enriched on ATG9A-positive membrane in nutrient-rich and amino acid starvation conditions and filtered as follows. Of all proteins identified, only those that were detected with a higher intensity in the specific ATG9A immunoisolation compared with IgM CTRL immunoisolation were considered as candidates. For each of these proteins, the log2 values of the normalized SILAC ratios were plotted against the log10 of the intensity to generate the scatterplot depicted in http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository.For SILAC experiments, HEK293 cells were grown in DMEM supplemented with 10% dialyzed FCS , and growth medium was supplemented with a combination of either 100 mg per liter of light , 0.2% Triton X-100 (ULK1 staining), or room temperature methanol (endogenous LC3 staining) for 5 min. Coverslips were blocked with 5% BSA  for 1 h, incubated with primary antibody in 1% BSA for 1 h, washed with PBS, and incubated with secondary antibody in 1% BSA for 30 min, before final washing with PBS and MilliQ water. Cells were stained with Hoechst 33258 solution  and mounted with Mowiol 4-88 .Cells were grown to subconfluence on coverslips and treated with FM or ES for 2 h. Cells were fixed by addition of 1 volume of prewarmed 4% PFA to the medium for 15 min at room temperature. Cells were then washed three times using PBS containing 50 mM NH4Cl and once with MilliQ water, and mounted.For PI4P staining, cells were permeabilized for 5 min with 20 µM digitonin in buffer A containing 20 mM Pipes, pH 6.8, 137 mM NaCl, and 2.7 mM KCl. Cells were washed three times in buffer A and then blocked for 45 min in buffer A containing 5% BSA. Anti-PI4P antibody was added in buffer A for 60 min at room temperature. After three washes with buffer A, secondary fluorophore-labeled antibodies were added in buffer A. Cells were postfixed for 5 min with 2% PFA, washed three times with PBS containing 50 mM NHFor conventional confocal microscopy, a confocal laser microscope  with a 63× NA 1.46 plan-Apochromat objective was used. Superresolution microscopy was performed with Zeiss LSM 880, Airyscan-equipped confocal microscope with a 63× NA 1.4 plan-Apochromat objective. After acquisition, images were processed using an Airyscan processing tool on the ZEN software provided by Zeiss. Image channels were acquired sequentially using appropriate filter sets for DAPI, EGFP, mRFP, iRFP, and Alexa Fluor 555, 488, and 647 and combined in the corresponding microscope software. All imaging was performed at room temperature.For conventional confocal microscopy, a confocal laser microscope  with a 63× oil-immersion lens was used. High-resolution confocal microscopy was performed with a Zeiss LSM 880, Airyscan-equipped confocal microscope with a 63× oil-immersion lens. After acquisition, images were processed using an Airyscan processing tool on the ZEN software provided by Zeiss.The experiments were repeated as indicated in the figure legends, and representative images are shown. The level of colocalization was determined by acquiring confocal images from ∼30 cells per condition. Pearson’s coefficient was then determined using Fiji software. LC3, WIPI2, and ULK1 puncta formation was quantified by Imaris image analysis software. Line scans have been made by using Fiji software. Association between GFP-ATG13 spots with the different markers , GFP-LC3 spots with PI4P, and endogenous LC3 spots with GFP-PI4KIIIβ and with WIPI2 were analyzed with Fiji software based on the Manders’ overlap coefficient. Additionally, lines were drawn crossing GFP-ATG13, GFP-LC3B, or LC3 spots associated with the different markers, and line plots of the fluorescence intensity were used to confirm the association. In all experiments, images shown in individual panels were acquired using identical exposure times or scan settings and adjusted identically for brightness and contrast using Photoshop CS5 (Adobe).2 and immediately imaged using a Zeiss LSM 880, Airyscan-equipped confocal microscope with a 63× NA 1.4 plan-Apochromat objective. Images were processed using an Airyscan processing tool on the ZEN software provided by Zeiss.Live-cell imaging was performed on HEK293A cells stably expressing GFP-ATG13 (HEK293A-GFP-ATG13) and mRFP-ATG9A cotransfected with iRFP-ARFIP2 or GFP-ATG13 and mRFP-ATG9A cotransfected with iRFP-PI4KIIIβ or on HEK293 cells stably expressing mRFP-ATG9A cotransfected with GFP-P4MX2 seeded on glass-bottom microwell dishes . Cells were placed into ES at 37°C and 5% COn numbers. Statistics were performed using GraphPad Prism 6 software, as detailed in the figure legends.The statistical details of all experiments are reported in the figures, including statistical analysis performed, error bars, statistical significance, and exact Fig. S1 provides evidence that in contrast to ARFIP2, ARFIP1 is not required for autophagy. Fig. S2 provides evidence that ARFIP2 is essential for autophagy. Fig. S3 shows the relationship between ATG9A and the PI4P pathway. Fig. S4 shows that the different stages of autophagosomes contain PI4P and PI4KIIIβ. Video 1 provides evidence that ATG9A vesicles containing ARFIP2 interact transiently with ATG13 vesicles. Video 2 shows that ATG9A vesicles contain PI4P. Video 3 shows that ATG13 vesicles interact with ATG9A vesicles containing PI4KIIIβ. Table S1 is a list of proteins associated with ATG9A-positive membranes under nutrient-rich and starvation conditions. Table S2 is a list of Golgi proteins associated with ATG9A-positive membranes. Table S3 is a list of proteins associated with, or depleted from, ATG9A-positive membrane upon ARFIP2 depletion.


By calculating the one-body carrier density probability measure of the graphene sheet, we show that the density fluctuation ( The understanding of the origin and the influence of disorder, as well as interactions in graphene seems to be essential in understanding of the experiments and also in designing graphene-based electronic devices. The effect of these quantities and the resulting screening are not as easygoing as ordinary simple metals5. There is a huge literature concerning the interplay of particle interactions and disorder in mono and multilayer graphene13 which cause variety of phenomenons. Examples of the effect of disorder on the properties of graphene are its effect on: the compressibility14, the electron-phonon interaction15, the Fano factor and conductivity5, the magnetoresistance of bilayer graphene16, the graphene Hall bars17, the optical properties of graphene quantum dots18 and the effective electronic mass in bilayer graphene19. Disorder-based spintronics in graphene20, and Levi-flight transport caused by anisotropically distributed on-site impurities21 are other examples for which the disorder plays a dominant role. The disorder-mediated Kondo effect22 and the formation of electron-hole puddles (EHPs) are examples for which the disorder and the inter-particle interactions play vital roles simultaneously. EHPs are believed to be responsible for the observed minimum conductivity of graphene and was predicted theoretically by Hwang et al.23 and Adam et al.24 and was also confirmed in experiments in the vicinity of the Dirac point34. They are characterized by the state in which some strong carrier density inhomogeneities with density fluctuations much larger than the average density (for low densities) emerge35. In this case  and the similar situations, a many body treatment is needed in which the effect of interaction and the disorder enter to the play with the same footing. Such an investigation can be found in36 in which it has been shown that for the gauge field randomness, related to the formation of ripples characterized by a disorder strength 36. This shows the vital role of disorder in electronic properties of graphene which even can change the role of interaction and cause the system to exhibit the non-Frmi liquid behaviors6.Since the discovery of graphene as a hybrid between metal and insulator, this material with relativistic energy spectrum of zero-gap Dirac Fermions38. For the undoped graphene in one loop approximation of the polarization function (for which the intra-band excitations are forbidden due to the Pauli principle), it is shown that the static dielectric function 39 and no collective modes are allowed within the PRA approximation. For finite μ however, the more general solution of Shung40 is applicable which predicts that the potential is screened with the screening length vF ≡ Fermi velocity, kF ≡ Fermi momentum, 40. Based on these results, in low-densities (in intrinsic or weakly disordered graphene) due to the lack of screening one should take into account the long-range part of the potential which leads to the issue of logarithmic phase shifts for Coulomb scatterers6. These results were obtained for disorder-free system and within the RPA approach which is questionable for the graphene as pointed out by Mishchenko41. Despite of this huge literature, there is a limited understanding concerning the properties of the polarization function in the general form and also the behavior of multi-body charge density probability measures, especially in the low-density case in which EHPs appear.The polarization function of the graphene as an important quantity which is sensitive to both the inter-particle interaction and the disorder has been obtained and analyzed in the cone approximation in the bubble expansion42. To obtain disorder corrections to the dielectric function we develop a diagramatic technique for the TFD theory. We obtain the two-body correlation function using some stochastic analysis and find the equation governing it. We show that, in addition to the screening length found by others, there is a characteristic length scale for the screening, namely qch which is related to the disorder strength and inter-particle interactions via ni is the disorder strength, rs is the dimensionless interaction factor to be defined in the text and d is the substrate distance. For small densities we show that there occurs an instability in which the imaginary part of the dielectric function becomes negative and some charge density oscillations with disorder-dependent wave length emerge. At these densities screening-anti-screening transition occurs along with changing sign of the real screened potential. The disorder significantly changes the Fourier component of the screened potential for large wave lengths showing the fact that the large-scale behaviors of the system is mostly affected by the disorder. We characterize the system in the vicinity of this transition in detail. In addition to the one- and the two-body (static) correlation functions, the equation governing the total probability measure of the system is introduced and analyzed and a closed expression is proposed for it. The proposed expression is valid for large densities.A more reliable approach should involve both interaction and disorder simultaneously which is the aim of the present paper. For this, the Thomas-Fermi-Dirac (TFD) is a proper candidate which has the capacity to bring the effect of all LDA energies in the calculations as well as the tunable disorder in the same time. We employ this approach involving exchange-correlation energies and white noise out-plane disorder. For the EHPs, TFD techniques have proved to be very useful and are widely usedThe paper has been organized as follows: In the next section we present the general construction of the problem and introduce the TFD theory for determining the ground state of graphene. The one-body probability measure is obtained in SEC. 2. Section 3 is devoted to finding the two-body probability measure and the dielectric function. The screened potential as well as the charge density oscillations are argued in this section. The total probability measure of the system is analyzed in SEC. 4. In the conclusion section we close the paper by highlighting the main findings of the paper. Appendix 41. The renormalization group analysis in the weak coupling limit in the first order approximation shows that the Coulomb interactions are marginally irrelevant due to the logarithmically divergent velocity6. In the strong interaction side however, it is shown that the graphene has non-Fermi liquid behaviors with power-law quasiparticle dispersion43, the limit which cannot be reached for finite densities away from the Dirac point. In the weak coupling side, it is known that in contrast to ordinary metals in which the screening makes the interactions short-ranged, in low-density (intrinsic or weakly disordered) graphene due to the lack of screening one should take into account the long-range part of the potential which leads to the issue of logarithmic phase shifts for Coulomb scatterers6. The effect of disorder is however of special importance since, as stated in the introduction, it may completely change the behavior of the Dirac Fermions36. TFD theory, as an approach which has the potential to bring the interaction and disorder in the calculations in the same footing is introduced and analyzed in this section. We mention some points concerning the TFD theory as a coarse grained method and the perturbation diagrams which are used in our analysis. The multi-point probability measures and their relations to the many body disordered response functions are of special importance in this paper which is described in the next sections.The effect of interaction and disorder in graphene has a long story in the literature. It is known that the random-phase approximation (RPA) fails to describe the interaction effects in graphene which was firstly pointed out by MishchenkoT is the time ordering operator, 44:Let us start by analyzing the zero-temperature polarization operator which is defined by 45. In this figure the single-line circles show the carrier density at the point x, i.e. n(x) and the shaded area shows all interaction effects, such as Coulomb lines and external (disordered) interaction lines. In the present paper we mainly deal with r and r′ are r are the full electronic density Consider the ground state, and define mentclass2pt{minimmentclass2pt{minimmentclass2pt{minim44  term of the Fig. mentclass2pt{minimThe determination of r and r′), the extra contribution comes from the quantities like f is an arbitrary function). In such cases we define the pair (or two-body) distribution measure46:n at r and n′ at r′. It is notable that For the more general case in which we are interested in calculating multi-point correlation functions with two insertion points . We turn back to this point at the end of this section.In this section we concentrate on calculating one-body probability density The differential form of the charge profile is:n(r). We consider a one-body function P be the probability measure of charge density to be n at the position r, so that f is defined as Now let us perform some Ito calculations to obtain the probability measure of Let us consider the above expression term by term. Firstly we note that fs, we can demand that the equality holds for the integral kernels and obtain:If we note that this equation should hold for all arbitrary Now let us concentrate on mentclass2pt{minimmentclasspt{minimar, the left hand side of the above equation equals to zero which results to:Therefore for the homogeneous system, which is independent of the observation point n(r) as follows:Fns. In fact Fn is everywhere non-zero and well-defined and finite except at n = 0 and n = 0 for which, as we will see, becomes divergent and n) |n|3/2 whose derivative is not well-behaved in n = 0.By equating the expression inside the bracket to a constant (which is zero for the symmetry considerations) we obtain the equation governing the distribution of ζ0 limit. In this limit, and considering S is the area of the sample and P0 is the normalization constant. This relation may seem to be unsuited, since it grows unboundedly for negative values of n. Actually there is no contradiction, due to the presence of Gn whose amount grows negatively for negative n values, which returns the above equation to the expected form. In fact the original charge equation has electron-hole symmetry for the case Pn. Our approximation (considering Gn as a constant) violated this symmetry. Re-considering this quantity as a dynamical variable retains the mentioned symmetry. It is also notable that the second term in the exponent Pn.With the above limitation in mind, let us first turn to the asymptotic behaviors of Eq. μ have been actually coded in ζ′. Large amounts of G favors large charge fluctuations. To see how G controls the charge fluctuations, we note that the limit G. In fact μ controls 〈n〉 which directly affects G. In the limit G becomes vanishingly small, so that In the above equation, the effects of disorder and interaction and The total solution of Eq. rsFn and Pn for positive ns for Pn approaches continuously to n〉. Before closing this section, let us turn to calculating the compressibility (κ). Knowing that the compressibility is proportional to 〈n2〉 − 〈n〉2 we can easily do the integrals and show that, for the un-gated intrinsic graphene n〉 → 0, In the above equation f caused by the infinitesimal growth of the charge density is In this section we consider a two-body functional. i.e. Pn,n′:Just like the calculations of the previous section we write . The result has been shown in Fig. ζ (Rmax is the amount of R in which Gx(R) shows a peak). It can be shown that Gx(R) has some interesting features. Firstly the tail of this function (for large R’s) behaves in a logarithmic fashion. To show this we have shown R/Rmean in a semi-logarithmic graph in the inset of Fig. ζs (ζ = 1 in this graph), the graphs fit properly to each other with a nearly the same slope. Clearly this behavior can’t remain for very large Rs, since Gx(R) should be positive in all R range. The logarithmic behavior in graphene has been observed in many aspects, e.g. the logarithmic enhancement of Fermi velocity39, or exchange-driven singularity in the Fermi velocity in intrinsic graphene49, but up to the author’s knowledge such a behavior in two-point correlation functions has not been reported before. The logarithmic behavior is the characteristics of free Boson systems that our effective model for intermediate spatial scales corresponds to. The other feature of Gx(R) is the narrowing of its peak as ζ increases, and that Rmax and Gx(Rmax) are decreasing functions of ζ. The change of these quantities has been shown in the Fig. ζ. The power-law behavior arises from the scale-invariance of the TFD equation which was analyzed at the end of SEC. 1. To characterize the behavior of Gx(R) let us analyze the width of the Gx(R) defined by Gx(Rmax) and Rmax in terms of ζ. The behaviors are A very important quantity is the density-density correlation function x〉 is small enough, i.e. ζ is small, Rmax becomes large and therefore δ increases unboundedly which leads to large density inhomogeneity, i.e. formation of EHPs.In the limit in which the interaction is strong enough and (or) the fraction 〈Gx(R), i.e. G(q) which is shown to be:Jn(x) is the Bessel function of the first kind, G(q) vanishes. The presence of disorder induces a characteristic wave vector ζ in a semi-logarithmic scale. For large ζ values this function is nearly constant for small values of q value which we name q0. For q values in the order of (and larger than) qch, we enter the phase in which q0 is ζ-dependent and increases as ζ increases. It is interestingly seen that q0 show power-law behaviors in terms of ζ with exponents shown in the figure, i.e. The other important quantity is the Fourier transform of n(r), i.e. Eq. To understand where these power-law behaviors come from, we should look at the main equation of G(q) on the screened potential is very interesting. The Fourier component of the screened potential (vsc(q)) in long wave lengths  is crucially changed, so that vsc is not changed with respect to the bare interaction potential for large and intermediate wave numbers. It has been shown in the inset of Fig. q for large q’s. The cross-over between these two behaviors occur in some q interval. It is seen that there is a characteristic q there (the peak), which results to some oscillations in the real space. The potential in the real space v(R) has some interesting features. For large R’s and large ζ’s this quantity falls off just like the bare potential, i.e. 1/R with some oscillations which is evident in Fig. R limit the potential tends to a constant value, which cause ζ increases, ζ decreases  shows a peak. The emergent oscillatory behavior is the effect of characteristic wave number for which the screened potential shows peak.The effect of G(q). It is well-known that for zero temperature case the polarization given by the RPA for mono-layer garphene is constant for q with a discontinuity in the derivative at 5. G(q)  for which Before closing this section, it is worth mentioning some points concerning the form of mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimP({n}) which is the probability measure for the charge configuration {n}. This function actually depends not only to n configuration, but also to its gradient: f is an arbitrary local or non-local smooth function of the density n, i.e. r0 is some reference point. Without loose of generality we set n  shows a peak. It was found that Gx(R) falls off logarithmically for large R’s, i.e. kF a disorder-driven characteristic momentum (qch) emerges in the system that controls the behaviors of the screened potential. The Fourier component of screened potential changes significantly for small wave numbers. for small enough ζ’s  we have observed an instability in which the imaginary part of the dielectric function becomes negative and some oscillations are observed for the charge density δn(r). The oscillations are absent for larger ζ values . Along with this change, a screening-anti-screening transition occurs in which δn(0) changes the sign. The observed instability is certainly affected by the higher order contribution, but we think that it should show itself for small enough densities, since the effect increases in a power-law fashion with ζ.In this paper we have investigated the properties of the disorder-averaged dielectric function by analyzing the one- and two- body charge density distribution functions of mono-layer graphene. For calculating them, we have used the Thomas-Fermi-Dirac (TFD) approach, taking into account the tunable disorder and inter particle interactions on an equal footing. To use the TFD theory for calculating the linear screening, we developed a diagrammatic technique and in the first order approximation in disorder strength, the polarization operator and charge modulation due to external potential have been obtained. The one- and the two- body charge density distribution functions were obtained using some stochastic analysis which carries both the effect of white-noise out-plane disorder and the interaction. The closed form of the functions were obtained for high density and low inter-particle interaction limits. By analyzing the one-body distribution function we found that the charge density fluctuations is proportional to ζ limit and was shown to be of the quadratic form. For smaller ζ’s we think that derivatives of charge densities should come to calculations which is the subject of our future research.In the last part of the paper we have calculated the total distribution measure of the mono-layer graphene sheets. The resulting equation has been analytically solved for large Interaction-disorder-driven characteristic momentum in graphene, approach of multi-body distribution functions