A clean and functional microscope is necessary for accurate diagnosis of infectious diseases. In tropical climates, high humidity levels and improper storage conditions allow for the accumulation of debris and fungus on the optical components of diagnostic equipment, such as microscopes.Our objective was to develop and implement a low-cost, sustainable, easy to manage, low-maintenance, passive humidity control chamber to both reduce debris accumulation and microbial growth onto the optical components of microscopes.Constructed from easily-sourced and locally available materials, the cost of each humidity control chamber is approximately $2.35 USD. Relative humidity levels were recorded every 30 minutes over a period of 10 weeks from two chambers deployed at the Belize Vector and Ecology Center and the University of Belize.The humidity control chamber deployed at the University of Belize maintained internal relative humidity at an average of 35.3% (SD = 4.2%) over 10 weeks, while the average external relative humidity was 86.4% (SD = 12.4%). The humidity control chamber deployed at the Belize Vector and Ecology Center effectively maintained internal relative humidity to an average of 54.5% (SD = 9.4%) over 10 weeks, while the average external relative humidity was 86.9% (SD = 12.9%).Control of relative humidity is paramount for the sustainability of medical equipment in tropical climates. The humidity control chambers reduced relative humidity to levels that were not conducive for fungal growth while reducing microscope contamination from external sources. This will likely extend the service life of the microscopes while taking advantage of low-cost, locally sourced components. Infectious diseases are among the most significant contributors to global morbidity and mortality. Despite improvements in clinical care, tuberculosis and malaria were responsible for a combined 2 million deaths worldwide in 2017 1. In thes467Microscopy-based organism identification remains vital for the accurate and time-efficient diagnosis of many infectious diseases. The high relative humidity in warmer tropical regions, often exceeding 85%, increases the likelihood of fungal growth on microscope optics. Controlled studies have demonstrated that fungi grow in relative humidity levels of 80%, while growth is not observed at levels below 50% . This fi910There is a substantial need for a strategy to improve the functional operating longevity of microscopy equipment within tropical regions and underserved regions of the world where proper maintenance is particularly challenging. To this end, the Mayo Clinic Initiative for Medical Equipment Sustainability (IMES) has partnered with the Ministry of Health in Belize and the University of Belize to develop and implement a low-cost, sustainable, easy to manage, low-maintenance, passive humidity control chamber. The objective was to both reduce debris accumulation and microbial growth on the optical components of microscopes. Herein, we describe the criteria for use, material list, and fabrication instructions. We also characterize chamber performance with the goal of facilitating medical equipment sustainability.A passive humidity control chamber Figure  was desiRelative humidity consistently above 65%Accumulation of debris  on equipment surfacesFungal growth on the surface of the optical components of microscopes and other medical equipment that is sensitive to moisture such as otoscopes and endoscopesImproper storage conditions that do not comply with manufacturer recommendationsThe main components of the humidity control chamber consisted of a plastic bag to cover the equipment Figure , a weighTwo humidity control chambers were deployed to separate locations in Belize; one in the University of Belize in Belmopan and the other in the Belize Vector and Ecology Center in Orange Walk. Belize was chosen as the initial test location due to consistently high relative humidity levels of approximately 85% and a warm climate. Internal and external chamber relative humidity levels were recorded every 30 minutes using DHT11 humidity/temperature sensors . An Arduino UNO  collected this humidity data over a 10-week period. Relative humidity data was saved to an onboard micro SD card and transferred to the IMES team via email at the end of the 10-week period. Microscopes were left undisturbed for the duration of the study. A time series autoregressive statistical model  was used to determine if significant differences (p < 0.05) existed between internal and external relative humidity values.Data were collected from June 6, 2018 to September 10, 2018. The humidity control chamber deployed at the University of Belize maintained internal relative humidity at an average of 35.3% (SD = 4.2%) over 10 weeks, while the average external relative humidity was 86.4% (SD = 12.4%) Figure . The humThe humidity control chambers effectively maintained an average internal relative humidity level below the recommended limit of 65% 513. The pThe design of the humidity control chamber is intentionally simple. It is made with components commonly available in most parts of the world that can be substituted with other locally accessible materials. The environmental barrier can be created from any plastic bag large enough to completely cover the microscope. Additionally, the weighted skirt can be constructed from any dry, heavy material  that keeps the plastic barrier flush with the table surface. It is recommended that the weighting material be contained using a bicycle tire tube. A punctured bicycle tire may be acquired at no cost from local repair shops. Rice or a similar desiccant could be used to dehumidify the internal environment in the absence of silica beads. Uncovered glass jars were used to hold the silica beads; however, a perforated lid, muslin bag, or similar vessel may be used instead to avoid spilling. Verification of new materials would be required to ensure equivalent functionality. Although our study ended before the silica beads lost efficacy, our data indicates that 500 g or reusable silica bead desiccant will control humidity levels around an undisturbed microscope for at least 10 weeks before recharging the desiccant becomes necessary. When the humidity control chamber is deployed around heavily used medical equipment, the service life of the beads may be reduced. We recommend the use of blue indicating silica beads which change color as they absorb moisture to notify laboratory personnel they must be recharged. An analog or digital hygrometer may also be deployed within the chamber to ensure relative humidity is kept below 65%.This is an initial effort to maintain humidity levels in a low-cost, low-maintenance storage device for microscopes in high humidity climates. While microscopes were initially selected for their sensitivity to high relative humidity, it is important to note that our humidity control chamber can be used to protect any small moisture-sensitive equipment; including basic research equipment, medical electronics, chemicals, or drugs. Preliminary results assessing humidity reduction potential are promising; however, continued longitudinal relative humidity data acquisition is necessary to fully characterize the humidity control chamber. Additional steps, such as assessment of fungal growth and demonstration of improved sustainability of microscopy equipment are needed. Field studies with additional humidity control chambers under typical usage conditions will provide information to further characterize the impact of the system.Sustainability of medical and diagnostic equipment is critical in clinical settings in tropical climates or underserved regions of the world. The low-cost humidity control chamber described herein reduced the relative humidity within the chamber to levels that were not conducive for fungal growth in the tropical climate of Belize. The physical barrier in this system also offered protection from debris. Together, these can extend the usable lifespan of valuable diagnostic equipment and reduce the need for allocating additional resources for repeated replacement of diagnostic equipment.


Shifts from coral to algal dominance are expected to increase in tropical coral reefs as a result of anthropogenic disturbances. The consequences for key ecosystem functions such as primary productivity, calcification, and nutrient recycling are poorly understood, particularly under changing environmental conditions. We used a novel in situ incubation approach to compare functions of coral‐ and algae‐dominated communities in the central Red Sea bimonthly over an entire year. In situ gross and net community primary productivity, calcification, dissolved organic carbon fluxes, dissolved inorganic nitrogen fluxes, and their respective activation energies were quantified to describe the effects of seasonal changes. Overall, coral‐dominated communities exhibited 30% lower net productivity and 10 times higher calcification than algae‐dominated communities. Estimated activation energies indicated a higher thermal sensitivity of coral‐dominated communities. In these communities, net productivity and calcification were negatively correlated with temperature , whereas carbon losses via respiration and dissolved organic carbon release more than doubled at higher temperatures. In contrast, algae‐dominated communities doubled net productivity in summer, while calcification and dissolved organic carbon fluxes were unaffected. These results suggest pronounced changes in community functioning associated with coral‐algal phase shifts. Algae‐dominated communities may outcompete coral‐dominated communities because of their higher productivity and carbon retention to support fast biomass accumulation while compromising the formation of important reef framework structures. Higher temperatures likely amplify these functional differences, indicating a high vulnerability of ecosystem functions of coral‐dominated communities to temperatures even below coral bleaching thresholds. Our results suggest that ocean warming may not only cause but also amplify coral–algal phase shifts in coral reefs. Community shifts and the ongoing loss of biodiversity , as the slope or rate of change in the rise and falling phases of a thermal performance curve before and after achieving the optimal temperature. Although activation energies are commonly assessed at the organism level  directly compare the magnitudes and directions of key functions of coral‐dominated and phase‐shifted algae‐dominated reef communities, (2) derive their functional responses to environmental changes induced by seasonality, and (3) describe their thermal sensitivity to seasonally variable temperature changes.−2·s−1; 400–700 nm wavelengths) by intercalibration and conversion as outlined in Roth et al. (NO3‐), nitrite (NO2‐), ammonium (NH4+), phosphate (PO43‐), and monomeric silicate (Si(OH)4) were taken in triplicates each month from 1 m above the seafloor. Details for sampling and analysis can be found in Appendix NO3‐, NO2‐, and NH4+ is termed “dissolved inorganic nitrogen” (DIN) henceforth.This experiment was carried out at Abu Shosha reef located in the central Red Sea on the west coast of Saudi Arabia  from January 2017 until January 2018. Key environmental variables were monitored at the sampling site and were previously reported in Roth et al.  Coral‐dominated communities were defined by having >40% coral cover but <10% algae cover; (2) algae‐dominated communities were defined by having >40% algae cover but <10% coral cover; (3) all communities had to fit into the incubation chambers . Among all suitable candidates in the study area, four coral‐dominated and four algae‐dominated communities were chosen randomly. These eight communities were revisited each month of sampling.P = 0.001; visualization in Appendix The community composition at the level of major functional groups was assessed for each community three times during the study period . Details on the assessment and statistical evaluation of the community composition can be found in Appendix Algae‐dominated communities were considered “phase‐shifted,” as complex structures and the occurrence of coral rubble indicated that branching corals were present previously. The co‐occurrence of coral and algal reef communities within small spatial ranges was reported as “mosaic‐dynamics” before  of the enclosed benthic community adapted after Roth et al. (Discrete water samples for dissolved inorganic carbon (DIC), total alkalinity (TA), DOC, and DIN were withdrawn from the sampling ports with acid‐washed syringes at the beginning and the end of each incubation  were calculated by changes in DIC concentrations, taking into account calcification and dissolution rates according to an adapted protocol by Albright et al. . The calculated photosynthetic (1.05 ± 0.02) and respiratory (0.97 ± 0.02) quotients agree with those obtained by various authors elsewhere, typically ∼1 mol of oxygen produced for 1 mol C fixed, and vice versa  was calculated by concentration differences in TA, which are primarily caused by calcification and dissolution of CaCO3, whereby TA is reduced (increased) by two molar equivalents for every mole of CaCO3 produced (dissolved)  that cause a change in TA unrelated to calcification and dissolution were accounted for according to Zeebe and Wolf‐Gladrow (−2·h−1) and DIN (in µmol N·m−2·h−1) fluxes were calculated from concentration differences between start and endpoints. Any temperature corrections that were necessary for seawater chemistry calculations were achieved by temperature readings from individual temperature loggers within each chamber.NCC  statistic software. Environmental variables and response parameters from incubations were grouped into spring (March–May), summer (June–September), fall (October–November), and winter (December–February) for statistical analysis. For the seasonal comparison and to derive GPP/CR ratios, hourly rates from light and dark incubations were used to calculate daily net fluxes. We acknowledge that there is a chance for a slight over‐ or underestimation because of associated changes in environmental conditions  during the course of the day. Thus, to minimize the error associated with extrapolating hourly rates, we chose a time window for daylight incubations (from around 09:00 a.m. to 11:00 a.m.) that is closest to daytime average irradiation and excludes the “ramping up” phase in the early morning hours and extreme values that can occur during midday. As all incubations during all sampling periods were conducted at the very same time of the day, incubations are comparable across community types and time points.t‐tests. Response parameters from incubations  were assessed by linear mixed models (LMMs) to test for differences in the respective response parameters with ‘treatment’  and ‘season’  as fixed factors, and the sampling dates (date) within seasons and the replicates of the communities (community ID) as random factors. Tukey’s Honest Significant Difference (HSD) test was used for pairwise comparisons if significant interactions (treatment * season) were found. Detailed statistical results, including significant post hoc comparisons, are presented in Appendix The full seawater carbonate system parameters were derived for each sampling period from measured salinity, temperature, nutrients, TA, and DIC data using the CO2SYS Microsoft Excel Macro by Pierrot et al.  and the Ea) based on Arrhenius equations  were estimated by fitting a linear regression equation between the natural logarithm of the metabolic rates and the reciprocal of temperature (1/kT), where k is the Boltzmann’s constant (8.62 × 10−5 eV/K) and T is the water temperature (K). To deal with observations ≤0 on log‐transformed data , a constant was added ) to shift all values above zero  was explored by calculating the activation energy . Integrating the hourly rates over 24 h, corals showed a 10‐fold higher NCC compared to algae‐dominated reef communities  but were net sinks of DOC in the dark (−0.37 ± 0.05 mmol C·m−2·h−1). When integrated over 24 h, net DOC fluxes in coral‐dominated communities were 3.5‐fold higher than those in algae‐dominated communities . In contrast, coral‐dominated communities released DIN at consistent rates during light and in dark incubations (65.7 ± 11.6 and 69.7 ± 8.9 μmol N·m−2·h−1).Both coral‐ and algae‐dominated communities were net sources of DIN , and NCP of algae‐dominated communities exhibited a positive  relationship with increasing temperature. Hence, coral‐dominated communities showed apparent negative Ea values for GPP and NCP, indicating them to be in the falling phase of the performance curve , while apparent Ea for CR was positive, indicating an opposite trend   Fig. , and posEa values  with increasing DIN concentrations of the ambient seawater  netSpecifically, algae‐dominated communities displayed a higher organic C metabolism, along with residual net calcification. Significantly higher GPP and >40% elevated NCP in algae‐dominated communities indicate a greater potential for autotrophic biomass accumulation per planar square meter of reef  as sponges and other filter feeders utilize dissolved organic matter , associated with a higher total amount of C available  to the ecosystem. The high NCP of algae communities facilitates rapid lateral and vegetative overgrowth of bare substrates  were in the falling phase of the performance curves, and thus, past the optimum temperature to peak rates. Thereby, the community metabolism of corals is pushed toward carbon losses because of high CR relative to GPP in summer. Likewise, the activation energy of CR was positive, as respiration continued to increase with temperature through the seasonal thermal range. Both the lower ratio of GPP to CR (GPP/CR) and reduced NCP integrated over diel cycles indicate that coral‐dominated communities shifted toward a more heterotrophic state with increasing temperature. It is thus apparent that summer temperatures exceeded the metabolic optima for coral‐dominated communities, which was previously suggested for individuals of Pocillopora verrucosa , which can be attributed to an increased release of cellular matter and/or mucoid exudates during thermal stress in corals , with a temperature threshold at around 30.5°C  indicate that these communities benefit from higher temperatures and that thermal optima were not reached in summer. In fact, some macroalgae species  from the central Red Sea have a reported thermal optimum (31.7°C) for gross primary production (Anton et al. In contrast, positive activation energies for algae‐dominated communities (Theoretical studies have provided a sound understanding of the relationship between biodiversity loss and ecosystem functions in tropical coral reefs (reviewed in Hughes et al. Pocillopora verrucosa and Stylophora pistillata (Anton et al. Pocillopora verrucosa (Roik et al. The sensitivity of corals and their symbionts to rising temperatures has been documented extensively (Hoegh‐Guldberg The frequency and intensity of climate‐driven stress events on coral reefs will inevitably aggravate in the near future. Our results suggest that the anticipated increase in the spatial footprint of algae‐dominated communities would exacerbate the magnitude of the functional changes described here. Ocean warming likely enhances the competitive advantage of algae‐ over coral‐dominated communities (Anton et al. Appendix S1Click here for additional data file.


Fungal mitochondrial (mt) genomes exhibit great diversity in size which is partially attributed to their variable intergenic regions and most importantly to the inclusion of introns within their genes. These introns belong to group I or II, and both of them are self-splicing. The majority of them carry genes encoding homing endonucleases, either LAGLIDADG or GIY-YIG. In this study, it was found that these intronic homing endonucleases genes (HEGs) may originate from mt free-standing open reading frames which can be found nowadays in species belonging to Early Diverging Fungi as “living fossils.” A total of 487 introns carrying HEGs which were located in the publicly available mt genomes of representative species belonging to orders from all fungal phyla was analyzed. Their distribution in the mt genes, their insertion target sequence, and the phylogenetic analyses of the HEGs showed that these introns along with their HEGs form a composite structure in which both selfish elements coevolved. The invasion of the ancestral free-standing HEGs in the introns occurred through a perpetual mechanism, called in this study as “aenaon” hypothesis. It is based on recombination, transpositions, and horizontal gene transfer events throughout evolution. HEGs phylogenetically clustered primarily according to their intron hosts and secondarily to the mt genes carrying the introns and their HEGs. The evolutionary models created revealed an “intron-early” evolution which was enriched by “intron-late” events through many different independent recombinational events which resulted from both vertical and horizontal gene transfers. Fungal mitochondrial (mt) genomes are abundant with group I and II introns, which usually carry genes (homing endonucleases genes [HEGs]) encoding homing endonucleases. These homing endonucleases play important role in mt intron splicing. Until recently, the evolution of these elements was never examined in a pan genomic level but only in certain mt genes. In this study, the coevolution of these composite elements is revealed, through phylogenetic approaches, analyses of their insertion target sites, the intronic secondary structure changes, and their distribution to the mt host genes. The fungal ancestor most probably had introns and HEGs independent, but HEGs and their introns coevolved through recombination, transposition, and horizontal gene transfer, as presented in our proposed “aenaon” model.Hanseniaspora uvarum  . HEs areLD endonucleases constitute the most widespread family of HEs in organelles and microbial genomes. The wide proliferation of LDs is due to their ability to recognize a variety of target sequences including group I and group II introns, Archaeal introns, and inteins . LD prot10–11)-YIG. This motif has been detected in some repair systems and in restriction enzymes  from certain fungal orders which included species with a medical and/or biotechnological interest . Howevertococcus , in orderps3 gene  , four independent MCMCMC searches were performed. For each data set, different random starting points were used and after setting the number of generations to 40M and 10M and sampling every 4,000 and 1,000 generations for the LD and GIY, respectively. The burn-in was set to 40,000 and 10,000. Convergence was checked visually after plotting likelihood scores versus generation for the four runs, in both cases.The protein sequences of GIY and LD endonucleases were aligned using the ClustalW algorithm  in Megaler. 1.3)  was usedatp6, atp8, and atp9), the oxidative phosphorylation, that is, genes of NADH dehydrogenase subunits (nad1–6 and nad4L), apocytochrome b (cob), cytochrome C oxidase subunits (cox1–3), and in the mt ribosomes, that is, genes of large and small rRNA subunits . From the 487 identified introns carrying HEGs located in 132 fungal mitochondrial genomes , half of the introns (50%) in this study were located in cox1 gene and the majority of them (81%) are group IB introns. The gene carrying the second more abundant intronic distribution is the cob gene with a contribution of 17% of the total examined introns and those introns were identified as subtype ID with a frequency of 45% (nad5 gene (7.5%) with mostly IB introns (48%) and introns of the rnl gene (6.5%) mostly typed as IA (28%) follow in abundance. Genes nad2, nad4, and nad4L contain IC introns exclusively, but their allocation is <2% when the total distribution is taken into account . The free-standing GIY and LD genes represent the 20% and 7% of the total, respectively  online. ob genes  online. ing exon  online. The total characterized introns with GIY and LD ORFs are 107 and 337, respectively. Less than 10% of the examined introns failed to be characterized due to their short size and/or their degenerate sequence  online.Most HEGs were located in IB introns followed by ID introns in all fungal phyla. Subtypes IA and IC carrying HEGs are less commonly found. Subtype IC as intron host was absent from EDF species. For GIY genes, IC host introns were only observed in the Pezizomycotina subphylum and for LD genes only in Ascomycota and Basidiomycota A.cox1 and in rRNA genes. Group II introns hosting HEGs were totally absent from the examined mt genomes of species belonging to Pezizomycotina , followed by LD1 (29%), LD2 (20%), and LD3 (2%)  online. cox1 . Conversely, almost one-third of the LD ORFs in cob gene belong to the LD2 type . Genes nad4 and atp6 have only LD1(2m) in their introns. In all fungal subphyla but Pezizomycotina, ID introns tend to host LD2 endonucleases  where the free-standing GIY gene seems to be duplicated, because there are two neighboring copies of the gene within the same genome. The copies have different sizes, but the duplication is supported by the significant identity percentage of those, which is between 31% and 60% and their neighboring topology  and Phlebia radiata (Basidiomycota). In both cases, partial duplications of the neighboring genes atp6 and cox1 have also occurred in each case, creating new respective pseudogenes . Seven other GIY genes were located in the proximal region of the rns gene. In Pezizomycotina and Basidiomycotina, two cases were found, in which the free GIY of the mtDNA of P. omphalodes and Microbotryum lychnidis-dioicae were spotted nearby the atp9 gene and it seems likely that those cases constitute evolutionary relics of the atp9-GIY-YIG batch  in which l events  online.The Bayesian-based independent phylogenetic trees of 129 GIYs and 339 LDs showed several important clades which determine the evolution of the HEGs. In detail, the strongly supported (>87% PP) clades of the trees contain endonucleases within the same gene and the same intron subtype with a few exceptions  online. A. macrogynus. Other free GIYs of EDFs are also located basally. Each free-standing GIY gene is at the base of a cluster with intronic GIY ORFs usually located in introns of the same mt gene , but they were distinct (Clade II). Clades III and IV include representatives in IB introns of cox1 from all fungal phyla   . LD2 in omycetes .In this phylogenetic work, LD3 are grouped with one motif LD1 (or LD that could not be characterized using BlastP). This shows a phylogenetic relationship between LD3 and LD1 endonucleases. The intron insertion sequences even in the case of LD3 remain conserved and identical to insertion sites of LD1 .Microbotryum lychnidis-dioicae, Ustilago maydis, Fusarium oxyporum, and Phaeosphaeria nodorum), entomopathogenic , saprophytic (P. radiata and A. macrogynus), and symbiotic species like lichens .Horizontal gene transfer (HGT) events were examined using LD and GIY protein sequences from the mtDNA of representative phytopathogenic . This domestication of the ancestral introns showed an adaptation to their host gene which may be explained as a result of lacking mobility  origin of LD2 endonucleases, because LD2 endonucleases are grouped separately from LD1 and there was a tendency to switch from LD2 endonucleases to LD1 (or LD1(2m)) during the fungal evolution. The one and two motifs of LD1 show a scattered distribution in the different tree clades, which suggests that those two LD versions originate from each other, by either duplication of the one motif or loss of the second motif. LD1 gave rise to LD3 in the lineage of Saccharomycetes (and in a single case of a species from Basidiomycetes) by accumulating mutations and changing their protein sequence. LD2 seem to be transposed between same intron types of different genes, whereas LD1 and LD1(2m) intermingle with no clear distribution . Additioes pombe . After acox1, rnl, and rns genes of mt genomes from all fungal subphyla, whereas there were no group II introns hosting GIY genes. This is the result of the LD mobility with a mechanism similar to the intronless homing of HEGs as proposed for group I introns (Rhizophydium) to LD2 in group IB introns of cox1 gene of Pezizomycotina, Basidiomycota, and Saccharomycetes (Group II introns carrying LDs were scarce and only in  introns . This pacox1 and cox2) and “expansion” that is, their actual structure evolved from the ancestral compact form to a modern “expanded” one with new added hairpins  model . This moGenome Biology and Evolution online.evaa126_Supplementary_DataClick here for additional data file.


Brain-derived neurotrophic factor (BDNF), a major focus for regenerative therapeutics, has been lauded for its pro-survival characteristics and involvement in both development and recovery of function within the central nervous system (CNS). However, studies of tyrosine receptor kinase B (TrkB), a major receptor for BDNF, indicate that certain effects of the TrkB receptor in response to disease or injury may be maladaptive. More specifically, imbalance among TrkB receptor isoforms appears to contribute to aberrant signaling and hyperpathic pain. A truncated isoform of the receptor, TrkB.T1, lacks the intracellular kinase domain of the full length receptor and is up-regulated in multiple CNS injury models. Such up-regulation is associated with hyperpathic pain, and TrkB.T1 inhibition reduces neuropathic pain in various experimental paradigms. Deletion of TrkB.T1 also limits astrocyte changes in vitro, including proliferation, migration, and activation. Mechanistically, TrkB.T1 is believed to act through release of intracellular calcium in astrocytes, as well as through interactions with neurotrophins, leading to cell cycle activation. Together, these studies support a potential role for astrocytic TrkB.T1 in hyperpathic pain and suggest that targeted strategies directed at this receptor may have therapeutic potential. Neuropathic pain, which can occur from trauma to or dysfunction of the peripheral or central nervous systems (PNS or CNS), affects approximately 8–10% of the adult population i in astroglial cells do not require TrkB.FL [2+]i in neurons, proper TrkB.T1 levels (or the relative ratio of TrkB.FL to TrkB.T1) are critical for maintaining neuronal Ca2+ homeostasis [One of its identified binding proteins is the rho GDP dissociation inhibitor (RhoGDI1) . RhoGDI1ignaling ,80. The  TrkB.FL . The funeostasis . TrkB.T1eostasis ,81. Thiseostasis ,82. It ieostasis . TrkB reeostasis ,85. Wheteostasis ,36,39. Teostasis . Howevereostasis . TrkB.T1eostasis . Finallyeostasis . We haveeostasis ,41 that Therefore, active signaling by TrkB.T1 has profound and varying effects. Targeted deletion of TrkB.T1 in muscle that has been subjected to acute injury, showing increases in neurotrophin-dependent activation of Akt, a downstream signaling target of TrkB.FL, and subsequently improved neuromuscular function . In the Although a number of neurotrophins, including BDNF, are up-regulated in response to CNS insults, the up-regulation of truncated TrkB receptors participates in the inhibition of axonal regeneration, likely through competitive binding in the extracellular space and at the cell membrane ,75. The In other neurodegenerative disease models, increased TrkB.T1 expression also correlates with disease progression. For example, expression levels of BDNF and TrkB.FL are decreased, whereas TrkB.T1 is increased in Alzheimer’s disease , with suIn amyotrophic lateral sclerosis (ALS), BDNF has long been a drug candidate. Both in vivo and in vitro models show that BDNF, as well as BDNF analogs and TrkB agonists, such as 7,8-Dihydroxyflavone, can prevent motor neuron cell death and development of ALS ,96. HoweFollowing moderate contusion injury in mice, the upregulation of TrkB.T1 expression is observed at 24 h post-injury, with sustained elevation in white matter through 8 weeks ,100. TrkSimilar to SCI, TrkB.T1 receptor expression is increased in the brain after injury. In the rat hippocampus, following ibotenic acid-induced neuronal injury, TrkB.T1 mRNA is elevated with a time-course similar to glial fibrillary acidic protein mRNA expression increases . TraumatPain pathways in the adult spinal cord are primarily processed within the superficial SDH. After SCI and peripheral nerve injury, neurons in the SDH exhibit increased spontaneous activity, reduced activation thresholds, and an increased response to suprathreshold stimulation ,114. NeuAntiretroviral treatment causes mechanical allodynia in the mouse by increasing BDNF expression, which leads to hyperexcitability of wide dynamic range (WDR) neurons in the dorsal horn . HoweverWith respect to SCI-pain, there is evidence from rodent studies that central sensitization mechanistically contributes to sensory abnormalities and persistence of central neuropathic pain ,119,120.To examine the mechanisms underlying the decrease in nocifensive behavior in mice with SCI in the absence of TrkB.T1, a microarray analysis was done to compare gene expression in the spinal cord of TrkB.T1 WT versus KO mice at 24 h, 3 days, and 7 days after moderate contusion injury SCI . The majIn peripheral nerve injury or inflammatory pain models, inhibition of cell cycle activation by the CDKs inhibitors, flavopiridol and roscovitine, reduce hyperalgesia to heat and tactile allodynia ,126,127.2+ signaling, with oscillations in ion permeability through astrocytic networks [2+ can modulate neural network activity and activate other types of glia cells through the production of cytokines, chemokines, and growth factors [Activated microglia after SCI has been implicated in the maintenance of chronic hyperalgesia ,132, whenetworks . Followinetworks ,137. Thenetworks ,139. Rea factors ,133,134. factors ,140.+ astrocytes were reduced in the absence of TrkB.T1 at both the lesion area and the lumbar SDH. RNA sequencing of cultured astrocytes derived from TrkB.T1 KO and WT mice revealed down-regulation of migration and proliferation pathways in KO astrocytes. KO astrocytes also exhibited slower migration/proliferation in vitro in response to 10% serum or BDNF as compared to WT astrocytes. Reduced proliferation of astrocytes was confirmed after SCI in astrocyte specific TrkB.T1 KO mice, which also showed reduced hyperpathic responses in mechanical allodynia and pain-related measurements on the CatWalk, along with improved motor coordination [In a mouse spinal cord contusion model ,42, elevDespite being identified as a contributor to apoptosis and pain signaling, ablation of truncated BDNF receptors is problematic, as the complete elimination of TrkB can have severe detrimental effects. BDNF and its primary receptor TrkB are crucial to nervous system development and serve a protective function in adulthood, contributing to neuronal survival, maintenance of the central and peripheral nervous systems, and facilitating recovery from trauma. However, its truncated receptor TrkB.T1 has harmful effects when up-regulated in response to injury and inflammation, including but not limited to activation of cell cycle proteins, inhibiting axonal repair and facilitating the development of neuropathic pain. Experimental deletion of the TrkB.T1 receptor has successfully reduced the detrimental effects while improving functional recovery from injury and ameliorating pain. Some clues regarding the mechanism of action for TrkB.T1 signaling have been detected and, although more work is needed to fully understand the pathways involved, the truncated receptor appears to be a potential therapeutic target for a number of neurological disorders and for neuropathic pain. In the future, a greater understanding of the mechanistic pathways of TrkB.T1 will hopefully serve to narrow the focus of such therapeutic targeting so that the deleterious effects of the TrkB.T1 pathway are minimized and the favorable effects of BDNF are conserved or amplified.


Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at  Tao Zhu et al. present UMI-ATAC-seq, an improved ATAC-seq technique that uses unique molecular identifiers to avoid filtering out true independent - but identical - Tn5 insertion events that would otherwise be treated as PCR duplicates. They apply UMI-ATAC-seq to rice panicle samples and show that their approach improves the quantification of chromatin accessibility and footprinting. Thus, open chromatin regions are considered to be the primary location of transcription factor binding sites3. ATAC-seq provides an efficient way to analyze nucleosome-depleted regions of the genome and has been applied widely to identify transcription factor footprints6. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. In the assay, the density of Tn5 insertion events reflects the chromatin accessibility of a region, while some small regions with sudden drops of insertion events in a highly accessible region might indicate footprints of bound transcription factors.In eukaryotes, nuclear DNA is tightly packed into nucleosomes. The interaction of transcription factors with DNA and the exercise of transcriptional regulatory functions require or eventually lead to the dissociation of nucleosomes and form nucleosome-depleted regions or called open chromatin regions7. However, fragments are amplified with different efficiencies, resulting in an excessive number of specific fragments in the final library and bias for a number of analysis8. To eliminate this bias, we usually remove the PCR duplicates by identical genome coordinates before the advanced analysis. However, this deduplication mode is flawed in some situations. For example, as the sequencing depth increases, the probability of the same fragment being generated from different Tn5 insertion events will increase. For species with a small genome size or many tandem repeats, this phenomenon is exacerbated because they have a much higher probability of producing the identical fragments before PCR amplification7. In this case, removing PCR duplicates with mapping coordinates will remove the usable fragments that are not generated by PCR amplification, resulting in information loss, and introducing another kind of bias.As with most sequencing methods, ATAC-seq uses PCR amplification to ensure sufficient DNA fragments for sequencing on Illumina platforms12. The identical fragments with the same UMI are thought to be generated by PCR amplification. And the identical fragments with different UMIs, on the other hand, are assumed to be derived from different cells or different genome coordinates. UMI is demonstrated to be effective in counting the absolute number of individual molecules, and has been applied to various sequencing methods14.UMIs, typically random oligonucleotides ligated to each molecule, are widely used to accurately detect the PCR duplicates and quantitative transcriptsHere, we report a UMI-ATAC-seq technique by incorporating UMIs with standard TruSeq sequencing adapters into standard ATAC-seq procedures. The UMI-ATAC-seq library is compatible for sequencing on the Illumina platforms. Our results show that UMI-ATAC-seq could eliminate PCR deduplication bias especially in highly accessible chromatin regions. Finally, we demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying footprints.5 and human15, which are generated using standard ATAC-seq protocol. We divided the Arabidopsis and human genome into 250 bp bins separately and counted the total number of reads mapped in each bin and the number of reads marked as PCR duplicates by their identical mapping coordinates, respectively. When we evaluated PCR duplication rates in regions with different chromatin accessibility, we found that highly accessible chromatin regions usually include more PCR duplicates  to insure enough base diversity in the first 25 cycles  will start from the UMI sequence. Illumina sequencing systems utilize the first 25 cycles of Read 1 for cluster identification and quality metrics calculationsles Fig. .17 and all other steps are the same as for conventional ATAC-seq18, except that the PCR primers need to be replaced when constructing the libraries   , UMI-based deduplication (UD), and no deduplication (ND), respectively, and compared them with each other. The results show that the correlation between UD and ND is slightly higher than UD and CD (ly) Fig. ; we obtaly) Fig. . As the ly) Fig. . We furtly) Fig. . These r19 with different parameters reported by different studies21. We compared the number of peaks, P-values, and fold-enrichments obtained with datasets processed in CD and UD modes, respectively. And we found only small differences in the number of peaks between the CD and UD datasets for various peak calling parameters  is a major step of ATAC-seq data analysis. To evaluate the impact of incorporating UMIs on peak calling, we called peaks using MACS2P-value and the fold-enrichment ets Fig. . For UD-ets Fig. .P-value < 10−4)25. We averaged the Tn5 insertions across all predicted binding sites of transcription factor AP2/ERF and TCP1 that overlapped with the common pyDNase footprints identified in both CD and UD modes and located in the top 25% of the most accessible regions. Compared to the CD mode, we indeed observed better footprint patterning in the UD mode  values for ND, CD, and UD datasets respectively and compared the differences of FPD between each other. A bigger FPD value could be interpreted as decreased Tn5 insertions in the footprint region compared to its flanking regions.Fig.  The resuP-value < 10−5). The common and UD-only footprints overlapped with the FIMO motif sites by about 30 and 25%, respectively   Fig. , suggest28. However, both published data and our data show an increase in the presumed PCR duplication rates in highly accessible chromatin regions, suggesting that the conventional way to identify PCR duplicates has adverse effects on quantification and footprint identification in ATAC-seq. Footprint identification often requires deep sequencing of the libraries. It could be straightforward to infer that as the sequencing depth increases, the probability of generating independent but identical fragments would increase. Such “natural duplicates” would be erroneously removed as PCR duplicates in conventional ATAC-seq, diminishing the benefit of deep sequencing and flattening the footprint pattern. This might be one of the reasons why a previous study reported that the number of footprint would reach the limit at moderate library depths15. Our UMI-ATAC-seq technique has successfully adapted the standard ATAC-seq procedure to incorporate UMIs so that it could accurately distinguish between PCR and natural duplicates, which would be expected to improve the footprinting efficiency in deep sequencing.ATAC-seq has proven to be an efficient method to analyze open chromatin and transcription factor footprints17. Thus, the idea of our method can theoretically be adapted to these relevant technologies, such as methyl-ATAC-seq29, Trac-looping30, and ATAC-see31.In addition, many genomic assays construct sequencing libraries based on Tn5 transposaseFinally, an inconvenience to our method might be the extraction and processing of UMI sequences. To simplify this process, we have developed and made available a relevant pipeline. We anticipate that UMI-ATAC-seq could enhance the potential of ATAC-seq applications.17 with little modification. pTXB1 clones were transformed into BL21 cells (NEB) for production. Cells were grown in LB with ampicillin at 37 °C until OD600 = 0.6. The culture was chilled to 23 °C, and the expression of Tn5 was induced by adding 1 mM IPTG. The culture was then grown overnight at 23 °C and harvested by centrifugation. The cells were resuspended by HEGX , with complete protease inhibitors (Roche) and lysed by sonication. 10% Polyethyleneimine was added to remove nuclei acid. The supernatant was loaded on a 10-mL chitin column (NEB). The column was washed with HEGX, and Tn5 was cleaved from the intein by adding 100 mM DTT and incubated for 36–48 h. The elution was tested using a Bradford assay. The fractions with the strongest blue color were pooled and dialyzed versus two changes of 1 liter of 2X Tn5 dialysis buffer .Commercial Tn5 transposase is available from Illumina or many companies. Alternatively, in our study, in-house Tn5 was purified according to the procedure published by PicelliAll the oligonucleotides were synthesized in Shenggong Shanghai . After staining with DAPI, the sample was sorted by BD Aria SORP flow cytometry, and 100,000 nuclei were collected into a 1.5 mL centrifuge tube. The nuclei were centrifuged at 500 × 4 with little modification. The tagmentation reaction was carried out by adding 2 μL Tn5 complex, 8 μL 5x reaction buffer, and 30 μL ddH2O into the separated rice nuclei and incubated at 37 °C for 30 min. The transposed DNA fragments were purified by TaKaRa MiniBEST DNA Fragment Purification Kit (No. 9761). Adapters were added by PCR amplification using NEBNext® High-Fidelity 2X PCR Master Mix (M0541). Supplementary Table The library construction was adapted from human ATAC-seq protocolt No. 976. Adaptert No. 976. Adapter32, with parameters of a maximum of 2 seed mismatches, a palindrome clip threshold of 30, and a simple clip threshold of 10. Reads shorter than 30 bp were discarded. We then extracted the first 6 bp in each read pair as the UMI sequence and added it to the information line of the read pair. We next located and trimmed the sequence before the AGATGTGTATAAGAGACAG (ME) sequence in Read 1. Trimmed reads were aligned to the Oryza sativa L.ssp.japonica (cv.Nipponbare) reference genome (v.7.0)33 using BWA-mem with default settings, and the reads mapped to mitochondrial and chloroplast DNA were filtered. We only used properly paired reads with high mapping quality (MAPQ score > 30) for further analysis. Sorting aligned reads and removing PCR duplicates with mapping coordinates were conducted using SAMtools (v.1.9)34. We considered the reads with the same mapping coordinates but with different UMI sequences as rescued reads. The Tn5 insertion positions were determined as the start sites of reads adjusted by the rule of “forward strand +4 bp, negative strand −5 bp”4.Raw reads were first trimmed by Trimmomatic (v.0.36)The ATAC-seq raw data in human (cell line HEK293) were downloaded from NCBI SRA (SRX3511086) and aligned to the human genome assembly GRCh38. The ATAC-seq data in Arabidopsis whole seedlings were downloaded from NCBI SRA (SRX2000808) and aligned to the Arabidopsis Genome (TAIR10). Tn5 insertion events were counted with no deduplication (ND) and coordinates-based deduplication (CD) separately. The PCR duplication rate was calculated as (ND − CD)/ND.19 with the same following parameters: “--shift 100 --extsize 200 --nomodel --keep-dup all -B --SPMR --call-summits” except with different “-g” . Footprints were identified using “wellington_footprints.py” script in the pyDNase package23 with ATAC-seq mode (“-A” parameter) and a threshold of P-value < 10−30.Peak calling were performed on Arabidopsis and human data using the “callpeak” function in MACS2Oryza Repeat Database35 using blastn (v.2.5.0) with the following parameters: “-evalue 1e-10 -num_alignments 1”.The reads rescued by UMI were aligned to the 19 with the following parameters: “-g 3.0e8 --nomodel --keep-dup all -B --SPMR --call-summits”. To evaluate the impact of deduplication modes on peak calling, sets of “--shift” and “--extsize” parameters were tested. The specific parameters are marked in Supplementary Fig. Peak calling were performed on reads with coordinates-based deduplication (CD) and UMI-based deduplication (UD) separately, using the “callpeak” function in MACS2wellington_footprints.py” script in the pyDNase package23. Another script “dnase_wig_tracks.py” was employed to output the frequency of Tn5 insertion events, and IGV (Integrative Genomics Viewer)24 was utilized for visualization. The scripts were used in ATAC-seq mode (“-A” parameter) with other parameters in default values. For footprints identified only in the CD or UD datasets, we merged the two sets of footprints and then calculated the footprint scores with both UD and CD datasets using the same intervals of footprints. The footprint scores were transformed from Wellington P-value23 using the following formulas:lFP is the length of a possible footprint (11–26 bp); lSH is the length of shoulder on each side of the footprint (fixed at 35 bp); FP+ (forward strand) and FP– (negative strand) are the total number of Tn5 insertions inside the footprint. SH + (upstream shoulder in forward strand) and SH– (downstream shoulder in negative strand) are the total number of Tn5 insertions inside the shoulder of the footprint. F is the binomial cumulative distribution function.After calling peaks in CD and UD datasets separately, we merged the two sets of peaks and extended the peak intervals with the length <100 bp to 100 bp. The merged peaks were used to identify footprints by the “FKI is the total number of Tn5 insertions in the 50 bp upstream and the 50 bp downstream of the footprint; FPI is the total number of Tn5 insertions within the footprint, and L is the length of the footprint.We calculated footprint depth values with the following formula:25 (v.5.0.5) with the default parameters except that the parameter “thresh” differed for different applications. The position weight matrices (PWMs) were downloaded from JASPAR36 (http://jaspar.genereg.net/downloads/).We identified potential binding sites of transcription factors using FIMO37. Then we estimated the new overlapping rate with FIMO results and repeated the above steps 100 times to obtain a distribution. The distribution was used to perform Z-test for the observed overlapping rate.A shuffling-based approach was used to evaluate whether the overlap between the identified footprints and FIMO motif sites is random. Briefly, we randomized the footprint intervals in peaks using bedtools shufflez.test function in the TeachDemos package. Correlations between the CD, UD, and ND datasets are Pearson correlation coefficients. All UMI-ATAC-seq data can be downloaded from NCBI and the statistical results can be reproduced according to the parameters given in Methods.All statistics and analyses were performed using the software packages listed in Methods. Statistical Z-test were conducted in R using the Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


In >95% of cases of amyotrophic lateral sclerosis (ALS) and ∼45% of frontotemporal degeneration (FTD), the RNA/DNA-binding protein TDP-43 is cleared from the nucleus and abnormally accumulates in the cytoplasm of affected brain cells. Although the cellular triggers of disease pathology remain enigmatic, mounting evidence implicates the poly(ADP-ribose) polymerases (PARPs) in TDP-43 neurotoxicity. Here we show that inhibition of the PARP enzymes tankyrase 1 and tankyrase 2 (referred to as Tnks-1/2) protect primary rodent neurons from TDP-43-associated neurotoxicity. We demonstrate that Tnks-1/2 interacts with TDP-43 via a newly defined tankyrase-binding domain. Upon investigating the functional effect, we find that interaction with Tnks-1/2 inhibits the ubiquitination and proteasomal turnover of TDP-43, leading to its stabilization. We further show that proteasomal turnover of TDP-43 occurs preferentially in the nucleus; our data indicate that Tnks-1/2 stabilizes TDP-43 by promoting cytoplasmic accumulation, which sequesters the protein from nuclear proteasome degradation. Thus, Tnks-1/2 activity modulates TDP-43 and is a potential therapeutic target in diseases associated with TDP-43, such as ALS and FTD.This article has an associated First Person interview with the first author of the paper. Highlighted Article: TDP-43 interacts with Tnks-1/2 via a tankyrase-binding domain. This interaction promotes the cytoplasmic accumulation and stabilization of TDP-43. Amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) are fatal and incurable disorders at two ends of a disease spectrum called ALS/FTD . AlthougMounting evidence implicates polyADP-ribose) (PAR) activity in the neurotoxicity associated with ALS/FTD and other neurodegenerative disorders such as ischemic stroke, Parkinson's and Alzheimer's diseases (ose (PAR)The first known protein substrate of Tnks-1/2 was the telomere-binding protein TRF1 . PARylatDrosophila melanogaster that reduction of the Tnks-1/2 homologue mitigates the neurotoxicity of TDP-43, whereas upregulation exacerbates TDP-43-associated toxicity . Primary neurons were virally infected with 5 multiplicity of infection (moi) of an attenuated herpes simplex virus encoding either a control protein (LacZ) or human TDP-43. Cultures were maintained for 7 days post infection, then immunostained for the neuronal marker Tubulin β-III chain. Surviving neuronal cell bodies were quantified. TDP-43 expression resulted in a significant reduction in the number of cortical neurons compared with the LacZ control  , indicating that virally induced expression of TDP-43 leads to neuronal toxicity in rat primary cortical neurons.Our previous studies demonstrate that Tnks is a dose-sensitive modifier of TDP-43-associated toxicity in toxicity . To asceFig. S1B,C, Fig. S2), indicating that G007-LK had little to no effect on general neuronal survival. By contrast, neuronal loss induced by HSV-TDP-43 was significantly reduced by treatment with 1 or 10 µM G007-LK , indicating that G007-LK protects neurons from TDP-43-associated toxicity. These data demonstrate that treating rat primary cortical neurons with the Tnks-1/2 inhibitor protects against TDP-43-associated toxicity. Furthermore, they suggest that targeting Tnks-1/2 activity in TDP-43-associated disease is a potential therapeutic strategy.To determine whether Tnks-1/2 inhibition could protect neurons from TDP-43-associated toxicity, we examined the effect of the small-molecule inhibitor G007-LK, as it is highly selective for Tnks-1/2 and has no reported effect on PARP-1 activity . To deteFig. S3A,B). These data demonstrate that TDP-43 and Tnks-1/2 interact in mammalian protein lysates.To explore molecular interactions between TDP-43 and Tnks-1/2, including the possibility that TDP-43 and Tnks-1/2 function together in a protein complex, we established a robust series of co-immunoprecipitation assays in mammalian cells. We consistently observed that both endogenous TDP-43 and an exogenously expressed wild-type (WT) form of TDP-43 (TDP-43-WT-YFP) co-immunoprecipitate with endogenous Tnks-1/2 in COS-7 cells and HEK-293T cells , which we call the tankyrase-binding domain (TBD) . Collectively, these data suggest that the TBD is essential for the interaction between TDP-43 and Tnks-1/2.To determine whether there is a region within TDP-43 that may directly interact with Tnks-1/2, we computationally aligned the tankyrase-binding motif (RxxΦDG) to the human TDP-43 protein sequence. This highlighted an evolutionarily conserved region (amino acids 165-170) with 84% identity to the tankyrase-binding motif (in (TBD) C. To estin (TBD) F. ImportFig. S3E).To define further the Tnks-1/2 interaction domain in TDP-43, we mutated each amino acid in the TBD (RxxΦDG) individually to alanine G and tesAnalysis of the previously solved NMR structure of RNA recognition motifs (RRM1 and RRM2) of TDP-43  revealedP<0.01) in the levels of endogenous TDP-43 protein compared with the vehicle control . Together, these data indicate that loss of the Tnks-1/2 interaction leads to increased degradation of TDP-43 and suggest that under normal conditions Tnks-1/2 functions to stabilize TDP-43.The ADP-ribosylation activity of Tnks-1/2 has been established to promote proteasomal turnover of Tnks-1/2 substrates including APC2, axin, 3BP2, HIPPO, PTEN, TRF1 and YAP1 . To test control A,B. This control A,B, the Fig. S5A-C). The effect of Tnks-1/2 inhibition on MG132-induced ubiquitination of TDP-43 was not additive, as treatment with the Tnks-1/2 inhibitor alone did not lead to ubiquitination of TDP-43-WT-YFP . Collectively, these data demonstrate that loss of the Tnks-1/2 interaction or reduced Tnks-1/2 activity promotes MG132-induced ubiquitination of TDP-43 and increases proteasomal degradation of the protein. Thus, our data suggest that Tnks-1/2 maintains TDP-43 in a non-ubiquitinated and stabilized state.Given that Tnks-1/2 stabilized TDP-43 , we examTo gain an understanding of how Tnks-1/2 could lead to stabilization of TDP-43, we examined TDP-43 localization by immunofluorescence. Under normal conditions, both TDP-43-WT-YFP and TDP-43-ΔTBD-YFP localized diffusely to the nucleus. In response to MG132 treatment, both proteins formed nuclear foci that co-labeled with ubiquitin A,B. HoweFig. S7A); mutation of this region (TDP-43-ΔNLS/PBM) prevents nuclear import and binding to PAR . It is important to note that under the same conditions, the cytoplasmic protein G3BP1 formed cytoplasmic foci in response to MG132 treatment , which is consistent with previous reports  to the proline-tyrosine NLS (PY-NLS) from heterogeneous nuclear ribonucleoprotein A1  . The NLS from TDP-43 differs from the PY-NLS not only in amino acid sequence but also in the transport system used to direct proteins to the nucleus. Importin α/β  directs TDP-43 to the nucleus whereas transportin  directs hnRNPA1 to the nucleus  also rescued MG132-induced accumulation of the protein in the nucleus.To determine whether TDP-43-ΔNLS/PBM remained diffuse upon MG132 treatment because of its cytoplasmic localization or, alternatively, because the NLS/PBM mutation impaired the ability of the protein to respond to proteasome inhibition, we generated TDP-43-ΔNLS/PBM-GFP with an exogenous NLS sequence. We compared the bipartite NLS/PBM from the TDP-43 protein  or cytoplasm (-ΔNLS/PBM). Upon MG132 treatment, both nuclear forms of TDP-43 (-WT and -ΔNLS/PBMTDP-43) were ubiquitinated  reduced the percentage of cells with observable cytoplasmic TDP-43-WT  , the levels of cytoplasmic TDP-43 and of total TDP-43 protein are significantly higher than in control cells , suggesting broad impairment of protein turnover in neurodegenerative disease . Furtherol cells . Additiool cells , suggestThere are no effective treatments for ALS/FTD and related disorders such as Parkinson's disease. In ALS, nuclear PAR is elevated in motor neurons of post-mortem spinal cord tissue and total PAR is elevated in the cerebrospinal fluid of patients with Parkinson's disease . These f2. Three times per week, half of the medium was removed and replaced with fresh prewarmed medium. The primary neurons were infected at 14-17 days in vitro with HSV-TDP-43 or HSV-LacZ with either the Tnks-1/2 inhibitor G007-LK  or DMSO . Every 2 days, half of the medium was removed and replaced with fresh medium containing G007-LK or DMSO. The drug-containing medium was made up at 2× concentration so that when it was diluted twofold in the well it was of the appropriate concentration. At 7 days post infection, the neurons were fixed in 4% paraformaldehyde, blocked in 10% normal donkey serum (Sigma Aldrich) in Tris-buffered saline containing 0.05% Tween 20 (TBST) (ThermoFisher Scientific) for 1 h at room temperature, and immunostained overnight at 4°C with antibodies directed to the neuronal marker tubulin β-3 chain . After three sets of 5 min washes in TBST, neurons were incubated with mouse AlexaFluor 488  in the dark for 1 h at room temperature. Neurons were washed three times (5 min each) with TBST, counterstained for 15 min with 1 µg/ml Hoechst 33342 (ThermoFisher Scientific), washed in deionized H2O and mounted in ProLong Diamond (ThermoFisher Scientific). Five images (10× magnification) were captured from each coverslip and the remaining neuronal cell bodies in each image counted. Each condition was repeated three times on three independent cultures, each from a different pregnant rat.Rat cortical neurons were from embryos isolated from female Sprague Dawley wild rats that were 16-18 days pregnant . About 100,000 neurons were plated out on poly-D-lysine coated coverslips  in neurobasal medium supplemented with serum-free B27, penicillin streptomycin and Glutamax . Neurons were cultured at 37°C with 5% CO2; a water bath was used for humidification. Cells were washed with Dulbecco's PBS without calcium or magnesium (ThermoFisher Scientific) and trypsinized in trypsin with 0.25% EDTA (ThermoFisher Scientific). No commonly misidentified cell lines were used.COS-7 cells originally purchased by ATCC were a gift from Virginia M. Lee (University of Pennsylvania). Prior to purchase, the COS-7 cells were authenticated by ATCC. HEK293T cells were kindly provided by Aaron Gitler (Stanford University). COS-7 cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM) containing high glucose and L-glutamine (ThermoFisher Scientific), 10% filter-sterile FBS (Sigma Aldrich) and penicillin-streptomycin (ThermoFisher Scientific). HEK-293T cells were grown in DMEM with high glucose, L-glutamine and sodium pyruvate (ThermoFisher Scientific), 10% filter-sterile FBS (Sigma Aldrich) and penicillin-streptomycin (ThermoFisher Scientific). Cells were grown at 37°C with 5% COWe computationally aligned the tankyrase-binding motif (RxxΦDG) to the human TDP-43 protein sequence using the PATTINPROT search engine . To map Human TDP-43-WT-YFP in pcDNA3.2 was described previously . TDP-43-TDP-43-ΔTBD, -D169G, -R165A, -H166A, -M167A, -I168A, -D169A and -G170A were made by performing site-directed mutagenesis (QuikChange II XL) on TDP-43-WT-YFP using the following forward primers (FP) and reverse primers (RP): ΔTBD, FP 5′-CAAGTGAAAGTAATGTCACAGCGATGGTGTGACTGCAAACTTCC-3′ and RP 5′-GGAAGTTTGCAGTCACACCATCGCTGTGACATTACTTTCACTTG-3′; D169G, FP: 5′-TCACAGCGACATATGATAGGTGGACGATGGTGTGACTGC-3′ and RP: 5′-GCAGTCACACCATCGTCCACCTATCATATGTCGCTGTGA-3′; R165A, FP 5′-CATCGTCCATCTATCATATGTGCCTGTGACATTACTTTCACTTG-3′ and RP 5′-CAAGTGAAAGTAATGTCACAGGCACATATGATAGATGGACGATG-3′; H166A, FP 5′-CACCATCGTCCATCTATCATAGCTCGCTGTGACATTACTTTCAC-3′ and RP 5′-GTGAAAGTAATGTCACAGCGAGCTATGATAGATGGACGATGGTG-3′; M167A, FP 5′-CACACCATCGTCCATCTATCGCATGTCGCTGTGACATTACTT-3′ and RP 5′-AAGTAATGTCACAGCGACATGCGATAGATGGACGATGGTGTG-3′; I168A, FP 5′-GTCACACCATCGTCCATCTGCCATATGTCGCTGTGACATT-3′ and RP 5′-AATGTCACAGCGACATATGGCAGATGGACGATGGTGTGAC-3′; D169A, FP 5′-GTCACACCATCGTCCAGCTATCATATGTCGCTGT-3′ and RP 5′-ACAGCGACATATGATAGCTGGACGATGGTGTGAC-3′; G170A, FP 5′-TGCAGTCACACCATCGTGCATCTATCATATGTCGC-3′ and RP 5′-GCGACATATGATAGATGCACGATGGTGTGACTGCA-3′.TDP-43) or the NLS of hnRNPA1 (TDP-43-ΔPBM-NLSA1) was cloned immediately upstream of the stop site in TDP-43-ΔPBM-GFP in pcDNA3-C-eGFP by GenScript, Piscataway, NJ, USA. The following DNA sequences were subcloned: TDP-43-ΔPBM-NLSTDP-43, 5′-AAAAGAAAAATGGATGAGACAGATGCTTCATCAGCAGTGAAAGTGAAAAGA-3′; TDP-43-ΔPBM-NLSA1, 5′-AATCAGTCTTCAAATTTTGGACCCATGAAGGGAGGAAATTTTGGAGGCAGAAGCTCTGGCCCCTATGGCGGTGGAGGCCAATACTTTGCAAAACCACGAAACCAAGGTGGCTAT-3′.The DNA sequence corresponding to the NLS of TDP-43  and 10% FBS (Sigma Aldrich). For all experiments, cells were trypsinized in trypsin with 0.25% EDTA (ThermoFisher Scientific). Cells were pelleted, washed in Dulbecco's PBS without calcium and without magnesium (ThermoFisher Scientific) and resuspended in 1 ml ice-cold Pierce IP Lysis Buffer (ThermoFisher Scientific) containing Halt Protease Inhibitor Single-Use Cocktail (ThermoFisher Scientific). For all co-immunoprecipitations, lysates from each T75 flask (∼8.4×106 cells) were incubated on ice for 10 min, passed three times through a 20G1½ syringe attached to a 1 ml syringe , transferred to centrifuge tubes, rotated at 15 rpm for 10 min at 4°C and centrifuged at 16,873 g for 10 min at 4°C. The supernatant was collected, the pellet discarded and 25 µl of lysate removed for input. The remaining lysate was divided in half (500 µl) for incubation with IgG or anti-TDP-43-coupled beads. The reaction volumes were made up to 1 ml with lysis buffer containing protease inhibitor and incubated with the antibody-coupled beads for 18 h at 4°C with 15 rpm rotation. The beads were washed three times by removing the supernatant, adding 500 µl lysis buffer containing protease inhibitor, briefly resuspending the beads, placing the tube on the magnet and immediately removing the buffer. Elution was performed at 95°C for 5 min in 40 µl 1× LDS Sample Buffer (ThermoFisher Scientific) containing 5% β-mercaptoethanol (Sigma Aldrich). Input samples (10 µl) were denatured at 95°C for 5 min in 1× LDS Sample Buffer containing 5% β-mercaptoethanol in a total volume of 20 µl.To examine the interaction between endogenous TDP-43 and Tnks-1/2, 2.5 µg of control mouse IgG  and 2.5 µg mouse anti-TDP-43   were couEluates and inputs were electrophoresed  on NuPAGE 4-12% Bis-Tris gels (ThermoFisher Scientific) in NuPAGE MES or MOPS buffer (ThermoFisher Scientific). Protein was transferred onto 0.45 µm nitrocellulose membranes (BioRad) using the XCell II Blot Module (ThermoFisher Scientific), in 1× Transfer Buffer (ThermoFisher Scientific) at 20 V for 105 min. Membranes were blocked in TBST (ThermoFisher Scientific) containing 5% non-fat dry milk  for 1 h at room temperature, then incubated in primary antibody in TBST overnight at 4°C. Membranes were washed 5×5 min in TBST, incubated in secondary antibody in TBST for 1 h at room temperature, washed 5×5 min in TBST and covered in ECL Prime detection reagent  for 5 min in the dark. Rabbit anti-TDP-43 , rabbit anti-Tnks-1/2  and goat anti-rabbit HRP  were used.5 cells in a six-well plate overnight in DMEM with high glucose and L-glutamine (ThermoFisher Scientific), 10% FBS (Sigma Aldrich) and penicillin-streptomycin (ThermoFisher Scientific). Each well was transfected with 2.5 µg plasmid DNA, 2.5 µl PLUS reagent and 7.9 µl LTX in 400 µl OPTIMEM I  in DMEM with high glucose (ThermoFisher Scientific) and 10% FBS (Sigma Aldrich). Control IgG  and anti-GFP-3E6  were coupled to 50 µl Protein G dynabeads (ThermoFisher Scientific). For MG132-induced ubiquitination of TDP-43, cells were treated with 10 µM MG132 (Sigma Aldrich) or an equivalent volume of DMSO (Sigma Aldrich) at 23 h post-transfection for the indicated times. Treatment with the DMSO control always matched the longest MG132 treatment. For MG132-induced ubiquitination of TDP-43 in the presence or absence of Tnks-1/2 inhibitor, cells were treated with 10 µM MG132 (Sigma Aldrich) combined with either DMSO (Sigma Aldrich) or XAV939, G007-LK or IWR1-endo  at the indicated times and concentrations. Cells were lysed by adding 300 µl ice-cold RIPA buffer  containing Halt Protease Inhibitor Single-Use Cocktail (ThermoFisher Scientific) and 5 mM N-ethylmaleimide (NEM) (ThermoFisher Scientific) to each well and incubating the plate on a platform shaker at medium speed for 10 min at 4°C. Lysates were passed three times through a 20G1½ 1 ml syringe , transferred to centrifuge tubes and rotated at 15 rpm for 10 min at 4°C. Lysates were centrifuged for 10 min at 16,873 g and 4°C. Then, 25 µl of lysate was removed for input and the remaining lysate was made up to 1 ml and incubated with the antibody-coupled beads for 18 h at 4°C with rotation.COS-7 cells were seeded at a density of 2.7×10Beads were washed three times in 500 µl lysis buffer for 10 min at 4°C with 15 rpm rotation. Elution was performed at 95°C for 5 min in 40 µl 1× LDS Sample Buffer (ThermoFisher Scientific) containing 5% β-mercaptoethanol (Sigma Aldrich). Input samples (10 µl) were denatured at 95°C for 5 min in 1× LDS Sample Buffer (ThermoFisher Scientific) containing 5% β-mercaptoethanol (Sigma Aldrich) in a total volume of 20 µl. Eluates and inputs were electrophoresed  on NuPAGE 4-12% Bis-Tris gels (ThermoFisher Scientific) in NuPAGE MES or MOPS buffer (ThermoFisher Scientific). Protein was transferred onto 0.45 µm nitrocellulose membranes  using the XCell II Blot Module (ThermoFisher Scientific), in 1× Transfer Buffer (ThermoFisher Scientific) and run at 30 V for 75 min (note that gels to be transferred to membranes for blotting of ubiquitin were soaked in transfer buffer for 5 min prior to wet transfer). Membranes were blocked in TBST containing 5% non-fat dry milk (LabScientific) for 1 h at room temperature, then incubated in primary antibody in TBST overnight at 4°C. Membranes were washed 5×5 min in TBST, incubated in secondary antibody in TBST for 1 h at room temperature, washed 5×5 min in TBST and covered in ECL Prime detection reagents (Amersham) for 5 min in the dark. Rabbit anti-TDP-43 , rabbit anti-ubiquitin  and goat anti-rabbit HRP  were used.5 cells in a six-well plate overnight in DMEM with high glucose (ThermoFisher Scientific), 10% FBS (Sigma Aldrich) and penicillin-streptomycin (ThermoFisher Scientific). The following day, each well was transfected with 2.5 µg plasmid DNA, 2.5 µl PLUS reagent and 7.9 µl LTX in 400 µl OPTIMEM I in DMEM with high glucose and L-glutamine  and 10% FBS (Sigma Aldrich). Cells were treated with 100 µg/ml cycloheximide (Sigma Aldrich) in DMEM with high glucose and L-glutamine  containing 10% FBS (Sigma Aldrich) and penicillin-streptomycin (ThermoFisher Scientific) starting at 24 h after transfection. The media with DMSO (ThermoFisher Scientific) or 100 µg/ml cycloheximide (Sigma Aldrich) were replaced every 24 h during this period. Cells were lysed at 0, 24 and 48 h (see paragraph below for cell lysis). To examine the stability of endogenous TDP-43, a six-well plate was inoculated with COS-7 cells at a seeding density of 5.6×105 cells. Cells were grown in DMEM with high glucose and L-glutamine (ThermoFisher Scientific) containing 10% FBS (Sigma Aldrich) and penicillin-streptomycin (ThermoFisher Scientific) in six-well plates overnight. Cells were treated with 100 µg/ml cycloheximide (Sigma Aldrich) in DMEM containing high glucose and L-glutamine (ThermoFisher Scientific) and 10% FBS (Sigma Aldrich) with penicillin-streptomycin (ThermoFisher Scientific) and lysed (see paragraph below) 1 h after drug treatment.To examine the stability of TDP-43-YFP, COS-7 cells were seeded at a density of 2.7×10N-ethylmaleimide (NEM) (ThermoFisher Scientific) and incubated on a platform shaker at medium speed for 10 min at 4°C. Lysates were then passed three times through a 20G1½ 1 ml syringe (BD Biosciences), transferred to centrifuge tubes for 15 rpm rotation for 10 min at 4°C and centrifuged for 10 min at 16,873 g and 4°C. Inputs were denatured at 95°C for 5 min in 20 µl 1× LDS Sample Buffer (ThermoFisher Scientific) with 5% β-mercaptoethanol (Sigma Aldrich) and run (5 µl) on NuPAGE 4-12% Bis-Tris gels in NuPAGE MES or MOPS buffer . Wet transfer was performed using 0.45 µm nitrocellulose membranes (BioRad) using the XCell II Blot Module (ThermoFisher Scientific) in 1× Transfer Buffer (ThermoFisher Scientific), run at 20 V for 105 min. Membranes were blocked in TBST (ThermoFisher Scientific) with 5% non-fat dry milk (LabScientific) for 1 h at room temperature, then incubated in primary antibody in TBST overnight at 4°C. Membranes were washed 5×5 min in TBST, incubated in secondary antibody in TBST for 1 h at room temperature, washed 5×5 min in TBST and covered in ECL Prime detection reagent (Amersham) for 5 min in the dark. Rabbit anti-TDP-43  and goat anti-rabbit HRP  were used.Cells were lysed by adding 300 µl ice-cold RIPA Buffer  containing Halt Protease Inhibitor Single-Use Cocktail (ThermoFisher Scientific) and 5 mM 2; a water bath was used for humidification. The following day (∼18 h later), each well was transfected with 500 ng of plasmid DNA, 1.75 µl lipofectamine LTX and 0.5 µl PLUS reagent in 100 µl of OPTIMEM I . At 21 h post transfection, cells were treated with 10 µM MG132 (Sigma Aldrich) for the indicated amount of time. Treatment with the DMSO control always matched the longest MG132 treatment. For localization studies in the presence of Tnks-1/2 inhibitors, cells were transfected in the presence of the indicated amount of G007-LK or IWR1-endo (both SelleckChem) and fixed for 21 h post-transfection. Cells were fixed in 4% paraformaldehyde  in 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2 and 1 mM EGTA . Fixed cells were permeabilized by three treatments of 3 min in 100 mM PIPES, 1 mM MgCl2, 10 mM EGTA pH 6.8 and 0.1% Triton-X 100 (PEM-T buffer) . Cells were blocked in 10% normal donkey serum (Sigma Aldrich) in TBST (ThermoFisher Scientific). Primary antibodies in TBST (ThermoFisher Scientific) were applied overnight at 4°C in a humidified chamber. Cells were washed three times in PEM-T buffer (3 min each); then, secondary antibody in TBST (ThermoFisher Scientific) was applied for 45 min at room temperature and in the dark. Cells were washed three times in TBST (3 min each), stained with 1 µg/ml Hoechst 33342 (ThermoFisher Scientific) for 15 min, washed in deionized H2O and mounted in ProLong Diamond (ThermoFisher Scientific). All experiments were performed at least three independent times.Immunofluorescence was carried out in a 24-well format. Cells (COS-7 and HEK293T) were seeded at a density of 60,000 cells per well onto glass coverslips (Neuvitro) in DMEM with high glucose and L-glutamine (ThermoFisher Scientific) and 10% FBS (Sigma Aldrich) with penicillin-streptomycin (ThermoFisher Scientific) and incubated overnight at 37°C with 5% COPrimary antibodies used were rabbit anti-G3BP1  and mouse anti-ubiquitin . Secondary antibodies  were goat anti-rabbit Alexa Fluor 647 ; goat anti-rabbit Alexa Fluor 594 ; goat anti-mouse Alexa Fluor 647 ; and donkey anti mouse Alexa Fluor 594 (#21203 RRID: AB_141633).TDP-43-GFP and TDP-43-ΔNLS/PBMA1-GFP, 129-514 transfected cells were quantified per condition. To quantify the effect of Tnks-1/2 inhibition on TDP-43-GFP localization, 4-5 independent images at 20× were captured at the same exposure time. Cells with diffuse cytoplasmic GFP and/or GFP foci in the cytoplasm were scored as cells with cytoplasmic TDP-43-GFP. Up to 540 transfected cells were quantified per condition. All experiments were repeated at least three independent times and the mean±s.e.m. calculated.All imaging was performed on fixed cells at 18-23°C on a Leica DMI6000, widefield epifluorescent microscope . To quantify MG132-induced foci, four or five independent images at 20× were captured. The number of transfected cells was quantified in each image; the number of transfected cells with either nuclear or cytoplasmic foci was counted and the number of cells with nuclear or cytoplasmic TDP-43 foci that co-labeled with ubiquitin was counted and the percentage calculated. To compare the effect of MG132 on TDP-43-WT-YFP versus TDP-43-ΔTBD-YFP, 139-298 transfected cells were quantified per condition. To compare the effect of MG132 on TDP-43-WT-GFP, TDP-43-ΔNLS/PBM-GFP, TDP-43-ΔNLS/PBMn is the number of biological repeats and is indicated in each figure legend. Student's t-tests, one-way ANOVA, two-way ANOVA and multiple comparison tests were performed, as indicated in each figure legend. Significance was set at P<0.05; values for asterisks are presented in each legend. All statistical analyses were carried out using Graphpad prism6 software .Each graph gives the mean (±s.e.m. or s.d.).


The coatings were prepared by dispersing graphene in waterborne epoxy with the addition of carboxymethylcellulose sodium. The structure and composition of the coatings were characterized by scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared and Raman spectroscopies. The corrosion resistance of the composite coatings was investigated by potentiodynamic polarization measurements and electrochemical impedance spectroscopy. Composite coatings with more uniform surfaces and far fewer defects than blank waterborne epoxy coatings were obtained on 1020 steel. The 0.5 wt% graphene/waterborne epoxy composite coating exhibited a much lower corrosion rate and provided better water resistance properties and long-term protection than those of the blank epoxy coating in CO2-satarated NaCl solution.This study investigated the corrosion resistance of graphene/waterborne epoxy composite coatings in CO CO2 corrosion is the main form of internal corrosion failure in oil and natural gas pipelines [2 flooding test in the Little Creek Oilfield, USA, the wall of the oil extraction pipe was corroded and perforated within five months with a corrosion rate of 12.7 mm yr−1 [The COrecovery . In this mm yr−1 . Pipelin mm yr−1 ,5.2 have promoted studies on corrosion prevention methods in the oil and gas industry. Coatings [These losses associated with COCoatings , inhibitCoatings ,8 and adCoatings ,10 are tTraditional organic coatings are mainly solvent based. The release of volatile organic compounds during the coating and service processes can be harmful to the environment and the health of construction workers. Unfortunately, the protective effects of waterborne coatings, such as their film-forming properties, water resistance properties and shielding properties, are significantly lower than those of solvent-based coatings. We therefore considered adding graphene to improve the corrosion resistance of waterborne epoxy coatings for application in oil and gas industry environments.2-hybridized carbon atoms. Graphene has high thermal and chemical stability and can isolate the substrate from the corrosive medium to provide effective corrosion inhibition [et al. [2 and H2O molecules will penetrate through the defects and corrode the metal substrate [Graphene is a flat film carbon material that is composed of monolayer sphibition . Chen et [et al.  first st [et al. –16, howeubstrate .et al. [et al. [Many reports have indicated that graphene composite coatings can provide considerable corrosion inhibition. The addition of graphene and functionalized graphene in organic coatings can further improve the corrosion performance in marine environments –20. Ye eet al.  intercal [et al.  synthesi2 and sometimes high concentrations of Cl− are both present. In this study, we investigated the corrosion resistance of graphene/waterborne epoxy composite coatings in CO2-satarated NaCl solution and discussed the corrosion mechanism of the composite coatings.Although graphene-reinforced epoxy coatings have shown improved corrosion resistance in many applications, there is still a lack of relevant research in oil and gas production environments where aggressive CO2.2.1.2 (10 mm × 10 mm) for electrochemical measurements. The chemical composition of 1020 steel used in this study is shown in Around 1020 steel specimens were cut into 10 mm × 10 mm × 2 mm and then sealed in epoxy resin with an exposure area of 1 cmF0704 two-component waterborne epoxy was purchased from Dongguan Rongzhong Chemical Co., Ltd. Graphene powder  was purchased from Beijing Forsman Technology Co., Ltd. Diamino-functionalized polyethylene glycol  was purchased from Dongguan Aoda Co., Ltd. Carboxymethylcellulose sodium (CMC) , fumed silica (purity: 99.8%) and other reagents were supplied from Shanghai Macklin Biochemical Co., Ltd.2.2.− concentration in formation water can be as high as 60 000–100 000 mg l−1. Therefore, electrochemical experiments were conducted in high chloride environment aqueous solutions with 10 wt% NaCl at 25°C and atmospheric pressure. The electrolyte solution was purged with CO2 for 8 h to deoxygenate and saturate with CO2 before the specimen was inserted into the solution. CO2 was continuously introduced into the electrolyte solution during the electrochemical experiment.According to the composition data collected from a gas production plant site, the Cl2.3.The composite coatings were prepared by the dispersion of different mass fractions of graphene in waterborne epoxy matrix. CMC was used as the dispersant and fumed silica was used as the anti-sediment agent. Different weight ratios  of graphene and a small amount of diamino-functionalized polyethylene glycol were added into the waterborne epoxy and the composite was then dispersed by ultrasonication for 0.5 h. Epoxy curing agent was added with a 2 : 1 weight ratio of epoxy matrix and curing agent. Then, the composite was dispersed by ultrasonication for another 0.5 h and uniformly coated on the test surface of 1020 steel using a wire bar coater (20 µm). The sample was then cured at 25°C for 24 h.2.4.An infrared spectrometer (Nicolet iS50) was used to record the Fourier-transform infrared (FTIR) spectra. Raman spectra were collected on a laser Raman spectrometer (LabRAM HR Evolution). Scanning electron microscopy (SEM) images were recorded on using an MAIA3 LMH microscope. In order to characterize the microscopic morphology of the graphene and composite coatings, an FEI Tecnai G2 F20 microscope was employed to collect the transmission electron microscopy (TEM) images.2.5.2-satarated NaCl solution were studied by measurements of polarization curve and electrochemical impedance spectroscopy (EIS). A glass cell equipped with a three-electrode system was employed in this study. The working electrode was a bare or coated steel 1020 specimen. A platinum electrode was employed as the counter electrode and a saturated calomel electrode was used as the reference electrode. The polarization curves were collected from −0.45 V to 0.45 V versus open circuit potential at the scan rate of 0.5 mV s−1 and fitted by Cview 2.6. The EIS data were recorded from 100 kHz to 0.01 Hz with a 40 mV amplitude signal and fitted by Zsimpwin 3.30d. Electrochemical workstation (CS350) was used to carry out all electrochemical tests.The anti-corrosion properties of different coatings in CO3.3.1.a, the pristine graphene exhibited two characteristic peaks corresponding to the G band at 1583 cm−1 and the D band at 1345 cm−1, respectively [2-bonding carbon atoms, while the D-band is owing to the breaking mode near the K zone boundary [Raman spectra were recorded to identify if interactions between the CMC and graphene sheets occurred during coating preparation. In figure 1ectively ,24. The boundary .Figure b. The G band for graphene (1583 cm−1) was blue-shifted to 1586 cm−1 for CMC–graphene, indicating strong interaction between the CMC and graphene sheets. The integral areas value of the D band to G band (ID/IG) was used to evaluate the quality of graphene sheets. The ID/IG ratio of pristine graphene was 1.12 which increased to 1.25 for CMC–graphene. This suggested that the degree of disorder for the CMC–graphene sheets was higher than that for the pristine graphene.Graphene dispersed by CMC is referred to as CMC–graphene in the rest of this paper. The Raman spectrum of CMC–graphene is shown in figure 1a shows the FTIR spectrum of the blank waterborne epoxy coating. Absorption peaks at 1606 cm−1 and 1508 cm−1 are owing to stretching vibrations of the phenyl ring. The peak near 2800–3000 cm−1 is owing to the C–H vibration of the aliphatic group. The peak in 1244 cm−1 and the broad absorption at 3382 cm−1 are assigned to C–O–C and –OH respectively. Absorption at 916 cm−1 and 827 cm−1 are assigned to epoxy groups. Figure 2b shows the FTIR spectrum of the 0.5 wt% graphene/waterborne epoxy composite coating. Introducing graphene did not evidently result in new functional groups. This indicated that graphene was physically dispersed in the epoxy matrix without chemical modification.FTIR spectroscopy was also used to analyse the interaction between the graphene sheets and epoxy matrix. Figure 2a. The flake structure of pristine graphene was clearly observed. Agglomeration and stacking were also observed, which may result in degradation of the properties of graphene. The dispersion of graphene was investigated by TEM. Figure 3b and c shows TEM images of the dispersion of untreated pristine graphene. The untreated graphene sheets were highly agglomerated and showed many wrinkles. TEM images of graphene dispersed by CMC are shown in figure 3d−f. A thin film was observed with no obvious aggregation and far less wrinkled surfaces. The high-resolution TEM image in figure 3f shows the average thickness of the CMC–graphene is about 6–7 nm. This indicated that monolayer or few-layer graphene sheets were obtained by CMC dispersion.The microscopic morphology of pristine graphene was investigated by SEM, as shown in figure 3Based on the above characterization, waterborne epoxy coatings with graphene dispersed by CMC were expected to exhibit better corrosion resistance performance than those with pristine graphene. Therefore, composite coatings mentioned in the following discussion refer to waterborne epoxy coatings containing CMC–graphene. The graphene/waterborne epoxy composite was coated on the test surface of the steel 1020 using a wire bar coater. The thickness of the coating was investigated by SEM. The SEM image in a shows a surface topography containing many pores and inhomogeneous defects that is owing to the rapid water evaporation during the curing process of waterborne epoxy coatings. These pores and defects will serve as active channels for aggressive species, and it is not surprising that the corrosion resistance of waterborne epoxy coatings are significantly lower than those of solvent-based epoxy coatings. The SEM image of the waterborne epoxy coating incorporating graphene in figure 5b showed a more uniform and smooth surface with far fewer cracks. This is because the graphene was well dispersed in the epoxy matrix, which blocked the diffusion pathways and slowed the evaporation of water [figure 5of water . The gra3.2.2-satarated 10 wt% NaCl solution for 2 h at 25°C are shown in Icorr and Ecorr stand for the corrosion current density and the corrosion potential respectivelya. B and cB represent the slope of the anodic and cathodic polarization, respectively. η stands for the inhibition efficiency calculated by equation (3.1):Ecorr of the 0.5 wt% graphene/epoxy composite coating (−0.604 V) was more positive than that of the blank waterborne epoxy coating (−0.676 V), which showed that adding graphene lowered the corrosion tendency. The anodic and cathodic polarization current densities of the 0.25 wt% graphene/epoxy composite coating were around 2–3 orders of magnitude lower than that of bare 1020 steel with η = 99.96%. When graphene was added with a mass fraction of 0.5%, the corrosion rate was further reduced compared with that of the 0.25 wt% graphene/epoxy composite coating with η = 99.98%. aB increased with the addition of graphene while cB did not change significantly. The increase of aB indicates the transition of anodic reaction. With the addition of graphene, the intensity of the Fe element of graphene/epoxy composite coating at the interface of the substrate metal and the composite coating is stronger than that of the pure epoxy coating, and the increase in the intensity of Fe element indicates the deposition of iron ions at the interface [The polarization curves of bare steel 1020 and steel 1020 coated with blank waterborne epoxy, 0.25 wt% and 0.5 wt% graphene/waterborne epoxy composite after immersion in COlectrode . The Econterface . Therefo3.3.EIS measurements were used to study the resistance and porosity of the coatings. a. It shows that the corrosion medium has penetrated into the interface and forms a double layer between the blank waterborne epoxy coating and the substrate metal after 90 min of immersion time.With the increase of immersion time, the second time constant associated with the capacitance of a double layer appears in the low-frequency range, as shown in figure 7In comparison, the appearance of the second time constant in the low-frequency range for the 0.25 wt% graphene/waterborne epoxy composite coating was delayed, while the second time constant for the 0.5 wt% graphene composite coating hardly appeared. This indicates that the permeation of the corrosive medium was inhibited.a, the radii of the two capacitive loops decreased dramatically with increasing immersion time and then stabilized after 72 h. This indicated poor water isolating properties of the blank epoxy coating.figure 8b, the radii of the two capacitive loops were larger than those of the blank waterborne epoxy coating. However, the impedance modulus still decreased significantly during the 96 h of immersion.For the 0.25 wt% graphene/waterborne epoxy composite coating in figure 8c was around two orders of magnitude higher than that of the blank waterborne epoxy coating. The radii of the two capacitive loops remained relatively constant during the overall immersion time. This indicated the anti-corrosion properties of the composite coating were improved with the addition of graphene content. The composite coating provided effective corrosion protection for the substrate metal during long-term immersion.The impedance modulus of the 0.5%wt graphene/waterborne epoxy composite coating in figure 8sR and cR stand for the solution resistance and the coating resistance, respectively. ctR is the charge transfer resistance. cQ is the capacitance of the coating. dlQ is the capacitance of the double layer. The fitting data are shown in cR values for all coating systems decreased and the cQ values for all coating systems gradually increased with increasing immersion time. This was mainly owing to the gradual penetration of water molecules and corrosive Cl− with strong polarity and high dielectric constant into the coating. The ctR values decreased with increasing immersion time, because water molecules, CO2 and Cl− penetrated through the interface between the coating and the base steel, accelerating the corrosion of the steel during immersion.cQ of the pure epoxy, 0.25 wt% and 0.5 wt% graphene/waterborne epoxy composite coatings were 6.11 µF cm−2, 6.34 × 10−4 µF cm−2 and 1.17 × 10−3 µF cm−2, respectively. This indicated that graphene enhanced the coatings compactness and prevented the diffusion of corrosive media into the coating. The cR of the blank epoxy composite decreased from 9.70 kΩ cm2 to 0.455 kΩ cm2, the cR of the 0.25 wt% graphene/waterborne epoxy composite decreased from 45.2 kΩ cm2 to 20.9 kΩ cm2, while the cR of the 0.5 wt% composite coating decreased from 227 kΩ cm2 to 117 kΩ cm2 within 96 h of immersion. This indicated that corrosion resistance of the composite coatings gradually increased and remained relatively constant with addition of graphene content. The ctR of the 0.5 wt% graphene/waterborne epoxy composite coating (896 kΩ cm2) was much higher than that of the pure epoxy coating (24.6 kΩ cm2). ctR reflects the difficulty of electron transfer across the interface between electrodes and electrolyte solutions. The addition of graphene increased the resistance of the corrosion reaction of the metal.After 96 h of immersion, the vX is the water absorption volume percentage of the organic coating, and cC(0) and c(t)C are the coating capacitances after immersion for time 0 and time t, respectively. In this study, the value of cC(0) was obtained by the fitting results of EIS measurements for an immersion time of 30 min. The stability of the water absorption volume percentage can be used to judge whether the performance of the coating is stable. The calculation results are shown in vX (t = 96 h) for the blank epoxy coating, 0.25 wt% and 0.5 wt% graphene/waterborne epoxy composite coating were 53.7%, 15.6% and 10.8%, respectively. The water absorption of epoxy coating decreased significantly with increasing graphene content, and the performance of all the coating systems stabilized after 96 h of immersion. We therefore concluded that the composite coating prevented the diffusion of water into the coating and provided durable and effective anti-corrosion protection for the substrate metal.According to the theory of Brasher and Kingsbury, equation (3.2) can be applied to calculate the water absorption volume percentages of the different coatings :3.2Xv%=14.2-satarated NaCl solution. Composite coatings with more uniform surfaces and far fewer defects than blank waterborne epoxy coatings were obtained on 1020 steel. The electrochemical characterizations proved improved corrosion resistance of waterborne epoxy coatings by adding graphene. The 0.25 wt% graphene/epoxy composite coating provided an inhibition efficiency η = 99.96%, while further improvement was achieved by increasing the content of graphene additive to 0.5 wt%. The anodic dissolution reaction was retarded by the graphene distributed inside the composite coating, which acted as an effective barrier and suppressed the diffusion of Fe ions. The increase in coating resistance and decrease in water absorption volume percentage of the graphene/waterborne epoxy composite coatings showed that they provided durable and effective protection for the substrate metal in CO2-satarated NaCl solution.Graphene/waterborne epoxy composite coatings with different mass fractions of graphene were prepared to investigate their corrosion resistance in CO