The all-flexible TENG has been employed as a self-powered humidity sensor to investigate the effect of raising humidity on the output voltage and current by applying mechanical agitation in two forms of using a tapping device and finger tapping. Due to the presence of superficial functional groups on the GO paper, water molecules are inclined to be adsorbed, resulting in a considerable reduction in both generated voltage (from 144 V to 14 V) and current (from 23 µA to 3.7 µA) within the range of relative humidity of 20% to 99%. These results provide a promising applicability of the first suggested sensitive self-powered GO TENG humidity sensor in portable/wearable electronics.Triboelectric nanogenerators (TENGs) offer an emerging market of self-sufficient power sources, converting the mechanical energy of the environment to electricity. Recently reported high power densities for the TENGs provide new applications opportunities, such as self-powered sensors. Here in this research, a flexible graphene oxide (GO) paper was fabricated through a straightforward method and utilized as the electrode of TENGs. Outstanding power density as high as 1.3 W.m In the last decade, nanogenerators have been introduced as sustainable self-sufficient power sources, converting energy from the environment into electrical energy2. They can be categorized to piezoelectric3, triboelectric4, and pyroelectric5 nanogenerators. Triboelectric nanogenerator (TENG) works on the basis of accumulation of electrostatic charges produced on the surface of two dissimilar materials while they are brought into a physical contact4. When the surfaces are separated by a mechanical force, the induced triboelectric charges generate a potential drop, leading to produce electric current6. Since their invention in 20127, TENGs have shown a rapid development in diversity of structures, amount of output power, and range of application, such as self-powered sensors9, biomedical monitoring10, and electronic skin11.Energy harvesting is an area of tremendous attention because of the huge worldwide energy demands motivating considerable research on self-powered and autonomous systems12. It has been demonstrated that the reactive oxygen functional groups of graphene oxide (GO), beside its high specific surface, makes it a good candidate as an energy-related material13. Moreover, high intrinsic mechanical strength of GO provides the ability of producing a flexible and stable thin layer, which let the induced charges achieve to the back contact in TENGs. Tian and his co-workers reported a flexible electrostatic nanogenerator based on GO film, with a multilayer structure of Al/PI/GO/PI/ITO with the output power of 60 nW14. The fabricated GO-TENG was able to generate a peak voltage of 2 V and a current of 30 nA upon the applying of a 15 N force at a rate of 1 Hz. Very recently, Wang’s group proposed a GO single-electrode-based TENG with maximum power of 5 mW (power density = 3.13 W/m2) at 3 Hz15. The short circuit current (Isc) of 55 μA and the open-circuit voltage (Voc) of 1100 V were obtained with latex-gloved hand patting. Moreover, the dynamic force sensing and the sterilizing performance of the aforementioned TENG were investigated. There are also several researches on designing novel TENGs containing GO which demonstrate weaker results and utilize more sophisticated fabrication processes17.Among the various energy-related materials, graphene-based materials have been studied extensively in the field of energy harvesting in recent years18. Humidity sensing plays a key role in domestic control, medical care, agricultural moisture monitoring, and industrial applications19. GO-based humidity sensors have been widely under attention due to notable properties such as simple, low-cost, and large-scale preparation of GO material along with high proton-conductivity in exposure to water molecules20. GO humidity sensors operate on the basis of detecting the variation of impedance or capacitance due to the inclination of the water molecules to adsorb on the surface of GO26. Both types possess the ability of fast response humidity sensing with high sensitivity in a wide range of relative humidity (RH). Impedance sensors detect a relatively low humidity over a wide frequency range, but generally require professional high-performance impedance spectroscopy. On the other hand, the capacitive sensors detect the humidity by measuring the capacitance change which consists of the interdigitated conductive electrodes and GO-based sensing materials as the dielectric connected to the LCR meter. For practical usage of a humidity sensor, not only the sensing performance such as fast response and high sensitivity, but also low cost, easy fabrication, facile integration, and good flexibility are significant factors, which are generally depends on the sensing materials and fabrication methods20. In spite of various investigations demonstrated the effect of utilization of polymers28, metal oxides31 and 2D materials33 alongside GO, with the purpose of enhancement of humidity sensing, the fabrication method has become relatively more sophisticated. Moreover, the recent development of the Internet of Things (IoT) provides a progressive research field on integrating numerous sensors, which demands a new approach to the power supply issue19.Beside energy harvesting field, detecting humidity is from promising applications of GO because of the strong interactions between water molecules and oxygen functional groups on the surface2. In spite of the fact that TENGs are considered as a promising option for self-powered sensors2, limited numbers of self-powered humidity sensors based on TENGs have been reported36. In the reported TENG-based humidity sensors, the TENG is generally used as an external power source connected to a humidity-sensitive electrode, analogous to a resistive sensor. In spite of being battery-free, there are some limitations in utilizing these sensors, such as low sensitivity and confined range of RH39. Moreover, the only value which demonstrates the amount of RH is the output voltage and no current variation was observed35. Beside the sensing properties, flexibility is a key factor for utilizing a sensor in wearable electronics, which was not considered in many previous studies of TENG-based humidity sensors. Therefore, the direct implementation of a humidity sensitive flexible material as one of the TENG electrodes may offer a self-powered humidity sensor with a less complicated structure as well as higher sensitivity and wider range of detection.In order to eliminate the drawbacks of utilizing the batteries as power sources in sensors , considerable investigations have been launched into the fabrication of self-powered sensorsIn this research, a sustainable GO-based TENG has been introduced, which shows superior power density and high sensitivity of humidity sensing, in comparison with the previous studies. Using the modified Hummer’s method, as well as drop casting technique for the synthesis of GO sheets and construction of the electrode, the whole procedure of production is extremely straightforward. Besides, utilizing GO paper and Kapton film as the electrodes, makes the GO TENG an appropriate candidate for wearable electronics, due to its flexibility and low weight. Applying the as-fabricated GO TENG as a self-powered humidity sensor is reported for the first time in this paper. Here, the GO electrode directly senses the amount of relative humidity, while corporates in signal generation as well. The results demonstrate considerable variation in the amount of both voltage and current at different values of RH. Investigations of humidity sensing were performed on large and small sizes of the electrodes, which present the fabricated TENG as an applicable option for portable devices, as well as industrial implementations. Therefore, our suggested GO TENG provides flexibility and high sensitivity of dual sensing in a wide range of RH and can be conveniently applied as a routine battery-free humidity sensor in desired places.The SEM and TEM images of the produced GO suspension Fig. , obvious40 as well as various kinds of sensors. The thickness of the layer is obtained to be around 32 µm /V, while V0 and V correspond to the voltage value at RH = 20% and the desired RH, respectively. Both of the response diagrams  of the GO surface through double hydrogen bondingime Fig. .21. In that case, a continuous water layer creates a uniform barrier layer for induction of positive charges on the GO surface, which results in the formation of a continuous depletion region and would lead to a more rapid decrease of current at higher RH. Calculated amount of the induced charge on the surface under various RH  has been fabricated, through a straightforward method. The outstanding Isc and Voc with the values of 90 µA and 870 V, respectively, at a frequency of 4 Hz are obtained. Applying the fabricated TENG as a self-powered humidity sensor demonstrates promising results. Increasing the relative humidity of the environment, gradually reduced the generated voltage from 144 V at RH = 20% to about 14 V at RH = 99%, while short-circuit current reduced from 23 to 3.7 µA. Performing sensing tests under finger tapping presents a lightweight self-powered humidity sensor. This first-suggested wide-range GO TENG humidity sensor with a high sensitivity of 500% (V/V-%RH) can be utilized in portable electronics, as well as industrial applications.In summary, a sustainable GO-based TENG which shows superior power density (1.3 W/m4), sulfuric acid (H2SO4), H2O2 (30 wt%), sodium nitrate (NaNO3) and hydrochloric acid (HCl) were bought from Chem Lab Co., Belgium. All chemicals were of analytical grade and used as received without further purification. The aqueous solutions were prepared using deionized water .Graphite powder with particle size <45 μm was purchased from Sigma Aldrich. All other reagents including potassium permanganate  and then stirred for 1 h at 80 °C. Subsequently, 80 mL of H2SO4 was added to the mixture and the beaker was placed in an ice-bath followed by the addition of NaNO3 (2 g) while stirring for 10 min. Afterwards, 8 g of KMnO4 (4 wt %) was slowly added to the solution under vigorous stirring for additional 15 min. The green paste was then transferred to an oil bath (38–40 °C) followed by 90 min stirring. The resulted brownish solution was diluted with 160 mL of deionized (DI) water and heated at 95 °C for 30 min. Finally, the oxidation process was stopped by the addition of 400 mL DI water and H2O2 . The orange-golden suspension was filtered and washed with diluted HCl and then DI water until the neutral pH (~5–6) was achieved. The filtered brown dough was redispersed in a certain amount of DI water and cleansed by centrifugation at 2500 rpm for 15 min and then 4000 rpm for 30 min to remove any un-exfoliated and tiny graphite sheets, respectively. Finally, GO nanosheets were prepared by sonication of the filtered graphite oxide suspension using an ultrasonic bath for 30 minutes.Graphene oxide was synthesized from natural graphite powder via a modified Hummers method as previously reported in the literatureLarge scale GO papers were prepared via a facile method. For this purpose, a plastic mold with desired dimensions was made. 20 mL of concentrated GO suspension (~7 mg/mL) was then poured into the mold and let it dry under ambient condition without any disturbance. Using this straightforward approach, one can prepare large scale GO papers with demanded sizes and control the thickness by adjusting the concentration as well as the volume of the GO suspension.2 and spacing of 2 cm between two electrodes. For humidity sensing, the size of electrodes reduced to 2 × 2 cm2 in order to enhance the accuracy of the GO TENG sensor.Aluminum tape was used as the back-contact for GO paper as well as for the Kapton film to construct lower and upper electrodes, respectively. Two electrodes can be connected to the gauges of a tapping device , by which can regulate the frequency and the force of tapping, as well as the spacing between two electrodes. The tests were performed under the vertical force of 8.3 N at frequencies from 1 up to 4 Hz. Electrodes had rectangular shape with the size of 8 × 8 cm2 and 2 × 2 cm2. The vertical tapping was applied using two approaches, utilizing a tapping device at the frequency of 2 Hz and the force of 8.3 N and also tapping by a human finger.The ambient humidity was regulated by a typical humidifier. To report the amount of relative humidity (RH), a hygrometer was situated quite close to the surface of GO electrode. Humidity sensing tests were performed on the GO electrodes with the size of 8 × 8 cmTEM images were obtained with a Zeiss (EM10C-80KV) instrument. The morphology of the prepared GO nanosheets and also GO paper was studied by a field emission scanning electron microscope . UV–Vis spectroscopy was performed on a Lambda25  spectrophotometer using a 1.0 cm quartz cell. The X-ray diffraction (XRD) pattern was recorded using a STOE (STADI P) instrument operating with Cu–Kα radiation (λ = 1.54178 A°) at 40 kV/30 mA. An Ivium Compactstat and an oscilloscope (DSO1022A) were used for current and voltage recording, respectively.Supplementary Information.Supplementary Information 2.Supplementary Information 3.


Coleochaete scutata strain SAG 110.80M. This mitogenome is the largest among the streptophyte green algae examined to date. At 242,024 bp, it is 4.3-fold larger than the mitogenome of Chaetosphaeridium globosum, the only other mitogenome available for the Coleochaetophyceae. This size difference is mainly explained by differences in the abundance of introns and in the length of intergenic regions containing vestiges of coding sequences thought to be of foreign origin. With 31 group I and 26 group II introns, the C. scutata mitogenome is the most intron-rich organelle genome known among streptophyte algae.We have sequenced the mitogenome of  Chaetosphaeridium globosum) had been examined . Paired-end reads were generated on the Illumina MiSeq sequencing platform by the ‘Plateforme d’Analyses Génomiques’ of Laval University. Reads (3.4 million) were assembled using Ray version 2.1.0  is 4.3-fold larger than its Chaetosphaeridium homolog, but its gene repertoire is essentially the same. It possesses 67 distinct genes, three of which  and trnP(ugg)) are duplicated or triplicated. The rpl6 gene is missing from C. scutata, which represents the main difference in gene content between the two coleochaetophycean mitogenomes. Gene order is highly scrambled between these genomes. The larger size of the C. scutata mitogenome is mostly accounted for by a higher abundance of introns (57 versus 11 in Chaetosphaeridium) and by the presence of longer intergenic regions near rns (56.1 kb versus 8.3 kb). In both algae, the latter regions harbor ORFs showing similarities  with recognized domains of phage/plasmid DNA primase and phage integrase. Considering that genes with these functions are not usually found in green algal mitochondria, we suggest that they were acquired by lateral transfers before the emergence of the Coleochaetophyceae. The C. scutata mitogenome contains 31 group I and 26 group II introns, a significant proportion of which (11 group I and 20 group II introns) encode LAGLIDADG homing endonucleases and reverse transcriptases/intron maturases, respectively. The cox1 and rnl genes alone exhibit a total of 19 and 14 introns, respectively. Twenty-four of the C. scutata introns are inserted at sites where no introns have been observed in any of the currently available mitogenomes of streptophyte algae, supporting the view that mitochondrial introns proliferated in the Coleochaetaceae.At 242,024 bp, the A maximum-likelihood phylogeny was inferred from 40 concatenated mitogenome-encoded proteins of 22 streptophyte taxa using RAxML version 8.2.3 (Stamatakis


Azadirachta indica leaves by 40 and 41% atEjura-Sekyedumase district as well as 45 and 20% at Atebubu-Amantin district for2016 and 2017 respectfully. Plots of seed yams treated with neem recordedreduced pests and damages in terms of parameters measured and subsequentlytranslated into yield. It is paramount to seed yam producers to adopt neem leafpowder as pre-treatment for higher productivity.Pests and diseases infested material of seed yams have led to sub optimal yieldlevels. Neem was applied as substitute for chemical pesticides and asenvironmentally safe bio-pesticide to reduce the crops annual loss due toherbivore pests and their resulting diseases. Field trials were conducted duringthe 2016 and 2017 major planting seasons in four communities each ofEjura-Sekyedumase and Atebubu-Amantin districts of the Ashanti and Brong Ahaforegions respectively. Yam minisetts sizes were pre-treated with Mancozeb at arate of 100 g and Lambda cyhalothrin at 40 ml in 10 L water as cocktail. Yamminisetts of 30 g each were planted on ridges at 100 cm between and 30 cm withinrows on a 20 m × 20 m plot size. Neem leaf powder was applied on fiverows per plot and another five served as check for assessment. Harvesting wasdone approximately 6 to 7 months after planting. Scale insects, mealybugs,beetles, termites and millipedes infestations. Galling due to root-knotnematodes, cracks, soft and wet rot were assessed on a scale of 1-5. Tuberyields were also assessed. Neem treated plots were observed to havesignificantly reduced arthropod pest populations and nematode galling as well asdamage signs compared to the control plots. Yields of seed yam were higher onthe neem treated plots than the control, probably due to the reduced damage onthe treated plots. Seed yam yield increased for plots treated with Dioscorea spp.) is one of the most important root and tuberbearing crop in the world and an important source of energy. More importantly, yamtubers have organoleptic qualities which make them the preferred carbohydrate stapleand can contribute up to 350 dietary calories per person each day  notonly enrich the diet of consumers but also make them ethnomedicinally important as traditionalproducts for crop protection  seed hasinsecticidal and medicinal properties  and Lambdacyhalothrin (insecticide) at 40 ml in 10 L of water mixed as cocktail. Mini sett sizes, 30 g each were planted on ridges at 100 cmbetween and 30 cm within rows on a 20 m × 20 m plot size. Neem leafpowder was applied to the soil on five rows per plot and another five served ascheck (control) for assessment. Air-dried neem leaves were milled using cornmill  and applied at 20 g perhill, which was placed in the planting hole and covered with a little soilbefore placing the yam minisett. The trial was weeded three times manually  before harvesting. Harvesting was done approximately sixto seven months after planting with the aid of hoe and cutlass.Aspidiella hartii), mealybugs(Planococcus citri), beetles (Heteroligusmeles), termites (Isoptera: Termitidae) and millipedes (Plethocrossus sp)(Diplopoda: Odontopygidae) infestations as well as nematode galling, cracks,soft and wet rot were assessed on the scale of 1-5; where 1 = free from attack,2 = 1-25%, 3 = 26-50%, 4 = 51-75% and 5 = 76-100%. The tuber yield ton perhectare (t/ha) was also assessed and recorded.Twenty seed yams were randomly selected and harvested from the neem treated andthe control plots. Scale insects (x +1) transformation. Percentage datawas transformed using arcsine of x prior to analysis. Meanswere separated using Tukey´s test at 0.05 probability level.Data obtained on number of feeding holes created by scale insects, mealy bugs,beetles, termites and millipedes infestations on tubers and tuber weightexpressed in yield were analyzed by one-way analysis of variance (ANOVA) withSAS, PROC GLM  Cockerel, the mealy bug P. citri (Risso)(Hemiptera: Pseudococcidae), yam tuber beetle (H. meles Billberger)holes, termites  and millipedes(Plethocrossus sp) holes.Major soil arthropods and their damages recorded in this study from bothEjura-Sekyedumse and Atebubu districts included scale insects The results from Ejura-Sekyedumse district in 2016 indicated that, neem applicationhad no significant effect (p > 0.05) on the scale insects and millipedeholes. However, a significant difference (p<0.05) on the number of mealybugs, termites and tuber beetle holes was observed when neem was applied . Considep > 0.05) effect on the scale insects, termiteand millipede holes. However, significant (p < 0.05) difference on the numberof mealy bugs and tuber beetle holes was observed when neem was applied (Results from Atebubu- Amantin district in 2016 indicated that neem application had nosignificant ( applied . At Ejur applied .P > 0.05) effect on the scale insectsand mealy bugs.The results from Ejura-Sekyedumse district in 2017 indicated that, neem applicationhad no significant  difference for three parameters;termite tuber beetle holes and millipede holes when neem was applied . For nemp > 0.05) effect on the scale insectsand tuber beetle holes. Mealy bugs, termite and millipede holes, however, showedsignificant (P < 0.05) difference as parameters when neemwas applied  between the neem treated plots and the control. Yieldsof seed yam/tubers were higher on the neem treated plots than the control . GeneralS. bradys, Pratylenchus spp. andMeloidogyne spp.) were responsible for tuber damages. Theyprobably might have served as primary invaders which facilitated fungal  pathogen tuber infection in the field. Theresulting damages that occurred in the field might have led to reducing the quality,quantity of food and planting material if neem were not applied. Similar findingshave been earlier reported by Amusa et al. (Meloidogyne spp. (root-knot nematodes),Rhizoctonia root-rot fungus and rice stunt virus , as well as increasing the food security in Africa.We also observed that late harvesting predisposes tubers to bruises and attack soseed yam producers are advised to be early to attain maturity.


This review acmes the latest developments of composites of metal oxides/sulfide comprising of graphene and its analogues as electrode materials in the construction of the next generation of supercapacitors (SCs). SCs have become an indispensable device of energy-storage modes. A prompt increase in the number of scientific accomplishments in this field, including publications, patents, and device fabrication, has evidenced the immense attention they have attracted from scientific communities. These efforts have resulted in rapid advancements in the field of SCs, focusing on the development of electrode materials with features of high performance, economic viability, and robustness. It has been demonstrated that carbon-based electrode materials mixed with metal oxides and sulfoxides can perform extremely well in terms of energy density, durability, and exceptional cyclic stability. Herein, the state-of-the-art technologies relevant to the fabrication, characterization, and property assessment of graphene-based SCs are discussed in detail, especially for the composite forms when mixing with metal sulfide, metal oxides, metal foams, and nanohybrids. Effective synthetic methodologies for the nanocomposite fabrications via intercalation, coating, wrapping, and covalent interactions will be reviewed. We will first introduce some fundamental aspects of SCs, and briefly highlight the impact of graphene-based nanostructures on the basic principle of SCs, and then the recent progress in graphene-based electrodes, electrolytes, and all-solid-state SCs will be covered. The important surface properties of the metal oxides/sulfides electrode materials  will be described in each section as per requirement. Finally, we will show that composites of graphene-based electrodes are promising for the construction of the next generation of high performance, robust SCs that hold the prospects for practical applications. To obtain molybdenum hexacarbonyl, a halide precursor (MoCl5) and H2S as a chemical reactant were deposited on the MoS2 films at an elevated temperature of 300 °C [2S plasma as the chemical reactant. After a very comprehensive investigation of the properties of these nanomaterials and of their growth on the 3D Ni foam, the electrochemical studies were carried out, to realize the potential of plasma-enhanced atomic layer deposition (PEALD) MoS2 as an electrode in asymmetric SCs. The efficiency of the materials developed in terms of capacitance retention was reported, as shown in Very recently, Nandi et al. reported a direct growth of MoSications . They adf 300 °C . Therefo2 for SC applications, therefore we selectively summarize some excellent investigations on MoS2 based electrode materials, in addition to other graphene-based and hybrid composites, as enlisted in It is difficult to review all published work related to MoSCompared with metal/metal oxide nanocomposites, conducting polymers (CPs) show some advantages as electrode materials, such as low cost and ease of large-scale production . Such hy2 nanosheets as a cathode and carbonized iron cations adsorbed on polyaniline (C-Fe/PANI) as an anode [2 nanosheets using a hydrothermal method, and then the fabrication of the C-Fe/PANI electrode material by direct pyrolysis of the Fe-PANI mixture coated onto a 3D Ni foam, in a tube furnace under N2 atmosphere. The synthesis procedure is depicted in Additional hybrid CPs with metal/metal oxides as pseudocapacitive materials for SCs are also very promising. Rantho et al. successfully synthesized an asymmetric SC using VSan anode . Using 6an anode . In theian anode . They ad2 sample was composed of a large number of nanosheets. However, in the case of C-Fe/PANI, as shown in The surface morphology of the nanostructured material was revealed by SEM analyses. 2SO4. However, the results revealed that the composites had a better current response in 6 M KOH electrolyte, and the relevant explanation for such an observation was also suggested by Rantho et al. They reported the specific capacitance of 516.5 F/g for VS2 and 486.5 F/g for C-Fe/PANI. To further explore the electrochemical performance of VS2 and C-Fe/PANI electrodes, an asymmetric device was constructed by Rantho et al. The VS2/C-Fe/PANI asymmetric device offered a high-energy-density of 27.8 Wh/kg and a power density of 2991.5 W/kg at a current density of 2 A/g, as discussed in details elsewhere [The electrochemical characterization of the composite materials was carried out in 6 M KOH, and in Nalsewhere .In the same line, Adhikari et al. reported a polyaniline (PANI)-stabilized structure containing ferrocene (Fc) and graphene as the electrode material for SCs . Their wLeakage;Corrosion of electrodes;Limited geometry shape and high flammability.It is very important to note that supercapacitors using liquid electrolytes can provide better ion conductivity, but they also encounter several disadvantages, such as volatility, narrow potential window, and high flammability. Further, supercapacitors involving a liquid electrolyte do not offer the flexible feature and are generally bulky. For modern electronic gadgets, mobility and flexibility constitute the two essential characteristics. Hence, researchers are keen to seek suitable forms of electrolyte that offers high efficient, good flexibility, light in weight, and high mobility for SCs. The SC devices based on liquid electrolytes suffer from different disadvantages that limit their applications. These disadvantages include :Leakage;Improved stability, safety, flexibility and mechanical stability.Significantly streamline the packaging and make the specific separator needless.Improved gravimetric or volumetric energy densities of the devices.Gel polymer electrolytes however can partly assuage the disadvantages of the liquid electrolytes, but the issues related to the liquid component in the electrolyte still exist, including the leakage and ionic reduction due to the solvent evaporation. The strategy that can be used to tackle these issues associated with the solvent based electrolytes is to develop solvent-free polymer solid electrolytes, which offer advantages, such as :ImprovedThe increasing demands for wearable and lightweight energy-storage devices have brought about the necessity to design novel, efficient, and compatible energy-storage modes. Solid-state SCs (SSSCs) are proven to be very apt for applications in modern electronic gadgets. SSSCs are characterized by high power density and very stable cyclic performance with considerably fast charge/discharge rates ,158,159.(i) Facile electrode preparation: The electrodes can be easily fabricated by several approaches.(ii) Easy electrolyte preparation that allows for the device compactness: Conventionally, liquid-electrolyte-based SCs are not practical options for mobile and compact electronic applications, as they are problematic to house the other elements and to achieve flexibility. In this context, the SSSCs are much more advantageous, since the all-solid-state property is not as itinerant as the liquid electrolytes, and the solid electrolyte can be easily kept separate from the other device parts.(iii) SSSCs devices are perfectly compatible with modern-day flexible, lightweight, and compact electronic gadgets.(iv) SSSCs can be fabricated into various flexible forms, such as thin films or fiber-shaped forms, or can be imbued with other novel properties. All these properties can widen the applications of SSSCs in many specific fields.(v) Finally, and most importantly, SSSCs are equipped with properties like environmental friendliness, portability, mobility, and cost effectiveness, which can broaden the application area of SCs . TherefoRecently, abundant carbon-based materials have been studied and evaluated for possible developments of SSSCs, in order to meet the demand of requisite energy storage. Example attempts include activated carbon ,161, porAmong the above long list, graphene-based electrodes are much more attractive and have been widely investigated. In addition, the yield of the aforementioned method is very low, and the cost involved in making graphene on a large scale remains high. A possible solution to the problem of large-scale synthesis of graphene is to use graphene oxide as a precursor. However, the performance of graphene oxide and reduced graphene oxide as electrode material is still limited, because of the limited surface areas of the electrode as a whole . Zhang e2O3)/graphene aerogel hybrids as a flexible SSSC electrode, and achieved an impressive capacitance of 440 F/g [3O4 composite aerogel electrode, and reported a high areal energy density of 35.92 μWh cm−2 [Various methods have been reported to assemble graphene into films, to prevent serious stacking aggregation, and to maintain their large specific surface areas while being lightweight. Khattak et al. reported the use of 3D . Other attempts included the fabrication of 3D graphene aerogel first and then combining it with other redox materials, such as combining PANI and MnO2, to create all-solid-state flexible asymmetric SCs, which exhibited a specific capacitance of 111 F/g at the current density of 1 A/g [To eliminate the drawbacks of aerogels  in SSSCs, several attempts have been documented to develop 3D hydrogels ,190. Forof 1 A/g .Despite their various advantages, graphene-based electrode materials suffer from limited charge-storage capacity, owing to the surface storage mechanism, as seen in other carbon-based electrode materials. Moreover, the charge storage is largely limited by the unused electrode active surface of the graphene and by the large resistance to ion transport within graphene-based electrodes. Consequently, the value of the gravimetric capacitance of the reported SSSCs lies mostly below 300 F/g, far below its theoretical value . The fab2 nanorods spread over the graphene surface, and reported a specific capacitance of 759 F/g [−2 at a current density of 1 A cm−2 [3O4 nanocrystal hybrid electrodes and used a scalable wet spinning approach to achieve the flexible electrodes. By adopting this process, Chen et al. further revealed that the Mn3O4 nanocrystals were strongly anchored over the rGO nanosheets, which effectively avoided further aggregation of the nanocrystals. The regular fibrous-network-like structure was found to be favorable for flexible SCs [3O4 nanocrystal-based SCs is schematically presented in To counter the above issues and boost the performance of graphene-based SSSCs, solutions including integrating graphene with other pseudocapacitive materials or doping it with various heteroatoms have been proposed ,175,176. 759 F/g . Using f1 A cm−2 . Chen et1 A cm−2 . They stible SCs . The pro3O4 nanocrystals, the tensile strength, flexibility, and capacitive performance of the electrode materials were thoroughly investigated [3O4 nanocrystals. The CV and EIS studies showed that the synergistic interaction between rGO sheets and the optimal amount of Mn3O4 crystals led to an excellent capacitive performance (3O4 crystals also offered an electrochemically active surface area with more electronic conduction channels. The flexible electrode offered a maximum volumetric capacitance of 311 F cm−3 in 1 M KOH electrolyte and 45.5 F cm−3 in PVA/H3PO4 gel electrolyte. It has good cycling stability of 85% over 10,000 cycles, and a high tendency to incur bending fatigue. Therefore, this approach can be considered as a successful example to achieve highly efficient energy-storage for the next generation of flexible electronics.By varying the amounts of the Mnstigated . It was formance . The Mn32SO4, and PVA/H3PO4, and they are summarized above in Numerous reports are available on SCs that use ionic, organic, and aqueous electrolytes. In the case of SSSCs, the electrolytes used are mostly in an aqueous or aqueous ion gel state, such as PVA/KOH, PVA/HGao et al. reported a direct laser writing technique for the fabrication of rGO/GO micro-SCs in which GO was used as a solid electrolyte . Yang et2SO4-intercalated GO as electrolyte/separator by coupling with pseudocapacitive rGO electrodes [−2 and a volumetric energy density of 1.24 mWh cm−3.The use of rGO as a separator has been gaining research attention. Shulga et al. reported that they achieved a specific capacitance of 200 F/g by using such a separator ; their dectrodes . This deThis review accounted for the recent progress in graphene and its 2D analogues based metal oxide/sulfide nanocomposites for applications in supercapacitors, with special focus on the developments incurred within the last 3–4 years. A comprehensive discussion has been included to link the surface morphology of the composites consisting of metal oxides, sulfides, metal foams, and metal containing polymers, with the performance for them acting as the electrode materials in SC applications. We have shown that these new nanocomposites have exhibited huge commercial applicability with easy processing features. A brief introduction to the basic principles for the supercapacitors has also been comprised, elaborated along with the current scenario in supercapacitors research. The properties of electrodes, separator, and electrolyte can be tuned separately and optimized together to realize ultimate improvements in the performance of the SCs. Furthermore, by using important schematic diagrams, figures, and tables, we created a comparative account on the state-of-the-art in SC research, so to help to achieve a deepened understanding and promote further thinking. By citing the most recent references in each section of this review, we have shown the ongoing cutting-edge research around the globe on supercapacitors, and also pointed out the potential directions where challenges remain for further exploration.2SO4 or ionic BMIPF6 electrolytes have resulted in maximum capacitance values of 348 and 158 F/g, respectively, projecting the excellent performance of rGO in aqueous and/ionic electrolytes [Graphene-based porous nanostructures are a class of materials of the utmost importance in the future development of high performance, portable, and flexible SCs. Since they can enhance the ion transportation rate in supercapacitors, consequently augmenting the power and energy density and the stability of supercapacitors. As recorded previously, rGO nanosheets working in aqueous Htrolytes ,27,28,29However, the cyclic stability remains a challenge, and it is prophesied to be improved along with the specific capacitance by the use of hybrid composites consisting of the newly discovered two-dimensional materials such as MXene and inorganic graphene ,155,156.Nevertheless, it has been reported that graphene and other 2D material-based separators suffer from damages with time, which opens another big challenge for future study. Research in this field should also be more focused on their commercial viability and provide technical details towards their commercialization. Most importantly, flexibility based on nanostructures of graphene and its analogues, in the form of nanocomposites, paper sheets or aerogel, becomes possible, however commercial production poses a big task of the near future, mainly owing to the cost and technical barriers. Over-claim of their advantages and selectively ignore their shortcomings and realistic viability could be detrimental for building up consumer confidence and therefore for their wide commercialization.Given the huge amount of joint efforts globally, the great investments in R&D, and the rapid paces of development in this area, we believe that these technical barriers and challenges could be solved in the short term. By combining the experimental selection of materials and parameters refinements with high-performance computer simulations at the molecular level, solutions to the challenges and optimizations to the performance of the SCs could be achieved much faster than working alone by materials scientists and chemists. We believe that SC, particularly low cost, lightweight, high power and energy density, flexible, and robust SCs will be realized very soon, not only in the lab but also produced in a commercial scale. We also believe that these next-generation SCs would contribute significantly to the future sustainable society.


Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form ‘electrical hot spots’ in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures. Field effect transistors based on graphene hold promise for sensing applications. Here, the authors report a millimeter-sized transistor based on deformed graphene as a biosensor that can detect nucleic acid molecules having detection limit of ~18 molecules of DNA in physiological buffer solution and ~600 molecules in human serum. These methods also offer considerable promise for forensics7, environmental monitoring8 and global personalized medicine9. In particular, field effect transistor (FET) based detection of nucleic acids has drawn great attention as label-free and highly sensitive biomolecular sensing platform, which can be readily integrated with other electronic components, such as data analyzers and signal transducers. 2D materials, such as graphene, are attractive due to their unique properties, such as ambipolar field effect, high carrier mobility, biocompatibility, mechanical strength, and flexibility10. 2D materials intrinsically exhibit high sensitivity in detection of charged biomolecules due to their ultimate thinness and extremely high surface to volume ratio. Compatibility to the conventional CMOS fabrication process is another potential advantage of using 2D materials, which carbon nanotube, Si-nanowire, nanoparticles do not have. Especially, large area graphene, which is grown through chemical vapor deposition (CVD) method, has been utilized in electrical systems, such as FET device for bio-sensing5 including detection of pH11, microorganisms12, blood sugar12, and more specifically, proteins at concentrations of 10 fM13, and nucleic acids (DNA or RNA) at the 100 fM concentrations14. There are a few reports that showed DNA and RNA detection at aM level, however, had significant level of background noise and lacked robust controls16. Further sensitivity would be highly desirable for detection of very rare molecules, such as micro RNA (miRNA) or cell-free DNA (cfDNA), from unamplified samples17.All-electrical detection of biomolecules and specifically nucleic acids are of great interest for gene-expression investigations18. Unfortunately, PCR is susceptible to interference by the inhibitory factors in biological samples, therefore not suitable to analyze miRNA directly from blood or serum samples19. Moreover, the result can be misinterpreted by bias and artifact due to the amplification efficiency of different sequences20. Therefore, there is an urgent need to develop amplification- and purification- free method to directly detect miRNA from biological samples such as serum.It is important to detect DNA/RNA, such as miRNA circulating in serum or plasma with high ionic strength. Such detection could enable liquid biopsy, which can replace invasive tumor-tissue biopsies in many diagnostic applications. The existing approaches to monitor cancer-related miRNA is based on the polymerase chain reaction (PCR)22. Outside the Debye length, which is <1 nm in physiological solutions, the charges are electrically screened. An increase in the Debye length can result in reduced screening effect and allow for a more sensitive electrical detection of charged biomolecules. While methods have been proposed to overcome this intrinsic limitation of FET biosensors22, these have focused on detecting biomolecules which are larger than the Debye length itself. None of the reports has tried to overcome the concentration limit of detection by modulating the Debye screening. Moreover, none of the works have modulated Debye screening in clinical solution such as serum or plasma22.One of the major hurdles to lower the detection limit of FET-based biosensor is shielding of the molecule charge by the counter ions in solution (termed Debye shielding)23. Thus, we hypothesized that if the surface of the sensing channel can be curved or bent at the micro- and nano-meter scale, the Debye length could be modulated resulting in higher sensitivity. Previous works have shown that 3-dimensional “crumpled” graphene can be created by deformations at the micro- and nano-scale on pre-strained thermoplastics by relieving the stress and inducing buckle delamination of the graphene24. This approach can be used to engineer curving and bending of 2D materials and thin films. Several applications have been investigated using the mechanically-tunable crumpled graphene such as stretchable photosensors25, nanoplasmonic sensor26 and strain gauges27, and the in-plane strain can change the electronic properties of graphene, opening a bandgap with a 1% stretch29. Moreover, crested 2D materials FETs recently showed large increase in the mobility as compared to standard devices30. It has also been separately shown that flat 2D semiconducting material such as MoS2 can be 10 times more sensitive than flat semi-metallic 2D graphene for biosensing applications31. Hence, we hypothesize that nanoscale bending of 2D graphene in 3-dimensions could result in high sensitivity due to modulation of the Debye length (or volume), and possibly due to strain induced band-gap opening in the graphene channels. Such deformed graphene (curved or bent 2D) layers have not yet been used for biosensor applications.Computational reports have also predicted that the curved morphology of sensing materials can affect the Debye length (or volume), which can increase in concave regions of a nanowire sensor32. The process does not require electron beam lithography to fabricate the nano-confined devices. The realization of the bent and crumpled graphene is achieved by macroscopic manipulation of the 2D layer and the resulting “nano”-sized features exhibit the superior sensing performance, while allowing facile fabrication and reproducibility. We demonstrate detection of 22-mer single and double stranded molecules by adsorption and hybridization experiments, respectively. We also showed that the target miRNA (let-7b) and a DNA probe hybridization was detectable as low as 600 molecules in 50 µL of buffer and undiluted human serum directly without amplification. The performance of the sensor was further enhanced using peptide nucleic acid (PNA) probe, which showed 600 zM of LOD, ~18 molecules in an hour of incubation time. We show via molecular dynamics simulations the formation of electrical ‘hot spots’ at the nanoscale crumpled graphene regions where the Debye length can increase and also result in a local high potential due to the charge of DNA/RNA. Furthermore, the bending of the graphene monolayer at these hot spots can result in opening of a bandgap and provide for an exponential increase in the conductivity in vicinity of the biomolecules. These effects combined can allow for a measurable current change in millimeter scale channels even with 600 zM concentration of the target molecules.Here, we report the use of these deformed and bent (crumpled) graphene FET-based electrical biosensors for ultra-sensitive detection of DNA/RNA molecules down to 600 zM of limit of detection (LOD) on millimeter scale structures. To the best of our knowledge, this is the highest sensitivity reported so far using any electronic biosensor for detection of DNA. Because of the simple fabrication process, the presented approach has several benefits over structures such as nanoribbons or nanopores24. To produce the crumpled FET device, the graphene on polystyrene substrate was annealed at 110 °C for 4 h. This annealing process induces the shrinkage of the underlying pre-strained thermoplastic substrate which results in buckling of graphene24. The optical image of the crumpled graphene device is in Supplementary Fig. The scheme for the graphene FET DNA sensing is illustrated in Fig. 24. AFM derived topography data  and atomic force microscope (AFM). Disorganized herringbone-like structures were observed Fig.  and the 34. The conductance of the graphene channel was modulated by liquid-gate potential applied to the solution reservoir. The accumulated ions modulate the conductance of the graphene channel by either p- or n-doping effects because of the ambipolar characteristics of graphene. The ambipolar transport characteristics of the FETs are illustrated in Fig. Id vs. Vgs curves, the Dirac points shifted as Vds changed. Dirac points were positioned between Vgd of 0 and 0.5 V, which is mainly related to the work function difference of Ag/AgCl gate electrode and the graphene. The measurements were repeated over time and confirmed that the Dirac voltage values were stable in PBS buffer solution before using the devices for measurements  , we measured a clear Dirac point shift, but about half of the earlier measurements in PBS. We measured shifts at 20 aM compared to the negative control tests as shown in Fig. n) of graphene and therefore a shift in Dirac point (ΔVD) given by refs. 46CT is the total gate capacitance, and Δn is directly proportional to the charge density of the unscreened DNA molecules 45. The counter-ion screening of the DNA molecules lowers the net charge of adsorbed DNA and affects the detection sensitivity. As shown in Fig. To investigate the phenomena underlying the experimental results and the effect of ionic screening of DNA molecules, we studied the electrostatics and charge distribution of DNA and ions near flat and deformed graphene surfaces using molecular dynamics (MD) simulations (see the methods section for the simulation details). The presence of the unscreened charges (acting as dopants) carried by DNA molecules near the surface of the graphene produces long-range electrostatic potential leading to a change in the charge carrier density  and σ is the graphene surface charge density. As shown in Fig. n) leading to a larger Dirac point shift. In the flat, convex and across configurations, because of weaker confinement, mobile ions are less restricted and freely present in the vicinity of the DNA molecule, screening its charge to a larger extent  in MD simulations with a diameter of 2.45 nm and a length of 8 nm as shown in Fig. 24. As a proof of principle, we created uniaxial crumple and probed the surfaces by AFM. The image in supplementary Fig. n) of the graphene. Such a deep and narrow trench, referred to as an “electrical hot-spot”, could provide very low ionic screening for an adsorbed DNA molecule by exposing most of DNA to the graphene surface. Therefore, The bending used in Fig. n) to explain our measured detection of aM concentrations of DNA. Sarkar et al. showed that the existence of a bandgap in a single-layer MoS2 leads to a higher sensitivity of charge detection compared to its graphene counterpart31. Here, we hypothesize the creation of a band-gap in bent graphene and next calculated the bandgap (Eg) for flat and crumped graphene in the absence and presence of DNA bases using density functional theory (DFT) and GW methods. Typically, GW methods are more accurate for graphene than DFT. Supplementary Table μ) in graphene decreases with increasing bandgap 47. As n is inversely related to μ, the change in n (and the corresponding Dirac point shift) due to bandgap widening upon addition of DNA can be obtained48. To achieve the observed Dirac point shift for aM concentrations used in our experiments, the change in bandgap from 0.4224 eV (crumpled graphene with no DNA) to 1.7641 eV (crumpled graphene with base A) must occur in at least ~10−7% of the area of our graphene sensor  DNA from the surface by a distance of 3 ntIn future, the devices could be miniaturized to micro- or nano-sized sensor in an array format. There are many fabrication and integration process challenges towards this goal. Some of these include; (i) maintaining a high quality crumpled surface if lithography is to be performed after shrinking, (ii) performing lithography first to form smaller flat graphene islands and then performing the heating or local shrinkage to cause crumpling while keep those smaller islands attached to the underlying surface, (iii) integration of silicon FETs at each pixel for row and column addressing to form larger arrays in a silicon substrate, etc.We have demonstrated nucleic acid molecule detection on crumpled graphene FET electrical biosensor with unprecedented sensitivity using DNA and PNA as a probe. DNA adsorption and hybridization tests were demonstrated using cancer-related biomarker miRNA let-7b sequence in buffer and in human serum. The limit of detection was found to be 600 zM for crumpled graphene FET biosensor and 2 pM for flat graphene. We show via experiments and simulations that the nanoscale bending and deformations increases the Debye length in ionic solution to decrease the screening of the DNA/RNA molecules, thus contributing to the dramatic enhancement of sensitivity as compared to flat graphene FET sensors. To explain the results, Molecular Dynamics simulations revealed generation of large electrical potential due to DNA molecules in the nanoscale crevices and deformed regions that exclude ions, compounded by the formation of electrical bandgap in the deformed graphene regions. These attributes coupled with increased molecular residence time due to increased roughness can result in “electrical hotspots” allowing for a change in local conductivity, hence allowing atto-molar detection of DNA in millimeter scale sensors. We demonstrate the applicability of the technology by target molecules detection in undiluted human serum for cancer-related miRNA. This technology can open opportunities for the development of more reliable and efficient diagnostic tools, and electrical point-of-care and implantable biosensors for early detection of biomolecules for various human diseases.2) gas flow continuously. Monolayer graphene growth was initiated when 50 sccm hydrogen and 100 sccm methane (CH4) were introduced to the CVD chamber at annealing temperature of 1030 °C, and the synthesis came to its end when two reaction gases were turned off. CVD furnace was then cooled down with flow of argon (Ar) gas, which completes the monolayer graphene synthesis process. To compare the performance among different source of graphene, graphene was also purchased from Grolltex and Graphenesquare. All the graphene showed the same LOD.Monolayer graphene was grown on a copper foil via chemical vapor deposition (CVD). Before placing into the CVD furnace, copper foil was degreased with acetone and IPA, followed by nitrogen blow drying. The foil was then annealed at elevated temperature for 3 h, while 50 sccm of hydrogen  (PMMA). Undesired graphene formed at the back side of copper foil was removed by oxygen plasma etching. The sample was cut into 1 mm × 15 mm pieces with scissors or a razor blade. PMMA serves as a supporting layer while copper foil was etched by floating on 0.15 M sodium persulfate for about 5 h. The PMMA/graphene was rinsed by moving from the sodium persulfate solution to deionized (DI) water. The PMMA/graphene was then transferred onto a polystyrene substrate. The PMMA layer was removed by soaking in acetic acid for 5 min. The sample was annealed at 110 °C for 2 h to shrink the polystyrene substrate into ¼ of the original size and crumple the graphene. To fabricate transistor, conducting silver paste was used as source and drain electrodes at both ends of the graphene. Silicone rubber (DOWSIL™ 3140 RTV Coating) was used to insulate source and drain electrodes from liquid and construct a solution reservoir.Pyrenebutanoic acid succinimidyl ester (PASE) (20 mM) in dimethyl sulfoxide (DMSO) was treated on the graphene overnight and rinsed with pure DMSO, ethanol and DI water; 50 μM of probe DNA solution was added on PASE-modified graphene for 3 h. The graphene FET with probe DNA functionalization was rinsed with 1× PBS. 200 mM ethanolamine solution was treated for 1 h to saturate the possibly unreacted amino group on PASE and rinsed with ethanol and 1× PBS solution. The volume of all treated chemicals and samples was 50 μL.The target DNA or RNA incubation was conducted by dropping complementary and negative control strands with concentrations that are indicated in the legends in Fig. Vgs) to the 0.1x and 1× PBS buffer solution. For the human serum test, the tests were performed in in serum. In case of DNA absorption, the graphene chips were incubated in PBS overnight because of wettability issue of hydrophobic graphene. For the serum test, the graphene chips were incubated in serum overnight and the blank measurements were repeated till there was no shift only with serum. Then target and NC RNA in serum was treated on the chip. Vg was swept from −0.5 to 1 V and drain–source voltage (Vds) was picked between 0.03 and 0.1 V. Drain–source current (Ids) was measured at an assigned Vds.I–V curves and resistance were measured in a semiconductor parameter analyzer equipped with a probe station. Ag/AgCl electrode was used to apply gate voltage (CT) based on CV is shown in Fig. The capacitance measurements of flat and crumpled graphene on PS substrate were carried out using a CS 350 potentiostat  with three electrodes, including reference, counter, and working electrode. Here, silver chloride (Ag/AgCl), platinum (Pt), and the surface of the graphene channel on PS substrate were used for reference, counter, and working electrode, respectively. Cyclic voltammetry (CV) was chosen for the characterization method, while all three electrodes were immersed in 1× PBS solution for the measurement. Normalized total capacitance by measured graphene area 53. To study the effect of the crumples on the EDL formation near graphene, different simulations with different graphene surface topologies were created  for all the systems. The systems are non-periodic in z direction . The Lennard-Jones (LJ) potential with a cutoff distance of 1.2 nm is used. The long-range electrostatic interactions are calculated using the PPPM54. SPC/E water model is employed. The SHAKE algorithm, which maintains the rigidity of each water molecule, is applied. Sodium chloride salt solution of various concentrations (1.2 and 0.6 M) is considered. The LJ parameters between atoms of graphene and water are modelled by the forcefield developed by Wu et al.55. The CHARMM forcefield56 is used for the DNA strands and ions.Molecular dynamics simulations were performed using the LAMMPS packageted Fig. . Flat an58 with a time constant of 0.1 ps. The production simulations were carried out in NVT ensemble for 10 ns. Trajectories of particles were dumped every picosecond to study the structure of DNA and ions near the graphene sheet.Before starting the equilibrium and production simulations, the energy of each system was minimized for 15,000 steps. The equilibrium simulations were then performed in NPT ensemble for 2 ns at a pressure of 1 atm and a temperature of 300 K. This ensures that the water reaches its equilibrium density. The systems were further equilibrated in NVT ensemble for another 2 ns at 300 K. Temperature was maintained at 300 K by using the Nosè-Hoover thermostat61. The local density approximation (LDA)62 functional of Ceperley-Alder is employed based on the projector augmented wave (PAW) method60. The cutoff energy for the plane wave basis set is 550 eV for all calculations. All ionic positions are optimized by a conjugate gradient method until the forces on each ion are less than 0.01 eV/Å. The size of flat graphene and crumpled graphene model is about 12 Å × 12 Å and 8.9 Å × 12 Å, respectively. A vacuum separation of 40 Å between graphene and its periodic replicas is employed to eliminate the interaction between them. For accurate calculations of the electronic structures, the Brillouin zone is sampled using 18 × 18 × 1 k-point grid. The lattice parameter of flat graphene is computed to be 2.445 Å which is consistent with previous theoretical and experimental results64.The optimized geometry of the adsorbed DNA nucleobases on flat and crumpled graphene are obtained using DFT calculations. All DFT calculations are performed using the Vienna Ab initio simulation package (VASP)65 was performed to obtain the band structures using VASP61. Each system has been initialized using DFT with the LDA66 exchange-correlation functional, energy cutoff of 400 eV, and Gaussian smearing of 0.05 eV. The number of total bands is set to 512 in all structures to ensure a significant number of empty bands is available as required by GW method. The energy cutoff of the response function and the number of frequency grid points are respectively set to 90 eV and 32 to control the high memory demand by the calculation of GW. Gamma-centered k-points are selected to be 6 × 1 × 1 for structures without a DNA base and 4 × 1 × 2 for structures with a DNA base. Finally, the vacuum space is maintained large enough (>20 Å) in the periodic directions to avoid unphysical interactions between the periodic images.Single-shot approximation of GW (G0W0)Supplementary InformationPeer Review File


In idiopathic pulmonary fibrosis (IPF), several factors may have a negative impact on the nutritional status, including an increased respiratory muscles load, release of inflammation mediators, the coexistence of hypoxemia, and physical inactivity. Nutritional abnormalities also have an impact on IPF clinical outcomes. Given the relevance of nutritional status in IPF patients, we sought to focus on some critical issues, highlighting what is known and what should be further learned about these issues. We revised scientific literature published between 1995 and August 2019 by searching on Medline/PubMed and EMBASE databases including observational and interventional studies. We conducted a narrative review on nutritional assessment in IPF, underlining the importance of nutritional evaluation not only in the diagnostic process, but also during follow-up. We also highlighted the need to keep a high level of attention on cardiovascular comorbidities. We also focused on current clinical treatment in IPF with Nintedanib and Pirfenidone and management of gastrointestinal adverse events, such as diarrhea, induced by these antifibrotic drugs. Finally, we concentrated on the importance of pulmonary rehabilitation program, including nutritional assessment, education and behavioral change, and psychological support among its essential components. More attention should be devoted to the assessment of the undernutrition and overnutrition, as well as of muscle strength and physical performance in IPF patients, taking also into account that an adequate clinical management of gastrointestinal complications makes IPF drug treatments more feasible.  In recent years, nutritional status has increasingly gained attention in the evaluation of patients with chronic respiratory diseases since their clinical course is often characterized by progressive weight loss and reduction of muscle mass ,2,3. Idiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial lung disease of unknown etiology characterized by rapid progression and poor prognosis ,5.As shown in Furthermore, given the rapidly progressive nature of the disease, with a median survival time of only two to five years, all these factors often develop and worsen in a short period of time.It is worth noting that not only may the “downward spiral” of the underlying pulmonary disease affect the nutritional status, but also nutritional abnormalities may have an impact on clinical outcomes. For instance, lower body mass index (BMI) and body weight loss have been associated with increased mortality ,11,12. CGiven the relevance of evaluating nutritional status in IPF patients, a topic which remains still largely understudied, we sought to focus on some critical issues, highlighting what is known and what should be further learned about these issues.clinicaltrials.gov databases including observational and interventional studies. Keywords used for each database are reported in We revised scientific literature published between 1995 and August 2019 by searching on Medline/PubMed, EMBASE, and Box 1Nutritional status AND (IPF OR acute exacerbation of IPF), nutritional assessment AND (IPF OR acute exacerbation of IPF), nutrition AND (IPF OR acute exacerbation of IPF), body mass index AND (IPF OR acute exacerbation of IPF), nutritional interventions AND (IPF OR acute exacerbation of IPF), dietary habits AND (IPF OR acute exacerbation of IPF), body composition AND (IPF OR acute exacerbation of IPF), metabolic rate and (IPF OR acute exacerbation of IPF), body weight AND (IPF OR acute exacerbation of IPF), weight loss AND (IPF OR acute exacerbation of IPF), weight gain AND (IPF OR acute exacerbation of IPF), malnutrition AND (IPF OR acute exacerbation of IPF), overweight AND (IPF OR acute exacerbation of IPF), obesity AND (IPF OR acute exacerbation of IPF), cardiovascular comorbidities AND IPF, cardiovascular risk AND IPF, dyslipidemia AND IPF, metabolic syndrome AND IPF, diabetes mellitus AND IPF, gastroesophageal reflux disease AND IPF, nintedanib AND , pirfenidone AND , IPF AND pulmonary rehabilitation.Studies targeting children and conference abstracts were excluded. In addition, given the aim of the present review, we included only studies on IPF excluding those on other interstitial lung diseases (ILDs). Topics and structure of the review were discussed and approved by the expert panel. Reviewing main topics were based on the author’s expert view on the current status of the field under discussion.Specific nutritional counseling and dietary advices for IPF patients are not yet available, with the exception of dietary recommendations to reduce the side effects of antifibrotic treatments (see the following sections). The implementation of a nutrition care process may be useful especially in those patients with IPF who are at risk or already malnourished . AccordiFrom a practical point of view, nutritional status may be assessed by evaluating nutrient intakes, energy expenditure, body composition, laboratory data, and body functions. Overall, only a few studies  have so far evaluated the nutritional status of IPF patients.To the best of our knowledge, there are no consistent results available on nutrient intakes in IPF. Two studies carried out in Japan ,15 showe2 in most studies from Western Countries [2) in those carried out in Japan and South Korea [Main anthropometric data were reported in many papers. Mean BMI was around 26–28 kg/mountries ,19,20,21th Korea ,23,24,25Thus, a high proportion of overweight/obese patients (overnutrition) may be expected ,21. As aThe assessment of health-related physical fitness provides relevant information on body functions by assessing muscle strength, physical performance, and exercise tolerance. There is a small amount of literature on this issue for IPF patients. An inverse association between handgrip strength and age or dyspnea was observed ,28. GaitConsidering that there are no guidelines about the nutritional follow-up in IPF patients, in our opinion, nutritional status should be re-evaluated at regular intervals in all IPF patients with regard to formal diagnosis, weight loss, and nutritional problems. According to the best clinical practice, the periodic evaluation should consider  dietary Previous studies have reported that body weight changes with time have a strong correlation with clinical outcomes in several diseases ,31. BodyFinally, as far as body composition is concerned, changes in muscle mass body composition, i.e., a reduced cross-sectional area of erector-spinae muscles, are associated with a worse prognosis in IPF patients .Acute exacerbation is characterized by acute respiratory worsening, being a severe event in the course of IPF and leading to high mortality . In this.Malnutrition is a highly prevalent consequence of hospitalization, especially in critically ill patients admitted to intensive care units (ICU) . MalnutrNutrient deficiency has been correlated with a prolonged length of ICU/hospital stay and is strongly associated with increased morbidity and mortality among critically ill patients ,37. ConvCardiovascular comorbidities, particularly coronary artery disease and arrhythmias, have been frequently observed in patients with IPF ,44,45. SPrevalence of diabetes mellitus also appears higher than in general population, even after the exclusion of individuals treated with systemic corticosteroid therapy. However, the prognostic significance of such finding is unknown . MetformAs already mentioned, observational studies reporting BMI in patients with IPF showed a presence of overweight or obesity in the majority of patients ,11,18.Although in IPF patients lower BMI and weight loss show a negative impact on disease outcomes more than overweight and obesity (see above), these latter issues may have a major impact in advanced stages as demonstrated by the fact that obesity is associated with an increased mortality risk in patients receiving bilateral lung transplantation and that obese patients may not be eligible for transplant . InteresA recent meta-analysis confirmed that gastroesophageal reflux disease (GERD) and IPF may be associated with each other; however, this association was found in case-control studies with smoking being a possible major confounder . The cauAlthough dietary intervention might play a role in effective treatment and management of GERD  and reflThe two currently available anti-fibrotic drugs, nintedanib and pirfenidone (PFD), have offered a therapeutic chance to IPF patients. However, as reported below, both of them cause adverse events that may interfere with food intake and absorption, potentially leading to weight loss and malnutrition. To the best of our knowledge, the relation between nutrition and anti-fibrotic therapies has not been carefully investigated and needs special attention. There is evidence that a weight loss > 5% occurs in about 20% of IPF patients, even in the absence of anti-fibrotic treatments . MoreoveIn IPF patients, the treatment with Nintedanib was not associated with serious adverse events , but was associated with increased gastrointestinal events, particularly diarrhea (62.4% in treatment group vs. 18.4% in placebo group). These episodes were mild-to-moderate and led to a low 4.4% incidence of discontinuation in the clinical trial . AdditioGastrointestinal adverse events can be managed by reduction to 100 mg twice daily of Nintedanib dose or temporary suspension as needed. For diarrhea control, concomitant usage of loperamide ,73 as weMoreover, Ikeda et al.  observedA retrospective comprehensive analysis of safety outcomes of treatment with pirfenidone (PFD) in 1299 IPF patients from five clinical trials  has shown that overall PFD was well tolerated over a 9.9 years follow-up. Mild to moderate nausea (37.6%), diarrhea (28.1%), dyspepsia (18.4%) and vomiting (15.9%) were the most frequent adverse events . PFD has2) had 418 treatment-related adverse events with nausea, vomiting, and diarrhea being the most common [In real-life studies, anorexia and gastrointestinal disturbance are the most common reasons for cessation of PFD ,81. Ikedt common . In ordeThe benefit of specific dietary recommendations other than those for oral hydration has not been well established in controlled trials.However, a diet can play a role in treating diarrhea avoiding foods that are high in fat, high in fiber, milk and dairy products , spicy foods, alcohol, caffeine-containing products, some fruit juices  . Main rePulmonary rehabilitation (PR) has been widely used in the treatment of chronic respiratory diseases, playing a fundamental role in improving exercise capacity, health-related quality of life, and breathlessness.PR is one of the most effective nonpharmacological treatments, and it is recommended by guidelines for patients with IPF, even if the evidence is still inconclusive.As summarized in in the review performed by Xuequing et al., pulmonary function was evaluated only in three studies, showing a significant positive effect of PR on FVC in IPF patients, with no statistically significant improvement was reported for DLCO .A randomized controlled trial, conducted by Perez-Bogerd et al. showed that exercise capacity, health status, and muscle force improved significantly after PR, maintaining such results after one year . In addiMoreover, Chehere et al. reported a 53% non-responder rate, related to the severity of exercise-induced desaturation and the impairment of DLCO before PR ; such isThe selection of patients for rehabilitation is of paramount importance because specific characteristics may be associated with a greater response to PR. Subjects with a higher FVC, less exercise desaturation, less disability, and more severe breathlessness demonstrated a more remarkable improvement in rehabilitation outcomes .The pulmonary rehabilitation program should consist of a comprehensive intervention of patient-tailored therapies . In partThe other components of PR include education, psychological support, and training in behaviors  that will assist in disease management.Morisset et al. investigated educational needs of IPF patients and provided some topics for educational programs such as disease education, symptoms management, medications use, oxygen therapy, and end-of-life counseling .Psychological support is another component of rehabilitation programs since depression and anxiety are frequent comorbidities in IPF patients. Uncontrolled data suggest that pulmonary rehabilitation might improve psychological health in patients with IPF through improvements in symptoms such as dyspnea, exercise tolerance, and sense of control over the disease .All of these components of PR must work together to achieve optimal outcomes for patients affected by IPF . StudiesTo date, evaluation of nutritional status and physical fitness, the nutrition care process, and physical activity are all largely unexplored issues related to the clinical management of patients with IPF.The increased interest in body composition and in the assessment of nutritional status in COPD over the last few years has led to the establishment of different nutritional risk profiles, based on different patients’ metabolic phenotypes . Such asRecently, more attention has been focused on comorbidities in IPF, especially from a prognostic point of view. A prediction model, named TORVAN, assessed the ability of comorbidities to improve prediction of survival in IPF patients beyond the variables included in the GAP model. The TORVAN model also suggests that GERD, pulmonary hypertension, lung cancer, valvular heart disease, and atrial arrhythmias were the comorbidities with a higher impact on IPF patients survival . There hClinicalTrials.gov Identifier: NCT03770845) and nutritional intervention  in IPF patients are ongoing, and their results are expected in the near future.However, epidemiological studies performing a complete nutritional evaluation  and overnutrition, as well as of low muscle strength (dynapenia) and low physical performance, taking also into account that an adequate clinical management of comorbidities, particularly gastrointestinal complications, makes IPF drug treatments more feasible.To the best of our knowledge, there is a paucity of longitudinal studies which should evaluate the prognostic impact of some comorbid conditions that affect IPF patients, such as obesity and cardiovascular diseases. Moreover, the current lack of nutritional intervention studies does not allow to assess their efficacy in undernourished or over nourished IPF patients.Hence, the clinical guidelines for IPF management ,61 shoulIn light of the above, it seems therefore quite clear that a multidisciplinary approach is needed not only in the diagnostic process, but also in follow-up and advanced phases of the disease—so that various specialists should be involved in multidisciplinary care of IPF, including nutritionists (physicians and dietitians) and rehabilitation specialists in addition to pulmonologists, nurses, and psychologists. Such holistic care might improve IPF patients’ quality of life and at the same time may be also a clear reference point for patients, who often feel alone with their disease .


Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in The online version contains supplementary material available at 10.1186/s40478-021-01148-z. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of motor neuron function, culminating in death due to respiratory failure , 79. MutTDP-43 is a nucleo-cytoplasmic shuttling, DNA/RNA binding protein which is predominantly localized to the nucleus where it regulates transcription  and splifutsch and hsc70-4 mRNAs (in axons)  v[+ t1.8] = TRiP.HMS01846}fCRattP40 (VDRC v38377) and y v[+ t1.8] = TRiP.HMS01848}attP40 (VDRC v38379). These lines were crossed with the D42 GAL4 driver  = UAS-dlp.WT}3 (Bloomington Stock #9160) and for RNAi knock-down we used y v[+ t1.8] = TRiP.GLC01658}attP40 (Bloomington Stock #50540). As a control for the RNAi experiments we used y = CaryP}attP40 . CRISPR TDP-43 knock-in models, TDP-43WT and TDP-43G294A in which the endogenous Drosophila TDP-43 (TBPH) had been replaced by human TDP-43 (WT or G294A) were previously described [w[*]; P{w[+ mC] = UAS-fz2-2}16/TM6B, Tb[ +] . Expression of dominant negative Fz2 was achieved with w[*]; P{w[+ mC] = UAS-fz2(ECD)-GPI}3 (Bloomington Stock #44221). For G6PD overexpression, we utilized w1118; UAS-G6PD  v[ sev;P{y[+ t7WT-YFP or TDP-43G298S-YFP were collected and flash frozen in liquid nitrogen. Frozen larvae were homogenized in lysis buffer  and RNAsin Plus 400 units/ml (Fischer Scientific PRN2615). Lysates were centrifuged at 10,000 × g for 10 min and then pre-cleared with magnetic beads  for 1 h at 4 °C. Magnetic beads bound to chicken anti-GFP antibody (Life tech A-11122) were added to the supernatant followed by rotation at 4 ºC for 2 h. Next, beads were washed 3 times with a sugar-rich buffer  and 2 times with a low-density buffer  followed by resuspension in Qiagen RLT buffer  with 1% 2-mercaptoethanol (Sigma Aldrich M6250).100 third instar larvae expressing TDP-43w1118; UAS-RpL10-GFP/CyO, w1118; UAS-TDP-43WT UAS-RpL10-GFP/CyO and w1118; UAS-RpL10-GFP/CyO; UAS-TDP-43G298S/TM6B Hu Tb and the buffers for the immunoprecipitation of RpL10-GFP also included 100 μg/mL cycloheximide.RNA associated with ribosomes was immunoprecipitated using a protocol similar to RIP (see above) except that the genotypes were RNA was isolated from the ventral cord lysates, whole larvae inputs, or immunoprecipitations using a Qiagen RNeasy Mini Kit (Qiagen 74104). For fractionation experiments, dissected ventral nerve cords (VNCs) or neuromuscular junctions (NMJ), RNA was isolated using Trizol (Thermofisher Scientific 15596026). With both isolation methods, RNA was eluted into 20 μl of molecular grade water with quality and quantity determined by a nanodrop spectrophotometer, measuring absorbance at 260/280 nm.Isolated RNA was prepared for RNA sequencing using the SMART-seq v4 Ultra Low Input RNA kit (Takara Bio USA 634888). The Nextera XT DNA Library Prep Kit (Illumina FC-131-1024) was used to tag, clean, and pool the samples with quality assayed by an Agilent 2100 Bioanalyzer. The cDNA libraries for RIP/TRAP were sequenced by the Beijing Genomics Institute using an Illumina HiSeq 4000 and the Illumina Nextera XT Kit (Illumina 15032350) with 100-bp paired end reads. Human fractionation RNA-seq were performed at the Translational Genomics Institute (TGen) using similar protocols.WT and TDP-43G298S complexes, respectively. In TRAP experiments, 9711, 9347, and 9669 genes were detected in RpL10 GFP complexes in the control, TDP-43WT, and TDP-43G298S models, respectively. To identify the “normal” motor neuron translatome we calculated the Log2FoldChange between RpL10 IP and RpL10 VNCs. Next, to identify alterations resulting caused by TDP-43 proteinopathy to the motor neuron translatomes, we subtracted the Log2FoldChange between RpL10 IP and RpL10 VNCs from the Log2FoldChange between TDP-43WT or G298S RpL10 IP and TDP-43WT or G298S RpL10 IP VNCs. Gene ontology (GO) analyses were conducted using David 6.8 [Reads were quality filtered to > Q30 then aligned to the Flybase FB2018_05 genome using MAFFT  with difavid 6.8 , 43. Humavid 6.8  then undavid 6.8 , STRING avid 6.8 , and theg for 30 min. The supernatant became the soluble fraction and the pellet was solubilized in urea buffer , 0.5 mM PMSF, RNAsin Plus 400 units/ml (Fischer Scientific PRN2615), pH 8.5) to generate the urea/insoluble fraction. For human fractionation samples, post-mortem tissue  was homogenized and subjected to the fractionation protocol described above.Fractionations of both overexpression and CRISPR Drosophila models were conducted as previously described . In brieThird instar ventral nerve cords were dissected as previously described . In brieThird instar larval NMJs were dissected as previously described . In brieParaffin-embedded post-mortem spinal cord tissue sections were obtained from the Barrow Neurological Institute ALS Tissue Bank and Target ALS Postmortem Tissue Core. All tissue samples were collected after informed consent from the subjects or by the subjects’ next of kin, complying with all relevant ethical regulations and approved by the appropriate Institutional Review Boards. Clinical neuropathological diagnoses were made by board certified neuropathologists according to consensus criteria. Subject demographics are listed in Additional file dlp mRNA was quantified using Taqman assay (Thermofisher Scientific Dm01798597_m1) using GPDH (Thermofisher Scientific Dm01841185_m1) for normalization. Fold change was calculated using the standard ΔΔCT method [All cDNA was synthesized using Fisher First Strand cDNA synthesis reagents (Thermofisher Scientific K1641). qPCR reactions were conducted in three biological replicates using Taqman Fast Advanced Master Mix (Thermofisher Scientific 4444556) and conducted on either an ABI 7100 or Analytik Jena 844-00504-4 qTOWER qPCR machine. T method .Third instar wandering larvae were placed on a grape juice agar plate. After being given 1 min to acclimate, the larvae were turned on the ventral side and timed until they uprighted themselves and began the first forward motion . Data podlp and Gapdh1 in dissected VNC and NMJs as previously described [FISH was performed using RNAscope (ACD Bio). A custom probe generated by ACD bio for dm-dlp and a control probe dm-Gapdh1 were used to detect escribed . Images For RNA seq analyses, significance was determined using the P adjusted value calculation embedded within Deseq2. All other statistical analyses were performed using GraphPad Prism 7.0 using two-tailed analyses. Normality was evaluated using the Kolmogorov–Smirnov test. All data shown are mean ± standard error of the mean.Drosophila models of TDP-43 proteinopathy that recapitulate key aspects of the disease including locomotor defects, cytoplasmic aggregates and reduced lifespan [Drosophila larvae expressing human TDP-43 protein specifically in motor neurons via the GAL4-UAS system (D42 GAL4 > UAS-TDP-43-YFP) and performed immunoprecipitation experiments to pull down TDP-43 and associated mRNAs (RIP). The mRNAs associated with TDP-43 (WT or mutant G298S) were isolated and subjected to RNA sequencing . Additionally, about 25% of mRNAs enriched with TDP-43 were unique to TDP-43WT or TDP-43G298S  highlight neuronal pathways previously linked to TDP-43 such as NMJ development [futsch [WT, Log2FC = 2.44, Padj = 8.07E−37; TDP-43G298S, Log2FC = 2.68, Padj = 6.86 E−12) and CSNK1E [WT, Log2FC = 0.721, Padj = 3.32E−3; TDP-43G298S, Log2FC = 0.847, Padj = 3.08E−3). Furthermore, we found that of the 834 TDP-43 interacting mRNAs recently identified in murine neurons using TRIBE (targets of RNA-binding proteins identified by editing) [Drosophila RIP data sets   about 1/WT, or TDP-43G298S. Following ribosome immunoprecipitations we isolated total RNA and conducted RNA seq, detecting ~ 9,500 mRNAs . These comparisons identified several genes enriched with or depleted from motor neuron ribosomes in both of the ALS models relative to RpL10-GFP controls (ΔLog2FC > 1 or < −1). Subsequent GO term analyses using David 6.8 [WT, spliceosome components were translationally dysregulated . Interestingly, oxidative phosphorylation genes encoding components of the electron transport chain showed both increased and reduced association with ribosomes , one of 14 genes that exhibited increased translation in a human cell model of TDP-43A315T proteinopathy [Interestingly, genes salient to translation exhibited altered translation Fig. f. In botWT, 37.6% for TDP-43G298S) suggesting that these may be direct targets of translation inhibition. GO term analyses of these TDP-43 enriched, TRAP depleted genes using David 6.8 [WT 27.6%, TDP-43G298S 34.9%) of the genes enriched with ribosomes in the context of TDP-43 proteinopathy were also associated with TDP-43 complexes . This suggests that in addition to specific pathways, a plethora of individual genes might represent salient targets of TDP-43 mediated translational inhibition or activation.To distinguish between translational changes that may be caused directly by TDP-43 versus compensatory alterations, we compared genes altered in their association with ribosomes to those enriched in TDP-43 complexes, in the context of TDP-43 proteinopathy. These analyses show that approximately 1/3 of the genes depleted from ribosomes were also enriched in TDP-43 complexes  and significantly depleted from ribosomes in the context of TDP-43 proteinopathy  family, localized to the plasma membrane via a GPI anchor or secreted in the extracellular matrix. HSPGs consist of a protein core and several heparan sulfate (HS) glycosaminoglycan (GAG) linear polysaccharide chains which exhibit complex sulfation patterns that dictate the ability of the HSPGs to interact with ligands such as wingless [Drosophila, Dlp was shown to function as a co-factor and competitive inhibitor for binding to the Wg/Wnt pathway receptor Frizzled 2 [frizzled 2 mRNA  and ovo/shavenbaby mRNA   and TDP-43G298S  relative to input  gene has been replaced with human TDP-43 [dlp mRNA is a novel TDP-43 candidate target that becomes insolubilized in the context of TDP-43 proteinopathy and are consistent with previous reports of Wg/Wnt alterations in disease.Further substantiating this finding, dlp mRNA enrichment with TDP-43 complexes, insolubilization and decreased association with ribosomes is inhibition of Dlp protein synthesis. To test the impact of TDP-43 proteinopathy on Dlp protein expression we conducted immunofluorescence experiments in the larval neuromuscular system. We examined Dlp expression at the neuromuscular junction (NMJ) and the ventral nerve cord (VNC), which comprises motor neuron cell bodies and compactly packaged neurites, particularly dendrites [WT or TDP-43G298S, and RNAi knock-down of TBPH (TBPHRNAi) are sufficient to deplete Dlp protein within NMJ boutons relative to the w1118 control  and cumulative granule area relative to the w1118 controls. In contrast, TBPHRNAi did not cause significant Dlp granularity phenotypes  we performed RT-qPCR experiments using dissected VNCs and larval NMJ preparations and found no significant changes in dlp mRNA levels between TDP-43 proteinopathy models and controls  to examine dlp using the motor neuron specific driver D42 GAL4 on its own and in the context of TDP-43WT or TDP-43G298S and measured its effect on TDP-43 induced locomotor defects. Using larval turning assays to measure locomotor function we found that although dlp overexpression (dlpOE) on its own resulted in a higher turning time , in the context of either TDP-43WT or TDP-43G298S disease models, it caused a lower turning time, indicating a rescue of locomotor dysfunction , dlp overexpression in this context is not sufficient to mitigate TBPHRNAi induced locomotor dysfunction  enhanced locomotor deficits in both ALS models based on TDP-43 overexpression  or co-overexpressed with TDP-43WT, TDP-43G298S or TBPHRNAi  in Dlp levels at the NMJ compared to w1118 controls . Together with our findings that dlpOE mitigates TDP-43 proteinopathy but not TBPHRNAi induced locomotor defects, these results indicate that Dlp levels at the NMJ are critical in the context of TDP-43 proteinopathy but not TDP-43 loss, and support a cytoplasmic gain of function rather than a nuclear loss of function mechanism. It is surprising that dlpOE in the context of TDP-43WT causes a statistically significant increase in Dlp levels at the NMJ; this suggests a potentially complex regulatory relationship between Dlp and TDP-43 proteinopathy that warrants further investigation.We next asked whether the rescue of TDP-43 induced locomotor dysfunction by NAi Fig. c-n. FirsTo further probe the relationship between TDP-43 induced neurodegeneration, Dlp, and Wg/Wnt signaling we used genetic interaction approaches between TDP-43 and the Frizzled 2 (Fz2) receptor, an established Dlp interactor . These eDrosophila in human patient derived tissues, we evaluated the expression and sub-cellular distribution of the Dlp human ortholog GPC6 [Pvalue = 0.0020, see Fig. Drosophila VNCs overexpressing TDP-43  [TDP-43 proteinopathy is a hallmark of ALS , 78 and  disease , 15. Rec disease . In contDennd4A) . Taken tDrosophila model of TDP-43 proteinopathy that recapitulates key features of the human disease including locomotor dysfunction and reduced lifespan [e.g., translation, mitochondrial function [dlp and the glutathione metabolism pathway. Although glutathione dysregulation has been shown to be deficient both in patient tissue [Here, using a lifespan , 24 we ifunction , 96) andt tissue  and patit tissue , it has t tissue .dlp as a novel target of TDP-43. dlp is translated into a GPI anchored glypican that interacts with the Wg/Wnt pathway receptor Fz2, serving as both a co-factor and competitive inhibitor for substrate binding [WT and TDP-43G298S disease models suggesting that the Wg/Wnt signaling pathway may be compromised by TDP-43 proteinopathy and consistent with published expression studies in patient tissues [i.e., motor neuron) and non-cell autonomous  aspects of Wg/Wnt signaling [dlpOE and dlpRNAi cause locomotor dysfunction suggest that Dlp levels must be exquisitely regulated in motor neurons. Indeed, Dlp overexpression mitigates but does not fully rescue TDP-43 dependent locomotor defects, perhaps due to an inability to restore wild-type Dlp levels. An alternative explanation for this result is that TDP-43 regulates additional mRNA targets.In addition to identifying motor neuron specific alterations to the translatome induced by TDP-43 proteinopathy, we identified  binding . RNAi kn tissues , 35. Altignaling  may be aignaling  and hippWT and TDP-43G298S both associate with dlp mRNA, and correlate with dlp depletion from ribosomes, the severity of some phenotypic alterations was variant dependent. While overexpression of dlp mRNA was sufficient to rescue locomotor dysfunction and Dlp protein levels were altered in the context of both TDP-43 variants, dlp mRNA was insolubilized only in the TDP-43G298S model. Additionally, different pathways were shown to have altered translation between the TDP-43WT and TDP-43G298S models. Differential results have previously been observed between the TDP-43WT and TDP-43G298S models [G298S stability [WT [We show that although TDP-43S models , which atability  and granlity [WT .dlp mRNA from ribosomes using TRAP, an approach that reflects translational changes in the entire motor neuron rather than specific compartments. It is possible that the synapse specific alteration in Dlp levels could also result from transport deficits, trapping NMJ-bound dlp mRNA/Dlp protein in the soma or neurites. Although our qPCR data show no change in steady state dlp mRNA levels, we cannot eliminate the possibility of mRNA and/or protein transport defects leading to the accumulation of Dlp in the VNC and reduction at the NMJ.In both ALS models, analyses of the whole VNC, which contains motor neuron soma and neurites, show that Dlp forms distinct puncta whereas staining at the NMJ revealed a significant reduction in Dlp protein. These findings suggest that TDP-43 dependent translational inhibition may be localized to axons and NMJs, which can explain the relatively low magnitude depletion of RNAi was sufficient to deplete Dlp from the NMJ, but not induce significant neuropil puncta, suggesting that the two phenotypes can occur independently of each other and might result from different mechanisms. Loss of nuclear function versus cytoplasmic gain of function remains an open question, with both mechanisms likely contributing to TDP-43 proteinopathy [RNAi suggests that this phenotype is the result of nuclear TDP-43 function whereas the neuropil puncta are likely the result of toxic gain of function as they are significantly increased only in the context of TDP-43 proteinopathy and not in the context of endogenous TDP-43 loss of function. Furthermore, the fact that restoring Dlp expression at the NMJ is not sufficient to mitigate TBPHRNAi induced locomotor defects while it improves TDP-43WT and TDP-43G298S dependent phenotypes suggests that Dlp levels at the NMJ are critical in the context of TDP-43 proteinopathy and that other factors may be at play in the loss of function caused by TBPHRNAi knock-down. Collectively, our findings suggest a model in which the mislocalization of Dlp found in flies and patient spinal cords results from a combination of translation inhibition and transport defects as previously reported for Futsch/MAP1B [Interestingly, TBPHinopathy , 110. Dlch/MAP1B   howeversmission , furthersmission  and for smission  further dlp as a target of TDP-43 proteinopathy highlights specific strategies including restoration of synaptic specific protein expression, intracellular transport as well as Wg/Wnt and other Dlp linked signaling pathways in ALS.Here we report several novel translational consequences of TDP-43 proteinopathy including effects on the spliceosome, metabolic pathways and the translational machinery itself. Our combined RIP and TRAP approach can distinguish between direct consequences of TDP-43 proteinopathy and compensatory changes in translation, which can inform future therapeutic strategies. Our identification of the glypican Additional file 1. RNAs enriched in TDP-43 complexes, GO term analyses and comparison with mammalian TRIBE targets.Additional file 2. RNAs associated with ribosomes in controls, RNAs with altered ribosome association in TDP-43 proteinopathy and TRAP GO term analyses.Additional file 3. RIP/TRAP overlap in TDP-43 proteinopathy and GO term analyses.Additional file 4. Demographic information for post-mortem patient spinal tissue.Additional file 5. Supplemental information.