Graphene and its derivatives combine a numerous range of supreme properties that can be useful in many applications. The purpose of this review is to analyse the photoelectrochemical properties of pristine graphene, graphene oxide (GO) and reduced graphene oxide (rGO) and their impact on semiconductor catalysts/quantum dots. The mechanism that this group of materials follows to improve their performance will be cleared by explaining how those properties can be exploited in several applications such as photo-catalysts (degradation of pollutants) and photovoltaics (solar cells). Global warming and the increment of pollutant concentrations are just two of the environmental issues associated with societal development. One possible way to address those and other existing environmental problems could be through the development of highly-efficient photocatalysts; exploiting processes that are based in solar energy. Semiconductor-based photocatalysis relies on the active absorption of a photon from a semiconductor to create an electron-hole pair. This process depends on the band gap of the material . Then, t2, ZnO, CdS, ZnS and Fe2O3 3+/2+ as a redox couple. This experience has been tested with other redox couples and it has always indicated that the electronic structure and the surface physicochemistry are extremely enhanced in electron transfer processes on rGO. In the basal plane it is inert electrochemically, with a transfer constant below 10−9 cm/s , hydroxyl and oxygen radicals can be produced by the electrolysis of water. Then, the oxygen radicals corrode the graphite anode on defect sites, grain boundaries and edge sites. This induces the separation of the edge sheets and the intercalation of RTIL anions within the sheets. The electrode is expanded and provokes the precipitation, which makes the sheets precipitate, generating a graphene solution. This is a relevant technique since it can produce rGO with reduced sheet resistance  and greater transparency  will be considered again in this review in solar cell devices. However, the contribution of graphene and derivatives has been remarkable in improving these systems. Therefore, a brief and more general review shall be undertaken.3−/I−) and finally, a counter electrode coated with platinum where the redox couple is restored. In this case, the photo-induced electron-hole pair is tightly bonded together, forming an exciton with higher energy than thermal agitation [DSSCs are formed by three main parts. A semiconductor with a dye that is deposited on top of a transparent conducting oxide , a redox couple in an organic electrolyte  are loaded into an autoclave and react at high pressure and temperature during several hours. Depending on the growth conditions, rod-shaped TiO2 [2 [There are four main preparation methods to for G/rGO/GO-TiOped TiO2 , nitroge TiO2 [2 , nanopar TiO2 [2  and othe2 can be produced by spin-coating graphene oxide and TiO2 and posterior UV radiation to reduce GO and attach TiO2 [Solution mixing is started with an ultrasonic mixing and then UV-assisted photocatalytic reduction of GO . Layer-bach TiO2 . Anotherach TiO2 . Finallyach TiO2 .2 can be formed in the last three methods by not proceeding with the reduction of GO to graphene. Besides, also different morphological structures can be produced with GO, such as TiO2 nanoparticles wrapped in graphene oxide [2 can be self-assembled under several conditions, such as in water/toluene interfaces or anionic sulphate surfactants [GO/TiOne oxide . In bothfactants . Self-asfactants . It is r2 as a photocatalyser. It has an electron-hole recombination time of 10−9 s, with a chemical reaction response of only 10−8–10−3 s; and it requires UV radiation since it has a too wide band gap (3.2 eV for anatase TiO2 and 3.0 eV for rutile phase). Therefore, G/TiO2, GO/TiO2 and rGO/TiO2 composites should be designed to have visible-light catalytic activity [There are two main limitations concerning the use of TiOactivity .2. They provide a way to enhance the separation between the electron and the hole that are produced in a photoexcitation thanks to very high electron mobility. They also enlarge the absorption range, including the visible region, in which the semiconductor operates by narrowing the band gap of the semiconductor to 2.8 eV with Ti-O-C bonds and nano-sized Schottky interfaces and acting as a sensitizer (it directly captures visible light). Finally, they also increase the interaction area and adsorption of pollutants and dyes with the photocatalyser by creating a π-π interaction with the delocalized electrons of graphene-based compounds [There are three many reasons that make of graphene and derivatives an excellent material to combine with TiOompounds ,60.2 particles have affinity for epoxy and carboxylate groups, where charge transfer is produced by the reduction of those compounds [2 in the contact between those compounds in a process known as percolation mechanism [2 but GO/rGO, this may be, experimentally, the mechanism followed by GO/rGO-TiO2 composites.Regarding the mechanism of electron transfer, the TiOompounds . Oxide gechanism ,76. SincThe reaction mechanism is shown in the following ,10:(1)2 and water to form superoxide and hydroxyl radicals, respectively [These electrons are mostly delocalized. Both electrons and holes react with Oectively . Thus, t2 photocatalyst. In order to do that, Zhang et al. studied how to decrease defects and improve interfacial contact between the carbon-based materials and TiO2. GO is usually prepared with Hummer’s method, which produces a large number of defects on GO surface. This means that alternative methods to produce this material should be used. The technique used by the group was solvent exfoliation (SEG). SEG/TiO2 has, indeed, better properties than GO/TiO2 obtained with Hummer’s method by reducing the number of defects but also the number of oxygen groups [Not only enhancing the electron-hole pair production is important but also to design ways to produce a more efficient G/rGO/GO-TiOn groups .et al. anchored Ag and TiO2 to rGO to produce reduction of silver ions into silver nanoparticles. rGO shows excellent properties to store and shuttle electron. Besides, they established that, given the conduction band of Titania at –0.5 V vs. NHE and the Fermi level of rGO at 0 V vs. NHE, the electron transfer is quick and efficient [Lightcap fficient .2 and GO/TiO2 have a promising future in photocatalysis, it still needs more evidence to prove the superior properties of this composite in comparison to, for example, other carbon-based materials combined with TiO2 and other semiconductors [Although it seems that rGO/TiOnductors . However2 with graphene and its derivatives has large number of applications. We can divide them into three main groups: Quantum dot sensitized and dye sensitized solar cells; degradation of organic, ionic and biologic pollutants and water splitting to produce H2 [The composite TiOoduce H2 .2 was proved to have better hydrogen production than TiO2-P25 nanoparticles [2-P25-rGO, was produced. This material showed a synergetic effect between the different components [An efficient way to produce photocatalytic hydrogen from water could be vital in the development of future energy sources. The main problem concerns the fast recombination rate produced in the photocatalyst excitation. Since the semiconductor produces a charge transfer to graphene or graphene oxide, it promotes oxygen and hydrogen splitting. Besides, the increase in the absorption range is again a key contribution of G/rGO/GO in the enhancement of the photocatalytic process. rGO/TiOarticles . Then, amponents ,89.2 and rGO. The second is that the potential rGO/rGO− in comparison to a standard hydrogen electrode (E°(H+/H2) = 0 V), turns to be −0.08 V; which is less negative than the conduction band of anatase TiO2, ≈ −0.24 V. This promotes the flow of electrons from TiO2 towards the rGO sheet and reduce a proton producing hydrogen gas [3 [There are two main reasons attributed to the enhancement of the performance. The first is the close interaction in face-to-face orientation of TiOogen gas . Howevern gas [3 .et al. conducted an experiment of controlled addition of O2 into the atmosphere for controlled production of superoxide radicals. Those molecules can re-oxidize the planar surface of rGO produced by rGO/TiO2 and that small quantity of oxygen promotes the hydrogen evolution. Therefore, the utilization of totally reduced graphene oxide is, in this case, unfavourable and the use of partially reduced GO is beneficial [Finally, Gao neficial .2 with graphene or any of its derivatives have been developed for water splitting, such as ZnxCd1−xS/rGO, CdS/rGO or WO3/rGO. For instance, Jingdong et al. designed a WO3/rGO photoanode to split water. WO3 has stronger absorption and a longer hole diffusion length. The potential of the semiconductor CB is more positive than the H+/H2 pair. The electrode generates 2.4 times higher quantity of hydrogen and oxygen than WO3 on its own and 2.5 higher photocurrent density. The overall photoreaction is limited by the charge separation and it is only effective at a larger bias than 0.7 V vs. Ag/AgCl. In darkness the work function rGO is much lower than that for WO3 and, as a consequence, the electrons in the CB cannot be injected. Only through interface states the transfer is possible. However, under illumination, the Fermi energy level in the semiconductor material rises and the electrons can be transferred directly into rGO. The recombination is then avoided and the electrons are quickly transported through an external circuit [Many composites, besides TiO circuit .2 composites is the photodegradation of ionic, organic and biologic pollutants. Kemp et al. reviewed the applications of graphene composites for water remediation, where rGO-based Titania compound turned out to be highly useful [2 was proved to have 3.46 times more efficiency in photocatalysis under visible light than commercial P25-TiO2 materials. This is produced by the higher conductivity of rGO and the uniform distribution of nanoparticles achieved by the self-assembly technique used [2 composite modified with P25 and produced by hydrothermal reaction was also studied by Zhang et al.2 [One of the most important uses of graphene, rGO and GO/TiOy useful . Regardique used ,94. The et al.2 . Thus, tet al.2 .E. coli was eliminated with GO/TiO2 thanks to the properties exhibited by GO, which is biocompatible and antibacterial [E. coli decreased to 13%. This is produced by the oxygen groups contained in the GO sheet that react with cell membranes creating oxidative stress [As far as the eradication of biological pollutants is concerned, acterial . After ae stress .2 since their energy levels do not match. A photoexcited electron from MB flows into Titania’s CB via graphene (Path 1), where radical species are generated. Pollutants are usually aromatic compounds that create π-π stacking with rGO, raising the concentration of those molecules near the catalytic semiconductor nanocrystals. The production of oxidants and the reduction of radicals facilitate the reaction when the pollutant is closer. Therefore, the photodegradation is enhanced by π-π interactions. Moreover, as with previous pollutants, large surface area, extended light absorption range, high electron mobility and increased efficiency in charge separation improve the photocatalytic activity [2 flowing to the conduction band of the semiconductor. This mechanism is possible by the band gap narrowing produced by graphene sheets. Reactive species that will degrade the pollutant are then produced (Path 2) [However, the most important type of compound that can be degraded is organic pollutants. There have been many studies, with different efficiencies, that degrade compounds such as malachite green , methyl activity . There i(Path 2) ,81.2 composite with PbS QDs. Both PbS and TiO2 can get excited by different wavelengths, as a consequence the photocurrent efficiency was increased. The mechanism is analogous to the previous example [et al. worked with CdSe-rGO-TiO2 particles. CdSe, with a band gap of 1.6–1.8 eV can accept visible light. The electrons generated in CdSe are transferred to the conduction band of TiO2, whose band gap is 3.0 eV. Besides, rGO can also capture electrons, which are transferred to the CB of the CdSe and, in the same way, to TiO2. This is one case in which, by coupling semiconductor—quantum dots and rGO—better photocatalytic results can be achieved [2O3 is another example of sensitized quantum dot that decorates (001) TiO2 facets on rGO. Hou’s group proposed a photocatalytic mechanism. TiO2 has a higher reduction potential than H+/H2 and therefore more active CB edge potential than Bi2O3. Photoinduced electrons on TiO2 are transferred to Bi2O3 compound and the holes to Titania [Some of the pollutants studied are dyes, which are degraded by the use of quantum dot—Titania–G/GO/rGO systems. In these cases, the photoabsorption can be produced by light excitation in the QD or in Titania. That is what Zhang and his work-team reported in a rGO/TiO example . Ghosh eachieved . Bi2O3 i Titania .et al. and other groups that are researching other types of ternary composites based on rGO-TiO2 and Fe3O4. This photocatalyst can degrade many different organic dyes , has enhanced photocatalytic activity, and can be recollected with a magnet. Besides, photodissolution of Fe3O4 is inhibited, thus, it has a high stability and can be reused many times. However, its catalytic activity is not as good as pure rGO/TiO2 and GO/TiO2 composites [An interesting study was conducted by Lin mposites .2 systems, the photocatalytic oxidation reactions can degrade organic contaminants. In order to be valid for that task, photo-induced catalytic properties play a key role, and rGO/TiO2 would be a perfect candidate for that task [To conclude, some of the developed systems could be useful in self-cleaning coating. Under UV irradiation for TiOhat task .2 can be very useful in degradation of organic dye pollutants. At the same time, it can also function as an effective charge collection layer for Solar cells.It has been shown that TiO2 graphene-based composites can enhance the overall performance. At the moment, the most common type of these devices are Si based solar cells; however, alternatives such as dye sensitized solar cells, quantum dot solar cells and organic polymer solar cells are increasing in popularity. The excellent conductivity of graphene, acceptability and mobility of electrons, transparency, wide band tenability and flexibility provided by graphene may improve the state-of-the-art devices [Solar cells are one of the applications where the inclusion of TiO devices .2, GO/TiO2 and rGO/TiO2 can be used in both dye-sensitized solar cells (DSSC) and quantum dots solar cells (QDSC). Dye-sensitized solar cells could substitute traditional silicon solar cells in the future, since they have high photon-to-electron efficiency and low cost [2, a conductive transparent electrode a counter electrode and an electrolyte. The dyes are photoexcited and produce an injection of an electron to the semiconductor film. The dye molecules are restored by the electrolyte, which is a redox couple [G/TiOlow cost . Generalx couple .2 electrodes with ruthenium-based dyes is approximately 12%. This low rate is caused by electrons trapping and random pathways. The high specific area and electron mobility of graphene and derivatives may allow longer lifetimes and better conversion rates. Kim et al. embedded rGO on the top layers of an inverse opal TiO2 structure. C-Ti bonds enhance electron transport, and, therefore, electron injection and collection efficiencies. Besides, the light harvesting efficiency depends on the dye absorption and the optical properties of the electrode film [The conversion rate achieved so far with TiOode film ,105.2 [2, making easier the electron transport [The incorporation of rGO sheets improved the electron lifetime by increasing the chemical capacitance and decreasing the resistance. rGO was used as electron acceptor layer that transports the negative charged particles, which increased the electrical conductivity. The direct contact of the semiconductor structure with rGO induced a reduction in the recombination loss. The fact that the embeddement was limited to some external layers, where the electrons have a higher potential for recombination loss, improved the electron transport. At the moment, a conversion rate of 7.5% is achieved, a 55% improvement over DSSC with pure TiO2 . In addiransport ,105.2. That electron is then transferred to graphene, which acts as a bridge between the semiconductor and the conductive substrate [ubstrate .et al. has reported rGO/TiO2 on top of ITO as photoanode for DSSC. The increase of electron transport manifested in an increase of the short-circuit current density. They achieved a power conversion five times higher than pure TiO2 and conductivity with two orders of magnitude improvement [Tang rovement .2 have the same characteristics of DSSCs’ and the main point would be to improve the electron transport between those QD and the composite. Regarding quantum-dot (QD) sensitized solar cells, they have the same structure of DSSC but with inorganic QD  instead of organic dyes. The advantages of QD in comparison to organic dyes are the high extinction coefficients, tuneable band gaps, large intrinsic dipole moment and good stability. Therefore, the enhancements made with G/rGO/GO-TiO2 with sensitized Quantum Dots of CdS have been improved by Zhu et al. by introducing reduced graphene oxide in its structure. The improvement shown in comparison to the pure photoanode without this material is of 56%. In this case, the conduction band of TiO2 (–4.2 eV) was better matched with the work function of the conductive transparent electrode FTO (–4.4 eV) by means of rGO, whose work function is –4.4 eV. This enhanced the overall conduction. Therefore, the caption of an electron by the quantum dot is coupled with Titania, as shown in 2 and rGO. However, some electrons are trapped in the surface states and band gap of TiO2 and alternative recombination pathways have to be considered It is remarkable that graphene and its derivatives can enhance the photocatalytic properties of different materials, apart from Titania. Good examples of that are quantum dots such as CdS and ZnS and other semiconductors such as ZnO combined with graphene, rGO or GO.2+ ions and has a fast electron-hole recombination, which limits its photocatalysis activity. There are several ways to improve the photocatalytic activity of CdS; for example, binding it to other semiconductors or noble metals. Another way is to bind it with a mesoporous or macroreticular material creating a composite were the electrons created by the photoexcitation can move freely while the hole is trapped in the CdS nanoparticles. For that purpose, large surface area and conductive materials are required [CdS is a photocatalyser under visible light that has a band gap of 2.42 eV, although it self-oxidises liberating Cdrequired ,117. Thiet al. used solvothermal/hydrothermal method to produce a graphene-based magnetic composite by generating CdS and Fe3O4 nanoparticles at the same time [Zhou ame time . In the ame time ,68.2− and hydroxyl radicals OH− by the electron and the hole respectively. Those compounds will ultimately react with Rh. B producing its degradation [Proof of the enhanced catalytic activity is that rGO/CdS exhibits better performance than GO and CdS on their own in the photodegradation of organic and inorganic compounds, such as Rhodamine B, as shown in radation .E. coli, and B. subtilis) and Rh. B, methyl orange to produce hydrogen [6+ [It was also used in the degradation of other compounds such as methylene blue, where an efficiency of 94% degradation was achieved by means of visible light; in comparison to pure CdS, that only achieved a degradation of 38% . The amohydrogen  and Cr6+ogen [6+ .Another example of graphene’s possibilities would be rGO/ZnS and GO/ZnS composites. ZnS is a II–VI semiconductor with a wide band gap (3.75 eV) that is of interest since it can be used, for example, in field effect transistors, LEDs, photocatalysis and solar cells ,124. RecThe good photocatalytic activity of this compound is a consequence of the rapid photo-excitation, combined with the highly negative reduction potential of the excited electrons . Hu and Pan and Liu assigned the charge transfer mechanism to a chemisorption interaction. The photons cause a resonant charge transfer between the semiconductor and the adsorbate, forming specific complexes. Once the electron is excited into the conduction band of ZnS, they interact with reduced graphene oxide and get energy from the excitation levels of this material. In this case, they are then recombined with the holes produced in ZnS leading to better photoluminescence results of the quantum dot. Moreover, if the quantum dots has reduced size, they also have more surface states that can easily coordinate with rGO .2, water splitting or photoreductive dehalogenation [Photocatalysis is also possible under visible light. This is a result of a narrowed band gap to 3.45 eV for 5%rGO/ZnS  and the contribution of reduced graphene oxide. rGO is photoexcited by visible light and the electron is then transferred to the conduction band of the ZnS. Thus, the carbon based material acts as a visible light photocatalyser in a similar way to organic dyes. This behaviour is different to the previously charge transfer mechanism, common for G/rGO/GO-semiconductor composites. Graphene and derivatives can be used, therefore, as photosensitizers . ZnS nangenation . GraphenAs a final example of the capabilities of graphene, reduced graphene oxide and graphene oxide in photocatalysis, it is interesting to mention their composites with ZnO. This is a semiconductor with a 3.2 eV band gap that is interesting since it is benign to the environment and has a low recombination probability, its valence band is only formed with d orbitals while the conduction band is formed by p-hybridized orbitals .et al. described the reasons that are attributed to the enhanced performance of the semiconductor in the degradation of the organic pollutant by reduced graphene oxide. As in previous cases, the π-π interaction between the semiconductor and the carbon based material improves the absorbance of MB. The electron ejected from ZnO is accepted by the rGO layer, which was then used to degrade the pollutant. Moreover, the short distance between rGO and zinc peroxide enables the fast transfer of the excited electron [The photocatalytic properties of rGO/ZnO have been proven by many groups through the degradation of methylene blue under UV light ,131,132.electron . This shet al., which is depicted in the It is interesting the in-depth study of the mechanism of electron transfer that could happen in the degradation of most pollutants done by Ahmad 2O4/ZnO immobilized on reduced graphene oxide ; which also has magnetic properties that enable an easier separation, and therefore reuse, of the catalyst [Since the energy levels of rGO enhance the electron transfer, avoid recombination and allow visible light absorption ; several photocatalytic activities have been performed to exploit this effect. For example, as well as other organic pollutants like Rhodamine 6G , also mecatalyst .−2 A/W since it has weak light absorption and absence of multiple photoexcitation. Nevertheless, its fast response time and broad spectral width are of great interest in photodetection. On the other hand, colloidal quantum dot films have poor carrier mobility and limited absorption range. Graphene, with an absorption range from UV to terahertz range overcomes the “long-wavelength limit”. Besides, the maximum operating bandwidth of photodetectors is restricted by their finite duration of photogenerated current. By creating a composite of monolayer or bilayer graphene with colloidal quantum dots, a responsivity of ≈107 A/W can be achieved by using the high charge mobility on graphene layers [Quantum dots combined with a graphene derivative seem to have great potential for future photocatalytic purposes. Within the potential applications, the possibilities provided by their fast photon detection response should not be forgotten. Graphene-based photodetectors have been limited to a photoresponse of ≈10e layers ,139.et al. claim that the key of the enhancement in the light absorption of graphene is the implementation of photoconductive gain, i.e., the ability to generate multiple charge carriers with a single photon. Thus, they developed a G/PbS composite with ultrahigh photodetection gain  and graphene oxide (GO) have shown great possibilities in composite generation. They can be combined with semiconductors and quantum dots to generate bi and tri-component composites. The wide range of superlative properties of graphene derivatives benefits the photoelectrochemical performance of many materials in multiple aspects. For example, the big surface area with delocalized bonds, in a similar way as “giant” aromatic compound, allows the π-π stacking of several components, which leads to intimate interactions between substrate and organic compounds such as pollutants.Moreover, the interactions between the oxide groups of GO or rGO and the semiconductor also lead to a close interaction between these two components. This close distance between them enhances the conductivity of photoexcited electrons and reduces the recombination rate of the electron-hole pair. Besides, the high electron mobility of the electron in a layer of graphene also contributes to that effect. This can be useful, for instance, as a bridge between the semiconductor and an organic compound to enhance its degradation.Many of the most important semiconductor catalysers have a wide band gap that is mainly excited by UV light. rGO narrows the effective band gap of the semiconductor material. It has a wide spectrum of light absorption, thus, it can also act as a sensitizer to capture visible light. However, this mechanism can either lead to enhance the photocatalytic activity or act as a competitor in light harvesting. This would reduce the performance of the semiconductor. A precise control of the parameters and an in-depth study of the reaction mechanisms are required. It seems feasible to introduce rGO and GO in next-generation photocatalytic structures since, a priori; those components lead to enhanced performance results. Although more research is required to fully understand and exploit graphene and derivatives possibilities, current investigation indicates that the performance achieved so far without these materials can be surpassed, and many combinations of different semiconductors, carbon structures, and graphene and its derivatives would be required to take full advantage of this material. By creating new, more complex composites, with tuned coordinated energy band gaps between the different components, we would be able to achieve a high performance photocatalytic response.However, it is remarkable that, in most cases, it is rGO and not pristine graphene that is used in the development of composites. This is a direct consequence of the different chemical properties of graphene and graphene oxide. The latter allows tailoring and functionalization of the layer, which is much more difficult in monolayer graphene. Nevertheless, it would also be interesting to control the position and concentration of oxygen groups to enhance the optoelectronic properties of graphene, which are more convenient and are affected by the oxygenation of the surface. For that reason, they should be treated as independent compounds and alternative production methods that reduce the number of defects, with intermediate properties, such as electrochemical exfoliation, should be developed.In conclusion, GO and rGO can be mixed with quantum dots and many other different semiconductors as composites while, on the other hand, pristine monolayer graphene is usually combined with fine-tuned quantum dots to enable a decent photodetector, which uses intrinsic physical properties from defect-free/less defect graphene. Different photoelectrochemical applications will have different requirements on properties and material costs, which is also determined from their different scalability of manufacturing. Graphene and its derivatives obviously provide us various options to explore and it will be exciting to witness how this new type of material will revolutionize/improve the materials used in our daily life.


Based on the importance of the Gln/Asn-rich 341–367 residue segment for efficient aggregation of endogenous TDP-43 when presented as a 12X-repeat and extensive spectroscopic and computational experiments, we recently proposed that this segment adopts a beta-hairpin structure that assembles in a parallel with a beta-turn configuration to form an amyloid-like structure. Here, we propose that this conformer is stabilized by an especially strong class of hypercooperative hydrogen bonding unique to Gln and Asn sidechains. The clinical existence of this conformer is supported by very recent LC-MS/MS characterization of TDP-43 from ex vivo aggregates, which show that residues 341–367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation. Its distinct pattern of SDS-PAGE bands allows us to link this conformer to the exceptionally stable seed of the Type A TDP-43 proteinopathy.TDP-43 is an essential RNA-binding protein forming aggregates in almost all cases of sporadic amyotrophic lateral sclerosis (ALS) and many cases of frontotemporal lobar dementia (FTLD) and other neurodegenerative diseases. TDP-43 consists of a folded N-terminal domain with a singular structure, two RRM RNA-binding domains, and a long disordered C-terminal region which plays roles in functional RNA regulatory assemblies as well as pernicious aggregation. Evidence from pathological mutations and seeding experiments strongly suggest that TDP-43 aggregates are pathologically relevant through toxic gain-of-harmful-function and/or harmful loss-of-native-function mechanisms. Recent, but not early, microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates  Based on this model, the efficient aggregation of the 12X Q/N TDP-43 construct in vivo suggests that these 12 repetitions are likely to assemble into two stacked beta sheets, each containing six beta-strands or 12 beta strands that form the nascent, quasi-amyloid core. Residues 358–366 act as a linker to connect 341–357 beta-hairpins and allow proper cross-beta spine formation. This segment could connect the beta-hairpins within each sheet, and link the two sheets, allowing them to pack against each other (Figure Using computational tools, we built up an amyloid-like structural model based on beta-hairpins composed of residues 341–357 of TDP-43 arranged in parallel and with a beta-turn topology. This arrangement was found to be stable in long MD simulations Figure . By contr Figure .ex vivo aggregates, we therefore propose as a working hypothesis that our conformational model for this segment (Figure ex vivo aggregates of patient 2 described by Kametani et al. (Based on the apparent protection of residues 341–367 against Ser phosphorylation, Gln/Asn deamidation and Met oxidation in TDP-43 t Figure  may corri et al. . Indeed,i et al. , our coni et al. . As TDP-i et al. , what re35VGGVV40 from Aβ (Mompeán et al., We have recently compared hydrogen bond cooperativity in a Q/N-rich amyloid, the paradigmatical GNNQQNY heptapeptide form the yeast prion Sup35, to the hydrophobic amyloid MAccording to this working hypothesis, hydrogen bond hyperpolarization provides a very favorable free energy change for binding monomers to the quasi-amyloid structure formed by the Q/N-rich segment, thus permitting the TDP-43-12X Q/N construct to efficiently sponge up endogenous TDP-43 Figure . HydrogeRecruitment of endogenous TDP-43 into aggregates within this cellular model can be tentatively attributed to two different processes. First, the tandem repetitions could form an amyloid fold that might sponge up and sequester nuclear TDP-43 through its Q/N segment. Second, heterodimerization via the NTDs of endogenous TDP-43 with TDP-43-12X Q/N would increase the local concentration of the Q/N rich segments and thereby favor their self-association (Romano et al., Recent studies strongly suggest that, once formed, pernicious conformers of TDP-43 could spread through the nervous system in a prion-like fashion (Nonaka et al., MM, MB, EB and DVL conceived the ideas, wrote the manuscript, critically revised it and approved the final version for publication. All four authors agree to be accountable for the contents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Atribacteria, Dehalococcoidia, and Aerophobetes. Sulfate-reducing bacteria comprised only 4% of the community across sulfate-bearing sediments from multiple cores and did not change in abundance in sediments from the methanogenic zone at the site with the lowest sedimentation rate. Microbial communities were significantly structured by sediment age and the availability of sulfate as an electron acceptor in pore waters. However, microbial communities demonstrated no partitioning based on the sediment type they inhabited. These results indicate that microbial communities in the SCS are structured by the availability of electron donors and acceptors rather than sedimentological characteristics.The deep marine subsurface is a heterogeneous environment in which the assembly of microbial communities is thought to be controlled by a combination of organic matter deposition, electron acceptor availability, and sedimentology. However, the relative importance of these factors in structuring microbial communities in marine sediments remains unclear. The South China Sea (SCS) experiences significant variability in sedimentation across the basin and features discrete changes in sedimentology as a result of episodic deposition of turbidites and volcanic ashes within lithogenic clays and siliceous or calcareous ooze deposits throughout the basin's history. Deep subsurface microbial communities were recently sampled by the International Ocean Discovery Program (IODP) at three locations in the SCS with sedimentation rates of 5, 12, and 20 cm per thousand years. Here, we used Illumina sequencing of the 16S ribosomal RNA gene to characterize deep subsurface microbial communities from distinct sediment types at these sites. Communities across all sites were dominated by several poorly characterized taxa implicated in organic matter degradation, including  Several non-exclusive theories exist to explain the factors driving the assembly of biological communities. Many current theories are rooted in the concept of patch dynamics  and seafloor location (space) and are known to influence the metabolic processes, and thus microbial communities, present in the marine subsurface . The SCS is an enclosed sea with highly variable sedimentation rates across its several subbasins ], U1432 , and U1433  using an advanced piston core. Cores were split lengthwise onboard to reveal geological interfaces and samples were collected where visible interfaces were observed throughout the cores, usually both from sediments above and below the interface. Exposed sediment was aseptically scraped away prior to sampling and samples were collected only from the center of the cores, avoiding the outside edges of the cores that may have contacted the core barrel. Fifteen samples representing eight interfaces were collected from site U1431, six samples representing three interfaces were collected from site U1432, and 20 samples representing 10 interfaces and two depths at which no interfaces were observed were collected from site U1433. Samples were stored at −80°C until analysis. Down-core measurements of sediment methane concentrations, alkalinity, and pore water sulfate, ammonium, and phosphate were made onboard , a tool that minimizes core disturbance and is designed to recover undisturbed sample material from unconsolidated sediments  and its isotopic composition for each sediment type. Prior to TOC analysis, any inorganic carbon (IC) in the form of biogenic, authigenic, and/or detrital CaCO3 was dissolved using 6% sulfurous acid applied to weighed samples in amounts and steps optimized for carbonate-rich sediments  since deposition. Measurable TOC (described below) in all the ashes is likely derived during the descent through the water column or during reworking on the seafloor, but was not visible in smear slide at 630x magnification. The lithogenic clays ranged in grain size and generally were characterized by fine grained mineral and/or lithic content, with no or trace amounts of biogenic shell fragments. All but one of the turbidite occurrences were dominated by lithogenic minerals and/or rock fragments, with minor amounts of biogenic fossil (carbonate or silica) materials. The remaining turbidite (U1432C 6H-1 47–49 cm) contained significant calcareous shell materials from foraminifera and calcareous nannofossils and is considered a calcareous turbidite in this study. Calcareous oozes were characterized by >60% biogenic calcium carbonate in the form of foraminifera and calcareous nannofossil shells and shell remains. Siliceous oozes were characterized by >60% biogenic silica in the form of diatoms, radiolarian, and sponge spicules.Sedimentological characterization of the samples from smear slides revealed several distinct sediment lithologies: volcanic ashes, lithogenic clays, lithogenic and calcareous turbidites, calcareous ooze, and siliceous ooze Figure . The voldf = 2, 37, F = 2.24, p = 0.12). However, TOC content differed significantly between sediment lithologies . TOC content was also significantly higher in the sulfate reduction zone than in the methanogenic zone at sites U1432 and U1433 (Wilcoxon rank-sum p = 0.02). Organic carbon C/N in all sediment horizons ranges from 9 to 16 . These OTUs represented 44 bacterial phyla and 3 archaeal phyla . Microbial diversity was also significantly higher in site U1433 than U1431 . Sediment age and pore water phosphate concentration predicted 34 and 36% of community variance, respectively, among sulfate reduction zone communities across all sites  and are not predictive of changes in community composition within any single site. Among communities inhabiting methanogenic zone sediments, the amount and isotopic depletion of organic carbon were significantly correlated with microbial community variance . At all sites, microbial richness in the sulfate reduction zone declined exponentially with sediment age . Within methanogenic zone sediments, microbial diversity was positively related to methane concentration  and inversely related to TOC content . In contrast to communities in the sulfate reduction zone, no relationship was found between sediment age and species richness .Microbial community diversity also varied across the gradient from sulfate-bearing to methane-bearing sediments. Within sites U1432 and U1433, microbial richness was significantly higher among sulfate reduction zone communities compared to methanogenic zone communities . The majority of these sulfate-reducing bacteria were classified as Desulfobacterales, a lineage of sulfate-reducing bacteria that have previously been detected in methane seep sediments in the presence of ANME . This was found to be the case across all sites and regardless of geochemical zonation . Considering broader classifications of sediment lithologies, for example grouping all turbidites, did not alter this result . Furthermore, no correlation was observed between community similarity and the vertical distance between communities within the sediment column regardless of the site or geochemical zone examined . These results contrast a previous study of microbial communities in the Sea of Okhotsk, a marginal sea similar to the SCS, which found that communities inhabiting layered ash and clay sediments in a single core were distinct between sediment types . Within methanogenic sediments, however, communities in adjacent sediment horizons were not significantly similar to one another compared to other methanogenic zone communities . This finding was confirmed by examining the calculated dissimilarity between adjacent communities in the sulfate reduction and methanogenic zones in sites U1432 and U1433. The average dissimilarity between communities from adjacent sediment horizons was higher among communities in the methanogenic zone than among those in the sulfate reduction zone . In addition, neither the difference in TOC content nor isotopic depletion between interfacing sediments lithologies was correlated with the dissimilarity between adjacent communities regardless of geochemical zone . The broad applicability of this finding remains to be seen given the limited number of adjacent communities that were sampled from cores that penetrated both the sulfate reduction zone and the methanogenic zone, the limited number of sites examined for this relationship, and the fact that several types of sedimentological interfaces were only sampled once in this study.Interestingly, the degree to which microbial communities inhabiting adjacent sediment horizons diverged was related to the geochemical zone in which the sediments were found. Within sulfate-replete sediments, communities in contacting sediment horizons were significantly similar to each other and distinct from communities from other sulfate reduction zone communities (PERMANOVA Atribacteria, Chloroflexi, Nitrospira, and Aerophobetes between sites U1432 and U1433 and site U1431. All of these taxa have previously been implicated in organic matter degradation in marine sediments, suggesting that differences in the availability or reactivity of organic matter across the basin may play a role in structuring the biogeography of the SCS.Microbial communities inhabiting seemingly homogeneous sediments within marginal seas have previously been found to be conserved across sites within the same basin yet distinct between basins (Liu et al., Sedimentation rate may serve as a proxy for the relative availability of organic matter in marine sediments. Sedimentation rate has previously been demonstrated to exert a primary control on microbial biomass and its decay with depth in marine sediments globally (Kallmeyer et al., Atribacteria, Aerophobetes, Dehalococcoidia, and Nitrospira, were negatively correlated with sediment age across the SCS (Figure Sediment age may also reflect the energetic potential of the sediment column, since microorganisms preferentially oxidize labile over recalcitrant organic matter in recently deposited sediments (Westrich and Berner, S Figure . Thus seEcological models of community assembly have not previously been applied to microbial communities in the deep marine subsurface. Microorganisms in this environment are thought to be dispersal-limited due to the presence of a sediment matrix as well as energetic limitations. Communities in vertically adjacent sediment horizons were more similar in sulfate reduction zone sediments than in methanogenic zone sediments, indicating a potential role for dispersal under moderately favorable energetic conditions. However, this is counterbalanced by the finding that changes in community composition were not correlated with the vertical distance between communities, which suggests that dispersal is not a limiting factor for microbial community distribution within the sediment column or that the community is under such a high degree of connectivity that vertical patterns are lost. Disturbance, which has been found to play a significant role in structuring macrofaunal communities (Supp and Ernest, MG, AT, CZ, HL, and FC contributed to study conception and design. GD and MB performed DNA extraction and amplification. JJ performed sedimentological characterization and TOC measurements. MG, GD, MB, and AT performed statistical analysis. MG, AT, JJ, HL, and FC contributed sections to the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C/EBPβ expression was reduced by α-MSH and Foxc2. FFA level was elevated by α-MSH and pc-Foxc2 treatment along with increased FAO in white and brown adipocytes. The expression of FAO key enzymes, medium-chain acyl-CoA dehydrogenase (MCAD) and long-chain acyl-CoA dehydrogenase (LCAD) were increased in α-MSH and pc-Foxc2 group. Combination of α-MSH and Foxc2 treatment synergistically promoted FAO through increasing the activity of CPT-1 and phosphorylation of ACC. We found C/EBPβ bind to MC5R and Foxc2 promoter regions and inhibited FAO. cAMP level was increased by α-MSH and Foxc2 individually treated or combined treatment. Furthermore, cAMP/PKA pathway-specific inhibitor (H89) blocked the FAO, despite in α-MSH and Foxc2 both added group. While forskolin, the cAMP agonist, promoted FAO and enhanced the effect of α-MSH and Foxc2. Collectively, α-MSH and Foxc2 mutual promote FAO in WAT and BAT via cAMP/PKA signal pathway. And C/EBPβ as a transcription suppressor inhibits α-MSH and Foxc2 expression and FAO.Alpha melanocyte stimulating hormone (α-MSH) and Forkhead box C2 protein (Foxc2) enhance lipolysis in multiple tissues. However, their relationship in adipose fatty acid oxidation (FAO) remains unclear. Here, we demonstrated that α-MSH and Foxc2 increased palmitate oxidation to CO Obesity, type 2 diabetes, cardiovascular and cerebrovascular diseases are increased risks for human health. Promotion of cellular lipid metabolism comes to be a valid therapeutic strategy to treat these diseases2357et al.2021Melanocortins also affects energy homeostasis by acting on peripheral tissues. Alpha melanocyte stimulating hormone (α-MSH), produced by proopiomelanocortin (POMC) neurons, act via the interaction with its receptors in reducing food intake and increasing energy consumption of the peripheral tissues and organs910161718Forkhead box C2 protein (Foxc2) is a member of the forkhead/winged helix transcription factor family in various organs and tissues, such as cardiac system, mammary gland, liver and adipose tissue. Our previous studies found Foxc2 enhanced mice pre-adipocytes proliferation and inhibited apoptosis via activating Akt/mTORC1 signaling pathway2426Here, we provide evidences for the profound effects of C/EBPβ in α-MSH-mediated and Foxc2-mediated fatty acid oxidation in both white and brown adipose tissues. Furthermore, we show that cAMP/PKA signal pathway which activated by α-MSH and Foxc2 is essential in stimulating FAO in adipose tissues.14C]-palmitate into different adipose tissues  showed α-MSH injection remarkably produced a 0.3-fold to 0.5-fold decrease in the palmitate incorporation into iWAT, eWAT and iBAT . α-MSH also elevated the activity of CPT-1, and increased fatty acid oxidation most effectively during lipolysis in white and brown adipose tissues. Meanwhile, α-MSH elevated the mRNA levels of MC5R and Foxc2. These data suggested α-MSH exhibits positive regulation on fatty acid oxidation in adipose tissue and Foxc2 effects during this process.α-MSH also control energy expenditure and lipid metabolism in peripheral tissues and organs, especially in adipose tissue by binding to specific receptors3435202014C]-palmitate incorporation into white and brown adipose tissues. CPT-1 activity and phosphorylation of ACC were increased in iWAT and iBAT. This effect was more obvious in iBAT which has more energy expenditure and thermogenesis. Our results indicated α-MSH and Foxc2 had the same effects on fatty acid oxidation in white and brown adipose tissues.Foxc2 is originally found in mice embryo trunk, head, and limbs in the mesoderm26402 production. By treating white and brown adipocytes with α-MSH and Foxc2, we observed that these two factors combined together to promote FFA release and increase palmitate oxidation to CO2. The mitochondrial UCP, a distinctive and specific marker for FAO, has been a major factor on the transcriptional control of MCAD, LCAD and other-oxidation enzymes in vivo44Mitochondrial β-oxidation is one of the major biochemical factors to maintain adipocytes function in both white and brown adipocytes42C/EBPβ plays an important role in pre-adipocyte differentiation and adipogenesis461949In summary, our study provided new insights into the mechanisms required for the regulation of α-MSH and Foxc2 on fatty acid oxidation in white and brown adipose tissues. Our data indicated α-MSH and Foxc2 were mediated by cAMP/PKA activation, and C/EBPβ was a novel transcriptional suppressor of MC5R and Foxc2 . Our finad libitum water and standard laboratory chow diet purchased from Animal Center of the Fourth Military Medical University. Animal room was maintained at 25 ± 1 °C, humidity at 55 ± 5%, and a 12 h light/12-dark cycle. Four-week old mice were injected either PBS or α-MSH (500 nM) intraperitoneally once a day for two days before dark cycle. Inguinal adipose tissue (iWAT) was removed at 0.5 h, 1 h, 2 h, 4 h and 6 h after the last injection from the treatment group. Vehicle, si-Foxc2 and pc-Foxc2 were administered as a daily intraperitoneal injection for 6 days. Tissue samples of epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), and interscapular brown fat (iBAT) pads were removed for future studies.Three-week old C57BL/6J male mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University . All mice handling methods and all experimental protocols were conducted following guidelines and regulations approved by the Animal Ethics Committee of Northwest A&F University. Mice were provided 4 cells/dish, and incubated at 37 °C under a humidified atmosphere of 5% CO2 and 95% air until confluence. Differentiation of white pre-adipocytes was performed as following. Cells grown to 100% confluence (Day 0) were exposed to the induction DMEM/F12 containing dexamethasone , insulin , IBMX  and 10% FBS. Four days after the induction (from Day 2), cells were maintained in the induction medium containing insulin  and 10% FBS until the day for cell harvest. And brown adipocytes were induced to differentiate using DEME/F12 with 15% FBS, 0.5 mM IBMX; 0.25 mM indomethacin; 2 μg/mL dexamethasone; 1 nM T3, 20 nM insulin for 2 days and subsequently maintained on differentiation media .For collected iWAT samples, the connective fiber and blood vessels were removed and washed three times with PBS buffer containing 200 U/mL penicillin  and 200 U/mL streptomycin . The adipocyte culture was carried out according to our previous publication. Briefly, pre-adipocytes were seeded onto 35-mm culture dishes at a density of 8 × 109 IFU/mL for Foxc2. The in vivo experiments were performed after 6–8 days of infection. H89 and Forsklin were bought from Selleck.cn . Thirty minutes before α-MSH stimuli, H89 (10 μM) or Forskolin (10 μM) were used to treat adipocytes. cAMP was measured using the FlashPlateTM method according to the FlashPlate  protocol.α-MSH was purchased from Sigma , and the working solution (500 nM) was prepared to treat adipocytes for 1 h. The recombinant adenovirus overexpression vector of Foxc2 (pc-Foxc2), recombinant lentiviral interference vector of Foxc2 (si-Foxc2), and lentiviral interference vector of MC5R (si-MC5R) were constructed in our lab. The overexpression plasmid vector of C/EBPβ (pc-C/EBPβ) and interference vector of C/EBPβ (si-C/EBPβ) were constructed by the Shanghai GenePharma Co., Ltd . The adenovirus and lentiviral recombinant vectors were infection in a titer of 1 × 102 by tissue homogenate and the incorporation of palmitate into lipids were measured according to previous published method51525314C] palmitic acid and measured for trapped CO2. For in vivo study of palmitate oxidation to CO2, we used the modified method from Sebastián and our pervious study552 measurement, a parafilm-sealed system was used. The reaction was stopped by the addition of 40% perchloric acid through a syringe that pierced the parafilm. Serum and adipocytes triglycerides (TG) and free fatty acids (FFA) contents were determined by using commercial ELISA kits  according to the instructions.Palmitate oxidation to CO3H]carnitine) with or without different levels of malonyl-CoA. Samples were incubated at 37 °C for 3 min. And then stopped by the addition of 0.5 ml of ice-cold perchloric acid. After mitochondrial centrifuged, the mitochondrial pellet was then resuspended in ddH2O and extracted with 600 μL of butanol 300 μL of the butanol-phase was counted by liquid scintillation.The CPT-1 activity assay was performed as described by Pawel and BremerSerum α-MSH, CPT-1, MCAD and LCAD were measured using commercial ELISA kits . Adipose tissues were collected as described previously. Cells were collected and disrupted by ultrasonication .Adipocytes were prepared for chromatin immunoprecipitation (ChIP) assay using a ChIP assay kit  according to the manufacturer’s protocol. Crosslinking was reversed, and purified DNA was subjected to Real-time PCR with SYBR green fluorescent dye .Four fragments containing Foxc2–5′ sequences from −1100 to −264 relative to the transcription initiation site were sub-cloned into pGL3-basic vector . HEK293T cells were cultured in 24-well plates and co-transfected with Foxc2 promoter plasmids and pGL3-basic plasmid (control reporter). Cells were harvested 48 h after transfection, and detected using the Dual-Luciferase Reporter assay system . The MC5R promoter reporter assay and luciferase reporter assay were performed as the same protocol. And the four fragments containing MC5R-5′ sequences were from −1200 to −210 relative to the transcription initiation site.−ΔΔCt.Total RNA from adipose tissues and cells were extracted with TRIpure Reagent kit . 400 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase kit . Primers were synthesized by Shanghai Sangon Ltd . Quantitative PCR was performed in 25 μL reaction system containing specific primers and SYBR Premix EX Taq . The levels of mRNA were normalized using β-actin. The expression of genes was analyzed by method of 2Mice pre-adipocytes and adipose tissues were solubilized in lysing buffer. The solubilization was preceded for 40 min at 4 °C, then the solution was centrifuged at 12,000× g for 15 min at 4 °C, and the supernatants were used for determination of protein concentration. Protein samples (30 μg) were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus, and transferred to PVDF nitrocellulose membranes , blocked with 5% skim milk powder/Tween 20/TBST at room temperature for 2 h. The membranes were then incubated with primary antibodies against CPT-1, p-ACC, ACC, Cidea, PGC1-α, FABP3, FABP4, UCP1, UCP2 and GAPDH  at 4 °C overnight. Following this step, the appropriate HRP-conjugated secondary antibodies  were added and incubated for 2 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate , and then the blots were quantified using ChemiDoc XRS system .p < 0.05 was considered to be significant.Statistical analyses were conducted using SAS v8.0 . Data were analyzed using either one-way ANOVA or two-way ANOVA. Comparisons among individual means were made by Fisher’s least significant difference (LSD). Data were presented as mean ± SD. How to cite this article: Gan, L. et al. α-MSH and Foxc2 promote fatty acid oxidation through C/EBPβ negative transcription in mice adipose tissue. Sci. Rep.6, 36661; doi: 10.1038/srep36661 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Social media is increasingly used by patients to source information for managing chronic disease. The aim of this study was to understand patient information needs about gout by a content analysis of questions posted on the social news website Reddit.We analysed questions posted onto the ‘Gout sufferers unite’ subreddit site. Two reviewers coded questions into categories (inter-reviewer kappa 0.70), with discordant coding resolved by a third reviewer. Data were analysed by calculating the frequency of questions within the categories. Where relevant, categories were further separated into sub-categories to allow organisation and interpretation of the data.We analysed 359 questions in 287 posts by 213 individuals. A wide range of questions arose. The single most common category related to uncertainty of diagnosis (22.3% questions), with questions about disease management common. Information-seeking about medications was generally cautious, with questions about side-effects, risk of flares after starting urate-lowering therapy, and decision to start urate-lowering therapy. Community users experiencing flares posted questions about flare management, including medications, sometimes in real-time. Dietary management questions included the effectiveness of dietary changes as a management strategy, choice of alcoholic beverage, and weight loss strategies. Questions about serum urate levels were rare (2.8% questions).Questions about gout posted on the subreddit site most often related to uncertainty about symptoms and disease management strategies, with infrequent questions about serum urate testing, results or targets. These findings may inform development of strategies to address the information needs of people with gout. Gout is a chronic disease of monosodium urate crystal deposition that typically presents as recurrent flares of painful inflammatory arthritis . It is tGout is poorly controlled worldwide, with low adherence to urate-lowering therapy and infrequent achievement of serum urate target . Strategreddit.com have appeared where registered users can submit content and seek advice. In 2016 Reddit had 542 million monthly visitors and was ranked the 25th most visited website in the world with 8 billion page views per month [Social media is widely and increasingly used by patients to source information about disease . As the er month . Within While it is clear patients are increasingly using social media platforms to inform disease management, very little research has examined the types of questions patients seek answers for on these platforms. The aim of this study was to understand patient information needs about gout by a content analysis of questions posted on Reddit.We analysed questions posted onto the ‘Gout sufferers unite’ subreddit. This open access subreddit has existed for five years, and had 1811 ‘Gout friends’ at the time of writing. We analysed questions in the “Hot” section of the subreddit posted from September 4th 2015 to August 23rd 2016. Posts were extracted from the website with information including title, text, username, and questions in the text. Posts that did not include a question were not extracted for analysis.A sample of questions were initially reviewed by four reviewers , and 13 categories (themes) were agreed upon. Two reviewers (CD and AS) then coded all questions into the categories. Kappa values were analysed using SPPS v23 . Kappa (SE) for inter-reviewer agreement was 0.70 (0.026). Discordant coding was resolved by review by a third author (ND). Data were analysed by calculating the frequency of questions with the categories. After coding and agreement, categories were reviewed and where relevant, further separated into sub-categories to allow organisation and interpretation of the data.According to the University of Auckland Human Participants Ethics Committee policy, this work did not require ethical approval, as it involved analysis of publically available data. Consent was not obtained from individuals or Reddit, as the posts are available in the public domain and were made voluntarily from individuals who could not be identified.We analysed 359 questions in 287 posts by 213 individuals. The majority of individuals  submitted a single post. The mean number of posts per individual was 1.34 (range 1–8). For most posts, it was not possible to determine disease duration of gout, but a new or very recent diagnosis was mentioned by 41/213 (19.2%) users. A wide range of questions arose. The single most common category was about uncertainty of diagnosis (22.3% questions), with management categories  also common  questions about medications. These included questions about specific named drugs, most commonly allopurinol (33 questions) and colchicine (15 questions). Questions about medications were generally cautious or negative, with key sub-categories of medication side effects, risk of flares after starting urate-lowering therapy, and deciding whether to start urate-lowering therapy. Community users experiencing flares posted questions about medications for flare treatment in real-time. Examples of questions are shown in Table % questioThere were 44 (12.3%) questions about dietary management. Sub-categories were questions about general dietary strategies, effectiveness of dietary changes as a management strategy, choice of alcoholic beverage, and the best weight loss strategies for people with gout. Examples of questions are shown in Table There were 37 (10.3%) questions about non-pharmacological management, including questions about the benefits of supplements, the role of activity during flares, choice of footwear, and topical treatments during a flare. Less frequent questions related to exercise strategies, devices and mobility aids during walking, and the value of blood donation or sex to improve gout.There were 13 questions that specifically related to supplements. These supplements included curcumin, theracurmin, multivitamin, black cherry, vitamin C, “enzyme” supplements, herb gel, and alkaline water. Questions about several supplement combinations marketed for urate or gout were also posted including: ‘Gouch’ , celery seed extract, stinging nettle root, ‘Urcinol’ , and ‘Uric Acid Support’ .Really open to suggestions... for anything, really…… I also know so little about gout, so any information is good information, tbh.”). Requests for non-specific advice were also made during the initial flare of disease There were 37 (10.3%) non-specific questions regarding gout management posted. These questions often included a detailed narrative about the course of the person’s experience, with a question requesting general advice from the community , and questions about financial and employment issues such as negotiating health insurance systems, disclosure of the condition to an employer, and the ability to work once a diagnosis of gout has been made. There were also questions about sourcing of expensive medications (“They gave me a script for colchicine and i don't have insurance. ….. Any idea on how to get more in the future for cheaper?”). Questions about reliable information primarily related to electronic sources or apps that can be used to help manage gout.A number of other categories were less frequently observed, with <10% questions per category (Table This analysis of questions posted on the social media website Reddit shows uncertainty about symptoms and management strategies for gout are common. The questions show that medications, particularly urate-lowering therapy, are often viewed negatively, with concern about side effects and the balance between risks and benefits. In this online community sample, there was a strong interest in non-drug strategies, including many different dietary solutions and supplements.The most common single category was uncertainty about symptoms, particularly for attacks that did not conform to typical patterns. A recent detailed symptoms analysis of patients with crystal proven gout has shown that many patients do not present with stereotypical attacks, and that flares may affect more than just ‘classical’ joints such as the 1st metatarsophalangeal joints . It is aManagement categories were also common, emphasizing the information needs for patients for general management advice, medication advice, and non-pharmacological advice. The community users demonstrated concerns about long-term medications, and it appeared that the balance of benefits and potential side effects for some users was uncertain. The concern about flares during initiation of urate-lowering therapy highlights the need for further discussion with patients about the long-term benefits of ULT and efforts to reduce flares during initiation using anti-inflammatory prophylaxis .There was a strong focus on non-drug management, including questions about the role of many dietary factors and supplements, including many with no reported benefits for gout. These findings are consistent with the lay view of gout as being caused by overindulgence in rich food and alcohol . It is aNotable from the analysis of the postings was that there were very few questions concerning serum urate testing, results or targets. Given that serum urate lowering is the central strategy for long-term effective management of gout, these data suggest that information about this fundamental aspect of gout management has not widely penetrated the gout patient community. Together, these findings highlight the importance of changing prevailing social narratives of gout from a self-inflicted condition that can be primarily managed through dietary modification, to contemporary concepts of gout as a chronic disease of urate crystal deposition that can be effectively managed through complementary pharmacological and non-pharmacological urate-lowering strategies .Study limitations include the lack of community user information. Reddit community users are known only by their usernames, and demographic and disease details are not available. Therefore, it is not possible to determine whether the questions are consistent with those of all people with gout, or indeed whether all the community users truly have the disease. Furthermore, disease duration was not available for all community users with gout, and we are unable to systematically determine whether the community user had a new diagnosis of gout, or had longstanding disease. It is possible that the information needs of Reddit community users do not represent all people with gout . This stThis analysis of questions posted on Reddit highlights a number of information needs for people with gout. The key findings are uncertainty about symptoms and questions about disease management, both pharmacological and non-pharmacological. These findings may inform development of strategies to address the information needs of people with gout, with the ultimate goal of improving patient and community understanding about the disease and its management.


The differential diagnosis of immune (ITP) and hereditary macrothrombocytopenia (HM) is key to patient management. The immature platelet fraction (IPF) represents the subset of circulating platelets with higher RNA content, and has been shown to distinguish hypo- from hyperproliferative thrombocytopenias. Here we evaluated the diagnostic accuracy of IPF in the differential diagnosis between HM and other thrombocytopenias in a population of patients with post-chemotherapy thrombocytopenia (n = 56), bone marrow failure (n = 22), ITP (n = 105) and HM (n = 27). TPO levels were also measured in HM and ITP matched for platelet counts. Platelet counts were similar in all patient groups. Higher IPF values were observed in both ITP  and HM  compared to hypoproliferative thrombocytopenias. IPF values were also higher in HM compared to ITP, yielding a diagnostic accuracy of 0.80  to distinguish these two conditions. Intra- and inter-assays reproducibility of IPF in HM patients revealed that this is a stable parameter. In conclusion, IPF is increased in HM compared to both ITP and other thrombocytopenias and contributes to the differentiation between ITP and HM. Further studies are warranted to understand the biological rationale of these findings and to its incorporation in diagnostic algorithms of HM. Recently, the feasibility of using parameters of the complete blood count (CBC) to support this differential diagnosis was illustrated by a series of studies which demonstrated and validated that the mean platelet volume (MPV) can help the segregation of patients with ITP and HM5.The differential diagnosis of thrombocytopenias includes a variety of conditions such as hematologic malignancies, bone marrow failure (BMF), hypersplenism, immune thrombocytopenia (ITP), microangiopathic hemolytic anemias and hereditary macrothrombocytopenia (HM). Among these, the differential diagnosis between ITP and HM can be challenging due to the absence of specific tests, particularly in patients with mild bleeding symptoms6. Initially, the IPF was measured by flow cytometry, and described as reticulated platelets7. Recently, studies have reported the clinical utility of measuring immature platelets in clinical settings using automated hematology analyzers10. The correlation between flow cytometry and hematology analyzers in quantifying this population has been previously demonstrated11.In recent years, new parameters were incorporated to the CBC, including the immature platelet fraction (IPF), which represents a population of newly formed platelets containing a greater amount of residual RNA12, less information is available about its use in the differential diagnosis between ITP and HM. In 2000, Fabris et al. evaluated reticulated platelets by flow cytometry in a population of 29 patients with HM, observing reduced IPF values when compared to ITP13. More recently, Miyazaki et al.14, in a study with 15 patients with HM in which the IPF was measured in an automated hematology analyzer observed significantly higher IPF values in HM compared to ITP. To further elucidate the value of IPF determination in the diagnosis of thrombocytopenias, with focus on the differential diagnosis between ITP and HM, we investigated the diagnostic accuracy and the precision of IPF measurements in a population of patients with different causes of thrombocytopenia, including a well-characterized cohort of patients with HM. In addition, we also evaluated whether thrombopoietin (TPO) levels could further facilitate this diagnosis.Although the utility of the IPF in the differential diagnosis between hypo- and hyperproliferative thrombocytopenias has been already reported15. The study population consisted of patients with thrombocytopenia confirmed in two independent samples and microscopic analysis, in regular clinical follow-up in the Hemostasis outpatient clinic of University of Campinas, or admitted to the hematology ward of the same institution. The inclusion criteria was a confirmed diagnosis of any of the following: (i) ITP (based on previously established guidelines)16; (ii) bone marrow failure (including aplastic anemia and myelodisplatic syndromes with a platelet count below 150 × 109/L)18; (ii) post-chemotherapy (Ctx) thrombocytopenia  with a platelet count below 150 × 109/L; and (iv) HM, with platelet counts below 150 × 109/L. Diagnosis of HM was established by clinical and laboratory criteria as determined by international guidelines21. These included the exclusion of all other causes of thrombocytopenia, objective confirmation of thrombocytopenia in first-degree relatives, platelet aggregation studies, molecular analyses and specific tests such as platelet glycoprotein studies and electron microscopy in selected cases. Exclusion criteria included: a platelet count above 150 × 109/L in ITP patients at the day of enrollment; (ii) the presence of conditions known to influence IPF values such as sepsis and other inflammatory diseases22, or (iii) the use of antiplatelet agents. The study was approved by the Institutional Review Board of University of Campinas (certificate number 411.620/2013) and performed in accordance with the Declaration of Helsinki. All patients provided written informed consent prior to enrollment.This was a cross-sectional diagnostic accuracy study, designed according to STARD (Standards for the Reporting of Diagnostic accuracy studies) guidelinesRecruitment occurred between July 2013 and February 2015. ITP, HM and BMF patients were included consecutively, aiming for an enrollment of 100 patients with ITP, 20 with BMF and all patients with HM. Post-Ctx patients were included using a convenience sampling strategy, which consisted in weekly visits to the hematology ward, with a target of 50 patients.Samples were collected by venipuncture by the same nursing team responsible for the collection of routine clinical samples, and using the same standard operating procedures in both clinical sites. Blood was drawn in Vacutainer® EDTA K2 tubes . IPF measurements were performed within four hours from sample collection, which is the threshold suggested in the equipment operational procedures. After IPF analysis, tubes were centrifuged , and plasma aliquots were frozen at −80 °C until analysis.10. Finally, we also evaluated the intra- and inter-assay reproducibility of IPF measurements in a subgroup of patients with HM. Platelet counts and mean platelet volume (MPV) were obtained from the same samples, by the same hematological analyzer, which measures the MPV by impedance. In the laboratory where IPF measurements were performed, commercial internal quality control samples  are run before the beginning of each 8 h-routine, as well as after any corrective or preventive maintenance of the hematological analyzer. In addition, the laboratory participates in a regular national external quality control program and is within the scope of the University laboratory quality management system.Whole blood samples were analyzed in a Sysmex XE 5000 hematology analyzer . Briefly, the IPF fraction was identified in the reticulocyte channel, employing a fluorescent dyes containing polymethine and oxazine. These dyes penetrate the cell membrane, staining residual RNA of red blood cells (reticulocytes) and platelets. In addition, these two populations are separated by cell size. A computer algorithm then discriminates mature platelets and the IPF, which was expressed as a percentage of total platelets (IPF%). In addition, we also calculated the absolute count of the IPF (A-IPF), which has been previously reported as superior to the IPF% in some clinical settings9/L were used as a control group for the measurement of TPO levels. All of these patients were off-ITP treatment for at least one year and had platelet counts above 250 × 109/L at the time of testing.Circulating TPO levels were measured in plasma samples using a commercial immunoassay , based on a sandwich ELISA technique, according to the manufacturer’s protocol, in all HM patients and in seventy-four ITP patients. Of note, none of these patients were using TPO receptor agonists. A group of eight patients with a history of ITP diagnosis that were not excluded from the study due to platelet counts above 150 × 10Results are expressed as median and range, or means ± SD, unless mentioned otherwise. Differences between quantitative variables were evaluated by Mann-Whitney or Kruskal-Wallis with Dunn’s post-test for comparison with 2 or more than 2 variables respectively. Associations involving categorical clinical variables were analyzed using the Fisher exact test. Correlation analyses were performed using the Pearson or Spearman correlation (rho) test, according to data distribution. The diagnostic accuracy of IPF for the segregation of patients by each diagnosis was estimated by the ROC (receiver operating characteristic) procedure, which allows the simultaneous analysis of sensitivity and specificity of each test in relation to selected clinical outcomes. Results are reported with confidence intervals and level of significance. A P value < 0.05 was considered statistically significant.9/L . In total, 210 patients were analyzed, divided in the following subgroups: Post-Ctx (n = 56), BMF (n = 22), ITP (n = 105) and HM (n = 27). Demographic and clinical characteristics are shown in Table 5. In all other samples, IPF was normally reported. Of note, no differences were found in platelet counts between ITP and HM patients.Two-hundred forty-eight patients were enrolled in this study, of which 38 were excluded due to platelet counts above 150 × 10Similar IPF values were observed between patients with Post-Ctx thrombocytopenia and BMF. All other comparisons yielded significant differences in IPF between different patient groups Fig. , with boAn inverse correlation between IPF and platelet counts was observed in BMF, HM, and ITP, but not in Pos-Ctx patients Fig. . IPF valThe diagnostic accuracy of IPF% and A-IPF measurements for the differential diagnosis of thrombocytopenia was estimated using ROC analysis. The AUC for the differential diagnosis between ITP and HM was 0.80 . Using an arbitrary IPF% cut-off value of 17.4%, which presented the best combination of sensitivity and specificity, ITP could be distinguished from HM with a sensitivity of 70% and a specificity of 90%. The positive and negative predictive values were 81.48% and 71.43 respectively. A similar result was obtained using the A-IPF, which yielded and AUC of 0.77 . Therefore, all additional analyses were performed using the IPF%.23 despite the former group having much lower platelet counts than the latter. Even when compared to HM patients with similar levels of thrombocytopenia, ITP patients presented significantly lower TPO levels  or Bernard-Soulier syndrome , lower IPF values were observed in the remaining patients classified as non-specified hereditary macrothrombocytopenia .Despite the low number of patients with HM, we performed a subgroup analysis to investigate whether IPF values differed between patients with different types of HM Table . While n4, a finding that was later validated in a multicenter study using different instruments to measure this parameter5. Using a population of 210 patients with different causes of thrombocytopenia, the main result of our study was that another platelet parameter derived from automated an hematology analyzer reliably differentiates HM from ITP, with an estimated diagnostic accuracy similar to that reported in the literature for MPV5.The differential diagnosis between ITP and HM represents a significant clinical problem since tests used to distinguish these two conditions are not available in most clinical laboratories, and even when available, cannot discern among all clinical etiologies. The development of hematological analyzers in the last decades has allowed the incorporation of new parameters in the diagnosis of hematological disorders; and the feasibility of this strategy for the diagnosis of HM has been shown by a series of studies evaluating platelet size in these patients. These studies demonstrated that MPV is significantly higher in patients with HM compared to ITP9/L for both groups). It should be noted that in contrast to other clinical settings, no relevant difference was observed in when IPF was reported as a percentage or in absolute values.In our population, IPF% values were significantly higher  in the HM group compared to hypoproliferative thrombocytopenias such as BMF and Post-Ctx. More importantly, IPF% values were higher in HM than ITP patients with similar platelet counts. An inverse correlation between platelet counts and IPF% was observed not only in ITP patients but also in patients with BMF and HM. The absence of such correlation in patients with Post-Ctx thrombocytopenia is most likely due to the blunted thrombopoietic response secondary to chemotherapy. The ROC analysis estimated significant diagnostic accuracies of IPF% and A-IPF in the differential diagnosis of ITP and HM even in a group of patients with similar levels of thrombocytopenia  compared to TPO. In regard to the fact that no correlation was found between TPO levels with either platelet counts or IPF in HM, this suggests that increased IPF is not associated with increased thrombopoietic activity in HM.25 is probably not sufficient to explain the 200–300% increase of IPF in these patients. In addition, formation of platelet aggregates was ruled out by the microscopic evaluation of blood smears, and by a review of the scatter plots of IPF measurements from all patients with HM. The strong correlation between IPF measurements performed one year apart suggests that the higher RNA content of these platelets could be a stable characteristic associated with the cellular mechanisms of thrombocytopenia in these patients. Cellular mechanisms responsible for thrombocytopenia in HM are complex and dependent on the molecular etiology of each disease entity. Increased counts of immature platelets were described in an animal model of HM due to RASA3 mutations in which platelet turnover is increased26. However, most of our patients presented with more classical forms of thrombocytopenia such as BSS and MYH9-related disorders in which thrombocytopenia is attributed to defects in proplatelet formation29. Interestingly, in MYH9 megakaryocytes, the physiological suppression of proplatelet extension exerted by the interaction with type I collagen is lost leading to premature or abnormal platelet release from the bone marrow29. We therefore speculate that the extremely high IPF values observed in most of our patients with HM are an expression of the premature release of platelets to the circulation. Interestingly, patients in whom the diagnosis of MYH9-related platelet disorders or BSS were confirmed by molecular or flow cytometry assays presented significantly higher IPF values than patients with a diagnosis of non-specified HMT. However, due to the limited number of patients in this subgroup analysis, these data should be considered preliminary. The confirmation of this hypothesis in future studies would provide a biological rationale to the use of IPF as a diagnostic tool in HM.The mechanisms underlying the increase of IPF in HM remain to be elucidated. The previously reported increase in platelet volume of around 50 to 100%23, our analysis demonstrated significantly lower TPO levels in ITP patients compared to control subjects with normal platelet counts. Nevertheless, suboptimal TPO increase did not preclude the demonstration of a marked negative correlation between TPO levels and platelet counts in ITP (rho = −0.50), which was even stronger when only patients using steroid were evaluated (data not shown). This suggests that in ITP, increase in TPO levels is blunted by yet unknown mechanisms which are apparently improved by steroid therapy.In accordance with the concept that ITP is a condition associated with suboptimal increases in TPO levelset al.13 or using automated hematological analyzers, as in the study by Myiazaki14 and ours. Few studies have formally evaluated the correlation of IPF measurements by these two methods but in two of the most recent ones, this correlation was limited to ITP patients30. Considering the challenges of standardizing platelet flow cytometry analyses compared to a CBC, we believe that it is fair to state that the former method should not be regarded as a gold-standard for IPF measurement. In fact, the vast majority of studies addressing other uses of IPF do not perform parallel flow cytometry analysis. Moreover, the concordance of our results with those from Myiazaki et al. in two independent populations supports the conclusion that IPF is increased in HM, possibly showing the premature release of platelets into the circulation. In addition, the analyzer used in our study measures MPV by impedance, which is a method that in patients with HM is known to report MPV results in less than half of the cases due to abnormalities in the platelet distribution curve5. This technical limitation was also present in our study, and precluded some interesting analyses such as the comparison of the estimated diagnostic accuracy of IPF and MPV for the differential diagnosis between HM and ITP, and the evaluation of the combined performance of IPF and MPV in this context. On the other hand, it illustrates the challenges of using the MPV as a diagnostic test for HM, highlighting the importance of additional assays such as the IPF. Interestingly, when MPV values from our patients obtained from the same sample in a different hematological analyzer (Advia 2120) were used, the estimated diagnostic accuracy for the differential diagnosis of HM and ITP  was lower than that of the IPF.Our study presents limitations that need to be acknowledged, with lack of flow cytometry data being possibly the most relevant. As mentioned earlier divergent results were obtained when the IPF was measured in HM patients by flow cytometry in the study by Fabris In conclusion, our results validate previous demonstrations that IPF is increased in HM in an independent and larger population of patients. We also demonstrate the reproducibility of this method, thus supporting the feasibility of its incorporation in the diagnostic approach of HM. Given the easy accessibility to this parameter for routine clinical laboratories, and the importance of differentiating HM and ITP, further studies are warranted to the incorporation of the IPF in diagnostic algorithms of HM, and to further investigate new questions raised by our study such as the mechanisms of IPF elevation in HM.