In this study we used a counterfactual approach to disentangle the effect of two closed areas on fishing location from other competing effects on the behaviour of the Indian Ocean tuna purse seine fishery. Our results revealed an inconsistent effect of the one of the closed areas between years, after taking into account the influence of environmental conditions on fleet behaviour. This suggests that the policy of closing the area per se was not the main driver for the fleet allocating its effort elsewhere. We also showed a marked difference in effect between the two closed areas resulting from their different locations in the fishery area. These findings highlight the need to account for other key fleet behavioural drivers when predicting or evaluating the contribution of area closures to achieving conservation and fishery management objectives.Spatial closures are widely used in marine conservation and fisheries management and it is important to understand their contribution to achieving management objectives. Many previous evaluations of closed area effects have used before-after comparisons, which, without controlling for a full range of factors, cannot ascribe changes in fleet behaviour to area closures  The use of area closures is commonplace in addressing problems of sustainability in fisheries management –3. ClosuIn the management of industrial tuna fisheries, area closures are most commonly used to reduce fishing mortality, either overall or for a particular component of the stock such as small or juvenile fish ,5. Shortper se. Policy change is only one of many factors that can cause or contribute to an observed management impact. Environment, economic and social forces also influence how fishing fleets behave. In offshore tuna fisheries, the physical environment is particularly important in determining the movement of target tuna in space and time, and safety and cost considerations play an important role in determining a fisher’s decision to visit a particular area. These forces, which are usually independent of any management action, may disrupt the intended effect of management. Thus, without accounting for these different effects and isolating the effect of policy, managers risk misinterpreting the relationship between an action and an outcome.This approach is helpful in acknowledging change but does not reveal the full picture of how a management action has performed. For instance, while a before-after analysis can identify whether a change in total catch has occurred, it cannot ascribe changes in catch to the use of an area closure http://ec.europa.eu/smart-regulation/guidelines/toc_guide_en.htm; accessed 27th February 2017) it has not been widely adopted for evaluation of fishery management interventions. Here, a counterfactual approach can be achieved by developing a predictive model of fishing effort allocation that accounts for a range of influences on fleet movement. By comparing the observed response of a fleet to a closure to predictions of how the fleet would have behaved if the closure had not been implemented it is possible to isolate the effect of closures on fishery dynamics. An interesting use of the counterfactual approach in a manner broadly similar to this is demonstrated by [A promising approach for diagnosing the reasons behind an observed change in a management indicator is to build up a counterfactual scenario. Counterfactual thinking considers what outcomes would have looked like in the absence of an intervention, yet while it is a common feature in some areas of policy impact evaluation  one-month fishery management closure off the Somalia coast, and the British Indian Ocean Territory (BIOT) marine reserve. We aimed to understand whether the policy of closing these areas Understanding the response of the purse seine fleet to the two area closures selected in this study is of interest for different reasons. In the case of the fishery closure, which was intended to temporarily reduce the fishing capacity of the purse seine fleet, it has been suggested that vessels may have reallocated effort in such a way as to maintain catches and therefore subvert the management outcome . In the http://www.iotc.org/English/resolutions.php; accessed 1st June 2013).The two closures examined differed in their size, location and intended management objectives. In November 2011 and 2012 the Indian Ocean Tuna Commission (IOTC) designated a one-month closure that extended from the Somali coast to 60°E and covered a large part of the productive northwest fishing grounds typically fished by the fleet during August-November. This region is a key fishing area for the purse seine fleet and is characterised by consistently very high catches, mainly around floating objects . The objIn late 2010 the British government designated their entire British Indian Ocean Territory a marine reserve. The BIOT reserve is positioned at the eastern periphery of the purse seine fishery area in a region typically fished during November-February and characterised by catches of free-swimming schools. Prior to the closure of BIOT, the importance of the area in terms of purse seine fishery production varied considerably from year to year, with the average proportion of total monthly catch taken within the Territory ranging from 0 to 23% . The designation of the BIOT reserve was not linked to regional fisheries management policy and whilst no formal management plan has yet been developed, several objectives of the reserve can be inferred, including the provision of a scientific reference site and near shore and pelagic biodiversity conservation .For the IOTC closure we focussed on the behaviour of the fleet during November, the month of the closure, in 2011 and 2012. For the BIOT reserve we focussed on the period December-January when the provision of fishing licences was historically highest . Fishing data were available until December 2012, allowing the analysis of two full fishing seasons. In both instances we chose to focus on the short term response of the fleet to the closures. Firstly, in a highly dynamic environment like the Indian Ocean it is difficult to accurately predict fleet behaviour in the long term (e.g. over a period of years), and any longer term effect of the closures on fishing location may be masked by other influences on fleet behaviour. Secondly, an understanding of the short term response of resource users is important for management decision making, which tends to operate over relatively short timeframes .We used the generalised additive model (GAM) described in  to generIn the absence of the closure the fleet was expected to have fished within the IOTC closure area in both years of its implementation. In this counterfactual scenario the predicted distribution of effort was noticeably different for the Spanish and French components of the fleet. The Spanish fleet component was expected to have fished across a large area extending from the Seychelles plateau in the south to the Omani coast in the north, with core fishing grounds predicted within the area of the IOTC closure. In comparison, the French fleet component was expected to have fished across a comparatively small area in subequatorial waters, with core grounds predicted on the Seychelles plateau to the south of the closure boundary . This diIn the observed scenario the fleet complied with the closure in both years of its implementation and fishing effort was allocated to the south and east of the closed area boundaries . There wIn the first year of the designation of the BIOT reserve the fleet was expected to have fished across a large area of the western Indian Ocean, with core grounds predicted along a subequatorial band extending as far east as BIOT . These cIn the observed scenario there was a marked difference in the allocation of effort by the French and Spanish components of the fleet in the first year of closure of BIOT. In the case of the Spanish fleet component, effort was mainly allocated in the Seychelles region as predicted, although some effort was allocated in the northwest Somali basin region . This suIn the second year of closure of BIOT the fleet mainly allocated effort throughout the Seychelles region, with very little effort allocated in the proximity of the BIOT closure. This largely corresponded with predictions of the model and thus suggests little disruption of fishing behaviour caused by the reserve.Retrospective analyses of the response of fishing fleets to closed areas are scarce, although from the handful of published examples a varied set of fleet responses has been demonstrated. For instance,  observedIn these examples, the reasons for the observed reallocation of effort around closed areas have generally been inferred and few studies have been able to disentangle the policy effect of a closure from other competing influences . This isper se caused the fleet to reallocate fishing effort. The construction of a counterfactual scenario proved to be crucial in isolating the effect of the closures from confounding environmental influences on effort allocation and our results showed a mixed and inconsistent closure effect on fleet behaviour. Our results also demonstrated the flexibility of the fleet in adapting to the closures, particularly its ability to explore new areas, and furthermore we identified varied responses within the fleet depending on flag nationality, suggesting that the impact of closures may vary for different components of the fleet.In this study we have demonstrated a counterfactual approach as a useful diagnostic tool in evaluating the performance of area closures. To our knowledge, this is the first use in the fisheries literature of a counterfactual approach for evaluation using model-based predictions, and therefore marks new territory. We did not focus on whether the closures achieved their stated fisheries and conservation objectives. Rather we focused on whether the policy of closing areas Of particular interest in our results was the inconsistent effect of the BIOT reserve on fleet behaviour. Comparison between the observed behaviour of the fleet and counterfactual predictions of effort allocation showed a response to the closure in the first year of its designation but not the second, most likely resulting from inter-annual differences in environmental conditions and hence the distribution of tuna schools. This result highlights the importance of placement of fixed closures in fishery systems characterised by high environmental variability where the complex suite of influences on fleet behaviour can make it difficult to predict or evaluate the contribution of a closure to achieving conservation and management objectives.A second key result was the varying magnitude of closure effect on the two fleet components observed for both closures. In the case of the IOTC closure, the Spanish fleet component showed the most marked change in behaviour, with a number of vessels fishing to the east of the closure in typically un-fished areas, whereas the French fleet component allocated effort mainly in traditional grounds to the south of the closure. This varied response is largely explained by differences in the seasonal movement pattern of the fleet components linked to fishing strategy, with Spanish vessels typically remaining in the northwest FAD-fishing grounds for a longer period than French vessels, which move into subequatorial free schools regions earlier. A switch in the relative impact on two fleet components was observed for the BIOT closure in 2010/11, when the French component showed a more evident response to the closure with considerable exploratory behaviour around the closure boundaries. This variation in response is probably again linked to company-level strategy and differences in the targeting of free and associated schools, with Spanish skippers belonging to FAD-orientated companies less inclined to search for free schools in the vicinity of the BIOT closure and the opposite true for French skippers.In this study we have examined the short-term response of the fleet to the two closed areas. It remains unclear how these or other similar closures might affect fishing opportunities over a longer period, including how any cumulative impact on catches in the long term might influence decisions at a company level to participate in the Indian Ocean fishery. Furthermore, we did not look at the overall effect of the IOTC and BIOT closures on catches of tunas or bycatch species and as such we cannot offer a conclusion as to the conservation efficacy of large offshore closures. The effect of closures on fish stocks can take years or decades to be demonstrated  and, as S1 TableAll variables were aggregated at monthly intervals and at a spatial resolution of 1° latitude/longitude.(DOCX)Click here for additional data file.


Interestingly, microglia activation by IL4 has been described to promote neuroprotective and neurorestorative effects, which might be important to slow the progression of neurodegenerative diseases. In the present study, we addressed the role of endogenous and exogenous IL4 during MPP+-induced degeneration of mDA neurons in vitro and further addressed the impact of IL4-deficiency on neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Our results clearly demonstrate that exogenous IL4 is important to protect mDA neurons in vitro, but endogenous IL4 seems to be dispensable for development and maintenance of the nigrostriatal system as well as MPTP-induced loss of TH+ neurons in vivo. These results underline the importance of IL4 in promoting a neuroprotective microglia activation state and strengthen the therapeutic potential of exogenous IL4 for protection of mDA neurons in PD models.Microglia are involved in physiological as well as neuropathological processes in the central nervous system (CNS). Their functional states are often referred to as M1-like and M2-like activation, and are believed to contribute to neuroinflammation-mediated neurodegeneration or neuroprotection, respectively. Parkinson’s disease (PD) is one the most common neurodegenerative disease and is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons in the substantia nigra resulting in bradykinesia, tremor, and rigidity. Interleukin 4 (IL4)-mediated M2-like activation of microglia, which is characterized by upregulation of alternative markers Arginase 1 (Arg1) and Chitinase 3 like 3 (Ym1) has been well studied  Our results clearly demonstrate that IL4 is important to protect mDA neurons in vitro but seems to be dispensable for MPTP-induced loss of TH+ neurons in vivo.Microglia are the resident immune cells of the central nervous system (CNS), and thus are involved in a plethora of physiological as well as neuropathological conditions . Similarin vitro , the roland IgG1 . Moreoveand IgG1 . CNS-derand IgG1 . In the ad libitum access to food and water. C57BL/6-IL4tm1Nnt/J mice as described by C57BL/6 mice were housed at 22 ± 2°C under a 12 h light/dark cycle with n = 37, IL4 KO n = 27). All procedures involving MPTP were conducted in strict accordance with published safety and handling guidelines (Adult (10–12 weeks) male wild type (WT) and IL4 KO mice were intraperitoneally injected with 20 mg/kg MPTP hydrochloride (Sigma-Aldrich) dissolved in 0.2 ml phosphate-buffered saline (PBS) once a day for three consecutive days as previously described . Mice weidelines .2 culture flasks. Cultures were kept in a 5% CO2/95% humidified atmosphere at 37°C. After 10–14 days in culture, microglia were shaken off (250–300 rpm for 1 h) from adherent astrocytes and were plated according to the experimental purposes.Primary microglia cultures were generated as previously described . Brieflyg for 4 min. Cell pellets were resuspended in DMEM/F12 medium (Gibco) containing either 10% FCS, 10% horse serum (HS) and 1% penicillin/streptomycin (Invitrogen) to obtain mixed neuron-glia cultures, or N2 supplement (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) to obtain neuron-enriched cultures. Both cultures were plated on poly-D-lysine-coated (Sigma-Aldrich) glass coverslips (12 mm diameter) at a density of 1–2 midbrains/coverslip. On day in vitro 1 (DIV1) medium was refreshed and mixed neuron-glia cultures were maintained for additional 7 days to yield mature cultures. Neuron-enriched cultures were treated from DIV1 according to the experimental design. Cultures were kept in a 5% CO2/95% humidified atmosphere at 37°C. MPP+ (Sigma-Aldrich) treatments were performed as described recently (Neuron-enriched and mixed neuron-glia cultures from embryonic day 14 (E14) ventral midbrains of NMRI mice (Charles River Laboratories Inc.) were generated as described previously . Dissectrecently . IL4  obtained from Ebiosciences . Functionality and optimal concentrations were confirmed in IL4-treated primary microglia cultures and the IL4 nAB was used in mixed neuron-glia cultures at a dilution of 1:50.Total RNA was isolated from primary microglia using TRIzol reagent  according to manufacturer’s instructions. RNAs from SN and caudate putamen (CPu) were isolated after dissection and transfer to RNA later (Ambion). Tissues were homogenized in peqGOLD TriFast (PeqLab) using a Precellys 24 homogenizer (PeqLab). RNA concentration and quality was determined using the NanoDrop 2000 . 1 μg RNA from each sample was reverse transcribed to cDNA using RevertAid  according to the manufacturer’s instructions.TM system  in combination with the Quantitect SYBR Green PCR Kit . 1 μl of cDNA template was used in 25 μl reaction mixture. Results were analyzed using the Bio-Rad iQ5 Optical System Software and the comparative CT method. All data are expressed as 2-ΔΔCT for the gene of interest normalized to the housekeeping gene Gapdh and presented as fold change relative to controls. The following primers have been used throughout this study: Arg1for 5′- AACACTCCCCTGACAACCAG-3′, Arg1rev 5′-CTGAAAGGAGCCCTGTCTTG-3′ [NM_007482.3], Igf1for 5′-CTGGACCAGAGACCCTTTGC-3′, Igf1rev 5′-GGACGGGGACTTCTGAGTCTT-3′ [NM_010512], IL4for 5′-ATTTTGAACGAGGTCACAGGAGAAG-3′, IL4rev 5′-ACCTTGGAAGCCCTACAGACGAG-3′ [NM_021283.2], Gapdhfor 5′-GGCATTGCTCTCAATGACAA-3′, Gapdhrev 5′-ATGTAGGCCATGAGGTCCAC-3′[NM_001289726].Semiquantitative RT-PCR  was performed using the PCR Red-Mastermix 2x . PCR products were separated using agarose gel electrophoresis and visualized after staining with GelRed (Genaxxon Bioscience). Images were captured using a Biometra  gel documentation station. Quantitative RT-PCR was performed using the MyiQ+-treated groups after 48 h as well as in medium from primary microglia cultures from control and MPP+-treated groups after 24 h using the murine IL4 ELISA Development Kit . IGF-1 was detected in serum-free culture medium from control IL4-treated primary microglia cultures using the murine IGF-1 ELISA Development Kit (Peprotech). ELISAs were performed according to the manufacturer’s instructions. Color reactions were performed using 2,2 azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) substrate  for 30 min in the dark. Finally, absorbance was detected using Multiskan FC plate reader (Thermo Fischer) at the absorption of 405 nm. Concentrations of IL4 and IGF-1 were calculated from standard curves using the GraphPad Prism5 software (GraphPad Software Inc.).Interleukin 4 was detected in serum-free culture medium from E14 mixed neuron-glia cultures from control and MPPNumbers of tyrosine hydroxylase (TH) positive neurons in E14 ventral midbrain neuron-enriched as well as mixed neuron-glia cultures were determined after immunocytochemistry. After incubation of neuron cultures for different time points, coverslips were washed once with PBS and fixed using 4% paraformaldehyde in PBS for 15 min at room temperature (RT). Cells were washed three times with PBS for 5 min each, and subsequently blocked and permeabilised for 1 h at RT in PBS containing 10% normal goat serum  and 0.1% Triton-X-100 (Roche Diagnostics). Primary antibodies rabbit anti-TH , anti-Iba1  and anti-Gfap  were incubated overnight at RT. Afterward, coverslips were washed three times with PBS, and cells were incubated with goat-anti-rabbit peroxidase-conjugated secondary antibodies  for 1 h at RT. The immunoreactivity was visualized using diaminobenzidine  as described by + mDA neurons, Iba1+ microglia and Gfap+ astrocytes were estimated according to the optical fractionator methods using the Stereo Investigator software . Cell counting was performed using an oil immersion 63 × objective , counting frame of 50 μm × 50 μm and a grid size of 120 μm × 100 μm. The optical fractionator was optimized to reach a coefficient of error ≤0.1. For each animal four sections with an evaluation interval of three were selected and labeled cells were counted. The nucleus of each immunoreactive cell was chosen as the counting event. Cell numbers are either presented as total estimated cell numbers or percentages (MPTP model) compared to PBS-treated control animals.Numbers of THg for 5 min at 4°C and filtered through 0.2 μm filters. DA concentrations were detected using a HPLC pump , an autosampler  and an amperometric detector . The isocratic mobile phase consisted of sodium acetate (90 mM), citric acid monohydrate (60 mM), ethylene- diaminetetraacetic acid (2 mM), 1-octanesulfonic acid (3 mM), and 8% methanol and was adjusted to a pH of 5.3. DA was separated on a Prontosil 120-3-C18 AQ column  by using a fixed flow rate of 300 μl/min. Data were registered and analysed by using Chromeleon 6 software (Dionex).Dopamine levels were quantified as recently described . Striatat-test. Normality of samples was assessed using the D’Agostino and Pearson omnibus normality test (as recommended by GraphPad Prism) and non-parametric tests have been used where appropriate. Multiple-group analysis was performed using one-way ANOVA followed by Bonferroni’s multiple comparison post-test. P-values ≤0.05 were considered as statistically significant. All statistical analyses were performed using the GraphPad Prism5 software (GraphPad Software Inc.).Data are given as means ± standard error of the mean (SEM). Statistical differences between two groups were determined using Student’s +-induced mDA neurodegeneration, E14 ventral midbrain cultures were treated for 48 h with MPP+ (0.2 μM) in the absence or presence of recombinant IL4 (10 ng/ml). Figures 1A,B demonstrates that neuron-enriched cultures responded to MPP+ application with a significant loss of mDA neurons (39.39% ± 7.821). IL4 co-treatment was not sufficient to protect mDA neurons in neuron-enriched cultures from MPP+-induced neurodegeneration (40.59% ± 7.417). Next, mDA neurodegeneration was induced by MPP+ in mixed neuron-glia cultures. As shown in Figures 1C,D, MPP+ treatment alone resulted in a significant decrease of mDA neurons in mixed neuron-glia cultures (54.93% ± 6.361). Interestingly, IL4 co-treatment significantly increased the survival of mDA neurons in mixed neuron-glia cultures (77.98% ± 4.655). These results indicate that IL4 only exerts its neuroprotective effects against MPP+-induced neurodegeneration in the presence of glia cells.In order to address the neuroprotective potential of IL4 against MPP+ astrocytes were negative for IL4, microglia labeled with FITC-coupled tomato lectin displayed a strong immunoreactivity for IL4 . Using an IL4 ELISA, we further analyzed the levels of IL4 in supernatants of control- and MPP+-treated cultures. Figure 2B demonstrates that 0.821 ng/ml (± 0.179) IL4 was detectable after 48 h in supernatants from control cultures and a slight but not significant increase of IL4 levels (1.123 ng/ml ± 0.5164) could be determined after treatment with MPP+. Moreover, we addressed whether primary microglia respond to MPP+ treatment with changes in IL4 secretion. 24 h after treatment with MPP+ no change in IL4 secretion from primary microglia cultures was detectable. Interestingly, the data demonstrate that microglia in mixed neuron-glia cultures release higher levels of IL4 than primary microglia. We next aimed to inhibit endogenous IL4 in mixed-neuron glia cultures in order to determine the role of microglia-derived IL4 during MPP+-induced neurodegeneration. Thus, the functionality of a neutralizing IL4 antibody (IL4 nAB) was validated. Therefore, primary microglia cultures were treated with recombinant IL4 (10 ng/ml) in the presence or absence of IL4 nAB at dilutions of 1:100 and 1:50, respectively. Using semiquantitative RT-PCR, expression of the IL4-induced M2 marker Arg1 was analyzed. As shown in Figure 2C, Arg1 expression was induced after treatment of primary microglia with IL4 for 24 h. Almost complete abrogation of IL4-triggered Arg1 expression was observed in the presence of the IL4 nAB at a dilution of 1:50, which was used for further IL4 neutralization experiments. Next, mixed-neuron glia cultures were treated with MPP+ and endogenous IL4 was blocked . Figures 2D,E show that inhibition of endogenous IL4 resulted in a significant increase in MPP+-induced neurodegeneration (38.75 % ± 6.116) as compared to MPP+ treatment alone (54.08% ± 4.72). These data clearly demonstrate that microglia-derived IL4 in mixed-neuron glia cultures is important to protect mDA neurons from MPP+ intoxication.Since IL4 has been recently described to be expressed by microglia , we analFigure 3A, results of the array using the proteome profiler kit for inflammatory cytokines revealed that IL4 did not influence the secretion of the vast majority of cytokines/chemokines from microglia. Notably, MCP-5 and MIP2 were two candidates with increased secretion after IL4 treatment (10 ng/ml) for 24 h. We further used microglia-conditioned medium to treat E14 neuron-enriched cultures in order to determine the survival promoting effects of microglia-derived factors on mDA neurons. Figures 3B,C show that microglia-conditioned medium after treatment with IL4 for 24 h (MCM IL4) significantly increased mDA neuron survival (157.4% ± 11.13) compared to microglia control medium (MCM). The observed neuroprotective effect of MCM IL4 was not promoted by IL4 itself, since direct IL4 treatment of E14 neuron-enriched cultures did not result in an increase of surviving TH+ neurons (108.5% ± 2.849). This observation demonstrates that IL4 treatment of microglia induces the secretion of neuroprotective factors, which increase the survival of TH+ neurons in E14 neuron-enriched cultures. IGF-1 has been identified as a survival-promoting factor secreted by macrophages after IL4 treatment . Moreover, Figure 3E clearly demonstrates that secretion of IGF-1 from primary microglia was significantly increased after 24 h (4.919 ng/ml ± 0.2868) compared to untreated control cells (2.061 ng/ml ± 0.3321). As IL4 was able to protect TH+ neurons from MPP+-induced degeneration in mixed neuron-glia cultures, we wanted to address whether microglia-derived IGF-1 was responsible for this protective effect. Thus, recombinant IGF-1 was used in order to determine whether TH+ neurons in E14 neuron-enriched cultures could be protected from MPP+-induced neurodegeneration. As shown in Figure 3F, MPP+ treatment significantly reduced the numbers of TH+ neurons (38.2% ± 9.848) and a slight non-significant increase of neuron numbers in the presence of IGF-1 could be observed. Although IGF-1 was able to increase the survival of mDA neurons in the presence of MPP+ (56.24% ± 10.39), the protective effect did not reach significance. Together, these results indicate that IL4 treatment of primary microglia results in the release of protective factors including IGF-1, which might at least partially mediate the neuroprotective properties of microglia-conditioned medium.The abovementioned results indicate that IL4 might shape the functions and properties of microglia in mixed neuron-glia cultures to promote survival of mDA neurons. Hence, primary microglia were treated with IL4 and secretion of cytokines and chemokines were analyzed. As shown in reatment . Therefoin vivo, IL4-deficient mice were used. IL4 KO mice are viable and fertile, develop normally and show no phenotypical abnormalities. However, these mice show impaired secretion of IL5 and IL10 and reduced IL4-dependent expression of IgE and IgG1  were not altered in IL4-deficient mice. Quantification of Iba1+ microglia in SNpc and SNpr revealed a slight and significant decrease in microglia numbers in SNpc  but no impairment of Iba1+ microglia numbers in SNpr . Analysis of Gfap+ astrocytes showed that the numbers of reactive astrocytes are neither changed in the SNpc, nor in the SNpr . Similar results were obtained to the basal ganglia (CPu). The numbers of Iba1+ microglia in the CPu of IL4 KO mice were comparable to WT mice . Numbers of Gfap+ astrocytes in the CPu were increased in IL4 KO mice without reaching statistical significance . These data indicate that loss of IL4 does not impair development and maintenance of the nigrostriatal system in vivo.In order to address the role of endogenous IL4 during MPTP-induced degeneration of mDA neurons and IgG1 . As a pr+-induced neurodegeneration in vitro, we used the MPTP mouse model to address the role of IL4 in this model for PD in vivo. Figure 5A displays the injection scheme and the time points used for RNA isolation, immunohistochemistry, and analysis of DA levels using HPLC. We first determined the expression of IL4 in the MPTP model. As shown in Figure 5B, a slight but not significant increase in IL4 expression was observed in the SN 1 day after MPTP injection. All other time points analyzed revealed no increases in IL4 expression in SN or CPu after intoxication with MPTP. Noteworthy, the relatively high CT values (between 32–36) indicate that in contrast to E14 mixed neuron-glia cultures, IL4 is hardly expressed in the nigrostriatal system in vivo. Two different time points for analysis of TH+ neuron numbers and striatal DA levels were chosen according to the acute neurodegeneration phase (7 days) and the regeneration phase (90 days) of this model  and IL4 KO mice (56.67% ± 14.39) without showing significant differences between both genotypes. Similar results were obtained after analysis of striatal DA levels. IL4 KO mice displayed a significant reduction of DA levels (46.4% ± 10.51) comparable to WT mice . Analysis of neuron numbers and DA levels 90 days after MPTP administration revealed that WT and IL4 KO mice showed a similar restoration of the MPTP-intoxicated nigrostriatal system. As shown in Figures 5F,H, numbers of mDA neurons in the SNpc recovered similarly in WT (84.82% ± 10.86) as well as in IL4 KO mice (93.29% ± 10.4). Moreover, striatal DA levels increased in both genotypes  compared to 7 days after intoxication, but were still significantly reduced at 90 days compared to PBS-injected control mice . Taken together, these data demonstrate that loss of endogenous IL4 does not increase the susceptibility toward MPTP-induced degeneration of mDA neurons in vivo. Moreover, the regeneration of the nigrostriatal system after MPTP administration is not impaired in IL4 KO mice, indicating that endogenous IL4 is dispensable in the MPTP mouse model for PD.Since endogenous IL4 was sufficient to protect mDA neurons from MPPis model . Figures+-induced degeneration in the presence of glia cells in vitro. Moreover, we provide evidence that endogenous microglia-derived IL4 in mixed neuron-glia cultures is important to reduce mDA neurodegeneration after MPP+ application and might facilitate neuroprotection by regulating microglia-mediated secretion of neuroprotective factors such as IGF-1. In vivo, the expression of IL4 was hardly detectable under basal conditions or after MPTP administration, and the loss of IL4 did not alter the susceptibility of mDA neurons toward MPTP-induced degeneration. Moreover, the regeneration of the nigrostriatal system after MPTP intoxication, as evident by restoration of TH+ neuron numbers as well as striatal DA levels, was not impaired in IL4-deficient mice.In the present study, we have demonstrated that exogenous IL4 protects mDA neurons from MPP+ neurons seem to be mediated by other microglia-derived factors. However, IL4 has been reported to induce IL6-producing macrophages that inhibit neuroinflammation in vitro and in vivo (+-driven degeneration (in vivo. IGF-1 increased survival of TH+ neurons and reduced neuroinflammation after intoxication with MPTP (+-induced degeneration, by adding recombinant IGF-1 to neuron-enriched cultures. Although the number of surviving neurons increased, the protection was not as effective as IL4 treatment of mixed neuron-glia cultures. It is high likely that microglia-derived neurotrophic factors and neuron–microglia interactions contribute to the neuroprotective effects observed after IL4 application in neuron-glia cultures. The fact that IL4 secretion from microglia is not a direct effect of MPP+ suggests that challenged neurons in mixed neuron-glia cultures trigger IL4 release from microglia. Damaged neurons have been shown not be passive targets of microglia-mediated neurotoxicity but are capable of regulating microglia functions. Release of CX3CL1 or IL34 from neurons and the subsequent binding to microglia-specific receptors CX3CR1 and CSF1R are well described mechanisms to shape microglia functions toward a neuroprotective phenotype (+-challenged neurons regulate microglia functions.Interleukin 4 has been described to promote a microglia activation state, which is characterized by functional features such as tissue repair and cellular protection . Here, w in vivo . Interesneration  and reduneration . Neverthneration . IL4 hasneration . Moreoveneration  indicatiith MPTP  as well ith MPTP . We furthenotype . Such neIn vitro, neutralization of endogenous IL4, expressed by microglia, worsens MPP+-induced degeneration of mDA neurons. This result suggests that IL4 acts in an autocrine manner on microglia in order to silence their activation. In vivo, loss of IL4 did not change the extent of neurodegeneration and neuroregeneration, indicating that IL4 is dispensable in the MPTP mouse model for PD. One of the reasons for these observations, might be the fact that IL4 expression in the nigrostriatal system is hardly detectable under basal conditions as well as after MPTP intoxication. The main cell type expressing IL4 are Th2 T-cells (+ T-cells (+ T-cells to protect mDA neurons from MPTP-induced neurodegeneration has been described after adoptive transfer of CD4+/CD25+ regulatory T-cells ( T-cells . However T-cells  and also T-cells  are able T-cells  but the  T-cells . This is T-cells . Lack of T-cells  and high T-cells  might be T-cells . Microgl T-cells  and coulThe therapeutic potential of exogenous IL4 has been well documented in several CNS pathology models including models for spinal cord injury , APP/PS1in vivo but the presence of IL4 in vitro is important to limit degeneration of TH+ neurons after MPP+ intoxication. Our results strengthen the hypothesis that IL4-treated microglia are capable of facilitating protection of mDA neurons and underline the therapeutic potential of IL4 administration in models of PD.Taken together, the results presented in this study demonstrate that IL4 is able to shape microglia functions to promote survival of mDA neurons. We provided evidence that endogenous IL4 is dispensable for MPTP-induced neurodegeneration BS conceived the project. LH, JR, XZ, VM, and BS performed experiments and analyzed the data. RG performed HPLC analyses. BS wrote the manuscript. All authors have read and approved the final manuscript and further agreed to be accountable for the content of the work.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.


Graphene is currently at the forefront of cutting-edge science and technology due to exceptional electronic, optical, mechanical, and thermal properties. However, the absence of a sizeable band gap in graphene has been a major obstacle for application. To open and control a band gap in functionalized graphene, several gapping strategies have been developed. In particular, hydrogen plasma treatment has triggered a great scientific interest, because it has been known to be an efficient way to modify the surface of single-layered graphene and to apply for standard wafer-scale fabrication. Here we show a monolayer chemical-vapour-deposited graphene hydrogenated by indirect hydrogen plasma without structural defect and we demonstrate that a band gap can be tuned as wide as 3.9 eV by varying hydrogen coverage. We also show a hydrogenated graphene field-effect transistor, showing that on/off ratio changes over three orders of magnitude at room temperature. The absence of a band gap in graphene hinders its use in electronics. Here, the authors open a band gap as large as 3.9 electronvolts in graphene grown by chemical vapour deposition by treating it in hydrogen plasma, and then use this material to create a room temperature field- effect transistor. Graphene has opened a new arena in the field of two-dimensional condensed matter physics since its first isolation from graphite by means of micromechanical exfoliation124567912121591415161718Here, we show that monolayer graphene hydrogenated by indirect hydrogen plasma at room temperature acquires a band gap up to ∼4.0 eV that can be tuned by varying hydrogen coverage while preserving the structural integrity of the original graphene. We first demonstrate that the intrinsic band-gapped graphene we created fully functions as a field-effect transistor (FET), showing that conductivity at gate voltages changes over three orders of magnitude at room temperature.η on graphene increased. We found that the hydrogen coverage η saturated at ∼25% when we further increased the exposure time  graphene20time see . When thtime see  and 3.5 π and the π* orbitals, a fingerprint of graphene, disappeared and were significantly reduced, respectively, with the coverage. In other words, the sp2 orbitals rehybridized with the local sp3 orbitals as a result of hydrogenation. In addition, the C*=O orbital, located at ∼3.2 eV above the π* orbital, also disappeared as the hydrogenation continued, indicating that the hydrogen replaces oxygen chemisorbed on the CVD graphene. It is noteworthy that the pronounced π* band signifies topological ripples and corrugations, and that the intermediate states between π* and σ* contain residual adsorbates commonly observed in CVD graphene2021A close investigation of these spectra reveals that the band-gap opening indeed arises from the hydrogenation of graphene. The in-situ heating PES measurements of H-Gr directly prove that our indirect hydrogen plasma indeed inflicts minimal damage on graphene, that is, that H-Gr has no significant defects in the honeycomb basal plane after hydrogenation.  The indirect plasma technique is well known to significantly reduce damages to a surface, because the surface is not in direct contact with the plasma, which contains energetic particles. Both the band gaps of H-Gr created at an η of 12% and 25%, for example, returned to 0 (−10 Torr) at 550 K for 2 h. Besides, the recovery of the π orbital below the Fermi energy proves that hydrogen that had bonded with the pz-orbital of carbon was desorbed during heating and that H-Gr was reduced to metallic graphene. Such reversibility has been observed only when the graphene framework was not seriously damaged by surface treatments. Our observation of the full recovery of H-Gr to graphene bears a critical implication for the modulation of a band gap of graphene: we can control the band gap either by exposing the graphene to indirect hydrogen plasma or by thermal treatment of the resulting H-Gr.Our ned to 0  when H-Gπ plasmon located at ∼4.6 eV due to excitonic Fano resonanceπ plasmon peak is blue-shifted by up to ∼0.04 eV  band-to-band excitation. Therefore, the optical spectra often reveal the upper bound of the bandwidth in the case of indirect band-gap semiconductors. This is most likely the case in what we observed here—more of which later. Meanwhile, it is worth noting that the crucial reversible recovery from H-Gr to graphene was repeatedly confirmed through ex-situ optical absorption experiments. After we annealed H-Gr with an optical bandwidth of 4.6 eV at 550 K for 20 min under Ar atmosphere and carried out optical absorption experiments, the optical bandwidth decreased from 4.6 to 3.0 eV (Not only the transport band-gap measurements (PES and NEXAFS spectroscopy) but also optical absorption experiments support the wide tunability of a band gap of graphene induced by our plasma treatment. Initially, the absorption spectra of graphene acquired from near-infrared to ultraviolet at room temperature in air exhibited a general feature of a ∼0.04 eV . The opt04 eV  gradually increasedID/ID′) showed a similar saturation tendency and stayed at over 16, never dropping after saturation. The former ratio and its mapping imply that the defects are uniformly distributed and saturated at η of 25%, as the distance between defects no longer increases, and the latter ratio shows that the defect is not an atomic-vacancy type but an sp3 type in H-GrWe found that the hydrogenation or the formation of sp2 network can be ruled out by high-resolution transmission electron microscopy and by the evidence of the return of H-Gr to the pristine graphene state by the aforementioned thermal treatment. Neither structural damage nor disintegration of the innate symmetry of graphene was found in the micrographs  dependence of resistivity (ρ) of our H-Gr shows that the band gap stems from a long-range order at an η of 25%, whereas it can be opened by delocalized defect states at the lower hydrogen coverages (ρ−1/T of H-Gr (η=25%) showed three distinct behaviours: (1) intrinsic excitation, (2) variable-range hopping and (3) saturation towards the Mott minimum conductivity—all of which are essential features observed in crystal semiconductorsρ=ρ0 exp[Eg/(2kBT)]) in the intrinsic region was ∼3.8 eV, which is close to the band gap (∼3.9 eV) measured by transport band-gap measurements. On the other hand, when hydrogenation coverage was insufficient, the ρ−1/T showed the behaviour of variable-range hopping conductance, in which ρ is proportional to T−1/3 (not shown here) as others observedThe temperature  band-to-band excitation: optical absorption has been found to be very sensitive to the direct transitions at high-symmetry points, such as K, M and G, in the Brillouin zone of C4H-type hydrogenated graphene. The difference in the magnitude of the measured band gaps may also in part stem from the difference in the degree of hydrogenation or in the distribution of C–H bonds possibly caused by the different substrates used for transport measurements and optical absorption measurements—Si/SiO2 and sapphire, respectively (see Methods).Judging from the size of a band gap and the temperature-dependent resistivity of our H-Gr showing an C4H refs , 32, 33,2 gate dielectric showed a dramatic change in current of over 103 on/off ratio on the sweep of a back-gate voltage at room temperature as shown in 4. We believe such a limit can be ascribed to the high off-current due to leakage current through the thermally oxidized SiO2 of poor quality. I–V source-drain characteristics were initially nonlinear but became linear as the gate bias was increased over –20 V , indicating that the performance of FETs can be significantly improved by implementing a high-κ gate dielectric and an improved FET structure (20η∼12%), the on/off ratio was unexpectedly low. This is because the band gap was opened by random hydrogenationFinally, we demonstrate the functionality of a FET made from H-Gr . Our prof 3.9 eV . To showtructure . We noteThe very considerable advantage of our method is that a device can be built entirely of graphene: a conductor, that is, graphene, and a semiconductor and an insulator, that is, our hydrogenated graphene with small and large band gapsIn summary, we have demonstrated that the indirect exposure of CVD graphene to hydrogen plasma successfully modulated the band gap of graphene and, as a result, opened a band gap as wide as ∼4.0 eV without breaking the honeycomb structural symmetry of graphene; we have also shown that our hydrogenated graphene can fully function as a FET at room temperature. All of our hydrogenated graphene with a band gap retained very high structural integrity and was fully reduced to pristine graphene with a zero band gap simply by heating. Our study of monolayer graphene hydrogenated by indirect hydrogen plasma promises to provide answers to many controversial theoretical predictions and to trigger studies on new aspects of semiconducting graphene that have so far been hampered by experimental difficulties. Thus, this new form of graphene should play a significant role in enabling unique applications for long-envisioned carbon-based electronics and photonics.p-doped Si substrate with 300 nm-thick SiO2 for Raman, X-ray photoemission spectroscopy, PES and NEXAFS measurements. A sapphire substrate was used and CVD graphene showing ∼2.3% absorption in the visible range was chosen for hydrogenation and optical absorption measurements.Graphene was prepared under high vacuum by CVD on Cu foils and transferred onto a heavily Hydrogen plasma was generated in a separate gun with a microwave power of 180 W at 2.5 GHz. The flow rate of hydrogen was 25 s.c.c.m. and the working pressure was 10 mtorr. The as-prepared CVD graphene was downstream positioned 10 cm away from the end of the gun.p-doped Si substrate with 100 nm-thick SiO2. PMMA/HSQ bilayer resist on graphene was exposed by an electron beam and HSQ-uncovered PMMA/graphene layers were etched out by O2 plasma after developing. After removing them in acetone, FETs with graphene was obtained. They were annealed at 300 °C for 1 h in a gas mixture of Ar/H2 (10:1) to remove residual contaminations. Finally, FETs were treated by hydrogen plasma. We measured the electrical properties at various back-gate bias from 2 K to 300 K in vacuum. For a two-probe measurement, we applied constant source-drain voltage (Vsd) and FET mobility was extracted from the differential curve of the electric field effect. For a four-probe Hall measurement, constant source-drain current of 100 nA was applied and the Hall voltage (VH) was measured. The mobility and carrier density were estimated from VH measured by the four-probe Hall measurement.At first, to fabricate graphene-based FETs, electrodes were patterned by a lift-off process after Cr/Pt (5/25 nm) was sputter-deposited on the patterns prepared by photolithography. Then, CVD graphene was transferred onto a heavily The optical transmission and absorption coefficient of graphene and H-Gr were measured at room temperature via visible-ultraviolet spectrophotometry  over the wavelength range of 175–3,300 nm at normal incidence. Raman spectra were obtained with a Horiba Jobin-Yvon Lab Ram HR spectrometer with an Ar laser wavelength of 514.5 nm at room temperature. Diffraction patterns and atomic images were taken by Cs-corrected STEM  with 80 keV electron beam. PES and X-ray photoemission spectra were measured using the synchrotron source at the 4D beam-line and NEXAFS spectra were obtained at the 2 A beamline at Pohang Accelerator Laboratory. The NEXAFS spectra were obtained with partial electron yield at a 55° angle of incidence of the synchrotron photon beam with respect to the surface of the samples.The data that support the findings of this study are available within the article and its How to cite this article: Son, J. et al. Hydrogenated monolayer graphene with reversible and tunable wide band gap and its field-effect transistor. Nat. Commun.7, 13261 doi: 10.1038/ncomms13261 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-4, Supplementary Table 1, Supplementary Notes 1-4 and Supplementary References.


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.