Anopheles funestus is threatening recent gains in the fight against malaria. Here, we established the role of an over-expressed P450, A. funestus CYP6AA1 in insecticides resistance. Transcription profiling of CYP6AA1 across Africa using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that it is significantly more over-expressed in southern African populations compared to West (Benin) and East African (Uganda). Heterologous expression in Escherichia coli coupled with metabolism assays demonstrated that CYP6AA1 metabolises type I (permethrin) and type II (deltamethrin) pyrethroids, as well as bendiocarb (a carbamate). Transgenic Drosophila melanogaster flies over-expressing CYP6AA1 were significantly more resistant to pyrethroid insecticides, permethrin and deltamethrin compared with control flies not expressing the gene, validating the role of this gene in pyrethroid resistance. In silico modelling and docking simulations predicted the intermolecular receptor-ligand interactions which allow this P450 to metabolise the pyrethroids and bendiocarb. Validation of CYP6AA1 as a pyrethroid resistance gene makes it possible to monitor the spread of resistance in the field where this P450 is over-expressed. Its potential cross-resistance role makes it necessary to monitor the gene closely to inform control programs on molecular basis of multiple resistance in the field.Resistance to pyrethroids (the ingredients in bed net insecticides) in the major malaria vector  Anopheles gambiae Complex and the Anopheles funestus Group are increasingly common  compared with the negative control, –NADPH) and a t-test used for statistical analysis. Metabolism assays were conducted with permethrin (representative type I) and deltamethrin (type II) pyrethroids, DDT (an organochlorine), the carbamates-bendiocarb and propoxur, as well as malathion (an organophosphate). Protocols for incubations and high-performance liquid chromatography (HPLC) analyses for the above insecticides followed procedures previously published ,34. Brieications . All reamK and maxV were established from the plot of substrate concentrations against the initial velocities and fitting of the data to the Michaelis-Menten module using the least squares non-linear regression in the GraphPad Prism 6.03 Software .Steady state kinetic parameters were determined with permethrin and deltamethrin by measuring the rate of reaction for 20 min while varying the substrate concentrations (3–24 µM) in presence of 22.5 pmol recombinant CYP6AA1. Reactions were performed in triplicates both for +NADPH and –NADPH for each concentration. CYP6AA1 alone can confer resistance to the pyrethroid insecticides, transgenic D. melanogaster flies expressing this gene were generated using the GAL4/UAS system. The preparation of the transgenic flies followed the protocols described previously  by Genetic Services  to generate the transgenic line UAS-CYP6AA1. Ubiquitous expression of the transgene in adult F1 progeny  was obtained after crossing virgin females from the driver strain Act5C-GAL4 ), with polar metabolites eluting at the beginning of the chromatogram of incubation with NADPH+. These kind of putative metabolites have been described from in vitro assays of recombinant A. funestus CYP6Z1 with bendiocarb [HPLC analyses established that CYP6AA1 metabolises permethrin and deltamethrin respectively, with high depletion of 83.5% ± 4.6 (cubation A. No actndiocarb . p = 0.2) with no polar metabolites eluting at the beginning of chromatogram. Also, less than 5% of DDT (even following addition of solubilising agent sodium cholate) and malathion were depleted by the recombinant CYP6AA1, indicating lack of enzymatic activity toward the organochlorine and organophosphate insecticides. This is in line with the DDT and malathion susceptibility observed in southern Africa, Mozambique, for example [In contrast, very low activity was observed with propoxur  of 11.99 min−1 ± 2.17 and 15.65 min−1 ± 2.642, respectively for permethrin and deltamethrin (Km) for permethrin and deltamethrin were also comparable, 33.62 µM ± 9.180 and 30.01 µM ± 7.915, respectively. Though these Km values are within the ranges described for binding and metabolism by insect cDNA-expressed P450s [A. funestus CYP6P6P9a and CYP6P9b [Km values obtained from recombinant A. minimus CYP6AA3 and CYP6P7 with pyrethroids [−1 µM−1 ± 0.12 and 0.51 min−1 µM−1 ± 0.16 respectively, for permethrin and deltamethrin. Metabolism of pyrethroids follows the canonical Michaelis-Menten pattern with high maximal catalytic rate   at all the eight different exposure times compared with the control groups  A. p < 0.05) at 12 h and 36 h of exposure. These results confirmed that over-expression of CYP6AA1 alone is sufficient to confer resistance to permethrin and deltamethrin. A similar pattern was observed using deltamethrin B with a CYP6AA1 (1518 bp) from different regions of Africa revealed relative homogeneity within each geographic region, with haplotypes from each country forming a cluster in the maximum likelihood phylogenetic tree , from 10 haplotypes out of 22 sequences from four different countries). The very low Hd in the sequences, especially from Malawi and Ghana  suggests a directional selection (selective pressure) acting on CYP6AA1 in populations from these two regions. A neutrality test of all sequences revealed Li and Fu’s D* as positive but not statistically significant. When all sequences were analysed according to country of origin, statistics was only positive with FANG sequences, suggesting rare polymorphism and lack of background selection. Haplotype diversity is high , West (Ghana) and East Africa (Uganda and FANG) populations. High genetic differentiation estimates were observed between Ghana (0.82 < KST < 1), East African (0.53 < KST < 0.68) and the other two populations, whereas the two southern African populations exhibited a null level of genetic differentiation (KST = 0.0)   and clusT = 0.0) C.A. funestus CYP6AA1 to other closely related sequences reveals that it is 89% identical to its ortholog A. minimus CYP6AA3 (GenBank: AAN05727.1), 87% identical to A. gambiae CYP6AA1 (AGAP002862) and 57% identical to Culex pipiens pallens CYP6AA9 (GenBank: AKA45037.1) (Culex CYP6AA9 (515 amino acids) all the other three P450s are composed of 505 amino acids.Comparison of 45037.1) . Apart fAnopheles sequences , and SRS-6. Sequence-to-sequence mapping reveals that the WxxxR motif, the signatory oxygen-binding pocket (AGFETS)/proton transfer groove, the ExxR motif which stabilises the heme structural core, the cysteine pocket/heme-binding region (PFxxGxxxCxG), which forms the fifth axial ligand to the heme iron ,51 were equences . Major sA. funestus CYP6AA1 able to metabolise pyrethroid insecticides, a docking simulation was carried out using the homology models of A. funestus CYP6AA1 and A. gambiae CYP6AA1 with insecticides from three of the four classes used in public health control of malaria vectors. The binding parameters for each insecticide are provided in A. funestus CYP6AA1 model, deltamethrin exhibited the highest affinity (lowest binding energy) with high contribution from hydrophobic interactions and intermolecular hydrogen bonding. The insecticide docked into the active site of CYP6AA1 with the 4′ spot of phenoxy ring oriented above the heme at a distance of 3.6Å from heme iron, suggesting ring hydroxylation (A. gambiae [To understand the underlying mechanism which makes xylation B. 4′-hyd gambiae  and in o gambiae . A. gambiae CYP6AA1 model consistent with the pose of deltamethrin in the active site of this P450 (2 position of benzyl ring positioned 6.4Å from heme iron)  . Very hiectively . The traA. funestus CYP6AA1. The binding energy of −7.8 kcal/mol indicates moderate affinity to CYP6AA1, compared with A. gambiae CYP6AA1 with lower affinity (higher binding energy) of −7.2 kcal/mol. This is consistent with the moderate scores obtained from hydrophobic interactions, which is less than half the values obtained from the pyrethroids. The carbamate insecticide docked productively with the carbamic ester group above the heme catalytic centre, at 3.7Å  from heme catalytic centre  with permethrin, deltamethrin, and bendiocarb were compared from docking simulations of models from A. funestus and A. gambiae, respectively. For both permethrin and deltamethrin, Phe309 was predicted to enhance catalysis through π-stacking with the phenoxy ring in the case of permethrin, and with both phenoxy- and benzyl rings in the case of deltamethrin, in A. funestus model (307Phe308Phe309) from the substrate recognition site-4 (SRS-4) within the αI helix (109 (SRS-1) were predicted to enhance catalysis via π-stacking with the aromatic rings of both permethrin and deltamethrin in A. gambiae CYP6AA1 active site  was predicted to be involved in hydrophobic interactions with aromatic rings of deltamethrin and bendiocarb in the A. funestus CYP6AA1 models  to the ester oxygen of the carbamate moiety (A. gambiae: (i) donated by alcohol side chain of Tyr109 to benzodioxol moiety; (ii) donated by His120 to the amide nitrogen (-NH) of the methylcarbamate moiety. No hydrogen bonding between permethrin and redicted . In contredicted , both doe moiety . This co106 of A. gambiae CYP6AA1 with deltamethrin , and even down-regulated in East Africa (Uganda). Overall, the expression pattern of CYP6AA1 resembles that of the duplicated P450 genes CYP6P9a and CYP6P9b which have so far been found to be highly over-expressed in southern Africa and only moderately over-expressed in West (Benin) and East Africa (Uganda and Kenya) [CYP6AA1 is located on the chromosome 2R, together with CYP6P9a and CYP6P9b, on the same cluster of cytochrome P450s spanning the rp1 pyrethroid resistance quantitative trait locus (QTL) [d Kenya) ,53. CYP6us (QTL) . CYP6AA1 exhibited pyrethroid activities comparable to values observed from other P450s implicated in pyrethroid resistance in A. funestus, notably CYP6P9a and CYP6M7 [CYP6Z1 [A. gambiae P450s such as CYP6M2 [A. funestus, A. minimus, and A. gambiae particularly suggests that its detoxification function was retained even after speciation. Indeed, the ortholog from A. minimus was shown to metabolise type I and type II pyrethroids in vitro, with higher activity towards deltamethrin than permethrin [A. funestus and A. gambiae CYP6AA1 suggests possibility of multiple metabolites. Of course, the ortholog A. minimus CYP6AA3 is known to possess a very large substrate access channel [A. funestus CYP6AA1, the ortholog from A. minimus displayed no activity towards malathion and propoxur. The inability of CYP6AA1 to metabolise malathion further explains the field susceptibility to these insecticides in A. funestus [d CYP6M7 , and CYP [CYP6Z1  or others CYP6M2 . The conrmethrin . The dif channel  which ac channel . Like A.funestus ,56.A. funestus with potential metabolic activity towards bendiocarb, though with very low activity compared with A. funestus CYP6Z1 [A. funestus CYP6AA1 is not polymorphic like the A. funestus CYP6M7 characterised by [CYP6P9a and -b has been shown to be the major driver of resistance to pyrethroid insecticides across Africa in the same A. funestus species [It is important to monitor the spread and evolution of this potential cross resistance gene, as it is the second P450 found in s CYP6Z1 . A. funerised by , and is rised by . For exa species . CYP6AA1 exhibited lower activity in vitro and confers lower resistance to permethrin and deltamethrin in transgenic flies compared with the major pyrethroid resistance gene CYP6P9b [Though  CYP6P9b  it is anCYP6AA1 without hydrogen bonding contributions was as observed with A. arabiensis CYP6P4 model [A. funestus CYP6AA1. Metabolism of permethrin is possibly driven through hydrophobic interactions, as further supported by the presence of Trp219 of SRS-2 within its catalytic hotspot. This is the opposite of the situation with deltamethrin, in which hydrogen bonding plays a significant role in energetic contribution. Indeed, differences in the composition of amino acids lining catalytic hotspot are known to dictate presence or absence of intermolecular hydrogen bonding interactions with resulting differences in choice of substrates even among closely related proteins. For example, polar cages composed of hydrophilic residues in the active site of specialist CYP6B1 from Papilio polyxenes contribute to the P450’s hydrogen bonding capabilities which contribute to its ability to metabolise only two furanocoumarins [CYP6B8 from Helicoverpa zea is devoid of polar cages (hydrogen bonding network) in its active site and was established to metabolise diverse allelochemicals, and even a pyrethroid cypermethrin. In silico predictions using modelling and docking simulations have become important tools used to study insecticide resistance genes and their heterogeneities in the metabolism of insecticides ,34,57,58oumarins . In contA. funestus CYP6AA1 to metabolise DDT is consistent with docking predictions, with the organochloride insecticide binding unproductively. Indeed, the productive pose of DDT has been established as C-1 of the trichloromethyl group docking above the heme in A. gambiae CYP6Z1 [The inability of e CYP6Z1 .CYP6AA1 contributes to the cocktail of P450s responsible for pyrethroids resistance in southern African populations of A. funestus. But, unlike the major pyrethroid resistance genes CYP6P9a and –b, this P450 exhibited activity toward indoor residual spray insecticide bendiocarb as well, making it a potential cross-resistance gene which should be monitored closely in the field.Knowledge of insecticide resistance in malaria mosquitoes and the mechanisms driving it in the field is essential for design of appropriate malaria control tool and for management of the resistance. However, this requires a thorough understanding of the molecular basis of the resistance to inform control programs and influence policy decisions. Here, we established that


Control of both the regularity of a material ensemble and nanoscale architecture provides unique opportunities to develop novel thermoelectric applications based on 2D materials. As an example, the authors explore the electronic and thermal properties of functionalized graphene nanoribbons (GNRs) in the single‐sheet and helical architectures using multiscale simulations. The results suggest that appropriate functionalization enables precise tuning of the doping density in a planar donor/acceptor GNR ensemble without the need to introduce an explicit dopant, which is critical to the optimization of power factor. In addition, the self‐interaction between turns of a GNR may induce long‐range disorder along the helical axis, which suppresses the thermal contribution from phonons with long wavelengths, leading to anomalous length independent phonon thermal transport in the quasi‐1D system. With regard to ensemble regularity, both verticalAs an example of exploring material architecture, a recent theoretical study predicted that nanopatterning via chemical functionalization can enhance the optimized ZT of graphene significantly, due to the increased Seebeck coefficient and reduced thermal conductivity.In this work, we explore the impact of the material ensemble and architecture on TE properties, with emphasis on understanding the tunability of doping and thermal conductivity. Among the numerous derivatives of 2D materials, GNRs are chosen as our prototype systems because their electronic structure can be tailored by both geometry and passivation, which makes them especially attractive to provide desirable properties for electronic and spintronic applications.T = 300 K). Our results suggest that appropriate design of the GNRs leads to precise tuning of the electronic structure and modification of vibrational channels via self‐interactions. For example, with the single‐sheet architecture, type‐III electronic interfaces can be established to achieve controllable ground state doping while avoiding effects that can arise from an extrinsic dopant  of the donor and the lowest conduction band (CB) of the acceptor near the Γ point Figure a–d. Whil12 to 2.1 × 1013 cm−2 becomes attainable  has been observed in graphene encapsulated by hexagonal boron nitride,4–105 cm2 V−1 s−1 predicted by deformation potential theory assuming acoustic phonon is the dominant scattering mechanism.In order to tune the potential profile and attain a range of doping densities, two different groups of GNRs with various widths were used, with the end groups of their ligands switched between H, CN, and OH. Consistent with the previous study on graphene nanoroads,6 S cm−1, more than twice that of the optimal σ in graphene. Only the conductivity in the y‐direction  was computed as carrier transport should be restricted to the 1D domain as revealed from the flat bandstructure in the x‐direction , up to a value of 1.8 × 10S) and electrical conductivity  of the GNRs was calculated based on the fluctuation–dissipation theorem and rescaled to account for the long‐wavelength phonon contributions. The simulations were conducted on functionalized graphene sheets composed of a single type of GNR with the H end group, which accounts for the major impact of adjacent GNRs on κp while taking advantage of the efficient force field that is available only for C and H atoms. Consistent with the literature,p of GNRs are reduced by 5–6 times compared to that of pristine graphene (1810 W m−1 K−1)T = 300 K, with which the device ZT is estimated to be 0.55, much higher than the ZT of graphene (0.09) at its optimal doping level, mainly due to the reduction of κp . To further illustrate the importance of accurate control of the doping level, we computed the S2σ and ZT at various doping levels for graphene and the functionalized GNRs  can either be suppressed by the scattering of long‐wavelength phonons which account for a large proportion of κp, or enhanced by the additional transport paths through the side chain ligands in the helix architecture as illustrated by the cartoon in Figure Since the radius of a helical GNR is much larger than the sizes of the relevant atomic orbitals (≈2 Å), as perhaps expected the electronic properties of the helical GNR are similar to those of the corresponding planar GNR, with a slight drop in the computed conductivity due to the scattering induced by surface curvature  of the GNR—a nearly invariant κp, in contrast to the power‐law relation for the planar GNR 2) as defined in Section A in the Supporting Information) of the GNR helix with L = 54 nm is 40% lower than the κp of its planar counterpart . As expected, the reduction decreases with increasing spacing between turns due to the smaller curvature, while κp is still independent of L in most cases , implemented in the Vienna Ab Initio Simulation package (v5.3),p) of the functionalized graphene sheets and the planar/helical GNRs were calculated using equilibrium molecular dynamics (EMD) and nonequilibrium MD (NEMD) simulations, respectively, as implemented in the Large‐scale Atomic/Molecular Massively Parallel Simulator package (v2014).The phonon contributions to the thermal conductivities (κ7 MD steps (1 ns) within the NVE ensemble after a 100 ps equilibration period within the NVT ensemble. All interactions were described by the adaptive intermolecular reactive empirical bond order (AIREBO) potential.All simulations were carried out at 300 K with a time step of 0.05 fs. For the functionalized graphene sheets, the EMD approach based on the fluctuation–dissipation theorem was chosen to reduce finite‐size effects. The Einstein relation was applied,p, which reveals interesting physics for this architecture, could not be obtained by the EMD approach. Therefore, the reverse NEMD approach with the Muller–Plathe algorithmFor the helical GNRs, the EMD method is inappropriate because the direction of the microscopic heat flux is ill‐defined. In addition, the length dependence of κWe note that a number of approximations have been applied in this work in order to make the estimation of the thermoelectric figure of merit possible, which inevitably introduce uncertainties, for example, in the accuracy of the DFT energy levels, the phonon thermal conductivity based on finite‐size classical MD, and the absence of defects and boundary disorder. Thus, rather than interpreted in a quantitative way, this study is more appropriate to be considered as an attempt to qualitatively explore the relative potential benefits of architecture design in 2D materials for thermoelectric applications.SupplementaryClick here for additional data file.


GRN gene cause frontotemporal lobar degeneration (FTLD). The affected neurons display distinctive TAR DNA binding protein 43 (TDP-43) inclusions. How partial loss of PGRN causes TDP-43 neuropathology is poorly understood. TDP-43 inclusions are also found in affected neurons of patients with other neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. In ALS, TDP-43 inclusions are typically also immunoreactive for fused in sarcoma (FUS). Mutations within TDP-43 or FUS are themselves neuropathogenic in ALS and some cases of FTLD. We used the outgrowth of caudal primary motor neurons (MNs) in zebrafish embryos to investigate the interaction of PGRN with TDP-43 and FUS in vivo. As reported previously, depletion of zebrafish PGRN-A (zfPGRN-A) is associated with truncated primary MNs and impaired motor function. Here we found that depletion of zfPGRN-A results in primary MNs outgrowth stalling at the horizontal myoseptum, a line of demarcation separating the myotome into dorsal and ventral compartments that is where the final destination of primary motor is assigned. Successful axonal outgrowth beyond the horizontal myoseptum depends in part upon formation of acetylcholine receptor clusters and this was found to be disorganized upon depletion of zfPGRN-A. PGRN reversed the effects of zfPGRN-A knockdown, but a related gene, zfPGRN-1, was without effect. Both knockdown of TDP-43 or FUS, as well as expression of humanTDP-43 and FUS mutants results in MN abnormalities that are reversed by co-expression of hPGRN mRNA. Neither TDP-43 nor FUS reversed MN phenotypes caused by the depletion of PGRN. Thus TDP-43 and FUS lie upstream of PGRN in a gene complementation pathway. The ability of PGRN to override TDP-43 and FUS neurotoxicity due to partial loss of function or mutation in the corresponding genes may have therapeutic relevance.Progranulin (PGRN) is a glycoprotein with multiple roles in normal and disease states. Mutations within the  GRN suppresses the development of disease-like phenotypes in murine models of Parkinson’s disease (PD) [GRN, the gene encoding PGRN, results in 50% depletion of PGRN protein levels, and causes frontotemporal dementia (FTD), an early onset form of dementia [GRN have been reported (www.molgen.ua.ac.be/FTDmutations/) the majority of which result in nonsense mediated decay of the GRN mRNA transcript [MAPT), GRN and hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) [GRN mutations are responsible for 5–10% of all cases of FTLD and 13–25% of familial cases [MAPT [GRN or C9orf72 [TARDBP, the gene encoding TDP-43 or FUS can cause either ALS [Progranulin (PGRN) is a secreted glycoprotein that regulates many processes including cell proliferation, survival and motility . In mammase (PD)  and Alzhase (PD) . Haploindementia , 5. To danscript –6. FTD ianscript , 8. AbouC9orf72) . GRN mutes [MAPT , ubiquit C9orf72 , 16. TDP C9orf72 , and in  C9orf72 . In some C9orf72 . FUS-con C9orf72 . Mutatiother ALS , 21, 22 ther ALS  confirmihuntingtin gene. Both TDP-43 and FUS are required for full expression of polyQ toxicity in mammalian striatal neurons [C. elegans model of MN toxicity caused by huntingtin exon-1 polyQ toxicity. These results support a genetic interaction between TDP-43 and PGRN in evoked neuronal cell death. This conclusion is based upon the fact that PGRN blocks polyQ toxicity and TDP-43 blocks the cytoprotective action of PGRN. However, no corresponding interaction between PGRN and FUS was observed.Both TDP-43 and FUS are RNA-binding proteins. Other neurodegenerative conditions including spinal muscular atrophy are also caused by mutations within genes for RNA-binding proteins . Underst neurons . PGRN alC. elegans, can be readily performed in order to address such questions. The zebrafish genome carries four GRN genes [zfPGRN-A is the orthologue of mammalian GRN, and is a long-form PGRN [zfPGRN-B is also a long-form PGRN, whereas zfPGRN-1 and zfPGRN-2 are short-form PGRNs, with one and one half granulin motifs, zfPGRN-A [zfPGRN-A or zfPGRN-B alone or double mutants produced neither truncated CaP MN nor other neuropathological phenotypes suggesting the activation of compensatory mechanisms upon loss of GRN genes in the germline [It is unclear, whether the depletion of PGRN is by itself a direct cause of TDP-43 toxicity, or whether TDP-43 related neurotoxicity is a secondary response to a more generalized cellular dysfunction caused by PGRN depletion. Zebrafish provide a vertebrate model system in which gene complementation studies, similar to those undertaken in simpler invertebrate animals such as RN genes . Of thesorm PGRN , encodinzfPGRN-A  and whenzfPGRN-A . EctopiczfPGRN-A . In contgermline . Similargermline .zfPGRN-1 is able to rescue motor defects resulting from the depletion of long form zfPGRN-A.Here we take advantage of the zebrafish model of motor dysfunction to investigate the genetic interactions of PGRN with TDP-43 and FUS in a vertebrate system. We also determined whether the short form Wild type zebrafish were purchased from Aquatica Tropicals (Florida) and housed on a 14h/10h light/dark cycle at 28.5°C in a laboratory aquarium . The McGill University Institutional Animal Care and Use Committee (IACUC) specifically approved this study and Zebrafish were maintained according to Protocol Number 3935. Fish were fed twice daily. In the late afternoon of the day before embryos are required (approximately 3:00 p.m.), fish were transferred to a breeding tanks with a divider to maintain male and female separately. In the morning, immediately after the light cycle begins, divider removed to facilitate spawning. After spawning has stopped, the eggs that have fallen through the breeding tanks to the outer tank were collected from the bottom of the tank. Embryos to be used for experimental studies were collected and staged by hours post fertilization (hpf)  and stor5'GAGCAGGTGGATTTGTGAACAGCGG3' [Tardbp (GTACATCTCGGCCATCTTTCCTCAG3’5’) and Fus (5’GGCCATAATCATTTGACGCCATGTT3’) and standard control (5’CCTCTTACC TCAGTTACAATTTATA3’) were also used. For Morpholino injection the optimal concentration of gene- specific AMOs were determined initially and 10 ng zfPGRN-A, 10 ng of Fus, 1.5 ng of Tardbp or 10ng of standard control were used.Anti-Sense Morpholino oligonucleotides (AMO)  were obtCAGCGG3' . The seqFUS (WT or mutant R521H) or 25ng/ μl of Tardbp (WT or mutant G348C) or PGRN-1 100 ng/ μl mRNA was used. The optimal concentration of gene specific mRNAs was determined to avoid toxicity related phenotype.For zfPGRN-A mRNA over-expression and rescue experiments a full-length zfPGRN-A/pcDNA3 vector was generated . BrieflyThe znp1 immuno-labelled wild-type embryos were carried out according to previously described protocol . BrieflyTo analyse the rescue effect of PGRN in reversing MN defects due to altered expression of TDP43 or FUS at a functional level, we tested the embryo touch response and avoidance swimming behaviour at 52 hrs post injection , 57. TheCaudal primary motor axons in whole-mounted 48 hpf wild-type embryos labelled with Znp1 monoclonal antibody. Only the trunk CaP MNs (12 pairs) were scored . Embryosin vivo. Images were captured at 5x magnification and the hatched box was further subject to 4-5x zoom. MN axonal length from the specified areas in the spinal cord region were measured using imageJ by tracing the labelled MN axons. The spinal cord length or embryo length were also measured by tracing spinal cord or embryo using imageJ. For each treatment at least three experiments were performed and at least 50 MNs were scored per determination. Values were expressed as mean +/- standard error of the mean.Embryos obtained from the HB9:GFP transgenic fish expressing GFP within the MNs following microinjection of embryos with AMOs or mRNAs or co-injection of both were visualized and photographed Statistical significance among experimental groups was determined by one-way ANOVA followed by Student-Newman-keuls Multiple Comparisons Test  using GraphPad software  or one-way ANOVA followed by Holm-Sidak Multiple Comparisons Test (p < 0.05-*) using Sigma plot software . Error bars represent s.e.m.Zebrafish embryos were lysed using Laemmli buffer and extracts were boiled for 5 minutes. Embryo extracts were resolved using 10% acrylamide gels and transferred to nitrocellulose membrane. SDS/PAGE western blotting analyses were performed as described previously using a p5'-TGATGGAACCACATGCTGTAA-3'/ 5'-CAGATGACCTCTGACCTGCTC-3’ or 5'-CTCAACTCACTCACATCCGC3'/5'-GTTTATAGAGTTAGGGCTC-3’as described in [5'-CCCCATGTCTAAATGCTCTCA-3'/5'-CTTCTCACTCTTCGCCATCAC-3' or 5'-CGTCACCTTCGCAGACGATCAGGTT-3'/5'-GCCCACGATCCATCATTTGCCTACTATT-3' and 5'-CGTCACCTTCGCAGACGATCAGG TT-3'/5'-GCCTAAGCACAATAATATTCATCACCTCTTTTCCAATT-3', respectively as described in [5'- ATGGATGATGAAATTGCCGC-3' and 5'-TGTCATCTTTTCCCTGTTGG-3' primers and zfPGRN-A mRNA observed employing 5'-TGATGGAACCACATGCTGTAA-3'/ 5'-CAGATGACCTCTGACCTGCTC-3’ as described in [Zebrafish embryos were lysed in trizol and the extracted RNA was quantified. cDNA synthesis was carried out according to the manufacturer’s protocol (Fermentas) and used for gene specific PCR reactions (Invitrogen). Species-specific primer sets designed to detect zfPGRN-A mRNA employing ribed in . TDP-43 ribed in . Beta-acribed in . PolymerIn previous work, we demonstrated that administration of the antisense morpholinos against zfPGRN-A but not control morpholinos, knock down zfPGRN-A protein expression. The protein suppression is specific for zfPGRN-A since there was no effect on levels of zf-PGRN-B, and the zfPGRN-A phenotype, namely impaired touch-sensitive motor responses, truncation of MN development and aberrant branching of MNs, is reversed by co-injection of zfPGRN-A mRNA or its human ortholog, hPGRN mRNA, but not by GFP mRNA when co-injected together with the zfPGRN-A morpholinos . Here weWe have further characterized the phenotype of zfPGRN-A knockdown animals. They display reduced overall body length and a corresponding reduction in the length of the spinal cord . This inTardbp, which is the ortholog of the mammalian TARDBP gene and its paralog, Tardbpl (TAR DNA binding protein-like). Tardbpl lacks the glycine-rich RNA binding domain where the ALS and FTLD-U mutations occur [Tardbp can result in alternate splicing of Tardpbl, resulting in restoration of the glycine-rich domain allowing the alternately spliced tardbpl protein product to potentially compensate for the loss of Tardbp [Tardbpl  or FUS(R521H) mutants, resulted in motor neuron abnormality using HB9:GFP embryos as well as WT embryos. We have observed at the morphological level, TDP43  and FUS We have quantified the axonal length among different groups. Reduced expression of either TDP-43 or FUS resulted in reduction of axonal length while co-injection of either TDP-43 AMO or FUS AMO together with PGRN significantly increased axonal length . In addiG348C) or FUS (R521H) mRNA  mRNA  resultedTo determine whether there is complementarity between TDP-43 and FUS with GRN, we investigated whether enhanced expression of TDP-43 or FUS is able to compensate for the depletion of zfPGRN-A. Expression of WT TDP-43 or FUS was unable to reverse the touch evoked swim response or the MN structural abnormalities in the zfPGRN-A knockdown in both HB9:GFP embryos  as well GRN results in a form of FTLD-U that is characterized by the accumulation of cellular inclusions of ubiquitinated-TDP-43 in affected neurons [GRN. There is considerable potential for therapies based upon PGRN. In contrast, PGRN does not suppress the development of disease-like phenotypes in models of ALS that were caused by mutations of SOD1 (Superoxide dismutase-1) [The development of CaP MNs in zebrafish embryos provides a valuable tool with which to investigate the interaction of genes that influence vertebrate MN development and function , 67. In  neurons , 68, 69. neurons  and AD [utase-1) , 70. It TARDBP represent 1–4% of familial ALS cases while mutations in FUS represent 4% of familial ALS cases [TARDBP [Mutations in LS cases . FUS andLS cases , raising [TARDBP . We haveunplugged that interfere with AchR clustering, also disrupt the outgrowth of primary MNs beyond the horizontal myoseptum [Depletion of zfPGRN-A in zebrafish embryos resulted in delayed development, as indicated by shorter body length . In addiyoseptum . AchR clyoseptum . We founGRN genes Click here for additional data file.S2 FigS2B Fig TDP-43 knockdown and partial rescue with over-expression of PGRN mRNA in WT embryos. Average number of Truncated (B1) and Branched (B2) CaP MNs per group. S2C Fig. FUS knockdown and partial rescue with over-expression of PGRN. Average number of Branched (C1) and Truncated (C2) CaP MNs per group.S2A Top panel. PGRN rescues motor axon defects produced by TDP-43 Knockdown in WT embryos. TDP-43 knockdown produced shorter axons that were rescued by co-injection of hPGRN mRNA. Lateral views  of embryos labelled with znp1 mAb at 27 hpf in wild-type embryos, embryos injected with TDP-43 AMO, embryos co-injected with hPGRN mRNA (TDP-43AMO+hPGRN). Embryos co-injected with hPGRN mRNA (TDP-43AMO+hPGRN) partially reversed the truncation phenotype. Observed phenotypes were normal MN development (WT), increase in truncated and branched axons (TDP43 MO) and partial rescue of truncated MNs (TDP43 MO+PGRN). Dashed lines represent the horizontal myoseptum. S2A Fig bottom panel. PGRN rescues motor axon defects induced by FUS knockdown. FUS knockdown produced shorter axons that were rescued by hPGRN mRNA. Lateral views  of embryos labelled with znp1 mAb at 27 hpf in wild type embryos, embryos injected with FUS AMO, embryos co-injected with hPGRN mRNA (FUS AMO +hPGRN). Embryos co-injected with hPGRN mRNA (FUS AMO+hPGRN) partially reversed truncation phenotype. Observed phenotypes were normal MN development (WT), increase in truncated and branched axons (FUS MO) and partial rescue of truncated MNs (FUS MO+PGRN). Dashed lines represent horizontal myoseptum. Images were captured at 20X magnification and the hatched box was further subject to 4-5X Zoom. (TIF)Click here for additional data file.S3 FigS3A Fig PGRN rescues motor defects due to TDP-43 knockdown (A1) but not vice versa (A2) in WT embryos. Touch-evoked swimming is greatly impaired in embryos injected with TDP43 Antisense MO. The motility defect was partially but significantly rescued when the embryos were co-injected with hPGRN together with the TDP-43 antisense MO (A1). S3B Fig PGRN rescues motor defects due to FUS knockdown (B1) but not vice versa (B2) in WT embryos. Touch-evoked swimming is impaired in embryos injected with FUS antisense MO. The motility defect was partially but significantly rescued when the embryos were co-injected with hPGRN mRNA together with the FUS antisense MO (B1). S3C Fig PGRN rescues motor defects due to toxic gain of function induced by TDP-43(G348C) (C1) /FUS (R521H) (C2) in WT embryos. Touch-evoked swimming is greatly impaired in embryos injected with TDP43 (G348C) or FUS (R512H). The motility defect was partially but significantly rescued when the embryos were co-injected with hPGRN mRNA together with TDP43 (G348C) (C1) or FUS (R521H) (C2).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Single layer graphite, known as graphene, is an important material because of its unique two-dimensional structure, high conductivity, excellent electron mobility and high surface area. To explore the more prospective properties of graphene, graphene hybrids have been synthesised, where graphene has been integrated with other important nanoparticles (NPs). These graphene–NP hybrid structures are particularly interesting because after hybridisation they not only display the individual properties of graphene and the NPs, but also they exhibit further synergistic properties. Reduced graphene oxide (rGO), a graphene-like material, can be easily prepared by reduction of graphene oxide (GO) and therefore offers the possibility to fabricate a large variety of graphene–transition metal oxide (TMO) NP hybrids. These hybrid materials are promising alternatives to reduce the drawbacks of using only TMO NPs in various applications, such as anode materials in lithium ion batteries (LIBs), sensors, photocatalysts, removal of organic pollutants, etc. Recent studies have shown that a single graphene sheet (GS) has extraordinary electronic transport properties. One possible route to connecting those properties for application in electronics would be to prepare graphene-wrapped TMO NPs. In this critical review, we discuss the development of graphene–TMO hybrids with the detailed account of their synthesis. In addition, attention is given to the wide range of applications. This review covers the details of graphene–TMO hybrid materials and ends with a summary where an outlook on future perspectives to improve the properties of the hybrid materials in view of applications are outlined. Since the first report in 2004 [−1·K−1) [−1) [2·g−1) [Graphene consists of a single layer of carbon in a two-dimensional 2D) lattice. It is a densely packed, atomically thin layer of sp in 2004 , graphenD lattice in 2004 . Graphen in 2004 . It also[−1·K−1)  and elec−1) [−1)  and high [2·g−1) . Graphen [2·g−1)  and negl [2·g−1) . Due to  [2·g−1) , hydroge [2·g−1)  and sens [2·g−1) . DespiteIn the last few decades, the synthesis of transition metal oxide (TMO) NPs has attracted much attention, providing the advantages of controlled shape, size, crystallinity and functionality, as well as being ecologically benign, corrosion resistance, easily scalable and relatively cost effective –16. In pThe interest in preparing graphene–TMO NPs hybrids is increasing enormously due to the peculiarities in combining the required properties of building blocks for a specified application. In the recent review by Khan et al., the synthesis, properties and applications of graphene–metal oxide composite NPs are discussed in detail . The rev−1. However, this is not sufficient for applications requiring high energy capacity. Single layer graphene has a high theoretical lithium storage capacity of 744 mAh·g−1, but graphene has a tendency to stack due to van der Waals interactions between graphene layers [−1 with 100% capacity retention for up to 100 cycles [Graphite is commercially used as an anode material for LIBs due to its large lithium storage capacity of 372 mAh·ge layers . The ince layers . Poizot 0 cycles . For met0 cycles . These dGraphene is used as a supercapacitor because of its unique properties, such as high surface area, excellent flexibility, chemical inertness and good electrical conductivity . The praIn a photovoltaic cell, sunlight energy is directly converted to electricity. Graphene and graphene–NP hybrids have been investigated extensively in the field of solar cells because of their unique properties, such as high optical transparency, electrical conductivity, and mechanical flexibility. As mentioned before, graphene is an excellent electron-accepting and electron-transporting material. When graphene is integrated with semiconductor materials, it promotes photogenerated electrons through π–π bond interactions and suppresses the charge recombination in the semiconductor materials . As a co2/Si or glass [The first free-standing single-layer graphene was obtained in 2004 by the isolation of graphene from graphite by micromechanical cleavage . Later, or glass . For the2 which form amines, hydrazones or other similar functional groups covalently attached on the sheet of graphene [4 has been demonstrated as being more effective than hydrazine as a reducing agent of GO. NaBH4 is more effective at reducing the C=O group, while it has moderate efficiency in the reduction of epoxide and carboxylic acid [2 [The most common top-down approach for the synthesis of graphene is the reduction of GO and of graphite fluoride by thermal or chemical methods. The reduction of GO is a low cost, facile technique and yields bulk quantities of GSs. GO was first reported 150 years ago . It is ngraphene . NaBH4 hlic acid . Other rlic acid , gaseous acid [2 , alkalin acid [2 –59, and  acid [2 . Despite acid [2 –62, elec acid [2 –64, ther acid [2 ,65, phot acid [2 , solvoth acid [2 –68, ther acid [2 , or chem acid [2 . In termFor the preparation of graphene–NP hybrids, bulk quantities of GSs are required. So, for the synthesis of graphene–NP hybrid systems, in most cases, top-down approaches have been used. Different architectures of graphene–NP hybrids can be prepared by this one-pot synthesis method . In anot2 is an interesting material because of its unique optical and electrical properties. It has been used as a heterogeneous photocatalyst and it has outstanding advantages because of its low cost, scalability, nontoxicity, strong photo-oxidising power and stability in oxidative and acidic environments [2 nanomaterials has shown that the energy conversion efficiency of the photovoltaic devices that use TiO2 NPs critically depends on the morphology and size of the NPs [2–graphene hybrid systems, the morphology of TiO2 plays an important role in various applications.Nanocrystalline TiOronments . Extensi the NPs –80. Addi2 anode materials have outstanding cycling stability and almost no volume expansion occurs when TiO2 is fully lithiated. As the electrical conductivity of TiO2 is low, it has a weak rate capability in electrical devices. The modification of TiO2 with conductive materials like graphene improves its electrical performance significantly. The introduction of graphene into TiO2 results in increased conductivity of the hybrid material, higher transparency and efficient charge separation of the system which causes enhanced photocatalytic activity and other novel properties [2 anodes and is responsible for the improved photocurrent response of the hybrid materials [TiOoperties . Also, daterials .2–graphene hybrids, TiO2 is prepared by hydrolysis of Ti-containing precursors [2 NPs. The fabrication of TiO2–graphene hybrid systems results in a strong coupling between the components therefore resulting in superior activity as compared to the individual materials. The resultant hybrid material displays a synergistic effect of accumulated graphene and TiO2 NPs, exhibited by the extraordinary physical and chemical properties in comparison with pristine graphene and bare TiO2 NPs [In most cases, for the formation of TiOecursors –85 as thTiO2 NPs –87.2–graphene hybrid systems. Liang et al. prepared TiO2 NCs on the GO sheet by hydrolysis methods and then converted GO to graphene by hydrothermal treatment [2–graphene nanocomposite hydrogel (TGH) was prepared by Zhang et al. by a facile one-pot hydrothermal approach where the spherical nanostructured TiO2 NPs were densely decorated onto the GS [Different synthesis routes have been introduced; among them, hydrothermal methods are used extensively for the preparation of TiOreatment . A TiO2–o the GS .2 sandwich structure was prepared by using ethylenediamine/H2O solvent in a reduction–hydrolysis technique. The photocatalytic activity of the hybrid has been confirmed by the conversion of CO2 to valuable hydrocarbons (CH4 and C2H6) in water vapour  and GO nanosheets. The layer materials are spatially phase segregated to maintain unique 2D characteristics due to the functional separation of the layers at the nanoscale [An anionic surfactant-mediated growth of the self-assembled TiOge rates . When ananoscale . This hy2–Pt–graphene hybrid has been prepared by Xia et al. where the graphene was synthesised by the arc discharge method and the Pt–graphene hybrid electrocatalysts were prepared using a polyol process. This structure exhibits enhanced electrochemical performance due to the strong metal–support interaction and proposed synergetic effect [2 nanoparticle films for application in dye-sensitised solar cells. The conductivity of the film increases with the incorporation of the GSs, which in turn results in an enhancement of the power conversion efficiency [A highly efficient, stable, sandwich-structured TiOc effect . A molecficiency .2 intercalated between graphene layers as pillars which provide a 3D open space with distinct advantages when used as LIB anode materials [2 NPs, and then the TiO2 NPs were wrapped by graphene. These hybrids have high potential for photocatalytic application [2 nanospheres as efficient photocatalysts for the decomposition of rhodamine B with an efficiency up to 91% in 90 min, which is much higher than the efficiency of TiO2 nanospheres (65%) [2 nanospindles [2 nanorods [2 mesoporous [A unique three dimensional (3D) nanostructure was fabricated by Hu et al. with nanometre-sized TiOaterials . (3-Aminlication . Zhang ees (65%) . The graspindles , TiO2 nananorods  and TiO2soporous  shows im2 make use of its photocatalytic activity, there are also some applications of shape-controlled TiO2–graphene hybrids used in pollutant abatement [Although most of the applications of graphene–TiObatement , high-pebatement , and selbatement .2 has two crystalline phases, monoclinic and rutile. The monoclinic form of vanadium(IV) oxide can be transferred to the rutile form by a thermally induced, reversible treatment at 68 °C [2 (M) behaves as a semiconductor whereas VO2(R) behaves as a semimetal. Vanadium pentoxide (V2O5) has very low electronic conductivity due to its low d-band mobility. It also shows a thermochromic transition at 257 °C and this material is used as a catalyst for industrial processes, gas sensors and in LIBs [2O5 such as nanotubes, nanowires, nanofibers, nanobelts, and nanorods have been prepared by sol–gel processes, hydrothermal processes [2 is quite stable during lithium intercalation–deintercalation cycles and has been regarded as a promising electrode material for both organic and aqueous LIB owing to high capacity, unique structure and suitable electrode potential. However, its cyclic performance is limited by its high charge-transfer resistance. The incorporation of graphene solves its electrical resistance problem with the VO2 electrodes and it acts as a high-performance electrochemical capacitor [Vanadium has oxidation states ranging from −1 to +5. Binary vanadium oxides have already been proven as a potential material for studying superconductivity at high pressures and low-dimensional quantum-spin transitions . VO2 hasat 68 °C . VO2 (M) in LIBs . Variousrocesses , electrorocesses . Other vapacitor , shows eapacitor , and repapacitor .2–graphene architectures by using graphene as a substrate for the in situ growth of VO2 ribbons (2O5 by GO simultaneously in a Teflon lined autoclave [2 (M) nanotube–graphene hybrids cathode material is used in LIB, where the VO2 nanotubes are wrapped by and trapped between the GS [Yang et al. have prepared VO ribbons  via hydrutoclave . When thn the GS . Althoug2 nanosheet-built 3D flower hybrids [2O5 nanoribbons [2O5 which facilitates the Li+ diffusion, significantly improving the electrochemical performance. V2O5 quantum dot/graphene is a promising cathode material for use in long-life rechargeable Li batteries [2–graphene hybrid materials for the fabrication of thermochromic films for energy saving windows. VO2 crystals have been prepared on graphene and the graphene-supported VO2 is easily transferred to a plastic substrate which enables formation of a new type of flexible thermochromic film [To improve the rate capability, Nethravathi et al. use a hydrothermal method to prepare N-doped graphene–VO hybrids , where toribbons . These satteries –116. Kimmic film . This hy2O3, a trivalent chromium(III) oxide, is an important industrial material that has been used in abrading agents and pigments. Unlike other TMOs, Cr2O3 exhibits poor dispersion. It is insoluble in both acidic and alkaline media. The low electrical conductivity of Cr2O3 inhibits its application for practical use and electrical research fields. The poor dispersion results in a low surface area and a nonuniform porous structure which restricts its supercapacitive performance. Severe volume change happens for Cr2O3 during the charge–discharge cycles which results in a rapid capacity fading and the end of the cycle life.Cr2O3 to improve the properties of the materials and the other strategy is to develop Cr2O3 materials on the nanoscale. Graphene–Cr2O3 hybrids have been explored with respect to the oxygen reduction reaction (ORR) [2O3–rGO hybrid makes it a promising catalyst for fuel cells.Several strategies have been designed to increase the conductivity of the materials and also to improve the volume change. In that context, graphene has been hybridised with Cron (ORR)  and applon (ORR) . ORR is 2), hausmanite (Mn3O4) and bixbyite (Mn2O3) are important minerals of manganese. These oxides have attracted great attention because of their environmental benignity and the high abundance of Mn in nature. Among all the oxides of manganese, Mn3O4 has been studied widely as an anode material for LIB to achieve higher specific capacities than graphite [3O4 has a spinal structure and is a potentially interesting electrode material as an electrolytic supercapacitor because of its low cost, environmental compatibility and intrinsically high capacity [3O4 and Fe3O4, Mn3O4 is a much less conductive material, so it is a great challenge to explore the Mn3O4 graphene hybrids in LIB applications. As a bulk material, Mn3O4 has low electrical conductivity that limits its ability in terms of both capacitance and capacitance retention at high current density. The most and widely used strategy is to combine Mn3O4 to lightweight and electronically conducting graphene. Graphene–Mn3O4 hybrids have also been employed as active, stable and low cost cathode electrocatalysts used for high capacity anode materials for LIBs [2O4 is used for the vanadium redox flow batteries [3O4 is electrochemically oxidised to the more conductive MnO2. This explains the interesting phenomenon of increasing capacitance with cycling [Pyrolusite  [3O4 and N-doped rGO which shows pronounced electrocatalytic activity towards ORR in alkaline solutions [3O4 NPs on the GO sheets. The Mn3O4–graphene hybrid is being explored for high capacity, low cost, nontoxic anode materials for battery applications . The application of MnO in LIB is a great challenge because the low electric conductivity of MnO results in poor cycling stability and inferior rate capability. A long-term stable, nano-architecture of graphene-supported MnO NPs for LIB applications has been prepared by cycling where the oxidation of Mn(II) to Mn(III) and interfacial lithium storage upon cycling contribute to an enhanced specific capacity [The Mnlue (MB) , for thelue (MB) , for carlue (MB) , for ORRlue (MB) , for enhlue (MB) –133, andlue (MB) . Bag et olutions . This hyolutions , liquid olutions  or hydroolutions  for growications . MnO2 haeactions . Yu et afor LIBs . Dong et acidity . Ultrathpacitors . Graphenications . MnO hascapacity . N-dopedcapacity .2O3 has the advantages that it can be produced in high abundance, with low cost, and the nontoxicity of Fe results in a reduced environmental concern. Therefore, it is expected to meet the requirements of future energy storage systems. It is an attractive anode material for LIBs as it has a high theoretical capacity (1007 mAh·g−1) [2O3 electrode pulverisation and rapid capacity decay happens due to the large specific volume change and particle aggregation. These obstacles can be removed by creating hybrids of Fe2O3 and graphene which have superior performance regarding flexibility and electrical conductivity [2O3–graphene hybrids are prepared by hydrothermal methods without any reducing agent. Tian et al. have prepared α-Fe2O3 NP anchored graphene hybrid materials by hydrothermal methods which have good cycling performance and enhanced rate capability [2O3–graphene has been fabricated by vacuum filtration and a thermal reduction process. This network shows superior electrical performance because the porous nature of the hybrid accommodates the volume change [3O4 NPs (Fe3O4/N-GA) as efficient cathode catalysts for ORR [3O4/N-GA material shows excellent electrocatalytic activity for the ORR in alkaline electrodes. It is even larger than N-doped carbon black or N-doped GSs (N-GSs) due to its 3D macroporous structure and high surface area, in addition to exhibiting a higher current density, lower ring current, lower H2O2 yield, higher electron transfer, and better durability   which isuctivity . Most ofpability . A 3D nee change . Wu et a for ORR . This Ferability . Chen etormation . Fe2O3 smaterial . Fe2O3 g in LIBs –153. Li material . α-Fe2O3 of H2O2 . Graphennd Fe3O4 . Yang etnd Fe3O4 . Liang ene paper . In anotformance . One int storage . Anotherfor LIBs . The impe2O3 NPs ,162. Therocesses . Su et arocesses .2O3 NPs showed volume expansion and morphological changes upon lithiation, and the surface of the electrode was covered by a Li2O layer. They also found that the single crystalline Fe2O3 NPs were converted to polycrystalline NPs and the delithiated product is no longer Fe2O3 but FeO. A solvothermal process is introduced to construct 3D macroscopic Fe2O3 nanocube/N-doped graphene aerogels as an anode material for LIB applications [2O3 and GS hybrids, which have excellent prolonged cycling stability [3O4–rGO hybrids by one-pot solution chemistry which have good adsorption capability of various dyes , malachite green, and new coccine) [2O3 NPs. In addition, the materials could be regenerated and utilised via a simple annealing treatment.Feications . This aetability . Geng etcoccine) . These m3O4)–graphene hybrids can be synthesised by solution methods. Like other hybrid TMO–graphene materials, this hybrid is also used as an anode for LIBs [3O4–N-doped graphene exhibits similar catalytic activity like Co3O4–graphene hybrids but superior stability to Pt in alkaline solution [3O4 (Co3O4/N-rmGO than in Co3O4/rmGO) due to favourable nucleation and anchor sites for Co3O4 nanocrystals as N-groups help on rGO. In the ORR, the electronic effect of N-doping of graphene also plays a role. Mao et al. have prepared 3D crumbled cobalt–GO nanostructure hybrids which show both ORR and oxygen evolution reaction (OER) [3O4/flocculent graphene hybrids on Ni foam for supercapacitor applications as their nanocluster morphology synergistically results in an improved electrochemical performance [3O4 NPs on vertically aligned graphene. This hybrid has also been used for nonenzymatic glucose detection in microdroplets [Cobaltosic oxide  . Wu et aformance . Symmetrdroplets .3O4 hybrid electrodes constructed on a micropipette tip which can detect nonenzymatic glucose [3O4 has been used for selective oxidation of olefins and alcohols [3O4 NPs were anchored on conducting graphene and used as an anode for high-performance LIBs. This hybrid material exhibited large reversible capacity, excellent cyclic performance and good rate capability [3O4 NPs on N-doped graphene by using a hydrothermal method [3O4–graphene hybrid possesses catalytic performance for heterogeneous activation of peroxymonosulfate for the decomposition of phenol [3O4, homogeneous dispersion of the NPs, increased surface area of the catalyst, and most importantly, the synergetic interaction between the N-doped graphene and Co3O4 NPs. In another work, Singh et al. have prepared three different morphologies of Co3O4 nanocubes, blunt edge nanocubes and spherical particles supported on N-doped graphene by a hydrothermal method [3O4 NPs and their synergistic interaction between N-doped because graphene helps to tune the properties of the OER electrocatalyst. Yang et al. fabricated graphene encapsulated Co3O4 NPs which have a high reversible capacity of 1000 mAh·g−1 over 130 cycles and is superior to Co3O4 NPs with respect to capacitor applications [3O4-intercalated hybrid nanostructures using microwave irradiation [Wang et al. fabricated graphene–Co glucose . N-dopedalcohols , LIBs [1alcohols , and oxyalcohols , and as alcohols . Co3O4 Npability –179. Magpability . The Co–pability . Singh el method . Here, tf phenol . Moreovel method . Among tications . Kumar eadiation  and this2 for phenol degradation and it takes only 10 min for 100% phenol removal.CoO attracts extensive interest because of its high lithium storage capacity. CoO–NP-supported GSs effectively circumvent particle cracking, pulverisation, and aggregation upon cycling, thus serving as a high-performance anode material with long cycle life, high reversible specific capacity and excellent rate capability for LIB applications . Self-asII and NiIII. The NiO–graphene hybrids show good sensing capability for the reducing gases such as H2, NH3, H2S, NO2 [NiO, a p-type wide band gap semiconductor is extensively used as catalyst, battery cathode, electrochemical capacitor and magnetic material . NiO is H2S, NO2 . In anotH2S, NO2 . RecentlH2S, NO2 . This hyH2S, NO2 –208.Cu nanowire (NW) films and indium tin oxide (ITO) films have comparable sheet resistances and optical transmittance. A few drawbacks of Cu NW films, such as low oxidation resistance or weak adhesion to the substrate, can be compensated for by the addition of graphene. The resulting hybrid films have improved electrical conductivity as they provide 2D pathways for charge transfer . The gra2O) is a p-type semiconductor and is used for solar energy conversion, as sensors and for photocatalytic degradation. The controlled synthesis of Cu2O results in a vast palette of architectures including nanocubes, nanocages, nanowires, solid and hollow spheres. Graphene-wrapped Cu2O nanocubes exhibit higher electrocatalytic performance towards electro-oxidation of glucose with lower detection time, and this hybrid has improved electrochemical stability [2O microspheres on rGO by a one-step synthesis which have potential application for nonenzymatic electrochemical glucose and H2O2 sensors [2O and Cu NPs on copper foil by annealing at 200–1000 °C under an Ar atmosphere where the particle phase and particle size and shape strongly depend on the process temperature [2O NW mesocrystals by nonclassical crystallisation under hydrothermal conditions [2O NW–graphene hybrids, was used as a high-performance NO2 gas sensor  and partially encircle the ZnO NPs. The quasi-core–shell structure of the hybrid was prepared by a one-step chemical method [Most of the reports of graphene hybrid systems are related to ZnO NPs, as it is very easy to control the size and morphology of ZnO NPs as well as the properties of the material. ZnO is an important II–VI semiconductor with large direct band gap of 3.37 eV and large exciton binding energy of 60 meV. It has been extensively studied because of its potential application in solar cells, sensors, diode lasers, piezoelectric devices, as surface acoustic wave propagators, antibacterial agents and ultraviolet light emitters. Graphene-based ZnO hybrids proved to be promising materials having a much wider range of applications in the field of energy conversion and storage, such as catalysts and in optoelectronics. Comprehensive work has been done to prepare graphene-wrapped ZnO NPs for better performance in optoelectronics applications. Single-layer graphene-wrapped ZnO NPs  have beel method .For the formation of graphene-wrapped ZnO NPs, some other routes have been used, like chemical functionalisation of the ZnO surface. In this process there are three steps: surface modification of NPs mainly by amine groups, coating the NPs with a GO shell, and finally, conversion of GO to graphene by a reduction process. For the amine functionalisation of metal oxide NPs, poly (PAH) –227 and 3, NO [2 [2 [The optical properties of ZnO NPs change when they form hybrids with graphene. Pure ZnO exhibits a photoluminescence (PL) band at 373 nm, which is due to the exciton recombination corresponding to the band edge emission, and a green emission peak is most commonly observed that arises from the defect of ZnO NPs such as oxygen vacancies, zinc vacancies, oxygen interstitials, and zinc interstitials . Graphen3, NO , methane3, NO , NO2 [263, NO [2 , formald3, NO [2 –266, H2 NO [2 [2 , and alsNO [2 [2 . FurtherNO [2 [2 –270. TheNO [2 [2 .−1) but it has attracted less attention compared to the other metal oxides due to its poor cyclability resulting from huge volume change during charge–discharge cycles. Kushima et al. show that when ZnO NWs are partially lithiated, multiple cracks are induced and divided the NWs into multiple segments. This results in a lithium embrittlement effect in ZnO [ZnO can be used as an inexpensive anode material and has high theoretical capacity (978 mAh·gt in ZnO . Such stt in ZnO . ZnO–grat in ZnO .Excellent performance in lithium storage capability is observed by incorporation of a conductive nitrogen-doped graphene matrix with ZnO due to a synergistic effect . Boruah 4), has attracted increasing attention because it is a promising candidate for cathode materials [4 is a promising cathode material for LIBs because of its high stability, high power, environmental safety, and low cost. However, the main drawback for LiFePO4 battery commercialisation is their poor rate performance (at current >5 C) due to the low electrical conductivity and Li-ion diffusion rate which limits its usage for electrical applications. Those drawbacks are compensated for by hybridising LiFePO4 with graphene. Graphene-hybridised LiFePO4 has been largely used for LIBs [4 are mainly that the operating voltage and theoretical capacity can be increased by hybridisation with graphene. Shi et al. have prepared graphene-wrapped Li3VO4 microboxes by a one-step in situ hydrothermal method. This hollow structure could relax the stress and strain of Li+ insertion/extraction and provides extra space for the storage of Li ion. It also increases the surface area of the material, resulting in improved capacity, rate capability and cycling performance [Two TMOs can be hybridised with graphene for improved performance which increases the range of potential applications of the hybrid materials. Since the report of Padhi et al., the iron-based nanomaterial, olivine lithium iron phosphate Co(III)Mn(III)O4. Hybridisation of this material with graphene results in further optimisation of catalytic properties. Wang et al. prepared a MnCo2O4–graphene hybrid by a two-step solution method by using Co(OAc)2 and Mn(OAc)2 precursors [2O4–graphene has been used as a LiO2 battery cathode [2+ replacement in the Co3O4 matrix by M2+  for increasing the catalytic performance [2O4 hybrids have been used for the ORR [2O4 hybrids as a methanol-tolerant electrocatalyst for the ORR with improved performance [2O4–graphene has also been used in electrochemical pseudo-capacitor applications [2O4 was anchored on the N-doped graphene prepared by a hydrothermal method, used for LIB applications, where the N-doping further improved the ORR performance in the LiO2 batteries [2O4 nanostructures on a 3D graphene foam with in situ deposition by a facile hydrothermal method which is used as an enzyme mimic for glucose and calcium detection [2O4 nanoplatelets and graphene hybrids have been used for ORR and OER [Much effort has been attributed to the development of Co-based anodes for LIB applications by partially replacing Coecursors . The hyb cathode , an oxyg cathode , and in  cathode . Mohamedformance . For the the ORR . Zhang eformance . NiCo2O4ications . NiCo2O4atteries . Wu et aetection . NiCo2O4 and OER –304.2O4 outperformed Co3O4–graphene hybrids in terms of the onset potential and current densities. This property is attributed to the fact that the incorporation of Ni cations into the octahedral sites of the spinel crystal structure enhances the electrical conductivity as well as to the creation of new active sites with much lower activation energy [2O4, but can also offer effective buffering to accommodate the lithiation-induced stress which is beneficial to lithium storage and cycling stability [2O4–graphene is an attractive electrode material for supercapacitors [2S4–graphene hybrids synthesised by a one-pot solvothermal strategy are used for ORR and ORE [2O4 NPs on N-doped rGO. This was revealed to be an efficient electrocatalyst, produced by a two-step solvothermal method which exhibits ORR catalytic activity [2O4/N–rGO hybrid, GO was reacted with Co(OAc)2 and Cu(OAc)2 in a 2:1 molar ratio at 80 °C in ethanol/water/ammonia, which led to the selective formation of the hybrid  of CuCo2O4/N-rGO, Co3O4/N-rGO, CuCo2O4 + N-rGO, CuCo2O4, and N-rGO suggest that CuCo2O4/N-rGO outperforms the other catalysts in terms of disk current density and half-wave potential in an O2-saturated 1 M KOH solution at a rotational rate of 1,600 rpm  has attracted increased attention because of its large application as a catalyst. Spinal ferrites have interesting magnetic, magneto-resistive and magneto-optical properties. The high density magnetic storage is derived from the large surface area-to-volume ratio on the nanometre scale, and these materials have super-paramagnetic behaviour. These spinal ferrites are considered to be promising anode materials for LIBs given their high specific capacity which is typically 2–3 times higher than that of the graphite anode used in the commercial LIBs. Bai et al. have used a one-pot solvothermal method for the synthesis of MFe2O4  through the simultaneous reduction of GO and growth of MFe2O4 [2O4 microsphere can be controlled by adjusting the metal ion concentration. This hybrid displays a large adsorption capacity and photocatalytic activity towards RhB and MB and can be easily separated by a magnet. Among all the ferrites of the form MFe2O4 , manganese ferrite (MnFe2O4) has been found to exhibit usually large capacitance. A MnFe2O4–rGO hybrid which shows higher catalytic performance than pure MnFe2O4 to activate peroxymonosulfate (PMS) to various oxidatively degraded organic pollutants in water, has been prepared by a facile approach [Over the past years another spinal-type ferrite having the general formula MFef MFe2O4 . In thisapproach .2O4–graphene hybrids by immobilising the MnFe2O4 microspheres on the graphene nanosheets by a solvothermal route for supercapacitance application [2O4 nanohybrids are used for removal of lead and arsenic from water [Cai et al. have synthesised MnFelication . This hylication  and effilication . GO–MnFeom water .2O4) which was proposed for biomedical applications. The hybrid material shows good magnetism and could be separated from the solution by using a magnet. This hybrid has been prepared by a solvothermal method, a facial vapour diffusion method [2O4–graphene hybrids show improved electrochemical properties and are used as binder-free anode materials for LIBs [2O4–graphene hybrids by chemical precipitation of Fe and Co precursors and reduction of GO in a hydrazine hydrate solution; this hybrid has been used for the degradation of phenol [Another material in the family of spinal-type ferrites is magnetic cobalt ferrite  of phenol [Li et al. prepared graphene-functionalised CoFeom water . Yao et f phenol  (Figure 2Fe2O4–graphene hybrid shows that the hybrid shows better catalytic activity than pure CoFe2O4 (2Fe2O4–graphene hybrids can be easily removed from the reaction solution by applying an external magnetic field (2O4–graphene hybrids are used for photodegradation of MB [2O4 hybridised with N-doped graphene provides a simple and efficient way to configure hybridised electrode materials with high lithium storage capacity [2O4. Furthermore, this hybrid has been used for microwave absorption applications and as electromagnetic wave absorbers [2O4 superparamagnetic NCs [2O4 by the introduction of graphene is due to the synergistic effect between the remarkable magnetic loss from the superparamagnetic CoFe2O4 nanocrystals and high electric loss from the lightweight graphene. For LIB applications, graphene-hybridised CoFe2O4 [3Sn2@Co [A nitrogen absorption isotherm was used to estimate the pore size of the hybrid to be about 1.7–5.9 nm. The catalytic study of the Co CoFe2O4 . The Co2ic field . CoFe2O4on of MB –323, degon of MB  and the on of MB ,325. Thion of MB . The biccapacity . The autbsorbers . Li et aetic NCs . The enh CoFe2O4  and Co3S[3Sn2@Co  have bee2O4 is an important, promising anode candidate for LIB applications due to its low discharge potential and high theoretical specific capacity of 1072 mAh·g−1 [2O4-graphene hybrid films are directly usable anodes for rechargeable lithium half-cells without adding any polymer or conductive additives [2O4 is a magnetic semiconductor material, thus ZnFe2O4-based catalysts can be magnetically separated in the suspension system. ZnFe2O4–graphene hybrids prepared by the hydrothermal method show higher catalytic performance [2O4–graphene hybrid by a self-assembly method for application in LIBs [ZnFe mAh·g−1 . The ZnFdditives . ZnFe2O4formance –332. The in LIBs . The hyb in LIBs .In summary, graphene–TMO NPs hybrids have received particular attention because of their unique properties. Due to the advantageous properties of these graphene–TMO hybrids, they have wide-spread application potential as photocatalysts, anode materials in LIBs, solar cells, sensors, diodes and also for the removal of organic pollutants from water .A multitude of synthesis methods have been introduced by different research groups for the synthesis of the hybrids, among those, hydrothermal methods, sol–gel processes, chemical synthesis, and microwave-assisted growth have drawn the most attention in top-down approaches, while in bottom-up approaches, CVD and arc discharge methods have been mostly used. Depending on the application need, hybrids with different structures have been prepared by selecting the appropriate synthesis process .Since the first experimental demonstration of single sheet graphene in 2004, the field of graphene-related research has grown at an outstanding speed. Graphene has a unique morphology, large surface area, and extraordinary electronic properties. Graphene has been synthesised by various methods and its chemical structure can be modified by changing the fabrication methods and also by combining with various NPs. Such materials are produced to meet the increasing demands of high-performance materials in electronic devices.Enhanced knowledge on the surface chemistry and functionalisation of the graphene surface, and also on the improvement in the colloidal synthesis of NPs, will lead to a wide range of potential applications related to the preparation of graphene–NPs hybrid materials by the incorporation of various NPs. The synthesis of graphene-based composites also requires a large amount of graphene, preferably with surface modification. By tuning the surface chemistry of graphene, the fluorescent properties can be changed accordingly which enables its potential application in photovoltaics, sensors and also in bio-imaging. The number of publications related to graphene and graphene-based materials has increased exponentially in recent years. However, there is still need for the scientific community to develop graphene-based models, hybrid materials and devices, and their possible applications in various fields. By improving the quality of the synthesised graphene, better quality hybrids can be prepared for different applications.Knowledge of GO chemistry will provide valuable input about its reactive properties and the properties of the graphene derived from it. Controlled methods of graphene synthesis are particularly important for the controlled growth of layers, which still remains to be established. Regarding the fabrication of graphene NP hybrids, the key obstacle lies in the reproducibility of the structure. For the synthesis of graphene-encapsulated NPs, the difficulty is that the thin graphene layer is not ideally formed as a uniform layer on all the NPs. Thus, the design and synthesis of new graphene-based nanostructures and architectures with uniform structure will be an important task for the future. It is important to mention that no such unique analysis technique has been introduced which can confirm that all the NPs are uniformly coated. In most reports directed at graphene-encapsulated structures, a quasi-core–shell structure was obtained instead of the desired core–shell structure.By achieving a high level of uniformity and reproducibility of graphene–NP hybrids, the electrical properties of the devices can be optimised which will lead to higher sensitivity and selectivity. Inexpensive and large-scale fabrication of defect-free graphene is still a challenge due to the existence of defects or oxidation sites in graphene, which is partially restored in graphene derived from GO. However, there is no doubt that more efficient synthetic strategies for graphene and graphene-based hybrids will be developed in an economically feasible quantity, leading to the commercialisation of graphene in the near future. Some research shows that the surfactants and the reductants used for the synthesis of the hybrid materials have a detrimental effect. In this sense, the biocompatibility and toxicity of the chemicals should be investigated to enable application of the hybrid materials in biomedical applications. Most of the applications of TMO–graphene hybrid materials are in LIBs, although further study is required to gain control over the phase and morphology of the NPs for this application. A prerequisite is a well-mastered growth synthesis to obtain a well-defined uniform structure of graphene. Control over the interface and interaction between NPs and graphene is necessary to enhance the performance of LIBs. Much attention is devoted to the application of graphene–NP hybrids in supercapacitor applications but more research is necessary. During the charge–discharge process, the graphene layers become aggregated, which degrades the electrode material. In some cases, the electrode materials are contaminated by impurities introduced from the incomplete reduction of GO. When using graphene–TMO hybrids as a photocatalyst, the improved performance in the catalytic properties is generally ascribed to the extended absorption and faster charge transfer in graphene as compared to TMOs. Therefore, research on the interfacial status of the hybrid is important to obtain a detailed understanding of the origin of the improvement.Further challenges exist in the application of graphene–TMO NP hybrids on the industrial scale. Some advanced applications of the graphene-based hybrids require particular understanding of the contact between the surface of the graphene and the NPs, which will have a direct impact on the properties of the hybrid. Despite the enormous efforts and huge attention, a significant breakthrough application of graphene and graphene-based materials in energy harvesting systems has not been achieved to date, but solutions to the key challenges appear within reach. In view of the manifold of potential applications, each having different requirements regarding material properties, it can be anticipated that the research on graphene–TMO NP hybrid materials will have a very bright future.


A methyltransferase enzyme termed ‘MddA’, which catalyses the methylation of MeSH, generating DMS, in a wide range of bacteria and some cyanobacteria, may mediate this process, as the mddA gene is abundant in terrestrial metagenomes. This is the first study investigating the functionality of MeSH-dependent DMS production (Mdd) in a wide range of aerobic environments. All soils and marine sediment samples tested produced DMS when incubated with MeSH. Cultivation-dependent and cultivation-independent methods were used to assess microbial community changes in response to MeSH addition in a grassland soil where 35.9% of the bacteria were predicted to contain mddA. Bacteria of the genus Methylotenera were enriched in the presence of MeSH. Furthermore, many novel Mdd+ bacterial strains were isolated. Despite the abundance of mddA in the grassland soil, the Mdd pathway may not be a significant source of DMS in this environment as MeSH addition was required to detect DMS at only very low conversion rates.Dimethylsulfide (DMS) is an environmentally important trace gas with roles in sulfur cycling, signalling to higher organisms and in atmospheric chemistry. DMS is believed to be predominantly produced in marine environments via microbial degradation of the osmolyte dimethylsulfoniopropionate (DMSP). However, significant amounts of DMS are also generated from terrestrial environments, for example, peat bogs can emit ~6 μmol DMS m Microbes, particularly marine bacteria, drive the production of DMS, generating ~300 million tonnes er annum  through er annum . DMS oxier annum . Howeverer annum . When DMer annum .S-methyl-methionine, dimethylsulfoxide or methoxyaromatic compounds  in marine environments via DMSP lyase enzymes , nitrogen-fixing cyanobacteria and sediment-dwelling pseudomonads. The mddA gene is present in marine metagenomes such as the Global Ocean Sampling and the Monterey Bay microbial study, but, it is much more abundant in terrestrial environments and particularly in soils, where it is predicted to be present in up to 76% of bacteria , a forest soil and two agricultural soils  and MeSH added as sodium methanethiolate  as described in To study DMS production from MeSH by different environments, 1 g or 20 ml of sample was placed in a 125 ml serum vial containing distilled water supplemented with Y minimal medium 5% , succinat=0 and t=19 h by GC.Air-tight field chambers of 2 l volume were placed in the grassland soil studied and supplemented with MeSH (200 μmol) as described in −1 (Sigma-Aldrich) or with 100 μg ml−1 ampicillin, 50 μg ml−1 chloramphenicol, 5 μg ml−1 tetracycline and 400 μg ml−1 streptomycin (Sigma-Aldrich), incubated and assayed for Mdd by GC as described in Microcosm experiments were supplemented with cycloheximide 200 μg mlM). The second set of enrichments was supplemented with MeSH (20 μmol). The third set of microcosms was supplemented with succinate (5 mM) and MeSH (20 μmol). Vials were incubated at 22 °C for 14 days and supplemented daily with fresh MeSH to the corresponding samples as this gas disappeared after 24 h. MeSH and DMS in headspaces were monitored by GC as indicated on To study the effects of MeSH addition on the processes of DMS production and consumption, and on bacterial diversity, three different enrichments were each set up in triplicate. One set of enrichments was supplemented with succinate (5 mM) and MeSH (20 μmol) were set up in triplicate to estimate MeSH consumption and DMS production and consumption rates as detailed in Two sets of microcosms supplemented with succinate  and DMS (272 nmol) for 24 h at 30 °C before measuring MeSH and DMS concentrations in the headspace by GC, as detailed in GC was performed as described by t=0 and those enriched with succinate plus MeSH for 14 days were plated onto Y minimal medium supplemented with succinate (5 mM) and MeSH (1 mM) as carbon sources. Colonies with different morphologies were purified and selected for further characterisation. Isolates were taxonomically identified by sequencing the 16S rRNA gene and characterised for their MeSH and DMS production as detailed in Samples from For nucleic acids extraction from environmental samples, 16S rRNA gene amplicon sequencing, metagenomics and statistical analysis, see −1, which represents 0.1±0.01% of the added substrate. In control incubations with sterile soil supplemented with MeSH, MeSH disappeared but there was no DMS production. Chemical oxidation of MeSH to dimethyldisulfide (DMDS) was also studied, but there was no variation in DMDS in the soil samples  or sterile controls  over the incubation period. No other sulfurous compounds were detected in any of the samples. From these results, we conclude that the Mdd pathway is active in microorganisms in this grassland soil when MeSH is available.To study the significance of the Mdd pathway in the environment, we initially analysed the ability of a local grassland soil with neutral pH (6.7) to produce DMS from MeSH. Soil samples incubated in the absence of MeSH produced no MeSH or DMS above our detection limits (0.2 nmol for DMS and 4 nmol for MeSH), likely indicating that MeSH is not abundant in this environment or that these gases are quickly consumed by the microbial population. However, samples incubated with MeSH (20 μmol) consumed it all in 24 h and produced 15.2±1.5 nmol DMS·g soilHaving demonstrated that the Mdd pathway was active in the grassland soil samples in microcosm experiments, the sampling site was re-examined by placing two sets of 2 l air-tight field chambers over the soil. After 19 h, the results were congruent with those observed with serum vials, with the soil alone not making MeSH or DMS, but when it was supplemented with MeSH (200 μmol) the soil liberated 239.3±18.9 nmol of DMS (0.1±0.01% of MeSH added). Therefore, field measurements confirmed that microbes in the grassland soil had a functional Mdd pathway when MeSH is available.−1, 0.5±0.1% of the MeSH added) compared with the controls with no inhibitors added . In contrast, the eukaryotic inhibitor cycloheximide had no effect on DMS production , indicating that prokaryotes are probably the main contributors to DMS production from MeSH in this particular grassland soil.Grassland soil samples were supplemented with antibiotics effective against either prokaryotes or eukaryotes and incubated for 24 h. The vials with prokaryotic inhibitors added showed a ~50% reduction in DMS . Subsequently, DMS production steadily declined (−1), and then stabilising before DMS was more rapidly consumed after 11 days  to 7 days  and reached maxima of 3.3±0.1 μmol MeSH·h−1·g soil−1 and 6.1±1.8 nmol DMS·h−1·g soil−1 at 14 days, confirming that extended exposure to MeSH enhances the activity of the Mdd pathway and may enrich for microbes producing DMS from MeSH. As DMS production rates increased over the course of the MeSH enrichment, it was not surprising to see that net DMS consumption rates also increased. The DMS consumption rate at t=0 was 0.2±0.01 nmol DMS·h−1·g soil−1, but after 7 days it was 3.9±1.0 nmol DMS·h−1·g soil−1, reaching a maximum of 10.1±1.9 nmol DMS·h−1·g soil−1 after 14 days. The fact that at 14 days DMS consumption was greater than DMS production indicates that there has probably been an enrichment of DMS-degrading microorganisms.To study in greater detail how the addition of MeSH affected the processes of DMS production and consumption, we measured both rates at 0, 7 and 14 days. For this, we used samples enriched with succinate plus MeSH as they showed the highest DMS production levels (see above). We also measured MeSH consumption rates at the same time points. Both MeSH consumption and DMS production net rates increased from t=0 and from enrichments with MeSH alone, succinate alone and succinate plus MeSH after 7 and 14 days of incubation. Extracted DNA was used as template in 16S rRNA gene amplicon sequencing (see t=0 samples) was dominated by bacteria from the classes Actinobacteria (18.5±0.9%) and Deltaproteobacteria , and marginally non-significant increases in Betaproteobacteria to 12.4±0.9%  and Sphingobacteria to 12.8±0.5% , after 14 days. Deltaproteobacteria were still one of the most predominant classes (15.4±1.7%), but Actinobacteria were substantially reduced to 4.2±0.04%  in the 14-day succinate enrichments compared with t=0 samples (The addition of diluted Y medium with succinate as a carbon source (succinate-only enrichments) changed the profile of the natural microbial community with a significant increase in relative abundance of Gammaproteobacteria to 14.8±1.5% (Tukey honest significant difference (HSD),  samples .P=0.293) in the 14-day MeSH enrichments and to 5.2±0.6%  in the succinate plus MeSH enrichments compared with the 14-day succinate samples. MeSH increased the relative abundance of Betaproteobacteria to 17.9±0.1%  in the 14-day MeSH enrichments and to 23.6±1.2%  in the succinate plus MeSH enrichments. The relative abundance of Gammaproteobacteria also increased to 22.8±3.8% in the 14-day MeSH enrichments  and to 23.6±2.1%  in the enrichments with succinate and MeSH compared with the 14-day enrichments with succinate  was still one of the most abundant genera in the bacterial community of the 14-day enrichments with MeSH. However, it was notable that the presence of MeSH significantly increased the relative abundance of Methylotenera and Massilia in the 14-day enrichments with MeSH  and 14-day samples enriched with succinate plus MeSH , compared with the 14-day enrichments with succinate. Arthrobacter also increased in relative abundance, although not significantly, in the 14-day enrichments with succinate plus MeSH  compared with the 14-day succinate samples , but their relative abundance did not increase either in the MeSH alone or the MeSH plus succinate enrichments.Soil bacteria that are known to oxidise MeSH and DMS such as t=0 and from enrichments with succinate plus MeSH after 7 and 14 days of incubation was also used to perform metagenomics (see t=0 was dominated by members of the class Actinobacteria (51.4%), confirming the results of the 16S rRNA gene analysis. The most abundant bacteria in the t=0 community that MetaPhlAn could classify at a genus level belonged to Agromyces (13.7%), Thiomonas (8%), Rhodopseudomonas (6.7%) and Pedobacter (6.2%) genera (DNA extracted from samples at mics see . Phyloge) genera .Pseudomonas  and Betaproteobacteria (37.4%). The most abundant genus at this time point was s 41.6%, . MetagenM. mobilis JLW8T, which is >97% identical at the 16S rRNA gene level to the Methylotenera in our MeSH enrichments, was tested for its ability to degrade MeSH and DMS. Cultures of M. mobilis JLW8T were supplemented with MeSH or DMS and the amount of these gases in the headspace was quantified by GC at t=0 and 24 h. After 24 h of incubation, M. mobilis consumed 543.6±11.2 nmol MeSH  in the first 24 h .rst 24 h . These rt=0 and from enrichments supplemented with succinate plus MeSH after 14 days of incubation. These enrichments were chosen because the presence of both succinate and MeSH led to the greatest levels of DMS production and also the samples at 14 days had the highest rates of Mdd and consumption.To complement cultivation-independent work, we isolated bacteria from grassland soil samples at t=0 samples were Gammaproteobacteria (Pseudomonas spp.), Actinobacteria (Streptomyces spp.) and Bacilli .Strains isolated from the t=14 days were mainly Alphaproteobacteria , but also Gammaproteobacteria (Pseudomonas spp. and Acinetobacter spp.), with Pseudomonas being the most abundant genus isolated  or MeSH. Only ~5% of the isolates from  or MeSH . These rWhen isolates from the succinate plus MeSH enrichments (14 days) were analysed, ~42% were able to produce DMS from Y medium alone. This percentage increased to ~96% and 100% of the isolates generating DMS when Y medium was supplemented with Met or MeSH, respectively . These rt=0 and from enrichments with succinate plus MeSH after 7 and 14 days of incubation was subjected to metagenomic analysis  and, although increased up to 25.3% at 14 days, was still lower than t=0  and Thioalkalivibrio (12.9%) forms were still the most abundant mddA-like genes together with Nodosilinea mddA homologues , followed by Cyanothece (12.5%) and Bradyrhizobium (11.5%) mddA-like genes  and 25.8±4.7 nmol DMS·ml−1 , respectively, indicating that DMSP is probably a more effective precursor for DMS than MeSH in these environments.To determine whether the Mdd pathway was functional in a wider range of environments, we analysed different terrestrial, freshwater and marine samples . After 22S in the anaerobic layers of the soil and sediments  of the MeSH added to our microcosm experiments was captured as DMS despite all the MeSH disappearing within 24 h. Some of the MeSH added may have been enzymatically degraded to HdmoA, ddhA and tmm) was relatively abundant in the natural grassland soil bacterial population. However, the relative abundance of dmoA, ddhA and tmm did not increase over the succinate plus MeSH enrichments despite a 50-fold increase in DMS consumption rate, perhaps suggesting that there are as yet unidentified microbial genes for DMS degradation. In any case, it is clear that the DMS production levels reported here are an underestimation of the activity of the Mdd pathway in the grassland soil studied.The biotic and abiotic degradation of MeSH greatly affects the amount of this compound available for microorganisms that use the Mdd pathway to produce DMS. A further contributing factor to the low levels of DMS achieved in our microcosm experiments is the biotic and abiotic degradation of DMS. The rates of DMS consumption increased over the course of the succinate plus MeSH enrichments to levels above those for Mdd after 14 days. DMS can be photochemically  and enzymatically (in situ measurements of MeSH and none of them from soils (Although we have shown that the ability to generate DMS from MeSH is inherent in all of the different soils and marine sediments tested, it is still very difficult to estimate the exact contribution of the Mdd pathway to global DMS emissions, since there are very few om soils .Methylotenera significantly increased in 14-day enrichments supplemented with MeSH, when DMS consumption rates were maximal. This indicates that Methylotenera likely has a role in DMS degradation, as suggested by Methylotenera to remove DMS was confirmed when test cultures of M. mobilis JLW8T consumed both DMS and MeSH. To our knowledge, this represents the first proof that members of the Methylophilaceae family, which includes obligate and facultative methylotrophs, are able to degrade these volatile compounds. Increases in the relative abundance of Arthrobacter genera in the 14-day succinate plus MeSH enrichments, suggests that they may also be involved in DMS consumption. Indeed, some Arthrobacter strains are able to metabolise DMS to generate MeSH (Hyphomicrobium or Thiobacillus can oxidise MeSH and DMS (t=0 samples, and did not increase over the course of the MeSH enrichments, suggesting that they may not have a significant role in the removal of MeSH and DMS in our microcosms experiments. Finally, 16S rRNA gene amplicon sequencing data and metagenomics analysis also showed that the presence of MeSH increased the relative abundance of Massilia, some of which contain mddA-like genes (Massilia in DMS production.The addition of MeSH to the grassland soil samples not only increased DMS production and consumption rates, but also changed the diversity of the bacterial population. According to cultivation-independent methods, relative abundance of Pseudomonas, which is not surprising, given that many contain mddA (Acinetobacter, Gemmobacter, Phyllobacterium, Rhizobium, Ensifer and Sinorhizobium that converted MeSH into DMS. These bacteria were not previously suspected to have mddA or produce DMS from MeSH, since close relatives with sequenced genomes lack this gene. Thus, the Mdd pathway is more widespread among different bacterial taxa than previously thought. It is probable that other Mdd enzymes drive DMS production from MeSH in these bacteria. However, detailed molecular characterisation of these microorganisms is required to identify their Mdd enzymes and ultimately prove this. The presence of other Mdd enzymes in the bacterial population of the grassland soil could explain the observed discrepancies between metagenomics analysis and process rates, where the relative abundance of mddA did not increase throughout the enrichments despite the increase in DMS production rates. Such differences could also be due to the expression levels of the genes encoding the diverse Mdd proteins and/or their respective enzyme activity. Future studies will need to consider these factors to ascertain at a molecular level how and why Mdd levels change.Cultivation-dependent methods showed that the addition of MeSH to grassland soil samples selected for microorganisms containing the Mdd pathway. Most of the isolates that generated DMS from MeSH belonged to the genus In conclusion, we show that the Mdd pathway is present and active when MeSH is available in a wide range of terrestrial and marine environments, especially in soils. However, as only ~0.1% of MeSH added is transformed into DMS via this pathway, it is not likely a significant process in the tested environments under natural conditions. More microbiological studies and process-based measurements are required on environments where MeSH is likely abundant to make an accurate estimation of the contribution of the Mdd pathway to the global DMS cycle.