The reduction reaction was also carried out and the catalytic species isolated and fully characterised. The catalytic activity of a graphene oxide‐supported iron oxide pre‐catalyst converted CO2 into hydrocarbons at different temperatures , and its activity compared well with that of the analogues supported on graphite oxide, the 3‐dimensional material precursor to the graphene oxide. Investigation into the use of graphene oxide as a framework for catalysis showed that it has promising activity with respect to reverse gas water shift (RWGS) reaction of CO2 to CO, even at the low levels of catalyst used and under the rather mild conditions employed at atmospheric pressure. Whilst the γ‐Fe2O3 decorated graphene oxide‐based pre‐catalyst displays fairly constant activity up to 405 °C, it was found by GC‐MS analysis to be unstable with respect to decomposition at higher temperatures. The addition of palladium as a promoter increased the activity of the iron functionalised graphite oxide in the RWGS. The activity of graphene oxide supported catalysts was found to be enhanced with respect to that of iron‐functionalised graphite oxide with, or without palladium as a promoter, and comparable to that of Fe@carbon nanotube‐based systems tested under analogous conditions. These results display a significant step forward for the catalytic activity estimations for the iron functionalised and rapidly processable and scalable graphene oxide. The hereby investigated phenomena are of particular relevance for the understanding of the intimate surface morphologies and the potential role of non‐covalent interactions in the iron oxide‐graphene oxide networks, which could inform the design of nano‐materials with performance in future sustainable catalysis applications.We report on the design and testing of new graphite and graphene oxide‐based extended π‐conjugated synthetic scaffolds for applications in sustainable chemistry transformations. Nanoparticle‐functionalised carbonaceous catalysts for new Fischer Tropsch and Reverse GasWater Shift (RGWS) transformations were prepared: functional graphene oxides emerged from graphite powders via an adapted Hummer's method and subsequently impregnated with uniform‐sized nanoparticles. Then the resulting nanomaterials were imaged by TEM, SEM, EDX, AFM and characterised by IR, XPS and Raman spectroscopies prior to incorporation of Pd(II) promoters and further microscopic and spectroscopic analysis. Newly synthesised 2D and 3D layered nanostructures incorporating carbon‐supported iron oxide nanoparticulate pre‐catalysts were tested, upon hydrogen reduction  New functional two‐dimensional (2D) and three‐dimensional (3D) nanomaterials relevant for sustainable chemistry applications have been designed and tested. This work is of relevance to enhancing our understanding of the role of supramolecular aggregation on nanotechnology and on optimised designs for future materials for energy conversion/storage as a basis for the reverse gas water shift reaction and Fischer Tropsch chemistry. CO2 can be viewed simultaneously as an attractive option as a feedstock towards fuels synthesis, through the conversion to methanol2 and the promise of its used towards the sustainable energy production has sparked a renewed interest in Fischer Tropsch chemistry, as this could lead to the generation of higher hydrocarbons through the use of metal catalysts and facilitate the access to this large‐scale available feedstock for a sustainable energy production.The Climate Action Forum Paris 2015 and the recent societal concerns into the use of energy‐intensive processes highlighted that increasing environmental pressures have seen a shift in global societal attitudes and behaviours towards the use of fossil fuels for energy production, for economic reasons, as well as for communities’ livelihoods.2 concentration that has already been released into the atmosphere. The other approach that concerns our sustained recent research interest2 to CO and the subsequent step‐wise transformation of the carbon monoxide with hydrogen into hydrocarbons.i. e. once hydrocarbons are burnt, the resulting carbon dioxide is then turned back into hydrocarbons.2 can be used as a starting material for FT chemistry processes,2O3 iron species are the preferred FT catalysts for transformations concerning syngas from coal plants, whereas cobalt ions as catalysts are the main choice for applications towards the conversion of natural gas derived syngas, through Eq. (B), although it is widely accepted that supported Fe3O4 heterogeneous catalysts are also highly active in the FT process (B).There are multiple alternative energy sources being investigated and there is sustained interest in novel research, which is needed to create newer ways of energy production and storage.Metallic nanoparticles, free or anchored onto supports have been shown to be very active as catalysts due to their high ratio of surface atoms.2/Vs),−1K−1), electrical conductivity,2 extended network such as epoxides, tertiary alcohols and peripheral carboxylic acids.Several allotropes of carbon such as fullerenes, carbon nanotubes (CNTs), graphene and its functionalised derivatives such as graphene oxides have garnered considerable interest over recent years.Recent work highlighted the potential of graphene‐based frameworks as catalytic support as it has similarities to other carbon based systems such as graphitic carbon in terms of large surface area and excellent electronic conductivity, whilst also displaying some variation as the sheets have edges which have different electronic properties.et al. and others, and the activity was assigned to be due to the embedded and bridging nanoparticles emerging from CVD to as‐made carbon nanomaterials or local defects in the nanotube structure.Several new examples of nano‐carbon based catalysts for the FT chemistry with activity towards the RWGS reaction has previously been generated and tested by Jones 22O3 nanoparticles of consistent sizes (γ‐Fe2O3@graphene oxide) was synthesised and reduced in situ using H2 gas (at 200 °C) prior to the catalysis testing towards the CO2 conversion at the atmospheric pressure, as described below. A pre‐activation step using H2 aimed at generating Fe3O4 in situ, as these species are known to be active for both steps (A) and (B) shown above, which would render the Fe‐based nano‐hybrids as active nanocatalysts, by analogy previously‐tested and iron‐decorated carbon nanotubes.in situ hydrogenation denoted Fe@graphene oxide and tested for the CO2 to CO and hydrocarbon conversion via FT reactions, without isolation, and using CO2 : H2 2 : 6 ml/min feed‐stocks at 300, 335, 370 and 405 °C. In a parallel experiment the activated pre‐catalysts isolated after the hydrogenation step were collected and fully characterised.A new nanohybrid displaying embedded γ‐FeHere we report on our advances made in the structural and morphological characterisation of the 2‐dimensional, layered nanocatalysts. For a comparison, we also used their 3D hybrid nanomaterial precursors, i. e. the graphite oxide‐based iron nanocatalysts as a control tests for the catalytic performance to investigate the potential benefits of having an oxide‐functionalised carbon framework support for the iron NPs. Scheme . This ap2.12O3 nanoparticle incorporation into the functional carbon nano‐sheets, as discussed in the Experimental Section. The graphene oxide synthetic scaffold was first synthesised by the exfoliation of graphite. Subsequently iron nanoparticles  with well understood morphologies and dimensions , were introduced into the nanostructure thus yielding a novel pre‐catalyst scaffold, ready for a subsequent pre‐activation by H2 and CO2/H2 reactions for a rapid CO2 conversion.A series of iron oxide functionalised graphene and graphite oxide pre‐catalysts were synthesised via a rapid and facile γ‐Fe2O3 nanoparticles (NPs), showing consistency in morphology and NPs distributions on surfaces, prior to the pre‐activation under H2 and catalysis testing. Specifically, graphite oxide was synthesised using an adapted Hummer's method,2O2. The resulting slurry was centrifuged at 3000 rpm and the resulting solid was washed at least 10 times and freeze‐dried. The procedure was repeated at least three times generating different batches of graphite oxide with high batch‐to‐batch consistency as evidenced by TEM and spectroscopic data. Both graphene oxide and graphite oxide emerging were functionalised with iron oxide nanoparticles by combining 20 %wt. of iron nanoparticles with the solid sample of carbonaceous materials in toluene followed by further re‐dispersions and drying protocols followed by full structural characterisation (ESI). Furthermore, palladium acetate (3 %wt.) and the iron oxide functionalised graphite oxide hybrid were combined in toluene and heated gently, the excess solvent was removed, the sample was washed with THF and excess solvent was removed under vacuum. The resulting pre‐catalysts were followed by in situ generation of the iron active species through mild hydrogenations and then their testing as nano‐catalyst for the CO2 activation following existing protocols. In our hands, a surprisingly short exposure time of only 40 minutes was found to give a comparable conversion rate with silica supported catalysts, and this intriguing result was assigned to the layered morphology and the accessible encapsulated iron nanoparticles within the graphene supports, vide infra.To bypass possible drawbacks from batch to batch reproducibility, several different batches of graphene and graphite oxides were prepared as precursors and were functionalised with tailor‐made and fully characterised Fe2.22O3 nanoparticles and Fe‐decorated nanocarbon pre‐catalyst confirm the presence of pure crystalline γ‐Fe2O3 nanoparticles (see ESI). Interestingly, the γ‐Fe2O3 NPs were clustered on the graphitic network of GO‐a as well as GO supports, whilst maintaining their NP character in terms of shapes and size , prepared and isolated as described above and in the Experimental Section, was characterised by spectroscopic methods including Raman, XPS and IR spectroscopies and imaged by TEM coupled with SAED and EDX  synthesised as a new composite from the treatment of graphite oxide with γ‐Fe2O3 nanoparticles as described in the Experimental Section, was characterised by spectroscopic methods including Raman, XPS and FTIR spectroscopies in the solid state. The resulting morphology on the nanoscale was imaged by TEM  synthesised from the exfoliation of graphite oxide precursors described above and used as the synthetic scaffold for heterogeneous Fe‐based nanocatalyst support. Images are also consistent with those expected for the few‐layers of GO  as evident from imaging by TEM and electron diffraction .The TEM image suggested a very small depth and shows clearly a very thin several layer sheet of graphene. Energy dispersive X‐ray diffraction gave information on the local elemental composition this isolated material seems to show a clean sample containing mainly C, H and O elements, with minimum N‐based defects being present. The elemental analysis by EDX showed no significant impurities in either GO or its corresponding iron oxide pre‐catalysts, in addition to those already present in the graphite oxide support which suggests that an iron oxide is the most likely iron‐containing functionality present on the surface of the desired γ‐Fe−1. These Raman bands indicate that the functional graphene type material in question is thin in layer count as the peaks are fairly sharp and especially intense when concentrated, as shown by Raman mapping using the 830 nm excitation.The Raman spectroscopy showed the pattern assignable to GO in solution and when deposited on a metal surface . In this sense, as the concentration of Pd in the samples is around 1 %wt, it is not possible to be detected by XPS (below the detection limit of the equipment). Sulphur is also detected over the surface of all the GO‐a samples, as previously observed for sample originating from Hummer's treatments.XPS spectroscopy was performed to determine the surface chemical composition of the pre‐catalysts as a consequence of the interactions between the iron oxide and functionalise graphene sheets . Figure FeGO and FeGO‐a pre‐catalyst, detailed microscopy studies were carried out in order to observe the morphology of the starting materials and for a comparison with the desired adducts with iron‐based nanoparticles. The SEM image  of free graphene oxide support (GO) indicates a crystalline structure while its direct observation by TEM reveals a sheet‐like nano‐material. TEM micrographs of the Fe‐oxide decorated graphene oxide shows a heterogeneous distribution of the nanoparticles with various degrees of aggregation, consistent with the AFM investigations Figure . In all vide infra) was also investigated by TEM. In this case the TEM indicates the formation of crystalline presumably iron‐based nanoparticles of the relevant hybrid together with amorphous material. The presence of the iron, together with carbon species was confirmed by EDX analysis (see ESI). The morphology of the pre‐catalysts was also investigated on the micro‐scale by SEM together with EDX  (ESI). These results indicate that both graphene oxide and its precursor graphite oxide can be functionalised with Fe‐containing nanoparticles. Interestingly, single layers of functionalised carbon nanosheets can be observed for the graphene oxide‐based adducts. However, a certain degree of aggregation and a presence of amorphous carbonaceous materials can be seen for all samples, across all batches, which would make the assignment of functionalities present and level of oxygenation hard to estimate, but which would likely have an effect on the level at which the CO−1 endorses the observations on the nanoscale and are consistent with the AFM imaging  and partially exfoliated areas (lighter). SEM imaging showed Fe nanoparticles (bright spots) on graphite oxide (dark region). Interestingly, the electron diffraction of un‐modified graphene oxide showed the blurred rings indicated exfoliated layers rotating in opposite directions. Interestingly, the Raman spectroscopy performed indicated that the iron oxide was distributed over the surface, but not on the edges of the graphene sheets, as bands characteristic for the iron oxide were not observable with the laser focused on the edge, however the presence of iron oxide was apparent when a wider focus on the bulk nanomaterials sheets was used. The increase in ratio of D to G peak is in line with all functionalised graphenes Figure . The RamInsights into the morphology and nature of the reduced species post hydrogenation treatment were obtained through combined AFM, SEM andTEM techniques. Surface studies do not show dramatic differences between starting materials and the corresponding pre‐catalyst samples, post hydrogen‐treatment however this is not surprising as the hydrogen activation involved rather mild conditions. Morphology of the activated species, when compared to the pre‐catalysts above studied by TEM and AFM are similar, however, as expected, reduced GO samples appeared somewhat more aggregated upon inspection by AFM but this is sample and region dependent. A qualitative comparison of the results from EDX maps did not show significant differences in terms of the level of oxygenation, as expected, and these are dominated by contribution from the carbon support and copper from the grids, whilst the comparison of such heterogeneous areas in terms of their relative oxygenation is not reliable.Intriguingly, the metallic nanoparticles on the surface of the reduced [PdFeGO‐a] sample seem to present a coating, observed by TEM, yet not previously present in the starting material PdFeGO‐a or in the other hydrogen‐reduced samples such as [FeGO‐a] or [FeGO]. The resolution of EDX was insufficient to reveal local composition in detail, however previous studies indicated that Lewis acid promoters facilitate formation of iron carbide as the active species.2‐reduced species were carried out by nitrogen adsorption/desorption isotherms.Specific surface are measurements conducted for the pre‐catalysts as well as the corresponding H2O3 over GO‐a and GO supports produces an increase in the specific surface area of the material. The sample FeGO‐a shows the highest specific surface area, which can be justified due to GO‐a preserves the layer structure of the parent graphite, with interlayer porosity. By contrast GO is a 2D material without interlayer porosity. These values can provide an idea about the density of active sites. Although FeGO‐a and PdFeGO‐a show the highest values specific surface area .Given that the chemical modification of the surface of graphene layers leads to its decoration with oxygenated functional groups and introduction of defects into the planar graphitic layers (as evidenced by XPS and Raman spectroscopies), this in turn allows the anchoring of Fei. e. pristine graphene oxide and graphite oxide, vide infra. The active nano‐catalytic species for both were generated via an in situ hydrogenation procedure of the FeGO‐a and FeGO pre‐catalysis, and the resulting H2‐activated species denoted reduced[FeGO‐a] and reduced [FeGO] were used in the catalysis tests for the reactions (A) and (B) above. This approach was already developed as a rapid and efficient route to the in situ generated active nanocatalysts in the case of iron or cobalt pre‐catalysts supported on a variety of inorganic scaffolds and pristine or N‐doped multi‐walled carbon nanotubes.2 to CO and subsequently of CO to hydrocarbons. The products yielded using the iron modified graphite oxide catalyst indicated mainly the formation of CO.Spectroscopic investigations outlined above allowed a comparison between the pre‐catalyst morphologies and the starting materials, At the low temperatures tested hereby  all catalysts showed good conversion and selectivity Table . However2 in total led to mainly CO formation, and conversion of CO2 to hydrocarbons was rather low, typically in 1 % overall, with the exception of the case of [PdFeGO‐a] nanocatalyst , where the total conversion at 300 °C was due to RWGS transformation to CO only (16 %), unlike with the graphite oxide catalyst, the graphene support showed enhanced stability at higher temperatures (405 °C) as no aromatic compounds were formed as observed by GC‐MS. Also, no alcohols or oxygenated products have been observed at these rather lower temperatures tested, but extensive degradation of the graphene oxide support was observed above 405 °C. The most promising catalyst for this transformation was the Fe supported on graphite oxide with Pd as an activator, which is not surprising as Pd(II) is a known hydroformylation and hydrogenation catalyst.A comparison of conversion and hydrocarbon selectivity for Fe‐based Graphite (FeGO‐a), Pd‐promoted Fe‐based graphite (PdFeGO‐a) and Fe‐Graphene (FeGO) catalysts at different temperatures and atmospheric pressure is shown in Table 2 upon FeGO, FeGO‐a and PdFeGO‐a pre‐catalysis, XPS measurements were carried out on the isolated reduced pre‐catalyst species. Although some investigations into the use of graphenes in FT chemistry have been reported for related transformations, the synergetic role of Fe2O3/GO and their simultaneous reduction in heterogeneous catalysis has not been elucidated thus far. Several earlier publications reported the use and characterisation of reduced graphene oxide and partially reduced graphene oxide as catalyst support in a number of different catalytic processesIn order to probe the reduction effects of H2 at 50 sccm at 300 °C for 2 hours) seem only affects to the surface functionalities of GO, and not lead to the reduction of Fe2O3 particles ;. Table 2 total conversion of FeGO is lower than FeGO‐a both at 300 °C (6.7 % vs 9.1 %) and 335 °C (11.9 % vs 14.4 %). From GC‐MS there were indications that the graphite derivative denoted FeGO‐a decomposed at higher temperatures with formation of benzene among other unexpected peaks appearing. This was not seen with the PdFeGO‐a, which could suggest added stability, GC‐MS analysis also showed that Fe doped graphite oxide had a higher conversion of CO2 to CO and hydrocarbons and had a higher selectivity for long chain hydrocarbons at temperatures 300–370 °C, with both catalysts showing greatest conversion at 370 °C  was highly active in converting CO2 was high for all catalysts  and compared well with the similar systems such as those encountered for carbon nanotubes (ESI).Our catalysis tests also showed that further modification of the graphite oxide pre‐catalysts with palladium enhanced the catalytic activity of FeGO‐a, as expected,2O3 species after the hydrogen activation, or the degradation of the graphite oxide 3D structure, or the ineffective formation of Fe3O4 species due to the oxygenated functionalities of the graphite/graphene oxides used.This implies that the catalyst was most active for the first step of the reaction, the reverse water gas shift process (A), but only moderately active for the second step of the process (B) at these reduced temperature, possibly due to persistence of the Fe2O3 deposited onto 2D and 3D nano‐carbonaceous supports and also comparable in terms of the activity of graphene oxide with that of CNTs, as evidenced by earlier tests under analogous conditions. Work is underway in our laboratories we are now focused on scale‐up investigations as well as the further modification of graphene oxide using a number of different Lewis acids as promoters as well as new catalytic metals, such as doping of graphene oxides with cobalt and molybdenum nanoparticles of controlled dimensions. For the nano‐carbon materials to be successfully implemented as a promising nano‐catalytic supports, the major hurdle remains in the development of a large scale synthetic routes for processable graphenes. This is made particularly difficult due to the high batch sensitivity of most synthetic carbon‐based nanoparticles such as carbon nanotubes. As such, work on large scale synthesis using simpler, more versatile supports such as graphene oxide is fundamental in paving the way to a successful future application of graphene or carbon nanotubes in hi‐tech industries and we addressed synthetic challenges hereby.Taken together with the catalysis tests, these structural and morphological testing suggest that the particular features of the resulting Fe‐graphene oxide composite have the potential to act in a synergistic way, and operate in the rather less extreme regime compared to traditional carbon or silica‐supported FT catalysts of particular relevance as RGWS nano‐catalysts. The majority of heterogeneous nano‐catalysis investigations reported to date involving carbon nanomaterials have focused on the use of multi‐walled carbon nanotubes as supports: these results are unique in the field due to combination of nanoparticulate Fe32O3 nanoparticles of well‐understood shapes and dimensions. Work on large scale synthesis is fundamental to the successful implication of graphene and graphene oxide in hi‐tech industries. The graphene oxide modified with Fe2O3 nanoparticles showed the highest activity when considering mass of catalyst used. The majority of work on carbon nanostructures to date has focused on carbon nanotubes as a catalyst support, and we found that the activity of graphene oxide over only 40 minutes of testing under atmospheric pressure is comparable with that reported by us earlier for Fe@CNTs. These approaches could become a useful tool in the fabrication of catalytically active inorganic nanomaterials, as well as those carbon‐based and graphene‐like material employed nowadays.Hereby we report on our recent explorations regarding the structural features which render the graphene oxide as a promising framework for nanoparticulate and supported FT heterogeneous chemistry. Some novel iron‐oxide decorated nano pre‐catalysts have been synthesised hereby and their catalytic activities compared for the two step conversion of carbon dioxide and hydrogen into hydrocarbons. Nanotechnology approaches to sustainable catalysis are challenging due to the high batch sensitivity of carbon‐based nanomaterials such as carbon nanotubes, but it was mitigated here by the used of graphene oxide starting materials emerging from established and scalable synthetic protocols. Our microscopic imaging results across several different scales, from molecular‐level investigations by XRD, to nanometre scale imaging by AFM, TEM and SEM showed, on several different batches, that the particulate 3D vs 2D morphology is consistent and is maintained upon functionalisation with FeAll starting materials were purchased from Sigma‐Aldrich and used without further purification. All solvents were of reagent grade quality,purchased from Sigma‐Aldrich and used as received.Synthesis of Graphite Oxide (GO‐a): Graphite powder (2.0 g) and sodium nitrate (1.0 g) were added to 46 mL of concentrated sulphuric acid. The mixture was kept below 20 oC and potassium permanganate was added slowly (6.0 g). The mixture was then stirred at room temperature for 30 minutes, then 90 mL of distilled water water were added slowly. After diluting the solution to 300 mL with distilled water, 2 mL of 30 % (v/v) hydrogen peroxide was added whilst stirring. The mixture was allowed to settle and the aqueous layer decanted. The solid graphite oxide was then washed, via re‐dispersing in de‐ionised water and centrifuged at 3500 rpm for 30 minutes, the resulting supernatant was decanted. The washing procedure is repeated a minimum of 10 times, the final solid was then dried in a freeze dryer.Synthesis of Graphene Oxide (GO): Graphite oxide was dispersed in 100 mL of 2 : 1 distilled water : ethanol and sonicated 6 times for 30 minutes each, allowing for the solution to stand for 10 minutes between cycles. The mix was then centrifuged at 3500 rpm for 30 minutes, removing graphite oxide from solution, leaving a suspension of graphene oxide.General method for the incorporation of Iron nanoparticles within the carbon nanomaterial supports Graphite (FeGO‐a) and Graphene Oxide (FeGO): The solid support synthesised as above was dispersed in toluene and mixed with solid gamma‐Fe2O3 nanoparticles . This mixture was then sonicated for 30 minutes and left stirring for 48 hours. The resultant solution was then gently heated whilst stirring to remove the toluene initially added. The remaining black slurry was then heated to 270 °C for one hour and allowed to dry under air.Hetero‐bimetallic Palladium/Iron‐Modified Graphite Oxide (PdFeGO‐a): Palladium acetate (0.0063 g) was dissolved in toluene with the iron modified graphite oxide catalyst FeGO‐a synthesised as above (0.21 g). The mixture was then heated gently whilst stirring to remove the toluene, dried under reduced pressure and analysed by AFM, TEM and FTIR.Samples preparations for analysis: For TEM analysis, samples were dispersed in methanol or ethanol and sonicated mildly (less than 5 min) to ensure dispersion. The samples were then placed on a carbon film coated copper grid. TEM was performed on a JEOL JEM 1200 EXII operated at 120 kV. For Raman spectroscopy analysis: samples were dispersed in either ethanol or methanol and a background spectrum was run against pure solvent. Samples were analysed on a Renishaw InVia Raman Microscope. For SEM coupled with EDX and XPS analysis: Samples were attached to metal stubs using adhesive carbon tape. Samples were analysed using a JEOL JSM 6480 LV Scanning Electron Microscope and SPECS Phoibos150 XPS spectrometer equipped with 2D‐DLD detector. The specific surface area was determined by the Brunauer‐Emmett‐Telle (BET) method on an ASAP 2020‐Micromeritics  at 77 K . Samples were degassed at 30 °C during 48 h before analysisCatalysis testing: The catalysis rig used consisted of a Carbolite furnace 90 cm in length with an internal diameter of 4 cm. Catalyst bed length was a 10 cm Swagelok 1/4inch piping and gas flow controlled by digital omega mass flow controllers. Gas flow was controlled via Omega mass flow controllers. For GCMS analysis, gas samples were collected from catalysis rig using a gas syringe. Samples were analysed by Agilent Technologies, 7890A GC which was calibrated using the procedure detailed elsewhere.Catalyst Testing was performed according to the experimental setup published earlier1/4inch  Swagelok stainless steel tubing with quartz wool on either end of the sample. The catalysts were subsequently loaded into a catalytic fixed bed reactor. Before the catalytic testing against CO2 activation, it was necessary to reduce the catalyst under a pure flow of H2 at 50 sccm at 300 °C for 2 hours. Each activated nano‐catalyst batch was then exposed in situ to a flow of CO2 (2 sccm) and H2 (6 sccm) at four temperatures: 300 °C, 335 °C, 370 °C and 405 °C for 40 minutes. The product mixtures were then analysed using gas chromatography mass spectrometry (GCMS). In a parallel experiment, after the H2‐activation process as described above the catalyst samples were isolated and characterised by AFM, TEM/EDX and XPS, and results were compared to those of the as‐made pre‐catalysts.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The use of the adrenergic agonist epinephrine at micromolar doses may have supraphysiological effects. We found that pretreating β cells with micromolar concentrations of epinephrine differentially inhibited activation of receptor tyrosine kinases. We chose TrkB as an example because of its relative sensitivity to the effects of epinephrine and due to its potential regulatory role in the β cell. Our characterization of brain-derived neurotrophic factor (BDNF)-TrkB signaling in MIN6 β cells showed that TrkB is activated by BDNF as expected, leading to canonical TrkB autophosphorylation and subsequent downstream signaling, as well as chronic effects on β cell growth. Micromolar, but not nanomolar, concentrations of epinephrine blocked BDNF-induced TrkB autophosphorylation and downstream mitogen-activated protein kinase pathway activation, suggesting an inhibitory phenomenon at the receptor level. We determined epinephrine-mediated inhibition of TrkB activation to be Gi/o-dependent using pertussis toxin, arguing against an off-target effect of high-dose epinephrine. Published data suggested that inhibition of potassium channels or phosphoinositide-3-kinase signaling may abrogate the negative effects of epinephrine; however, these did not rescue TrkB signaling in our experiments. Taken together, these results show that (1) TrkB kinase signaling occurs in β cells and (2) use of epinephrine in studies of insulin secretion requires careful consideration of concentration-dependent effects. BDNF-TrkB signaling in β cells may underlie pro-survival or growth signaling and warrants further study.Adrenergic signaling is a well-known input into pancreatic islet function. Specifically, the insulin-secreting islet β cell expresses the G ERK1/2 tivation .2-adrenergic receptors in pancreatic islet β cells has been extensively studied and is well-known to suppress or completely inhibit insulin secretion through Gαi/o-dependent signaling  were separated on 10% gels by SDS-PAGE and transferred to nitrocellulose for immunoblotting. All membranes were blocked in Odyssey blocking buffer (Licor) for 1 h before overnight incubation with primary antibodies diluted in blocking buffer. After three 10-min washes in 20 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20 (TBS-T), membranes were incubated with fluorescent secondary antibodies for 1 h at room temperature. After three 10-min washes in TBS-T, membranes were imaged on a Licor Odyssey scanner.L-glutamine, and 50 μM β-mercaptoethanol . Cells were lysed in 25 mM HEPES, pH 7.4, 1% Non-idet P-40, 10% glycerol, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 137 mM NaCl, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin, and 5 μg/ml leupeptin and cleared of insoluble material by centrifugation at 10,000 × g for 10 min at 4°C for subsequent use.MIN6 β cells were cultured in Dulbecco’s modified Eagle’s medium (D6429), supplemented with 15% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml oethanol . MIN6 ce1. After three 10-min washes in PBS-T , slides were blocked for 1 h at room temperature in normal donkey serum (NDS) block solution . Sections were outlined with a barrier pen and incubated overnight at 4°C with primary antibodies. Primary antibodies were diluted in NDS blocking solution at the indicated dilutions   as well 2-adrenergic stimulation could prevent BDNF-TrkB signaling to ERK1/2, we probed the upstream phosphorylation state of TrkB itself. Typically, BDNF stimulates autophosphorylation of the TrkB receptor at several tyrosine residues   . While tsed cAMP , BDNF hased cAMP . Howeverignaling . Therefoignaling . TherefoWe also examined the involvement of calcium influx, which regulates ERK1/2 activation in β cells and is inhibited by epinephrine . Eliminai-dependent blockade of TrkB activation.Inhibition of the phosphoinositide-3-kinase signaling pathway is reported to abrogate the hyperpolarizing effects of micromolar epinephrine . We pret+ channels that can be blocked by low-dose (20 mM) KCl or 0.5–1 mM BaCl2. We tested these conditions and found that treating with KCl or BaCl2 prior to addition of micromolar epinephrine did not rescue BDNF-TrkB signaling to ERK1/2  have been correlated with increased fasting glycemia and type 2 diabetes risk (Future studies are required to place these actions of BDNF in an tes risk ; impairetes risk .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MK: conceptualization and formal analysis. MK, ZH, DB, and KM: investigation. MK and MC: writing—original draft, supervision, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules.Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy.Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment. Neurodegenerative diseases are a heterogeneous group of disorders characterized by progressive functional decline of the central nervous system accompanied by progressive loss of the structure and function of neurons in the brain and/or the spinal cord RNA-binding proteins (RBPs) are involved extensively in various forms of neurodegenerative diseases Here, we elucidated the interplay between TDP-43 and neuronal mRNP granules in TDP-43 proteinopathy. We provided evidence for the synaptic localization of cytosolic TDP-43 and for the first time directly visualized the postsynaptic localization of TDP-43 in mammalian synapses by far field super-resolution microscopy. We showed that TDP-43 was a component of neuronal mRNP granules and we delineated the neuronal activity-dependent granule dynamics in normal physiological conditions and in TDP-43 proteinopathy. Our work also provided insight into the involvement of neuronal mRNP granules in pathological TDP-43 inclusions in cellular and animal models, and FTLD-TDP patients.First, we analyzed the distribution of TDP-43 in the mouse CNS using immunofluorescence. We detected TDP-43 in brain areas including the cortical layers and the hippocampus A. The shNext, we analyzed the subcellular localization of TDP-43. Subcellular fractionation from forebrain of adult mice were performed. Lamin A and PSD-95 were used as internal controls of nuclear and purified synaptosome fractions, respectively. Tissue fractionation showed that TDP-43 was detected in all fractions including nuclear, cytosolic and purified synaptosome fractions. This result suggests that in addition to its primary nuclear localization, TDP-43 also showed cytosolic localization and a potentially enriched synaptic localization Figure A.in vitro (19 DIV) primary mouse cortical neurons. We observed a predominant nuclear localization of TDP-43, while cytosolic TDP-43 puncta were also present. High magnification images showed that cytosolic TDP-43 puncta distributed along neurites and were colocalized with mushroom-like structures of dendritic spines labeled by CamKII . TDP-43 and reference synaptic proteins including the presynaptic and postsynaptic makers Bassoon and PSD-95 were immunolabeled in 19 DIV cortical neurons, which contain morphologically mature synapses. Wide-field immunofluorescence images showed puncta structure of TDP-43, Bassoon and PSD-95 distributed along neuronal processes. However, colocalization of TDP-43 with Bassoon and TDP-43 with PSD-95 was both observed in wide-field images  between TDP-43 and Bassoon, while the distance between TDP-43 and PSD-95 was 134.1 ± 5.0 nm . These results indicate closer proximity of synaptic TDP-43 to the postsynaptic marker PSD-95 compared to the presynaptic marker Bassoon. In addition, the distance between Bassoon, and PSD-95 was also analyzed as control, showing a mean distance of 121.6 ± 9.5 nm , which is in agreement with previously published measurements with super-resolution microscopy The distances between TDP-43 and presynaptic/postsynaptic markers imply two possible locations of TDP-43: 1) in the synaptic cleft and close to the postsynaptic membrane or 2) in the dendritic spine and distal to the postsynaptic density. Importantly, the Bassoon-TDP-43 distance was close to the sum of Bassoon-PSD-95 distance and PSD-95-TDP-43 distance, suggesting a postsynaptic localization of TDP-43 in dendritic spines. Taken together, by combining super-resolved dSTORM microscopy and distance analysis, these results revealed for the first time the postsynaptic localization of synaptic TDP-43 in the dendritic spines.Cytosolic TDP-43 binds to numerous target mRNAs in the 3' UTR  in vivo, frozen sections from adult mice were prepared as described in Material and Method section followed by TDP-43, STAU2 and FMRP staining. Confocal microscopy showed a predominant nuclear localization of TDP-43 observed in the hippocampal CA1 area  and stress granules (SGs) interacted with each other and exchange components Since the binding of TDP-43 to mRNAs inhibits translation, we hypothesized that upon reactivation by neuronal activity, TDP-43-containing mRNP granules disassemble and mRNP complexes unbind from TDP-43, thus, local translation can be completed. To test this hypothesis, we performed activity stimulation on 15 DIV primary mouse cortical neurons by repeated depolarization with 90 mM KCl three times. We analyzed the distribution of TDP-43 and FMRP in mouse cortical neurons with or without KCl stimulation with immunofluorescence. In comparison to unstimulated neurons, neurons treated with KCl showed lower number of colocalized puncta of TDP-43 with FMRP along the neurites  promoter Because TDP-43 is involved extensively in neuronal mRNP granule metabolism and TDP-43 proteinopathy is sufficient to cause neurodegeneration and FTLD-related phenotypes, we asked whether TDP-43 proteinopathy may lead to impaired neuronal mRNP granule function. We used a TDP-43 overexpression transgenic mouse model (TDP-43 Tg) which mimics the FTLD phenotype including impaired learning/memory function and formation of cytosolic TDP-43 inclusions. The FTLD-TDP mouse model specifically overexpresses full-length TDP-43 in the forebrain under the control of the CaWe first investigated whether activity-dependent mRNP granule disassembly and TDP-43 unbinding were affected in TDP-43 proteinopathy. To this direction, 15 DIV primary mouse cortical neurons were prepared from P0 neonates from TDP-43 Tg mice and stimulated with repeated KCl depolarization. Unstimulated and stimulated TDP-43 Tg neurons were then analyzed with immuneofluorescence and confocal microscopy to investigate the distribution of TDP-43 and FMRP  assays to measure active translation in stimulated and unstimulated WT neurons, stimulation with KCl significantly increased the number of puromycin labeled puncta, suggesting an increase in local translation in the dendrites induced by neuronal activity  promoter and overexpressed TDP-43 in the forebrain. PCR genotyping was performed to identify whether the mice were transgene-positive. Six months old FTLD-TDP transgenic mice showed cytosolic TDP-43 aggregation accompanied by nuclear TDP-43 depletion in the brain, which mimics the hallmarks of TDP-43 proteinopathy in the brains of FTLD-TDP patients All experiments were in accordance with the guidelines of the Institute of Animal Use and Care Committee at National Cheng Kung University (NCKU), Taiwan. Mice were bred at the NCKU laboratory animal center. Water and food were provided Sub-fractionation of mouse brain tissue was performed as described previously with some modifications For protein extraction, mice were sacrificed with rapid cervical dislocation and the cortex was isolated. Forebrains containing cortex were resuspended in ice-cold RIPA lysis buffer  containing a protease inhibitor cocktail tablet (Roche) and the mixture of phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich). Homogenates were centrifuged and supernatants were collected as total cell lysates.® Plus-ECL (PerkinElmer).Proteins extracts (15-30 μg), quantified with the Bradford protein assay (Bio-rad), were resuspended in loading buffer and subjected to polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membrane (Whatman). After blocking, membranes were incubated overnight at 4℃ with primary antibodies at optimal dilution . Then, membranes were incubated with horseradish peroxidase-linked (HRP)-conjugated secondary antibodies (PerkinElmer) and western blot analysis was performed using Western Lightning2, and 90% relative humidity. Half of the growth medium was replaced every two days. Primary neurons were cultured for 15 to 19 days prior to immunostaining. For repetitive stimulation with KCl in vitro) were treated repetitively with 90 mM KCl for 3 min and a spaced recovery for 10 min. Neuronal cells were harvested or fixed 30 min after the last stimulation.Before culture, 20-mm glass coverslips were coated with 1 mg/mL poly-D-lysine at room temperature for 20 minutes; coated coverslips were then placed into 12-well culture plates. Mouse neonates at postnatal day 0 were sacrificed and their cerebral cortices were excised under a dissection microscope. Cortical tissues were triturated for disaggregation and plated on poly-D-lysine-coated glass coverslips in Neurobasal medium containing B27 serum-free supplement and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin). Cultures were incubated at 37°C in an 95% air, 5% COFor immunohistochemistry of the mouse brains, mice were anesthetized and transcardial perfusion was performed with 4% paraformaldehyde (PFA) in PBS. The brain was isolated and embedded in paraffin blocks. Paraffin sections were generated and brains were sectioned at 10-μm-thick for immunofluorescence. Sections were deparaffinized in xylene and covered with citrate buffer for antigen retrieval. Subsequently, sections were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) and blocked with 5% bovine serum albumin for 1 h at room temperature. After blocking, sections were incubated overnight at 4°C with primary antibodies , followed by incubation with secondary antibodies (Alexa Fluor® 594 and Alexa Fluor® 488) for 1 h. Nuclei were counterstained with DAPI . Coverslips were mounted with fluorescent mounting medium.For immunocytochemistry, cells were washed with PBS and fixed with 4% PFA for 15 min. Cells were then permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin for 1 h at room temperature. After blocking, sections were incubated overnight at 4°C with primary antibodies , followed by incubation with secondary antibodies for 1 h. Nuclei were counterstained with DAPI (1:5000). Images were acquired using an upright fluorescence microscope  with 20X and 40X objective lens and a laser Scanning Confocal Microscope  with 40X objective lens.We used the imaging buffer composition suggested in a previously reported protocol Alexa Fluor® 647 and Alexa Fluor® 568 secondary antibodies were used for dSTORM imaging. The microscope was constructed around an Olympus IX-83 automated inverted microscope. For illumination, an objective-type, total internal reflection fluorescence, oil-immersion objective  was used. A multiline laser source  was used for excitation and activation. Single-molecular localization signals were separated using appropriate filters (Andor Technology) and detected using electron-multiplying charge-coupled device camera . Before dynamic image movie acquisition, conventional fluorescence images were acquired to determine the region of interest. Specifically, 10,000 frame image series were recorded with an exposure time of 50 ms (20 frames per second). The acquired image series were then analyzed using MetaMorph® Super-Resolution System (Molecular Devices) to generate reconstructed dSTORM images.Cells/forebrain tissue were lysed in RIPA lysis buffer  containing a protease inhibitor cocktail tablet (Roche). Extracts were quantified with the Bradford protein assay (Bio-rad). Protein extracts (0.5 mg-1 mg) were diluted to 0.5 mg/ml with lysis buffer.Co-immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer's instructions with either 4 μg anti-TDP-43  or 4 μg of anti-FMRP . For western blot analysis, precipitated proteins were washed and subsequently eluted in denaturation buffer and heated at 94 °C for 3 min. For RNA-immunoprecipitation analysis, precipitated proteins were washed and subsequently eluted in non-denaturation buffer, and RNAs in the immunoprecipitates were purified using Quick-RNA™ Microprep Kit (ZYMO RESEARCH) according to the manufacturer's instructions.Map1b, 5'-TGGGACACAAACCTGATTGA-3' and 5'- TGAAAATCTCTATGAAGTTCT-3'; GluR1, 5'-CTAGGCTGCCTGAACCTTTG-3' and 5'-GGGAAGATTGAATGGAAGCA-3'; CamKII, 5'-AAAGTGCGCAAACAGGAAAT-3' and 5'-AGGTGGATGTGAGGGTTCAG-3'. Gapdh mRNA was used as internal control to normalize RNA levels.For quantitative RT-PCR analysis, first strand cDNA synthesis was performed using Superscript RT (Invitrogen). Real time PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) and specific primers for 25-33 cycles of 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C. The following primers were used: Ribopuromycylation was performed as described previously with some modifications For analysis of urea-soluble proteins, forebrain tissues were dissected and extracted with serial buffers with increasing reducing power as previously described. Briefly, forebrains were extracted sequentially at 5 mL/g (volume/weight) with low-salt (LS) buffer , high-salt Triton X-100 (TX) buffer [LS buffer + 1% Triton X-100 + 0.5 M NaCl], myelin flotation buffer [TX buffer containing 30% sucrose], and sarkosyl (SARK) buffer [LS + 1% N-lauroyl-sarcosine + 0.5 M NaCl]. The SARK-insoluble fractions were further extracted in 0.25 mL/g urea buffer dimethylammonio]-1-propanesulfonate, 30 mM Tris, pH 8.5). The urea-soluble proteins were then subjected to Western blot analysis.in vitro transfection reagent (Signagen Laboratories). Cells were seeded into a 6-well plate at a density of 5 × 105 cells/well and incubated overnight. 2 μg of DNA was diluted to 100 μl with serum-free DMEM with high-glucose (4500 mg/L) (Invitrogen). In another tube, 6 μl of PolyJet reagent was diluted with 94 μl of serum-free DMEM with high-glucose. Diluted PolyJet reagent was added immediately into the diluted DNA solution and gently mixed by pipetting. The mixture was incubated for 15 minutes at room temperature to facilitate the formation of transfection complex and was added to 6-well plate containing HEK293 cells. After 24 h, 10 μM of MG-132 (Sigma-Aldrich) was treated for 0, 4, 8 and 12 h.Human embryonic kidney (HEK293) cells were maintained in Dulbecco's modified eagle medium  containing 10% fetal bovine serum , 1 mM sodium pyruvate and 1% penicillin/streptomycin solution (Gibco). HEK293 cells were transfected with GFP-TDP-43 containing full-length TDP-43 cDNA subcloned into pFUGW vector, by PolyJet™ DNA t test. Differences were considered statistically significant at p < 0.05, as indicated by the asterisks: *P < 0.05, **P < 0.01 and ***P < 0.001.All data are reported as the mean ± SEM. The statistical significance of differences between means was evaluated by two-tailed Student's Supplementary figures.Click here for additional data file.


Mycobacterium tuberculosis and Propionibacterium acnes are the most likely microorganisms. Potential latent infection by both microorganisms complicates the findings of molecular and immunologic studies. Immune responses to potential infectious agents of sarcoidosis should be considered together with the microorganisms detected in sarcoid granulomas, because immunologic reactivities to infectious agents reflect current and past infection, including latent infection unrelated to the cause of the granuloma formation. Histopathologic data more readily support P. acnes as a cause of sarcoidosis compared with M. tuberculosis, suggesting that normally symbiotic P. acnes leads to granuloma formation in some predisposed individuals with Th1 hypersensitivity against intracellular proliferation of latent P. acnes, which may be triggered by certain host or drug-induced conditions. Detection of bacterial nucleic acids in granulomas does not necessarily indicate co-localization of the bacterial proteins in the granulomas. In the histopathologic diagnosis of sarcoidosis, M. tuberculosis-associated and P. acnes-associated sarcoidosis will possibly be differentiated in some patients by immunohistochemistry with appropriate antibodies that specifically react with mycobacterial and propionibacterial antigens, respectively, for each etiology-based diagnosis and potential antimicrobial intervention against sarcoidosis.Sarcoidosis may have more than a single causative agent, including infectious and non-infectious agents. Among the potential infectious causes of sarcoidosis,  Sarcoidosis is a systemic inflammatory disease that is characterized by the formation of noncaseating epithelioid cell granulomas at the sites of disease activity in multiple organs, including the lungs and lymph nodes . DespiteMycobacterium tuberculosis and Propionibacterium acnes (formerly known as Corynebacterium parvum and currently referred to as Cutibacterium acnes) .,50.P. acM. tuberculosis [P. acnes [P. acnes.Reactivation at the sites of latent infection may be triggered under certain host conditions, such as physical or mental stress, in sarcoidosis patients , as obserculosis  or P. acP. acnes  at sitesP. acnes , which mP. acnes infection with the bacterium reliably detected in granulomas. P. acnes and M. tuberculosis are the only microorganisms with sufficient evidence for an etiologic role in sarcoidosis. More of the available histopathologic data support P. acnes as a cause of sarcoidosis compared with M. tuberculosis, suggesting that an endogenous infection by normally symbiotic P. acnes may cause granuloma formation in some predisposed individuals with Th1 hypersensitivity against intracellular proliferation of latent P. acnes triggered by certain host or drug-induced conditions. In the histopathologic diagnosis of sarcoidosis, M. tuberculosis-associated and P. acnes-associated sarcoidosis will possibly be differentiated in some patients by IHC for each etiologic diagnosis and potential antimicrobial intervention of sarcoidosis.The results of many molecular and immunologic studies supporting mycobacterial and propionibacterial etiologies of sarcoidosis are complicated because of potential latent infection by both microorganisms in deep organs such as the lungs and lymph nodes. A mycobacterial etiology is based on hypersensitivity to some pathogenic antigens without active infection. A propionibacterial etiology is based on active


Due to its ubiquity and versatility in the human body, collagen is an ideal base material for tissue-engineering constructs. Chemical crosslinking treatments allow precise control of the biochemical and mechanical properties through macromolecular modifications to the structure of collagen. In this work, three key facets regarding the collagen crosslinking process are explored. Firstly, a comparison is drawn between the carbodiimide-succinimide (EDC-NHS) system and two emerging crosslinkers utilising alternate chemistries: genipin and tissue transglutaminase (TG2). By characterising the chemical changes upon treatment, the effect of EDC-NHS, genipin and TG2 crosslinking mechanisms on the chemical structure of collagen, and thus the mechanical properties conferred to the substrate is explored. Secondly, the relative importance of mechanical and biochemical cues on cellular phenomena are investigated, including cell viability, integrin-specific attachment, spreading and proliferation. Here, we observe that for human dermal fibroblasts, long-term, stable proliferation is preconditioned by the availability of suitable binding sites, irrespective of the substrate modulus post-crosslinking. Finally, as seen in the graphical abstract we show that by choosing the appropriate crosslinker chemistries, a materials selection map can be drawn for collagen films, encompassing both a range of tensile modulus and fibroblast proliferation which can be modified independently. Thus, in addition to a range of parameters that can be modified in collagen constructs, we demonstrate a route to obtaining tunable bioactivity and mechanics in collagen constructs is uncovered, that is exclusively driven by the crosslinking process.  The ubiin vivo  and densin vivo , collagein vivo  and freein vivo . Though in vivo  and protin vivo  stabilitin vivo  have beeCollagen is recognised by a range of cell receptors including integrins, discoidin domain receptors, LAIR-1, mannose receptors and glycoprotein VI, of which integrin receptors comprise the primary group . These rOne of the most common crosslinkers for collagen includes a combination of 1-ethyl-3(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) with N-hydroxysuccinimide (NHS), with early work by Olde Damink et al.  establisGenipin and tissue transglutaminase (TG2) present two alternative amine based crosslinkers that have been widely used with tissue engineering constructs: genipin is commonly used with chitosan  whereas in vivo and have shown exceptional cellular responses, including increased attachment of human osteoblasts to collagen scaffolds upon crosslinking [Transglutaminases are a family of transferases responsible for crosslinking of skin, hair and blood clots slinking . Transglslinking . In the slinking . Thus TGHowever, treating collagen with any crosslinker can alter the structure of collagen I across various length scales. Additionally, the wide variety of formats adopted by collagen-based medical devices gives rise to a range of two–three dimensional architectures that can be fabricated, which can also contribute to differences in cellular behaviour. The concomitant alteration of various biochemical and structural cues upon crosslinking creates great difficulty in attributing cell responses to a single property of the substrate. Two dimensional films of insoluble microfibrillar collagen-I can be used in place of neutralised collagen monolayer coatings or three-dimensional scaffolds as a surrogate in the assessment of cellular response without introducing the complexity of microarchitecture, macroscopic swelling and fluid flow that can be varied in three dimensional architecture . Using h22.1Bos Taurus cattle, 38.8 mmol amide N/100g, 76.1 Type I dermal microfibrillar bovine collagen (18–36 month 2.22.2.1A crosslinking solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride  and N-hydroxysuccinimide  was prepared using a 5:2:1 M ratio for EDC: NHS:Collagen, hereafter referred to as the ‘100% concentration’. For every 1 g of collagen, the 100% concentration standard of crosslinking solution consisted of 1.15 g EDC and 0.276 g NHS dissolved in 75% ethanol. Diluted crosslinking solutions were prepared at a molar ratio of 5:2:2 and 5:2:10 EDC:NHS:Collagen, hereby referred to as the ‘50%’ and ‘10%’ crosslinking concentrations for EDC-NHS. Films were immersed in 100%, 50% and 10% crosslinking solution for 2 h at ambient temperature.2.2.2Using a mass for mass equivalence for genipin to PBS adapted from the protocol by Zhang et al. , for eve2.2.3Using a mass for mass equivalence for TG2  and Tris buffer in the protocol adopted by Chau et al. , for eveAll crosslinked films were washed with distilled water . 200 l of the resultant Ruhemann's purple dye was transferred to a 96 well plate and the absorbance was read at 570 nm using a using a SPECTROstar Nano plate reader . A standard curve was prepared using glycine (Sigma-Aldrich) and the linear region of absorbance with concentration was used to convert sample absorbances to amine content. The degree of crosslinking was then determined according to the formula:2.4Fourier-transform Infrared (FTIR) spectra of the free standing collagen treated and untreated films were obtained in ATR mode, with a wavenumber resolution of 4 2.5Collagen films were cast and cut into 35 mm 2.6Human dermal fibroblasts were purchased from the European Collection of Animal Cell Cultures  and maintained in a humidified incubator at 37 °C with 5% 2.72 or 5 mM EDTA were added for 1 h at 37 °C. Plates were washed three times with PBS to remove loosely bound cells (200 l) and permeabilised with 100 l per well of 2% Triton-X lysis buffer (in water) for 90 min at ambient room temperature. Subsequently, 100 l of lactate dehydrogenase (LDH) substrate , prepared according to the manufacturer's instructions was added per well for 20 min and the absorbance read at 490 nm using a SPECTROstar Nano plate reader . Results are reported as means Crosslinked films prepared in 48 well plates (Corning) were pre-blocked with 200 l 5% (w/v) bovine serum albumin (Sigma-Aldrich) for 1 h at ambient room temperature. Wells were washed with 200 l of PBS (Invitrogen) and 200 l of human dermal fibroblasts (12.8Crosslinked films prepared in 24 well plates (Corning) were seeded with human dermal fibroblasts (2.9Spent media collected from the cell proliferation assay was spun down at 300 rpm to remove any cells and 100 l pipetted into a 96 well-plate (Corning). 100 l of LDH substrate (Roche) was added and colour developed for 20 min at 37 °C. The absorbance was read at 490 nm using a SPECTROstar Nano plate reader .2.10The viability of fibroblasts seeded on crosslinked collagen films on days 1, 4 and 7 was assessed using the LIVE/DEAD assay (Invitrogen). Wells were washed with PBS (2.112.11.1Crosslinked collagen plates (24 well) were pre-blocked with 800 l of 5% BSA  for 60 min at room temperature and seeded with 5000 cells suspended in serum free DMEM media for 2 h at 37 °C. The plates were subsequently washed with PBS . A 100 pixel rolling ball radius background subtraction was applied to the DAPI channel (350 nm excitation/450 nm emission) and a 300 pixel rolling ball radius to the phalloidin channel (590 nm excitation/611 nm emission). Brightness and contrast of each channel was then adjusted prior to merging the image channels to minimise signal from autofluorescent collagen fibres. Single cells were chosen and isolated using the colour threshold in the phalloidin channel.2.12Crosslinked films prepared in 24 well plates (Corning) were seeded with human dermal fibroblasts  and subjected to reverse transcription (using random hexamers) followed by quantitative polymerase chain reaction (PCR). The human Collagen-I alpha 1 gene on Chromosome 17 from exon 1 to exon 49 was reannotated (included as part of the Open Data). Collagen type I alpha chain forward and reverse primers were designed as 5′ CTGGCAGCAAAGGAGACAC 3’ (exon 20) and 5′ TCGCTTTCCTTCCTCTCCAG 3’ (exon 21), respectively. A TaqMan fluorogenic probe (5′-6FAM- ACCAACAGGGCCAGGCTCTCCC 3′) was designed for Col-I alpha chain quantification, binding to the 3′ end of exon 20 and 5′ end of exon 21. The probe contains a fluorescent reporter 6-carboxyfluorescein (FAM) at the 5′ end and a fluorescent quencher 6-carboxytetramethylrhodamine (TAMRA) at the 3′ end. Real-time quantitative PCR was performed on the cDNA of both the Collagen-I alpha chain and as an internal control, the GAPDH genet value, which correlates inversely with the concentration of target mRNA, was determined as the cycle number at which the fluorescence emission of the reporter probe increases above a threshold level. The results from the thermocycler (Applied Biosystems 7300) are reported as the mean GAPDH normalised Ct for Col-I expression The reverse transcription reaction was performed using the reverse primer and reverse transcriptase . Real-time PCR was performed in duplex for the GAPDH and Col-I primers and probe sets using TaqMan MasterMix (ThermoFisher 4370048). The cycle threshold or C2.13The collected cell culture supernatants from the PCR experiment were diluted 1:200 in media. The media was used to perform the Human MMP1 ELISA kit  according to manufacturer's instructions for cell culture supernatants. Results are reported as means 2.14Crosslinked collagen films were weighed then incubated in 5 mL deionised water for 24 h at 37 °C. The hydrated samples were blotted on filter paper, and the hydrated films were subsequently weighed. The water uptake of the material was calculated according to the formula:2.15The 24 well plates with cells seeded for 7 days and lysed for PCR were imaged using phase contrast microscopy to identify morphological changes or the presence of shrinkage in the films following cell culture. Images were taken using a Zeiss Axio Observer Z1 microscope with an AxioCam 503 camera , non-specific (EDTA) and specific  and a decrease in integrin specific binding. All genipin crosslinked films, and 40% TG2 crosslinked films have a small but statistically significant increase in non-specific binding in comparison with non-crosslinked and ethanol crosslinked films. The overall, integrin specific cell attachment remains largely unaffected across all concentrations of genipin and TG2 crosslinked films when compared with non-crosslinked control samples, consistent with an intact GXOGER motif on the collagen fibres with these two crosslinkers.The effects of crosslinking collagen using EDC-NHS, genipin and TG2 on HDF proliferation were assessed using the MTS assay b. Over tThe gene expression level of the ECM protein Collagen-I, by the seeded human dermal fibroblasts was found to be unaffected by the crosslinking of the substrates (Supplementary Information). The expression levels of matrix metalloproteinase-1 (MMP-1), a collagenase that can degrade ECM proteins was noted to increase with increasing concentrations of EDC-NHS and TG2-treatment (Supplementary Information). Interestingly, this was not observed for genipin crosslinking where MMP-1 levels were found to decrease with increasing crosslinking concentration.3.3To evaluate whether the decrease in cell number corresponded to cell death from encountering a cytotoxic environment, an LDH assay was performed on the supernatant medium from the MTS cell proliferation assay. While some metabolic activity was noted with ethanol treatment and crosslinking at Day 4 and 7, as seen in These observations were further supported by the use of the live/dead assay. 3.4A cell spreading study was conducted to characterise the morphology of HDFs on non-crosslinked as well as EDC-NHS, genipin and TG2 treated films as seen in Several qualitative differences in the cell morphology can be observed between the treatments. For the EDC-NHS treated films, some single cells were well-spread on the film surfaces, whereas others have begun their apoptotic detachment from the sample surface. c and d. For all other conditions of crosslinking, broad lamellipodia was observed on the cells, with some tethers extending from the periphery. Genipin crosslinked collagen fibres exhibited autofluorescence in the red  channel,The data obtained here were also quantified in terms of the area, perimeter, aspect ratio, circularity and roundness of a single cell (Details provided in Supplementary Information, 4Among the cues responsible for guiding cell attachment, surface biochemistry and mechanical properties represent two of the most significant stimuli presented by the substrate. In this study, we attempt to separate the effects of the ligands available and the mechanical properties of the collagen based constructs by characterising the chemical and mechanical properties of the crosslinked films and evaluating short-term and long-term effects of the treatments on human dermal fibroblast attachment and proliferation.4.1The EDC-NHS, genipin and TG2-treated films were characterised using the ninhydrin assay to characterise the changes to the free amines in the underlying collagen structure. By colorimetrically analysing the purple ‘Ruhemann's dye’ released in the process , the ninPrevious work on 100% EDC-NHS collagen films revealed that tensile modulus increased nearly six fold upon crosslinking  when meaAs first explored in the introduction, a reduction in free amines may occur either through the formation of amide crosslinks or the amine reduction may also occur via a conversion of glutamines to glutamates for the TG2 treated samples. Although TG2 has been noted to increase the modulus in freeze-dried neutralised soluble collagen samples , this woHowever, further reduction in modulus at higher concentrations of TG2 is unexpected when compared with the soluble collagen counterparts which are used in collagen-based hydrogel networks. By taking into account the reduction in free amines, this behaviour can be resolved through the hydrolytic pathway that can be adopted by TG2 in the presence of few amines ,41. From4.2From the HDF attachment assay, EDC-NHS crosslinking was noted to significantly ablate integrin-specific motifs in collagen, resulting in a reduced specific binding at high concentrations of EDC-NHS. This result is consistent with other observations of integrin specificity with the integrin I domain adhesion  and anchPrevious work on the integrin-mediating binding to genipin-crosslinked constructs have suggested a decrease in integrin-binding since genipin may crosslink to arginines, resulting in the loss of RGD motifs . In thisTG2-treatment of soluble collagen-I also did not result in any changes to integrin specific binding. Therefore, although it was hypothesised that an increase in glutamates may increase integrin specific binding, this result suggests that the number of glutamates created are either insignificant in their total number, or do not present a significant contribution to the initial short-term attachment of fibroblasts. In the TG2-crosslinked collagen constructs evaluated in the literature, β integrin expression has been reported to be increased after treatment . In thesIn addition to the glutamic acid residue of the MIDAS motifs, the accessibility of the integrins has also been shown to be affected by steric hindrance surrounding the integrin motifs in triple helix-mimicking proteins . While aThe long-term effects of the availability of ligands and the mechanical properties of a film were further investigated through the MTS cell proliferation assay b. The coThis behaviour is also consistent with the increase in proliferation seen with TG2 over the untreated samples, due to the increased glutamates available upon TG2 treatment. As a result, the long-term proliferation can be concluded to be dependent primarily on the availability of appropriate ligands, even if short-term proliferation may be affected by mechanical stiffness as evidenced by the high rates of proliferation from Day 1–4 for genipin and EDC-NHS crosslinked samples.Although this study characterised the proliferation of HDFs on crosslinked surfaces, there is further scope to investigate whether the observed behaviour arises from a direct interaction with the crosslinked collagen, or an indirect interaction through the ECM deposited on the treated surface. It is hypothesised that at Day 1, cells interact directly with the crosslinked films, but begin to deposit their own extracellular matrix by Day 4. In normal human wound healing, the cell attachment protein fibronectin can be detected histologically at Day 4, with collagen-I and collagen-IV detected at Day 7 .In this work, the data from Day 7 revealed no difference in the gene expression levels of collagen I production by fibroblasts on the crosslinked films , indicat4.3The cytotoxicity of the treatments was evaluated using the live/dead stain and LDH assay on the supernatants of the MTS proliferation assay. As seen from the representative images in The increase in cell proliferation, with little apoptosis observed with the stiffened treated films has been previously noted with fibroblasts in crosslinked substrates of tunable mechanical properties ,54. Alth4.4The lack of variation in the overall cell spreading behaviour, excepting the greater incidences of rounded cells and cellular projections at higher crosslinking concentrations of EDC-NHS, indicates that the contact behaviour of the cells are largely dependent on the availability of ligands on a substrate. This is akin to the behaviour seen when human gingivial fibroblasts are seeded on fibrillar collagen with localised integrins . The pre4.5As a result, we propose the mechanism shown in 5In this body of work, collagen constructs were crosslinked using EDC-NHS, genipin and tissue transglutaminase to explore three elements of the collagen crosslinking process. EDC-NHS results in an extremely high tensile modulus, at the expense of integrin-specific attachment and long-term cell proliferation through the loss of glutamates in the crosslinking process.At the same degree of crosslinking, genipin-treated films offer remarkably high cell activity, and a comparable tensile modulus with EDC-NHS treated films. Following this work, genipin therefore stands out in its potential to replace EDC-NHS where medium to high mechanical stiffness and excellent cell attachment and proliferation are desired. Tissue transglutaminase may promote the conversion of pre-existing crosslinks in insoluble collagen to glutamines at high concentrations, resulting in low stiffness substrates that maintain higher cell attachment and proliferation than untreated samples. The use of three different crosslinking chemistries bring to light the relative importance of mechanical and biochemical cues on human dermal fibroblasts. Although short-term proliferation may be driven by mechanical cues, long-term stable proliferation of HDFs are correlated with high levels of integrin-specific binding, irrespective of the modulus post-treatment.The crosslinking process therefore represents a pathway for access to different regimes within the property space of tensile modulus as well as cell attachment and proliferation. As a result, the potential to create structures of tunable mechanics and bioactivity in collagen can be exclusively facilitated through the choice of crosslinker chemistry, further expanding the versatility of collagen within tissue-engineering scaffolds.Malavika Nair: Methodology, Investigation, Formal analysis, Writing - original draft, Visualization. Ramneek K. Johal: Methodology, Conceptualization, Investigation, Supervision, Writing - review & editing. Samir W. Hamaia: Investigation, Writing - review & editing. Serena M. Best: Conceptualization, Writing - review & editing, Supervision, Project administration, Funding acquisition. Ruth E. Cameron: Conceptualization, Writing - review & editing, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.