Precise dating of diamond growth is required to understand the interior workings of the early Earth and the deep carbon cycle. Here we report Sm-Nd isotope data from 26 individual garnet inclusions from 26 harzburgitic diamonds from Venetia, South Africa. Garnet inclusions and host diamonds comprise two compositional suites formed under markedly different conditions and define two isochrons, one Archaean (2.95 Ga) and one Proterozoic (1.15 Ga). The Archaean diamond suite formed from relatively cool fluid-dominated metasomatism during rifting of the southern shelf of the Zimbabwe Craton. The 1.8 billion years younger Proterozoic diamond suite formed by melt-dominated metasomatism related to the 1.1 Ga Umkondo Large Igneous Province. The results demonstrate that resolving the time of diamond growth events requires dating of individual inclusions, and that there was a major change in the magmatic processes responsible for harzburgitic diamond formation beneath Venetia from the Archaean to the Proterozoic. Dating of inclusions within diamonds is used to reconstruct Earth’s geodynamic history. Here, the authors report isotope data on individual garnet inclusions within diamonds from Venetia, South Africa, showing that two suites of diamonds define two isochrons, showing the importance of dating individual inclusions. Precise dating of diamond growth events is a prerequisite for such studies but represents a challenge owing to the complex growth history of many gem quality diamonds2. Inclusion-bearing diamonds derived from the sub-continental lithospheric mantle (SCLM), preserve evidence of tectono-thermal events such as continental assembly and mantle melting5. Diamonds crystallise from metasomatic reactions involving C-H-O-S-rich supercritical-fluids and/or silicate melts and on rare occasions include minerals that carry the imprint of the environment of growth2. Diamonds, transported from the SCLM to the surface as xenocrysts in kimberlitic or related magma types, protect the mineral inclusions from secondary processes such as later mantle metasomatism and re-equilibration with the host magma6. Radiogenic isotope studies of inclusions in combination with compositional data can constrain the conditions and timing of diamond growth, and provide fundamental information about the tectono-magmatic processes that led to the formation and modification of the lithospheric keels that underlie the oldest parts of the Earth’s continents1. Inclusions in diamonds derived from the sub-continental lithosphere are typically subdivided in three principal suites that characterise their mantle source rocks; peridotitic, eclogitic, and websteritic (see ref. 2 for an extensive review). The peridotitic suite is further subdivided into harzburgitic, lherzolitic, and wehrlitic parageneses based on Ca and Cr contents of garnet inclusions. Thus far, with one exception7, the low trace element concentrations of peridotitic inclusions in diamond and their small size has necessitated pooling tens to hundreds of inclusions to obtain Sm-Nd isochron ages13. In consequence, fundamental concerns have been raised about common inclusion parentage and the validity of these ages as records of actual diamond growth events16.The apparent antiquity of many diamond suites means that they represent a window to both the Earth’s early geological record and deep carbon cycle13. Three of the pooled groups defined a nominal Sm-Nd isochron age of 2.30 ± 0.04 Ga (2σ) with an unradiogenic initial ratio (εNd = −8). The authors recognised that the results potentially recorded mixing of different components and presented a model wherein Venetia harzburgitic diamonds crystallised at ca. 2 Ga associated with modification of a > 3 Ga Archaean harzburgitic SCLM by Bushveld-related magmas. The implication of this interpretation is that harzburgitic garnets of highly variable composition were formed during a single diamond-forming event in the mid-Proterozoic.A previous study of inclusions from the Venetia diamond mine located within the Limpopo Mobile Belt in South Africa, examined 400 garnet inclusions of harzburgitic composition of which 140 were divided into four compositional groups for Nd and Sr isotope analysisTo provide constraints on how the tectono-magmatic conditions responsible for harzburgitic diamond formation may have evolved over time, and assess the possible temporal evolution of the Earth’s carbon cycle, we present coupled major and trace element and Sm-Nd isotope data for 26 individual garnet inclusions extracted from 26 individual peridotitic diamonds from Venetia, which were also measured for their carbon isotope compositions. The data allow the unravelling of the mixed signature recorded in previous pooled inclusion data, and produce new accurate ages that date regional, but contrasting, styles of magmatism that affected the mantle beneath the Limpopo Mobile Belt in the Archaean and Proterozoic. The data provide a tantalising glimpse of how processes associated with diamond formation may have changed over time.17. In addition, the term ‘synchronous’ has been proposed, describing protogenetic inclusions that owing to chemical equilibrium with the diamond-forming medium record the time of diamond growth16.The relationship between diamond and its inclusions is potentially key in interpreting absolute age information obtained from the inclusions. There has been significant recent debate as to whether inclusions are formed simultaneously with diamond (syngenesis), or are pre-existing objects incorporated during diamond growth (protogenesis)19. Moreover, epitaxial relationships between inclusions and diamond hosts have also been thought as strong evidence of the control by diamond on the crystallisation of the inclusion21. Recent studies from Siberian diamonds reported that epitaxy played both a strong23 and limited control24 on inclusion formation. Theoretical considerations of adhesion energy and interface energy, however, suggest that there is no energetic benefit for olivine-diamond interfaces to form an epitaxial relationship25. An X-ray topographic study of an olivine inclusion-bearing diamond reported an absence of volume distortion in diamond around the inclusions17. In addition, the surface morphology of olivine inclusions had stepped morphology. Both observations are consistent with growth or dissolution processes contributing to the final inclusion morphology. The recent report of epitaxy between clinopyroxene inside and outside a diamond in a lherzolite xenolith adds to the discussion16. These authors propose that the use of the term syngenetic may be inappropriate to describe inclusions. They argue that the term ‘synchronous’ is more applicable to explain isotopic data when minerals were formed prior to diamond growth (protogenetic), but owing to chemical equilibrium with the diamond-forming fluid minerals will record the time of encapsulation in the diamond16. Experimental studies have produced syngenetic growth of inclusions from hydrous fluids containing salts, silicates, and carbonates under upper mantle conditions. Such experiments provide unequivocal evidence that diamonds and silicates can form syngenetically, producing mineral assemblages comparable to those found in inclusion suites in natural diamonds26.All inclusions studied in this work show cubo-octahedral diamond imposed morphology and some show diamond inherited surface morphology Fig. . The occ27, however, argued that the sinusoidal rare earth element (REE) patterns that characterise harzburgitic garnet inclusions in diamonds worldwide are indicative of a complex genetic history for the garnets prior to diamond encapsulation. The above authors argued that the sinusoidal REE pattern with low absolute heavy rare earth element (HREE) implies an extensive melt depletion phase in the absence of residual garnet was followed by a metasomatic event that involved a highly fractionated light rare earth element (LREE) component, suggesting that the inclusion was protogenetic27. An equally plausible argument, however, is that the diamond-forming fluid interacted with the melt-depleted host peridotite to produce an environment with a sinusoidal REE pattern.Taylor et al.17. Irrespective of the origin of the inclusions, a key aspect in dating a mineral inclusion is the extent to which the inclusion reached chemical equilibration with the diamond forming media, that is, do protogenetic grains record synchronous age information. The closure temperature of the Sm-Nd system in garnets in the crust and mantle varies depending on elemental diffusivity, mineral size, composition and cooling rate but is considered to be typically between 750 and 900 °C29. In the case of a garnet in the mantle in contact with a metasomatic fluid/melt precipitating diamond, free diffusion of REE is thus expected at typical mantle temperatures (1000–1400 °C). Under such conditions, the time for a garnet of protogenetic origin to reach full Nd isotope equilibration with the fluid/melt will depend on the effective garnet grain size and elemental diffusivity. An upper limit of equilibration time can be determined by assuming a defect-free gem quality mineral under anhydrous conditions. Based on the typical size of our garnet inclusions (100 μm radius), full chemical equilibration of REE  with a diamond-forming melt/fluid would occur within less than 0.6 kyr at 1400 °C (diffusion data from ref. 29). At a lower temperature of diamond formation (1000 °C) and again assuming a defect-free 100 µm radius garnet, it is possible that full chemical equilibration with the diamond forming melt/fluid could take up to 250 kyr. Hence under such circumstances, instantaneous diamond formation could potentially entrain minerals partially recording a protogenetic age. Given the diamond imposed morphology and growth features seen on the crystal faces of analysed inclusions  and Nd (0.9-26 ppm) and Sm (0.01–5.8 ppm) concentrations, used to calculate depleted mantle model ages (TDM), are presented in Supplementary Table TDM age of 3.0 ± 0.1 Ga is defined by nine samples and an age of 1.1 ± 0.2 Ga (1 SD) is recorded by 10 samples. The 3.0 Ga population generally has CaO contents lower than 2.5 wt.%, whereas the 1.1 Ga age population has CaO contents mostly higher than 2.5 wt.%. We use 2.5 wt.% CaO as a threshold value to further define the two groups, now with eight samples each  and the 1.1 Ga age group has δ13C > −5 ‰ . CaO and Crach Fig. . Signifi143Nd/144Nd and reciprocal Nd  and younger (1.3–2.3 Ga) than the 3.0 Ga age group  plot on the isochron of 1.15 Ga at low 147Sm/144Sm  but have relatively flat HREE patterns. They plot just below the 2.95 Ga isochron  show a significantly smaller range in 147Sm/144Nd and 143Nd/144Nd compared with the individual inclusions, demonstrating that the pooling masked the original heterogeneity. More than 25% of the individual inclusions analysed here have higher Sm/Nd and 143Nd/144Nd than the composite inclusion data, but because of low Nd contents their presence in a pooled population is obscured by inclusions with higher Nd contents. The average of the TDM’s for individual inclusions  obtained from pooled peridotitic garnet inclusions from Premier (Cullinan), SA12 and Udachnaya, RU33 may also be coincidental. Based on Re-Os dates from sulphide inclusions at Udachnaya, it has already been shown that old and young peridotitic diamond populations are recovered from these mines34, further stressing the need to re-examine published diamond ages obtained from pooled diamonds.Our data establish that it is vital to utilise single inclusions to obtain accurate age constraints on the time of diamond growth and that pooling of inclusions may mix populations of different age, resulting in ages with diminished geological meaning. The data reported by Richardson et al.35). This equates to > 40 tons of C moved from the mantle to the upper few km of the crust with a far larger mass of C stored in the kimberlites at depth and lost due to erosion of the upper levels of the kimberlite.Below, we consider the potential coupling between diamond growth within the lithosphere and large scale magmatic events expressed by the presence of crustal intrusions in the area with similar ages as the diamonds. A correlation is made between diamond formation and large tectono-magmatic events due to the large amount of carbon recovered in the form of diamond from the Venetia mine. The Venetia kimberlite cluster comprises a surface area of only 28 ha but since its opening in 1992 has produced in the order of 100 M carats and comparable resources remain to be exploited  the two garnet inclusion groups show distinct behaviour related to fluid or melt dominated metasomatic diamond growth;  decreasing strongly from fluids to meltsth; Fig. . The old39. Key to linking the diamond-formation event to magmatism in the region is reconstruction of the crust-mantle architecture prior to assembly of the two cratons. The origin of the Central Zone of the Limpopo Belt, the location of the Venetia diamond mine, is debated40. De Wit et al.41, for example, argue using geophysical data that the SCLM beneath the Central Zone of the Limpopo Belt was part of the Zimbabwe Craton with the overlying crust belonging to the Kaapvaal Craton or to an allochtonous block that over-thrusted towards the north43. The low temperature peridotites from Venetia, however, show marked Si-enrichment more characteristic of the Kaapvaal SCLM, in contrast to the melt depleted diamond facies samples44. These data suggest potential decoupling of shallow and deep peridotite suites, a conclusion compatible with mantle-crust decoupling, which is supported by lead isotope data and the inferred derivation of crustal rocks from a common source with a long-lived high μ (U/Pb) value in the Central Zone and on the Zimbabwe Craton. This Pb isotope signature differs from the inferred low μ source below the Southern Limpopo Belt and the Kaapvaal Craton45. Based on the suggested decoupling, we expect the crustal rocks associated with the 2.95 Ga diamond growth event at Venetia to be located to the north in the Zimbabwe Craton. The southern part of this Craton contains multiple crustal units dated at 2.9–3.0 Ga48. The Chingezi Tonalites in the Belingwe greenstone belt, for example, yielded whole rock Sm-Nd ages between 2.95 and 3.05 Ga with an average initial 143Nd/144Nd of 0.5088447, within error of our 2.95 isochron initial ratio (0.50867 ± 13). Similarly, zircons from the Mashaba Tonalite, part of the Early Archaean Tokwe region  to a rifting passive continental shelf associated with asthenospheric decompression and melting of the mantle beneath the lithosphere51. The resulting magmatism across the region is inferred to have driven the fluid-dominated metasomatism at depth within the depleted lithosphere that was responsible for diamond growth at 2.95 Ga.Based on the regional geological evolution of the Zimbabwe Craton, we infer that the older, 2.95 Ga diamonds formed in a highly melt-depleted mantle residue from fluids mobilised by passive asthenospheric decompression during crust-forming magmatism Fig. . The diatio 0.5087 ± 13. S13C lower than −5‰ with a significant spread . The isotopic variation defines a broad positive relation with CaO in the garnet inclusions . Thus, these δ13C values are unlikely to involve a recycled organic carbon component53. The variable 13C depletion of this fluid event is considered to reflect Raleigh-style fractionation suggesting diamond formation from relatively small volumes of super-critical fluids. The range in δ13C values implies diamond precipitation involving oxidation of a reduced fluid (CH4) causing isotopic fractionation to lower δ13C55. The observed relation between CaO and δ13C possibly reflects the effect of decreasing solubility in reduced C-saturated fluids moving upwards along a geothermal gradient56.Importantly, the host diamonds of the Archaean inclusions generally have δ2 across the Kaapvaal and Zimbabwe Cratons57 . This observation is consistent with melt infiltration into the depleted lithosphere caused by impingement of the thermal anomaly on the base of the SCLM that led to the Umkondo LIP. The δ13C values of the 1.15 Ga diamonds record a restricted range with an average of −4.6 ‰, indistinguishable from asthenospheric carbon1. In addition, the initial ratio of the 1.15 Ga isochron (εNd + 3.5 ± 5.6) is in agreement with derivation from the convective depleted mantle.The 1.15 ± 0.11 Ga diamond growth event can be linked to the Umkondo Large Igneous Province (LIP) magmatism that occurred between 1.106 and 1.112 Ga over an area of 2 million km12 and Udachnaya, RU33 may also be incorrect, bringing into question an apparent increase in worldwide peridotitic diamond formation at  ~ 2.0 Ga2. Our data provides convincing evidence for at least two distinct diamond growth episodes beneath Venetia. Seven samples with TDM both younger and older than the 3.0 Ga age group may reflect additional diamond growth episodes. Further diamond and mineral inclusion investigations are required to assess whether the observed change in the tectonic processes responsible for the distinct harzburgitic diamond growth events at Venetia, is a continent- or even a planetary-wide occurrence.The Venetia data indicate that Proterozoic diamond formation was associated with melt metasomatism resulting from a major thermal perturbation of the SCLM by active asthenospheric upwelling related to a LIP event, whereas Archaean harzburgitic diamond formation can be related to relatively cool fluid-dominated metasomatism caused by rift-related magmatism. Resolving whether the observed change in the tectonic processes responsible for harzburgitic diamond growth is a consequence of planetary-wide temporal evolution requires detailed dating of more diamond inclusion suites. Data presented here demonstrate that dating by pooling of garnet inclusions in diamond may result in averaging of ages from multiple diamond generations and potentially obscure the actual geological events responsible for diamond formation. This conclusion means that ages obtained from pooled peridotitic garnet inclusions from Premier (Cullinan), SA59. The harzburgitic garnet inclusions studied here were selected primarily on the basis of their relative large size to allow precise and accurate Sm-Nd isotope analyses.The studied garnet inclusions and host diamonds were donated to J.W.H. by the Diamond Trading Company, a member of the DeBeers Group. The inclusions had been liberated from their host diamonds by J.W.H and co-workers in the 90s60. After EMPA analyses, carbon coating was removed by washing in ultrapure methanol and milli-Q water. The inclusions were weighed on a Mettler 7 decimal balance with a sensitivity of 0.1 µg and spiked with a 149Sm-150Nd mix aiming for a Nd sample/spike ratio of 20 with Nd concentrations estimated based on the Cr2O3-CaO compositions13. Samples were dissolved in a concentrated HF-HNO3 mix on a hotplate at 140 °C for a minimum of 3 days. Chemical separation of Nd and Sm from the dissolved matrix was performed in a two column procedure using 0.16 ml TRU-resin and 0.7 ml LN resin61. Matrix fractions were collected and combined for trace element analyses by inductively coupled plasma mass spectrometry (Thermo X-Series II) at the Vrije Universiteit (VU) Amsterdam. BHVO-2 (n = 5) was used to correct for the chemistry yields for each element. Three inclusions, too small to date, were dissolved and analysed for trace elements directly by ICPMS. Data from these extra inclusions overlap both the SIMS data for garnet inclusions from Stachel et al.38 and the dated inclusions processed through chemistry, thus confirming the yield corrections to be appropriate  following the methods of Timmerman et al.13 Ohm amplifiers installed in the instrument since 201262. Faraday-amplifier gains were determined using the La Jolla Nd standard following the method of Timmerman et al.63. Beams on 1013 Ohm amplifiers where kept below 3 × 10−13 A. Samples were run to exhaustion. Instrumental reproducibility for 100 pg samples is determined by routine analysis of an in-house Nd standard (CIGO) that yields 0.511332 ± 67 (n = 49). Long-term reproducibility of 100 ng loads of this standard measured on 1011 Ohm amplifiers, yields 0.511334 ± 10 (n = 28), which equates to 0.511834 for La Jolla. Isotope composition and isotope dilution Nd and Sm concentrations were determined using an iterative approach to strip the spike and perform an exponential instrumental mass fractionation correction (146Nd/144Nd of 0.721903 for Nd and 147Sm/152Sm of 0.56081 for Sm). Small USGS BHVO aliquots ( ~ 1 mg) processed along with the inclusions yielded average Sm and Nd concentrations of 6.026 ± 0.038 and 24.34 ± 0.66 ppm, respectively, and 143Nd/144Nd = 0.512956 ± 0.000074 . Total procedural Sm and Nd blanks averaged 0.4 and 0.3 pg (n = 5). Blank corrections using the isotopic composition of lab solutes (0.511856 ± 90) have a negligible effect on isotope ratios and calculated TDM, and were not performed. Depleted mantle model ages were calculated with the parameters of Michard et al.64.Sm and Nd were analysed by Triton Plus using four 10δ13C except for one diamond . External standards USGS24 (−16.05‰) and VICS ( + 1.35‰) were measured in triplicate with each batch of 30 samples. Fragments of host diamonds were selected based on morphology,  and in four cases the specific growth zone that housed the inclusion was identified. Interiors and external surfaces of diamonds have consistent All data presented in this manuscript are available as Supplementary InformationSupplementary Data 1Peer Review File


Understanding the biologic mechanisms underlying the development of lethal prostate cancer is critical for improved therapeutic and prevention strategies. In this study we explored the role of tumor metabolism in prostate cancer progression using mRNA expression profiling of seven metabolic pathways; fatty acid metabolism, glycolysis/gluconeogenesis, oxidative phosphorylation, pentose phosphate, purine metabolism, pyrimidine metabolism and the tricarboxylic acid cycle.n = 113) were men who experienced a distant metastatic event or died of prostate cancer during follow-up. Non-lethal controls (n = 291) survived at least 8 years post-diagnosis without metastases. Of 404 men, 202 additionally had matched normal tissue . Analyses compared expression levels between tumor and normal tissue, by Gleason grade and by lethal status. Secondary analyses considered the association with biomarkers of cell proliferation, apoptosis and angiogenesis.The study included 404 men with archival formalin-fixed, paraffin-embedded prostate tumor tissue from the prospective Health Professionals Follow-up Study and Physicians’ Health Study. Lethal cases (p < 0.007), and within these pathways, a number of novel differentially expressed genes were identified including POLR2K and APT6V1A. The associations were tumor specific as there was no evidence any pathways were altered in the normal tissue of lethal compared to non-lethal cases.Oxidative phosphorylation and pyrimidine metabolism were identified as the most dysregulated pathways in lethal tumors  contains supplementary material, which is available to authorized users. It is well known that proliferating tumor cells have different metabolic requirements from normal, differentiated cells . These mProstate cancer represents a particularly appropriate malignancy in which to explore this question. It is the second leading cause of cancer mortality in men in many Western countries, with more than 29,000 deaths in the USA in 2014 attributable to prostate cancer . Yet, atThe objective of this study was to investigate the metabolic pathways that underlie the development and progression of prostate cancer using an integrative molecular epidemiology approach. We characterized metabolic signatures at the messenger RNA (mRNA) level in prostate tumors and adjacent benign tissue among men with prostate cancer, and compared the expression profiles across Gleason grade and cancer outcomes. The analysis was focused on genes encoding enzymes involved in metabolic pathways to further characterize the role of dysregulated metabolism as a key driver in tumor aggressiveness and prostate cancer mortality. There is compelling experimental evidence to support this hypothesis; malignant cells are known to possess unique metabolic phenotypes that differ from the healthy tissues from which they originated –4, yet lThis study was nested among men with incident prostate cancer from the prospective US Health Professionals Follow-up Study (HPFS) or Physicians’ Health Study (PHS). The HPFS  is a cohProstate cancer diagnosis is first reported on questionnaires and confirmed through medical record review. Clinical and pathological data including age at diagnosis, prostate-specific antigen (PSA) levels, and tumor stage are abstracted through medical record review. Post-diagnosis, men with prostate cancer are followed through questionnaires to collect information on their cancers’ clinical course, including development of metastases. Cancer-specific and all-cause mortality is ascertained through mailings and telephone calls to participants, and periodic searches of the National Death Index. A committee of physicians assigns cause of death through medical record and death certificate review. Follow-up for mortality is available through 2011 and is >98% complete.n = 113) defined as men with prostate cancer who experienced a distant metastatic event or died of prostate cancer during follow-up, and non-lethal prostate cancer cases (n = 291) who neither died of prostate cancer nor presented any evidence of metastases during follow-up and lived at least 8 years post-diagnosis. Mean follow-up was 13.2 years (range 0.1–27.4 years). Of 404 men, 202 additionally had profiling for matched normal tissue . Hematoxylin and eosin slides from all cases underwent standardized histopathologic review, including for Gleason grade and perineural invasion, by study pathologists [This study includes 404 men with available archival formalin-fixed, paraffin-embedded (FFPE) prostate tumor tissue who were part of a whole genome expression profiling project of lethal prostate cancer: 150 men from PHS and 254 from HPFS. The study included lethal cases , year of diagnosis, and body mass index (BMI) at diagnosis were included as potential confounders. For within person tumor versus normal comparisons, conditional logistic regression was used.Pathway level associations were explored using the Global test , a scoret test statistic and the GSEA p values were calculated using gene permutations; for the other analyses, standard GSEA was used with p values calculated by permuting individuals.We also performed Gene Set Enrichment Analysis (GSEA) , a compeFinally, a shrinkage and selection method, Least Absolute Shrinkage and Selection Operator (LASSO), was used to identify the genes contributing to pathway level associations and to determine the effect size. This approach fits a penalized regression model including all genes from each pathway as potential covariates and forces the gene expression coefficients not contributing to lethal outcome to zero so that they are removed from the model. The amount of shrinkage applied to the coefficients depends on a tuning parameter, which was chosen by using leave-one-out cross-validation to optimize the likelihood.All analyses were conducted using the R software package.Table p ≤ 0.05) . The most significantly overexpressed genes in tumor tissue were CANT1 , CMPK1 , and GUCY1A3  annotated with the purine and pyrimidine pathways; FBP1  and GPI  annotated with the pentose phosphate and glycolysis pathways. The most significantly downregulated gene in tumor tissue was ALDH3A2  annotated with the KEGG glycolysis and fatty acid metabolism pathways. The differences in mRNA expression between tumor versus normal tissue were similar among the lethal and non-lethal cases (results not shown).A total of 247 (58%) genes in the metabolic pathways were differentially expressed in tumor vs. normal tissue (p < 1 × 10−10) (Table p = 0.002), purine metabolism (p = 0.006), and oxidative phosphorylation (p < 0.0001) were significant at a Bonferroni corrected threshold of p < 0.007  Table . Using G07 Table . All patLASSO regression was then used to identify the genes contributing to the observed pathway level associations. Most of the significant associations for each pathway under the individual gene tests were also identified by the LASSO regression, and several additional genes were suggested to be of importance , of which six genes retained significance after Bonferroni correction. HPRT1 , RRM2 , PDE4D , and NDUFC2  ranked as the most significant , glycolysis/gluconeogenesis (p = 1.37 × 10−5), pyrimidine metabolism (p = 7.56 × 10−5), and oxidative phosphorylation (p = 8.3 × 10−5) were significant under the Global test (Table p = 0.003) and oxidative phosphorylation (p = 0.004) were also significant under GSEA . The associated odds ratios and p values are described in Fig. n = 98, 76%) of these genes were more highly expressed in the lethal cases . RRM2 (OR 6.8 p = 7.72 × 10−9), POLE2  (both associated with purine and pyrimidine metabolism), and ATP6V1A  (oxidative phosphorylation) were also expressed to higher levels in lethal tumors, while ALDH2 , PDE4D , and ALDH1A3  ranked as the most significantly downregulated genes. The associations with lethal disease were attenuated but still significant when adjusting for Gleason grade  with the normal tissue from non-lethal (n = 140) cases. None of the pathways were identified as significant according to the Global test , and thus the relationship between lethal disease and altered metabolic gene expression appears to be a tumor-specific effect.To determine whether the metabolic pathway gene expression changes may be influenced by gene expression changes in the surrounding tissue, expression levels were compared in the morphologically normal prostate tissue from lethal cases (p = 0.006) and oxidative phosphorylation (p = 0.006) were significant under GSEA analysis  (Table p = 1.1 × 10−6), glycolysis/gluconeogenesis (p = 1.2 × 10−4), purine metabolism (p = 1.9 × 10−5), and pyrimidine metabolism (p = 3.5 × 10−4).We explored the association between expression of the metabolic pathways with biomarkers of cell proliferation, apoptosis, angiogenesis, and perineural invasion. The strongest associations of the pathways were with cell proliferation (7) Table . ConversCancer cells share a metabolic requirement to support inappropriate cell proliferation and maintain growth ; howeverConsistent with previous literature , in thisCANT1 [FBP1 [RRM2 [POLE2 [PDE4D [ALDH1A3 [Among the top differentially expressed genes, many were common between the tumorigenesis, lethal and Gleason analyses, while others appeared to be specific to disease aggressiveness. A number of the top genes identified in this study have previously been implicated in prostate cancer development and progression, Gleason grade, metastases and biochemical recurrence; including CANT1 , FBP1 [3T1 [FBP1 , RRM2 [3P1 [RRM2 , POLE2 [2 [POLE2 , PDE4D [2 [PDE4D , and ALD[ALDH1A3 . This suGPI and ALDH3A2 with prostate cancer in a human population. Mammalian GPI has been demonstrated to function as a tumor-secreted cytokine and angiogenic factor, while cells with high ALDH activity have been shown to display metastasis-initiating behavior [ALDH2, has previously been associated with the progression of benign prostatic hyperplasia [. Similarly, we report novel associations between Gleason grade with HPRT1 and NDUFC2. NADH dehydrogenase has been repeatedly implicated in prostate cancer risk [Additionally, we report a number of novel genes with biological plausibility; this is the first study to report on the association between behavior . A furtherplasia , but thicer risk , and botOn a pathway level, the predominance of gene expression changes associated with glycolysis and oxidative phosphorylation pathways provides support for a shift in metabolism toward increased aerobic glycolysis in tumors with lethal potential . ChangesThere are potential limitations to this study. A significantly larger proportion of the non-lethal cases had biopsy tissue available from transurethral resection of the prostate as opposed to radical prostatectomy (RP), and our recent study suggests that the expression levels of some genes differs as a function of zone of origin . HoweverNevertheless, transcriptional regulation is known to play an important role in the control of metabolism , and thuIn conclusion, prostate tumorigenesis is associated with dysregulation of key metabolic signaling pathways. Within tumor tissue, we offer evidence of specific alterations that differ by Gleason grade and lethal outcome and identify novel genes that may play an important role. Our findings add further support to the hypotheses that metabolic alterations in aggressive tumors may extend beyond an increase in aerobic glycolysis and encompass changes in gene expression for enzymes involved in other pathways supporting proliferation , 4, 41.


Gluconacetobacter xylinus in deuterated glycerol as carbon source dissolved in growth media containing D2O. The derivative of deuterated cellulose was prepared by trimethylsilylation(TMS) in ionic liquid(1-butyl-3-methylimidazolium chloride). The TMS derivative was dissolved in toluene for thin film preparation by spin-coating. The resulting film was regenerated into deuterated cellulose by exposure to acidic vapour. A common enzyme, horseradish peroxidase (HRP), was adsorbed from solution onto the deuterated cellulose films and visualized by NR. The scattering length density contrast of the deuterated cellulose enabled accurate visualization and quantification of the adsorbed HRP, which would have been impossible to achieve with non-deuterated cellulose. The procedure described enables preparing deuterated cellulose films that allows differentiation of cellulose and non-deuterated bio-macromolecules using NR.Novel thin and smooth deuterated cellulose films were synthesised to visualize adsorbed bio-macromolecules using contrast variation neutron reflectivity (NR) measurements. Incorporation of varying degrees of deuteration into cellulose was achieved by growing  Cellulose is a renewable biopolymer that can be produced in relatively pure form at low cost. It is, hydrophilic, biodegradable, biocompatible and easy to functionalize due to the six hydroxy groups on each of the anhydrocellobiose units that comprise the straight chain polymer. Apart from its traditional uses in the manufacture of textiles and paper, cellulose is finding new uses in bio-diagnostic devices and other functional surfaces that take advantage of its regular polysaccharide surface and well-characterised composition. Recently, the development of bioactive paper and the production of bioethanol from the cellulosic components of wood and agricultural residues have attracted wide interest. Paper has been engineered as a platform for low cost diagnostics in environmental and health applications where identification and/or quantification of specific biomolecules or chemical agents is essential134Despite the importance of the interactions between bio-macromolecules and cellulose surfaces, there is little useful information available concerning the structure, conformation and density of absorbed bio-macromolecules at cellulose surfaces. The scarcity of information stems primarily from two factors. Firstly, paper and wood both have complex three-dimensional hierarchical structures89101112et al. measured the adsorption kinetics of H. insolens, a cellulase, on thin cellulose films in terms of adsorption mass and film thickness using ellipsometry. They reported full enzyme coverage on cellulose forming a monolayer 25 Å thick adsorbed at Γmax = 5.5 mg/m2; the dry cellulose films were 300 Å thick with a roughness of 50 Å and swelled to 820 Å once hydratedTo date, cellulose model films have been prepared from either nanocellulose, or cellulose derivatives using two main approaches: Langmuir-Blodgett (LB) deposition15889Despite these advances, our current knowledge of the conformations adopted by biomacromolecules at the cellulose/water interface and of the properties of the adsorbed layers remains limited. Little is known about the interfacial configuration adopted by biomolecules such as enzymes and antibodies once they adsorb onto a cellulose surface, nor on the effect of biomolecule interfacial configuration on functionality. There have been many published examples of the conformation of adsorbed biomacromolecule at the interfaces strongly influencing properties such as reaction rate, selectivity and longevity19202H) and the light isotope of hydrogen (1H), between either the biomolecule or the substrate. Under the best conditions, NR can reveal specific important details concerning the adsorbed biomolecule morphology. NR does however suffer from two major drawbacks due to the poor information content of the measurement since it is essentially a ‘one-dimensional technique’, only probing layer thickness composition and uniformity at the Å to μm level. Partial or complete deuteration of either the surface or the biomolecule enhances both level of contrast and the information content of the measurement. Deuteration of cellulose may be achieved more simply and cheaply than deuteration of most bio-macromoleculesWhile SPR and QCM offer quantification of the adsorption kinetics in real time, atomic force microscopy (AFM), ellipsometryGluconacetobacter xylinus in media containing a deuterated carbon source and D2O as a solvent. There are no cellulose solvents having sufficient volatility suitable for spin coating and so a number of more soluble chemical derivatives of the deuterated cellulose were prepared and these were dissolved in volatile solvents suitable for spin-coating. Derivatization of deuterated bacterial cellulose into trimethylsilylyl cellulose (TMSC) and cellulose tri-acetate was investigated. Different regeneration methods are available for the different synthesis processes of cellulose film5et al.It has been the objective of this study to prepare smooth deuterated cellulose (DC) films from deuterated bacterial cellulose (DBC) suitable for NR studies. This objective has been achieved by growing cultures of The procedure used to prepare deuterated cellulose films is represented schematically in Gluconacetobacter xylinus strain ATCC 53524 grown on media containing deuterated glycerol as a carbon source dissolved in D2O as a solvent. Strains of Gluconacetobacter xylinus are prolific in bacterial cellulose production, forming a pad or a membrane of cellulose called a pellicle on the surface of the culture medium. Unlike most plant-derived cellulose, the bacterial cellulose is not biosynthesized as a composite with other cell wall polymers , but is extruded into the growth medium from where it can be isolated and purified readilyDeuterated bacterial cellulose pellicles were produced from the 4 and 1.15 g/L of citric acid in H2O initially for adaptation of the bacteria and then moved to the deuterated medium through several increments of increasing deuterated glycerol and D2O in the solvent. The pH of the medium was adjusted to 5.0 at the start of growth. Cellulose pellicles were cultivated with increasing amounts of deuteration before final harvesting. This procedure allows in principle production of cellulose films with multiple levels of deuteration. The final DBC pellicles were first rinsed with distilled water to remove excess medium, and then heated (90 °C) in 0.1 M NaOH solution for 30 min to inactivate attached bacterial cells and remove proteins. After boiling, the pellicles were purified by extensive washing in distilled water at room temperature and centrifuged  several times until the pH of the water reached 7.0. Small samples of DBC were collected before and after boiling and subjected to vacuum drying for two days for attenuated total reflectance Fourier transforms infrared spectroscopy (ATR-FTIR). −1 and amide II at 1,545 cm−1) after the treatment with NaOH, washing and freeze dryingThe details and characterisation of bacterial cellulose pellicles using different carbon sources containing hydrogen atoms in natural isotopic abundances have been were published elsewhereOven-dried weights of samples were determined after drying overnight at 105 °C. The remaining wet DBC samples were repeatedly solvent exchanged with 100% ethanol by placing the wet suspension in a 50 mL tube for 10 min with 100% ethanol (40 mL), and then centrifuging . Removal of the aqueous ethanol supernatant and repetition of the process five times gave a suspension of DBC in ethanol that was essentially anhydrous. Finally, the excess ethanol was removed from the DBC by pressing against a nylon fabric mesh on top of a blotting paper. This DBC sample was kept at 4 °C in a closed container and used without further drying for preparing cellulose acetates and trimethylsilylation (TMS) of DBC that could be dissolved in volatile solvents.−1 and 3,300 cm−1 in the samples. The majority of the bands corresponding to the CH-stretching modes are located between 3,000 cm−1 and 2,800 cm−1, and the expected absorption bands were in fact observed in the BC spectrum  and natural bacterial cellulose (BC) as a reference were compared in spectrum . These bC sample  indicatiDBC as purified above was used for preparing solution of the (deuterated) cellulose derivative, trimethylsilylyl cellulose (TMSC), for spin coating and subsequent deuterated cellulose film preparation. This process-required trimethylsilylation of cellulose28A 25 mL three-necked, round-bottomed flask equipped with a reflux condenser and a magnetic stir bar was charged with DBC  and BMIM chloride (2.5 g dried as above). The mixture was stirred at 100 °C under vacuum (500 Pa). A clear solution of deuterated TMSC in the ionic liquid was obtained after 6 h. The flask was then equipped with a silicone bubbler and an inlet for dry nitrogen and was flushed with dry nitrogen for 30 min. Following dissolution of the cellulose, hexamethyl disilazane (1.8 g) was added and the mixture stirred at 100 °C for 2 h. Following this period, anhydrous toluene (8 mL) was added and the mixture stirred at 100 °C for a further 16 h. At that point stirring was stopped and the solution allowed cooling to room temperature. The toluene phase was separated from the ionic liquid using a syringe and the ionic liquid was extracted twice with anhydrous toluene (2 × 8 mL). The combined toluene fractions were reduced to a volume of 4 mL by rotary evaporation under vacuum (500 Pa). The product was precipitated by pouring the toluene solution into anhydrous methanol (50 mL). The product was isolated by filtration, washed three times with anhydrous methanol (3 × 5 mL) and dried at 60 °C, 5 Pa to give trimethylsilyl deuterated bacterial cellulose as a white amorphous solid (0.15 g). The trimethylsilylyl cellulose (TMSC 100 mg), was dissolved in toluene (10 mL) and the solution was placed on a cleaned silicon block and then spin-coated at 4000 rpm for 30 sec. The deuterated TMSC films were hydrolysed to deuterated cellulose films by exposure to vapours above 1M aqueous HCl in a closed container at 20 °C for 15 minsThe quality of the DC films prepared by spin coating as described in the Experimental section was first characterised by Atomic Force Microscopy (AFM). The smoothness of the film before and after regeneration with acid vapours  was char2O. A further modelling parameter may be introduced, in the solvent penetration model, where the slab contains a homogenous mixture of solvent and polymer. In this case the SLD of the slab is determined by the volume fraction of water, since the SLD of this slab will be a linear combination of the solvent and cellulose SLD’s323334Specular neutron reflection (NR) is a very useful technique for direct investigation of phenomena at the solid/water and solid/air interfaces5312 layer on the bare Si block. The resulting fitted parameters for subsequent samples  are shown in −6 Å−2 which is similar to the theoretically calculated SLD of 5.58 × 10−6 Å−2 for deuterated cellulose having a chemical composition of C6H3D7O5 and density of 1.5 g.cm−3.In all cases, modelling the NR curve was underpinned by a pre-existing SiOet al.2O buffer. The number of fringes in the NR curve for natural isotopic abundance cellulose film (open circle) clearly shows that the films are smooth and thick. The curve indicated with filled squares shows the NR profile for the same film with HRP adsorbed on its surface. There is no observable difference in NR curves with and without HRP adsorption; both curves are indistinguishable. This is due to the lack of contrast between the adsorbed layer of HRP and the cellulose layer, possibly compounded by the expected very low film thickness of the adsorbed HRP layer. It was therefore necessary to use deuterated cellulose to gain contrast and clear visualization of biomolecule adsorption conformation.Adsorption and visualisation of bio-macromolecules on deuterated and natural isotopic abundance (non-deuterated) cellulose was studied by neutron reflectivity. Horseradish peroxidase (HRP) was used as a model bio-macromolecule to investigate the utility of NR in revealing adsorption conformation information for biomolecules adsorbed at the solid solution interface. HRP is an alpha-helical enzyme containing a heme group. It is considered a suitable model system because of its well-known structure, mechanistic understanding2O as a sub-phase. Comparing the NR curve of the DC film after HRP adsorption (red squares) to the original DC film (blue squares) – shows the appearance of pronounced fringes from the film formed by absorbed HRP (red squares). These fringes are attributable to the appearance of a layer with noticeable SLD difference from the DC film. For further analysis, the NR curve of the DC film at the D2O/film interface was fitted with a single layer model. An additional layer was included to fit the NR curve with the HRP adsorbed onto the DC film surface. In order to insure full or maximum surface coverage of HRP at the liquid-cellulose interface, HRP was adsorbed from solution and equilibrated onto DC film under conditions of excess HRP. 6H10O5) is typically about 1.7 × 10−6 Å−2. Here, for DC film in the presence of D2O, the resulting SLD is about 7.1 ×10−6 Å−2 which corresponds to the composition lying between C6HD9O5 and C6D10O5. These data provide strong evidence that the regenerated deuterated cellulose films in contact with the buffered D2O are highly deuterated (between 90–100%). Moreover, the thickness of DC film increased from 98 ± 10 Å in air to 280 ± 15 Å upon swelling in buffered D2O; this corresponds to a swelling factor of about three showing that cellulose at the liquid interface becomes a hydrogel.−6 Å−2. The measurements were performed in the presence of D2O and the results show an adsorbed volume fraction for HRP of about 20%. This suggests either that HRP does not adsorb as a full monolayer, or that the molecules are strongly hydrated and adopt an open conformation when adsorbed into cellulose. Given that the molecular function of HRP continues when the molecule is adsorbed on a cellulose substrate39Results from HRP adsorption onto the DC film reveals an HRP layer thickness of about 98 ± 10 Å with a SLD of 3.9 × 10et al.et al. and relying on the direct cellulose dissolution in DMAc/LiCl were also tested2O as the solvent for the media was fully dissolved into an ionic fluid, and then reacted to completion (degree of substitution (DS = 3) into trimethylsilyl cellulose. This derivative was readily soluble in toluene, which is an excellent solvent for spin coating. ATR-FTIR investigations  is replaced by heavy water (D2O), all of the hydroxy groups may exchange so that isotopic mixture reflects that of the subphase. 44Deuteration, achieved either by manipulating the contrast of water in sub-phase, or by manipulating the biosynthesis of the macromolecule, is useful to reveal the biomolecule adsorption conformation at the solid-liquid interface by using NR. When natural isotopic abundance water  methods may yield further insights into the types of interactions and molecular conformations involved. Bacterial biosynthesis of fully deuterated cellulose clearly opens up a many avenues for studying how cellulose interacts with biologically important molecules.Changing the H6H10O5) is 1.7 × 10−6 Å−2 and after exchange of the three labile hydroxy protons per anhydroglucose unit, the SLD 3.4 × 10−6 Å−2 (C6H7D3O5) in D2O. The SLD of fully bio-deuterated cellulose (C6D10O5) in equilibrium with D2O is 7.1 × 10−6 Å−2. 2O content. The labile protons on the hydroxy groups reflect the D2O content of the solvent. The average value of SLD for HRP was calculated based on the number density of atoms and 10% exchange using the program CRYSON2O content, there is only a small difference in SLD value between cellulose having varying H/D ratios (red line) and the corresponding ratios in HRP (green line). This hinders visualization of proteins adsorbed onto natural isotopic abundance cellulose surface. On increasing the deuteration level of cellulose (blue line), the SLD difference increases significantly and high contrast levels for proteins with respect to deuterated cellulose can be achieved. This contrast enables the precise measurement of the thickness of a very thin layer of proteins adsorbed on cellulose, such as the enzymes and antibodies used in paper diagnostics.The SLD of natural isotopic abundance cellulose  for spin coating: either acetylation or trimethylsilylation in ionic liquid (1-Butyl-3-methylimidazolium chloride). The trimethylsilylation of deuterated bacterial cellulose allows high yield of soluble product, and was used to prepare toluene solution for spin-coating onto silicon substrate. The resultant thin film was hydrolysed into deuterated cellulose in HCl vapour. Thin (100–200 Å) and smooth (RMS = 10–20 Å) cellulose films resulted. Adsorption of an enzyme, horseradish peroxidase (HRP) onto the surface of DC film surface could clearly be visualized and quantified by neutron reflectivity analysis. Data analysis showed the thickness of adsorbed HRP layer at the liquid-cellulose interface is 98 ± 10 Å, with roughness of 17 ± 5 Å and with a volume fraction of 20%; this corresponds to partial surface coverage of HRP aggregates. The method developed enables preparation of thin cellulose films with controllable deuteration levels. Control over the level of deuteration can provide a wide range of controllable scattering contrasts that greatly enhances the investigation of the interactions between cellulose and biologically important bio-macromolecules.In the present study, thin, smooth deuterated cellulose (DC) films have been prepared and these provide sufficient contrast using neutron reflectometry to visualize and quantify absorbed layers of horseradish peroxidase (HRP) as an example of an important class of bio-macromolecules. First, deuterated bacterial cellulose (DBC) was obtained by growing Gluconacetobacter xylinus strain ATCC 53524 in deuterated glycerol in Dr~29,000, acetyl groups substitution ~40%) were all purchased from Sigma-Aldrich. Polished silicon wafers  were purchased from El-Cat Inc. The wafers were cleaned by soaking in a mixture of ammonium hydroxide, hydrogen peroxide and water (volume ratio = 1:1:5) at 70 °C for 15 min, allowed to air dry in a dust-free environment and used as substrates for spin coating. A spin coater from Laurell Technologies Co., Pennsylvania, model WS-650HZB-23NPPB-UD-3 was used.1-Butyl-3-methylimidazolium chloride , hexamethyldisilazane , toluene , acetone , cellulose acetate  were obtained by the Fourier Transform Method in attenuated total reflectance geometry . The spectra were the result of the accumulation of 64 scans, at a resolution of 4 cmAFM measurements were made using a JPK Nanowizard 3 in AC (intermittent contact) mode. The piezoelectric scanner elements were monitored by capacitive sensors to ensure accurate reporting of height, z, and x-y lateral distances. Cantilevers used were Bruker NCHV model tapping mode levers, with nominal resonant frequencies of 340 kHz and spring constants of 20–80 N/m. Imaging was performed with a force set-point of <1 nN. In post-acquisition processing, images were ‘flattened’ to arrange the scan lines in a plane only by the subtraction of a linear equation from each scan line in the JPK image analysis software. Height images converted the lateral (z) scale acquired by the AFM z-piezoelectric drive capacitive feedback loop to a colour for ease of presentation.Neutron reflectometry (NR) measurements were performed at the PLATYPUS beamline of the Australian Nuclear Science and Technology Organization Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.How to cite this article: Su, J. et al. Smooth deuterated cellulose films for the visualisation of adsorbed bio-macromolecules. Sci. Rep. 6, 36119; doi: 10.1038/srep36119 (2016).


Drosophila melanogaster carrying either a functional or non-functional regulator of the JAK-STAT immune pathway (G9a) when challenged with a range of concentrations of Drosophila C virus (DCV). While classical linear model analyses indicated that G9a affected tolerance only in females, a more powerful nonlinear logistic model showed that G9a mediates viral tolerance to different extents in both sexes. This analysis also revealed that G9a acts by changing the sensitivity to increasing pathogen burdens, but does not reduce the ultimate severity of disease. These results indicate that fitting nonlinear models to host health–pathogen burden relationships may offer better and more detailed estimates of disease tolerance.The ability to tolerate infection is a key component of host defence and offers potential novel therapeutic approaches for infectious diseases. To yield successful targets for therapeutic intervention, it is important that the analytical tools employed to measure disease tolerance are able to capture distinct host responses to infection. Here, we show that commonly used methods that estimate tolerance as a linear relationship should be complemented with more flexible, nonlinear estimates of this relationship which may reveal variation in distinct components such as host vigour, sensitivity to increases in pathogen loads, and the severity of the infection. To illustrate this, we measured the survival of  IdentDrosophila infected with the bacterial pathogen Listeria monocytogenes, and this allowed to disentangle changes in fly health during infection that arose due to bacterial pathogenesis and host immunopathology [While the linear reaction norm approach is intuitive and has been useful in advancing the study of infection tolerance (reviewed in ), in somathology , which wDespite these analytical advances, many studies continue to infer tolerance phenotypes from separate measures of host health and pathogen burdens measured at a single infectious dose . Drosophila melanogaster, which has been used extensively as a model host to dissect the mechanisms underlying tolerance of bacterial and viral infections [Drosophila melanogaster infected systemically with Drosophila C Virus (DCV) develops pathology in the reproductive and digestive organs, severe abdominal swelling due to enlargement of the crop and eventually death [G9a, was previously identified as a mediator of tolerance to RNA virus infection in D. melanogaster [G9a showed higher mortality than those with a functional G9a, even though there was no difference in the viral loads of the two lines measured at a single time point. This work was notable in providing one of the first examples of immune-mediated tolerance of RNA virus infection, but because it focused on the functional basis of hypersensitivity to DCV, it was designed in a way that did not allow a comprehensive assessment of tolerance. First, infection tolerance was extrapolated from separate analyses of host mortality and viral loads, providing limited information on the role of G9a on host vigour, sensitivity to viral growth or the severity of infection that flies can withstand. Further, tolerance was only measured at a single viral dose, making it unclear if the observed tolerance phenotype is dose-specific. Finally, this work only assessed the tolerance phenotype of female flies challenged with a viral infection. Given the prevalence of sexual dimorphism in immunity [Using both linear and nonlinear analyses, here we examine the tolerance response to a systemic viral infection in fections ,17,19–21ly death –24. An eimmunity –27, and immunity ,29 it isDrosophila lines with identical genetic backgrounds, differing only in having a functional or non-functional G9a (G9a+/+ and −G9/−). We challenged these flies with a range of DCV doses and then quantified their tolerance responses using both the slope of linear reaction norm and a nonlinear sigmoid model. This allowed us to measure infection tolerance in the most comprehensive way, identifying which components of infection tolerance are affected by a single regulator of fly immunity (G9a), while also providing a useful comparison of current methodology to estimate components of infection tolerance.We employed systemic infections in both males and females of two 2.2.1.−/−G9a flies showed significantly higher mortality compared with +/+G9a, indicating that the protective effect of a functional G9a is no longer observed when flies were challenged with very high doses of DCV.Following infection with a range of doses of DCV, we found that overall 2.2.c,d, Overall, female flies achieved higher viral loads measured 5 days post-infection compared to males . Differences in tolerance between +/+G9a and −/−G9a are indicated by a significant interaction between the viral dose and the fly line for survival, which reflects that the rate at which survival changes with increasing viral doses (tolerance) varies between fly lines. We detected a significant interaction in females (b), where +/+G9a females showed higher tolerance (Slope: −1.2 ± 0.4) compared to −/−G9a females (Slope: −1.8 ± 0.3). In males, however, no significant difference in slopes was detected between +/+G9a and −/−G9a .Given that we found a significant positive relationship between DCV infection dose and the viral titre . We further used this model and equation (4.1) to extract parameters—‘c’ (inflection point: sensitivity) and ‘a’ (disease severity)  to increasing viral doses. The sensitivity to infection differed by 70-fold between +/+G9a and −/−G9a in females (p < 0.0001) while we detected a 10-fold difference in males (p < 0.0001) (figure 3e). We did not find any difference between +/+G9a and −/−G9a in the severity of infection (figure 3f).We then analysed the relationship between fly survival and viral dose using a nonlinear 4-parameter logistic model ,15,30. U pathway . A compaeverity) . In cont2.5.−/−G9a males and females in the sensitivity to increasing viral loads (e) prompted us to investigate if this may be due to sex-specific regulation of fly immunity by G9a. As G9a is known to be an epigenetic regulator of the JAK-STAT pathway, we measured the expression of JAK-STAT pathway genes in flies receiving the highest viral concentration, 5 days following DCV infection . We chose this dose since we detected high viral titres at 5 DPI, and were therefore more likely to detect changes in immune gene expression. We measured the expression of an extracellular ligand upd3 which is released upon stress or infection, its transmembrane receptor domeless, a suppressor of JAK-STAT pathway, i.e. socs36E, and a stress response element totA. Both socs36E and totA are downstream regulators of JAK-STAT pathway. Compared to flies with a functional G9a, −/−G9a mutants showed a significant increase in the expression of the JAK-STAT ligand upd3, and this effect was stronger in male flies , which is commonly expressed in response to stress.The differences between al loads e promptele flies . Males sa status . G9a−/− 3.Targeting mechanisms that promote greater tolerance of infection is a promising addition to our current arsenal of strategies to fight infection ,6–8. How+/+G9a females have higher tolerance than −/−G9a females, when measured across a range of DCV doses. However, the two models presented different results in the case of males. Additionally, we observed similar mortality between +/+G9a and −/−G9a at the highest dose, highlighting the importance of studying infection at a range of doses, as artificially high infectious doses can mask these protective effects.Both linear and nonlinear models consistently showed that Differences in infection tolerance between groups of hosts are commonly extrapolated from single infection doses and by performing independent analysis for host survival and pathogen burdens ,12,17. TIn this regard, using nonlinear sigmoid models offers greater flexibility to fit a range of possible relationships (including a linear approximation) between host health and pathogen loads . MoreoveWe chose to use viral dose instead of viral load as the measure of pathogen burden in our analysis of tolerance. This approach has the advantage that the covariate (dose) is measured without error, which is one of the assumptions of ANCOVA, and avoids common problems with underestimation of slopes in ANCOVAS when there is experimental variance in the covariate . Using dDespite all the stated advantages, one potential drawback of using nonlinear sigmoid models to study tolerance is that the slope of health decline is measured over a very small range of pathogen doses. Accurate estimates of that slope would therefore require many estimates of host health within a very narrow range of pathogen burdens, which may be experimentally challenging. For this reason, in the current analysis we fixed the slope to −1, which also increased the statistical power to estimate the two parameters of interest (sensitivity and severity). In this respect, we propose that using the linear reaction norm approach could be useful if the main variable of interest is the rate at which hosts lose health, which is estimated along the full range of pathogen burdens. Nonlinear approaches are more informative if the parameter of interest is the dose that causes the greatest shift in host health, or if the question relates to how hosts may differ in the ultimate severity of an infection.G9a-mediated tolerance of DCV in both male and female Drosophila. Our results support previous work using a single dose of DCV, which found that the lower survival of −/−G9a flies was not attributed to differences in their viral load [+/+G9a and −/−G9a males in their sensitivity to infection. Further analysis of the sensitivity and severity of infection parameters extracted from the nonlinear model also showed that the effect of G9a in mediating host sensitivity to DCV was greater in females. Our results therefore show that dysfunctional G9a accelerates the onset of infection-associated mortality (affecting sensitivity), without causing substantial effects on the disease severity ultimately experienced by infected flies. It is important to note that while previous work also found −/−G9a females were hypersensitive to DCV infection, this was only assessed by measuring survival at one dose and infection tolerance was not measured in male flies [A subsidiary interest of this work was to quantify ral load . Applyinle flies .While males and females are generally susceptible to the same pathogens, sexual dimorphism in immunity is present in a wide range of species ,34,35, aG9a-mediated effects on infection tolerance are not clear. G9a is a histone methyltransferase [G9a during viral infection has been previously shown to be driven by the regulation of the JAK-STAT pathway [G9a is known to alter the methylation state of the positive regulator domeless, the negative regulator socs36E and downstream pathway components  of the JAK-STAT pathway [G9a show increased mortality due to immunopathology caused by excessive expression of these downstream genes [G9a, domeless and its ligands upd1, upd2 and upd3 are all found on the X-chromosome in Drosophila [domeless , kindly provided by R. van Rij . −/−G9a was originally constructed by excision of P-element KG01242 from the 5′ UTR of the gene on the X-chromosome [G9a, referred to as +/+G9a. Both fly lines were maintained on standard Lewis Cornmeal medium under standard laboratory conditions at 25°C, 12 h : 12 h light : dark cycle.Experiments were conducted on romosome ,39. As a4.2.To set up the experiment, we collected eggs from 15 males and 15 females of each line, kept in vials containing 6 ml Lewis medium supplemented with dry yeast to encourage egg laying. We set up 10 replicate vials per genotype. Flies were left to oviposit in the vials for 24 h before being removed and allowing eggs to develop under standard rearing conditions. To control the larval density of these flies, egg density was maintained between 80–100 eggs per vial by removing excess eggs when required. Flies emerging from these vials were challenged with DCV and their mortality post-infection and viral load were measured.4.3.Drosophila C virus (DCV) is a ssRNA virus of the family Dicistroviridae. We obtained a viral stock by amplifying DCV in Drosophila S2 cells as described previously [−10. qRT-PCR was performed on an Applied Biosystems StepOnePlus system using Fast SYBR Green Master Mix (Applied Biosystems) and DCV primers containing 5′AT-rich flaps DCV_Forward: 5′AATAAATCATAAGCCACTGTGATTGATACAACAGAC 3′, DCV_Reverse: 5′ AATAAATCATAAGAAGCACGATACTTCTTCCAAACC 3′). The reaction conditions used were: 95°C for 2 min followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The first dilution where no viral cDNA was detected was taken as zero, and stock viral quantity was back-calculated from this reference point. Using this method, viral copy number in the stock was estimated to be around 109 DCV IU ml−1.eviously . Cell ho4.4.6, 107, 108 and 109 DCV IU ml−1—obtained by diluting the viral stock with 10 mM Tris-HCl (pH 7.3). Flies were infected systemically by intra-thoracic pricking with a needle immersed in DCV suspension under light CO2 anaesthesia. The effect of injury caused by pricking was controlled by including sham-infections performed using a needle dipped in sterile 10 mM Tris-HCl (pH 7.3).We exposed experimental flies to 5 viral concentrations—0 (control), 104.5.4.5.1.We systemically infected 3–4-day-old adults by intra-thoracic pricking with DCV. For each fly line × sex × dose combination, we infected 20 replicate individual flies . Following infection, flies were housed individually in a vial and flies were monitored daily for mortality. Flies were transferred onto fresh medium once a week. Survivorship was followed for 25 days post-infection and flies that were still alive at the end were censored in the analysis.4.5.2.rp49  and expressed as fold change relative to the control flies, calculated as 2−ΔΔCt [An additional five individuals for each genotype × sex × dose combination were infected as described above to quantify differences in viral growth following infection, using the expression of DCV RNA. Flies were individually placed in TRI reagent (Ambion) following five days of infection (5 DPI) and stored at −80°C. Total RNA was extracted from flies homogenized in Tri Reagent and exactly the same protocol was followed as described previously in §4.3 (Virus preparation and titration). The expression of DCV transcripts was normalized to transcript levels of the housekeeping gene s 2−ΔΔCt .4.5.3.G9a deletion, to test for genotype-specific and/or sex-specific differences upon infection. Given that G9a is a regulator of the JAK-STAT pathway, we focused on measuring the expression of the extracellular ligand upd3, its transmembrane receptor domeless, and downstream regulators socs36E, and totA. Gene expression was quantified in flies exposed to the highest dose treatment (109) relative to their expression in uninfected controls, as we expected the strongest effects on gene expression to be detected under elevated viral challenge.Using the same cDNA samples described above, we measured the expression of immune genes likely to be affected by a 4.6.10 viral titre measured at 5 DPI using ANCOVA and fitted ‘fly line’ and ‘sex’ as categorical fixed effects, ‘dose’ as a continuous covariate and their interactions as fixed effects. All the non-significant interactions were removed, and the minimal model was used to estimate correlation coefficient between viral load and viral dose.To analyse differences in survival of each host line according to the virus dose they were challenged with, we analysed post-infection survival data using a Cox proportional hazards model with ‘fly line’, ‘dose’ and ‘sex’ and their interactions, fitted as fixed effects. To assess differences in viral titres following infection with infection doses, we analysed the LogWe tested for differences in infection tolerance between fly lines in two ways. First, we used the classical approach and analysed tolerance as a linear reaction norm ,45,46. AAs a second approach to quantify infection tolerance, we fitted a nonlinear 4-parameter logistic model (equation (4.1)) which is commonly used to assess dose–response curves ,30,47 toa is the level of disease severity, b is a host's general vigour, c is the sensitivity to increases in pathogen dose , s is the slope of the logistic model, and x is the pathogen dose. Since the experiment was terminated 25 days post-infection, we constrained the upper limit of the model (host vigour) to 25 (DPI) .Using GraphPad Prism v. 6.0, we therefore fitted a 4-parameter logistic model described by the following equation:see also ). To tessee also . Using t


Local mRNA translation occurs in growing axons enabling precise control of the proteome in response to signals. To measure quantitatively the spatiotemporal dynamics of protein synthesis in growth cones, we further developed a technique for single molecule translation imaging (SMTI). We report that Netrin-1 triggers a burst of β-actin synthesis at multiple non-repetitive sites, particularly in the periphery. The response is remarkably rapid starting within 20 seconds of cue application. Given this complexity and the significant distance they travel, growth cones must possess a high degree of autonomy to react rapidly. Previous studies have provided evidence that local protein synthesis contributes to the fast and local responses in navigating axons9. Specifically β-actin, a key structural component of growth cones associated with their motility, was found to have its mRNA in axons10. β-actin translation in growth cones has been shown to occur within 5–10 minutes in response to an extrinsic cue, such as Netrin-1, and to polarise on the near-side of a gradient using immunocytochemistry and a live translation reporter9. This represents a potential shift in the central dogma of receptor-based signaling and investigating the precise spatiotemporal dynamics of local translation at the single molecule (SM) level could provide valuable insights. For example, it is not known precisely where in the growth cone new proteins are synthesised, how fast synthesis occurs in response to a cue and whether it occurs repetitively in the same spot or singly in diverse sites indicative of polysomal versus monosomal translation respectively.During neurodevelopment, the growth cone at the tip of a growing axon navigates to its remote target by sensing extracellular cues and responding to these guidance cues by turning, advancing, or pausing in a timely manner5 does not allow for the translation events to be probed at the SM (protein) level. Recently, a number of techniques have been reported allowing spatiotemporal protein translation measurements with single-molecule resolution. Fluorescence amplification via multi-epitope tagging with SunTag proteins13 and site-specific dyes14 offer dynamic readout to visualize translation. Alternatively, a direct readout is also possible using single molecule translation imaging, SMTI16. Here, we describe further developments of the SMTI approach through protocol optimization and analytical methods and demonstrate it to be a powerful tool for the quantification of translation dynamics in retinal ganglion cell (RGC) growth cones  Venus17 is fused to the protein of interest, β-actin, flanked with the 3′ and 5′ untranslated regions (UTR) of β-actin. For our experiment, the DNA of the Venus-β-actin construct is introduced into Xenopus RGC neurons  and analysis tools capable of exploiting the rich content of SMTI data. We note that the time at which the stimulation was observed (increase in event rate) is dependent on both the diffusion time of Netrin-1 in the solution and the variance in folding times of Venus. Better FPs featuring shorter folding and higher quantum yield may lead to improvements in the method’s temporal resolution and detection sensitivity. The ability to quantitatively measure local translation rates in space and time at the single molecule level in delicate cellular systems such as axonal growth cones paves the way to understanding signal-driven subcellular responses.Xenopus laevis embryos were obtained via in vitro fertilization and raised according to Leung et al.24 at 14–22 °C in 0.1x modified Barth’s saline 2, 41 μM CaCl2). All animal experiments were carried out with approval of the University of Cambridge Ethical Review Committee according to the University of Cambridge Animal Welfare Policy.Total mRNA extracted from stage 32 embryos using RNeasy Mini Kit (QIAGEN) was reverse-transcribed into a cDNA library with SuperScript III First-Strand Synthesis System (Thermo Scientific) using Oligo(dT) as primer. To obtain the Venus-β-actin fusion construct, full-length β-actin including the 5′UTR, the coding sequence and the 3′UTR obtained from the cDNA library by PCR was cloned into a pCS2+ vector with constitutive CMV promoter. The monomeric Venus coding sequence was inserted between β-actin 5′UTR and the coding sequence, followed by a short linker (KLEFK).25. Eye primordial of stage 26 embryos were injected with DNA followed by electric pulses of 50 ms duration at 1000 ms intervals, delivered at 18 V. The electroporated embryos then recovered in 0.1X MBS.Targeted eye electroporation was performed as previously describedGlass bottom dishes (MatTek) were pre-treated with 5 M KOH (Sigma) for 1 hour, followed by 5 rinses with deionized water (Sigma). Eye primodia from stage 32–35 embryos were dissected and cultured in 60% L-15 at 20 °C for 18 hours  on KOH-cleaned dishes coated with 10 μg/ml poly-L-lysine and 10 μg/ml laminin .2. Transfection was performed with jetPRIME (Polyplus transfection) following the manufacturer recommendations.HEK293T cells were maintained in DMEM medium (ThermoFisher Scientific) supplemented with 10% FBS (ThermoFisher Scientific) in a 37 °C humidified incubator containing 5% COImaging was performed on a custom-made inverted single-molecule fluorescence microscope built around a commercial microscope frame (Olympus IX73). The illumination laser wavelength was at 488 nm (Coherent Sapphire) for excitation of the YFP derivate Venus in combination with a 525/45 emission filter (Semrock) and a dichroic beam splitter (Chroma ZT405/488/561/640rpc). The laser beam was circularly polarized to excite fluorescent proteins homogeneously regardless of their orientation. The microscope was equipped with an EM-CCD camera (Andor iXon Ultra 897) with effective pixel size on the sample of 118 nm. A 100x NA = 1.49 oil immersion TIRF objective (Olympus UAPON100XOTIRF) was used.2 under episcopic illumination and Venus SMTI recorded with an exposure time of 10 ms for 25 s under TIRF illumination. We used the same intensity of 1 kW/cm2 for SMTI imaging as for bleaching due to the much higher translation rate in HEK293T cells near the nucleus. Imaging was performed with an additional EM gain of 200 to ensure single molecule sensitivity  for 30 minutes, or left untreated (control). The cells were then photobleached for 5 s with an irradiance of 1 kW/cm26 and, in the negative control case, also pre-incubated with 275 μM translation inhibitor puromycin (Sigma). An outgrowing fluorescent growth cone was then selected and, prior to the bleaching step, imaged with low irradiance (<2 W/cm2) in both fluorescence and bright field mode to generate an outline image. The growth cone was then photobleached for 10 s with an irradiance of 1.5 kW/cm2. Afterwards, the flash-like recovery of Venus fluorescence was recorded with an exposure time of 200 ms for 60 s to generate a basal baseline. We used a reduced intensity of 0.3 kW/cm2 to ensure survival of the axons 27. A threshold of ~6700 ADC , corresponding to ~500 photons per localization, was applied to filter out noise and non-Venus blinking events. This threshold was found by manual selection of Venus flashes and determination of the “average” photon budget of a single emitting Venus molecule (Supplementary Fig. 2. Note that this area normalization step was not performed in the primary retinal culture assay as the objective’s depth of field was large enough to capture events from almost every axial position in the growth cones. The developed code for SMTI data analysis is freely available online under https://github.com/laseranalyticsgroup/SMTI.Localizations of individual protein translation events were retrieved using maximum likelihood estimation with a Gaussian model fit via the software package rapidSTORMData are available upon request from the corresponding authors.Supplementary informationSupplementary Video S5Supplementary Video S6Supplementary Video S7


In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation. Among the many choices that are available, graphene-based materials are emerging as a promising candidate. The culture of neural stem cells (NSCs) derived from brain tissue  on graphene substrates has been reported. These studies demonstrate that graphene is biocompatible for the growth of NSCs, providing excellent attachment properties that then confer enhanced neuronal differentiation and maturation towards neural networks –8. In adThe cerebral cortex is mostly composed of glutamatergic excitatory and GABAergic inhibitory neurons both of which are critical in the development and regulation of neural networks . For thiA recent advance in the field of graphene scaffolds is the fabrication of graphene foam . Graphen2.2.1.d-(+)-glucose, Sigma-Aldrich, Australia), GlutaMAX™-1 (Gibco/Life Technologies), phosphate-buffered saline solution (PBS)  made according to the manufacturer's instructions, poly-d-lysine hydrobromide  reconstituted in double distilled water at 1 mg ml−1 and stored at 20°C, laminin supplied reconstituted at 1 mg ml−1 Tris–HCl , NaCl (0.15 M) and stored at 20°C (Invitrogen/Life Technologies), ITS solution , N-2 supplement (Gibco/Life Technologies), B27 retinol-free supplement (Gibco/Life Technologies) and Pen/Strep solution .Growth factors: bFGF  and EGF , DMEM/F-12 medium (1 : 1) and Neural Basal medium (Gibco/Life Technologies), glucose at 30% (w/v) in distilled water (ThermoFisher Scientific) and stored at 4°C (2.2.2. The coating of plates and medium preparation were according to the protocols provided by the manufacturer (STEMCELL Technologies). Every 7 days, colonies were mechanically dissected and transferred to freshly coated plates. Cell culture media were replenished daily.The H9 cell line  was cultured using organ culture dishes  coated with vitronectin  using mTeSR™1 defined medium (STEMCELL Technologies) and maintained at 37°C and 5% CO2.3.d-lysine solution (10 µg ml−1 in PBS) and kept at room temperature for 30 min. This solution was aspirated and this was followed by the addition of laminin solution (10 µg ml−1 in PBS) and incubated overnight at 4°C. The next day, the laminin solution was removed and replaced with neural induction culture medium, termed N2B27. This medium consisted of a 1 : 1 mixture of neurobasal medium with DMEM/F12 medium supplemented with ITS 1%, N-2 1%, B27 2%, glucose 0.3%, GlutaMAX 1% and Pen/Strep 50 U ml−1/50 µg ml−1 for 14 days. For differentiation towards glutamatergic neurons, cultures were maintained in N2B27 supplemented with the small-molecule inhibitor SB431542  and noggin  for 7 days followed by N2B27 supplemented bFGF (20 ng ml−1) for 7 days. For differentiation towards GABAergic neurons, the small-molecule smoothened agonist  was also supplemented in the media together with SB431542 and noggin during the first week of neural induction, followed by the addition of bFGF (20 ng ml−1) a further 7 days  and Chambers et al. ,26 was ecultures b. We concultures d.Figurea,b) already known to be suitable for the culture of rodent-derived neurons [b). The van der Pauw method was used to measure the conductivity of the foams by taking the ratio between the electrical current (I) and voltage (V) [–1 and the conductivity was evaluated to be 0.5 S cm–1. The EDX spectroscopy of the graphene foam has also been performed as an additional characterization tool to confirm the phase purity. In the EDX spectrum, the C-Kα line is clearly seen and no other spurious element is observed as shown in figure 2c. Furthermore, Raman spectroscopy was carried out under excitation laser (λ = 532 nm) on the graphene foam to provide complementary knowledge on chemical structure. Raman spectrum of graphene foam (figure 2d) reveals that the graphene material exhibits well-known G and 2D peaks approximately at 1581 and 2710 cm−1, which is consistent with the literature [Graphene foam is a highly porous, conductive structure a,b alrea neurons . The portage (V) . The sheterature –32. The a; solid red arrows), although we did not observe a capacity to grow and extend neurites across the pores of the scaffold. Glutamatergic neurons were additionally imaged using HIM imaging which provides superior contrast and resolution because of its increased depth of field (figure 3b). HIM imaging confirms that the neurons were capable of attaching to the scaffold and could extend complex neurite outgrowths along the scaffold. To confirm this, samples were examined using immunofluorescence staining with the neuronal marker, tubulinβ3, followed by confocal imaging . Both images show a similar pattern of attachment as observed using SEM and HIM.To assess the biocompatibility of graphene foams, neurospheres derived from either the glutamatergic or GABAergic neural induction protocols were mechanically disaggregated and cultured onto laminin-coated graphene foam for 21 days. Following this culture period, graphene foam scaffolds with either glutamatergic or GABAergic-fated neurons were subjected to SEM, HIM and immunofluorescence imaging  and were also capable of extending complex neurite outgrowth processes across the pores of the scaffold . Additionally, in contrast to glutamatergic neurons, GABAergic neurons were capable of forming 3D clusters within the porous cavities of the lattice (figure 4b). To confirm this, samples were also prepared for immunofluorescence analysis by staining with two cytoskeletal neuronal markers, tubulinβ3 and MAP2AB. Samples were assessed by confocal imaging and both images showed a similar pattern of attachment to that observed using SEM .SEM imaging of GABAergic-fated neurons showed that like glutamatergic neurons, GABAergic neurons also engraft onto the scaffold; however, we observed some differences in attachment . Like thfigure 43.2.a–d). To achieve this, two-week-old neurospheres derived from glutamatergic and GABAergic neural inductions were gently dissociated and plated either as a monolayer onto laminin-coated dishes (2D) or onto a laminin-coated graphene foam. Following three weeks in culture, the biocompatibility characteristics of neurons cultured in 2D and in the foam were compared using Q-RT-PCR analysis. Cell viability was examined by assessing the expression levels of Ki67 (a marker of proliferation) and Caspase-3 (a marker of cell apoptosis) . Neuronal maturation was examined by assessing the expression of tubulinβ3 (TUBB3) and MAP2AB (Map2) .Having established capacity to engraft onto the scaffold, next we assessed biocompatibility by comparing the viability and neuronal maturation of cells cultured as a 2D monolayer with cells cultured on the scaffold a–d. To aoptosis) a,c. NeurB (Map2) b,d.Figua). The analysis of GABAergic neurons revealed a slightly different pattern of expression, in that expression levels of Ki67 were lower when cultured on the foam (c). This was not matched by increased expression of Caspase-3, which was equivalent across both the 2D and foam samples. We propose that the lower expression of Ki67 may indicate progression towards post-mitotic and more mature neuronal populations. Finally, we examined expression levels of tubulinβ3 and MAP2AB . Analysis of these markers in 2D monolayer and graphene foam cultures did not reveal any marked differences in expression levels of these neuronal markers. This suggests that, for the 21-day culture period, graphene is equally supportive of neuronal differentiation as the 2D cell culture model for both the glutamatergic and GABAergic-fated neurons .Cell viability results obtained for glutamatergic neurons showed comparable expression levels of Ki67 and Caspase-3 between the 2D (monolayer) and the graphene foam culture conditions. This demonstrates that graphene foam supports the culture of glutamatergic cortical neurons equally as well as the 2D monolayer a. The anthe foam c. This wd MAP2AB b,d. Anal neurons b,d.4.The culture of NSCs on graphene has shown that this scaffold can improve neuronal maturation and enhance functionality and circuit formation ,9. In adIn this study, we investigated the use of graphene foam as a potential basis for culturing hESC-derived cortical neuronal cells, to date not reported. To achieve this aim, we adopted a well-characterized method protocol that directs hESC neural specification towards tissue that is enriched for forebrain neuronal populations . This prThe key features of graphene foam that facilitate such long-term cell culture are its porosity, which facilitates the efficient exchange of nutrients and high surface/volume ratio that provides the landscape. A major objective in the study of neuronal homeostasis is to advance our understanding of the mechanism underlying neuronal differentiation, maturation, functionality and underlying electrical connectivity. The ability to conduct long-term culture studies would allow human-derived cortical neurons to differentiate over the extended periods that are required to reach a more mature differentiation status. This will be crucial for functional studies and the analysis of neural network formation, which are known to establish over lengthy periods of time (greater than 100 days) ,33,34. A5.If our understanding of brain development and neurological disorders is to advance, the need to accurately model neuronal maturation and functionality is a priority. In this study, we demonstrate that it is possible to culture hESC-derived neurons on graphene foam. Our study shows that hESC-derived neurons can engraft onto the scaffold, showing minimal toxic effect for the first 21 days of cell culture, and is supportive of neuronal differentiation. The conductive nature of graphene foam means such a scaffold could be used to study neuronal connectivity and functionality. Our data provide the rationale for moving graphene foam forwards as a substrate for such studies. Additionally, it may be possible to establish hESC- and graphene-based organotypic models to study brain development that could represent better translational models for neuroscience discovery.