Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner–Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used  The ability of diazotrophs to use dinitrogen gas as the sole nitrogen source confers many ecological merits, but also incurs physiological constraints resulting from the high energy requirements of biological nitrogen fixation. The function of the nitrogenase enzyme requires not only reducing equivalents but also the supply of a minimum of 16 ATP per N2 fixed. Substantial energetic equivalents require a coordinated central carbon metabolism which uses a series of enzymatic pathways (i.e. the Embden-Meyerhof-Parnas (EMP) pathway, the Entner-Doudoroff (ED) pathway, pentose phosphate pathway, tricarboxylic acid (TCA) cycle etc.) to convert organic carbohydrates into metabolic precursors and release electrons and ATP. However, to date, there is little understanding of how diazotrophic bacteria quantitatively tailor central carbon catabolism to nitrogen availability and vice versa.Reduced forms of nitrogen are required by every living organism and are used to make many important biological components including nucleic acids, proteins, pigments, energy carrier molecules, etc. In nature, a diverse group of bacteria known as diazotrophs have a remarkable capacity to fix atmospheric nitrogen to ammonium under ambient conditions. This capacity is catalyzed by the nitrogenase metalloenzyme complexed with accessory proteins for proper cofactor biosynthesis and can be exploited for biological fertilizer production. This process is accomplished on an industrial scale by the chemical process (Haber-Bosch) that requires elevated temperature, high pressure and special catalysts and is considered an energy intensive and major greenhouse gas emitting approache.g. heterocysts in nitrogen-fixing filamentous cyanobacteria). Alternatively, the non-compartmentalized diazotroph, Azotobacter vinelandii, is believed to have a different protection mechanism4 that exhibits high respiratory activity, especially when exposed to high oxygen concentrations. In addition, in environments where a high rate of respiration does not prevail (e.g. A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture), cells produce extracellular alginate, a viscous polysaccharide that leads to the formation of a capsule surrounding the cell that prevents oxygen diffusion into the cell5. In order to adapt to different environmental conditions while accommodating nitrogenase activity and ensuring ammonium availability, intracellular carbon flux may need to be directed toward either respiratory metabolism or alginate biosynthesis.In addition to its substantial energy cost, biological nitrogen fixation is also highly oxygen sensitive. To protect the nitrogenase complex from damage, some aerobic diazotrophs have evolved the ability to form specialized cells that create a microanaerobic environment for nitrogen fixation to occur  that exhibits constant expression of nitrogenase, and thus increased ammonium production and excretion into the surrounding media8. However, this mutant exhibits metabolic mechanisms of nutrient balancing that could favor biomass production and may thus oppose the metabolic engineer’s goal of maximizing ammonia yield. Therefore, understanding how A. vinelandii coordinates the utilization of carbon with the rate of nitrogen fixation is critical for the successful reprogramming of this microbe’s metabolism. Therefore, we aim here to understand how ammonium excretion is adding a metabolic energetic burden to the cells’ metabolism. Using 13C-metabolic flux analysis, by coupling chromatography to mass spectrometry we are able to quantify the isotopomer patterns of metabolites throughout the metabolic network. This method has emerged as a powerful tool for generating the quantitative data needed to probe and model central carbon metabolism and is uniquely applicable to provide novel bioenergetic insights into carbon and nitrogen metabolism12.A. vinelandii DJ wild type (wt) and its ammonium-excreting strain, AV3 (ΔNifL)8. To our knowledge, this is the first study to quantify the relationship between central carbon and nitrogen metabolism in this model aerobic diazotroph.The primary goal of this work is to understand quantitative carbon catabolism tailored to nitrogen availability and in particular in relation to mutations that yield high levels of nitrogen excretion. In this work, we present a quantitative fluxome analysis showing how central carbon metabolism is reprogramed in relation to nitrogen perturbation in diazotroph A. vinelandii wild type (DJ) and the ammonium excreting strain (AV3) with and without 50 mM ammonium (NH4Cl) added to the culture medium. The kinetics of cell growth, glucose consumption and ammonium levels in the media are shown in Fig. To investigate the influence of ammonium level on central carbon metabolism under different nitrogenase activity and environmental availability, we grew −1 h−1, respectively). Interestingly, in linear and stationary growth phase the AV3 strain seemed to re-assimilate the ammonium it produced earlier, indicating that nitrogenase expression might be down-regulated by other unknown mechanisms, or the ammonium was consumed by spontaneous mutants (Nif−) that were unable to fix nitrogen.In cultures grown without added ammonium Fig. , both thA. vinelandii is forced to fix nitrogen (in diazotrophic growth) due to the unavailability of ammonium, biomass formation and growth rate are significantly impeded. In addition, further increase in nitrogenase activity in the case of AV3, impedes the cells’ growth rate even further.For cultures grown with 50 mM added ammonium Fig. , AV3 mai13C-metabolic flux analysis (MFA). We developed 13C-MFA using isotope tracer and GC-MS to resolve the primary pathways in the carbon metabolism of A. vinelandii. For this purpose, we first validated growth on a minimal medium, modified DSMZ Azotobacter medium, to eliminate potential interference with labeled amino acids from non-labeled amino acids present in complex carbon nutrients  that can be incorporated by the cells. Second, we found the fractional labeling (FL) of the GC-MS-analyzed proteinogenic amino acids to be generally consistent with that of the isotopic substrates when sampled from exponentially grown cultures  pathway. Although the complete gene sets for these pathways are found in A. vinelandii’s genome14, their metabolic contribution to glycolysis is not fully understood. Quantitative estimations of flux ratios through these pathways were based on the labeling patterns of serine and alanine as defined by Mass Distribution Vectors (MDV)15. If serine originates through the EMP pathway, half of the serine will be labeled at position C3 while the other half will be unlabeled , strongly supporting that the ED pathway is the dominant pathway for glycolysis. Interestingly, we found the ED pathway flux corelates with the specific growth rate as the fastest growing cells carried the strongest ED pathway, and vice versa  proportionally by PEP carboxylase as an amphibolic reaction. In comparison, the backward fluxes such as “PEP from OAA”, “Pyr from Mal (lb and ub)” are negligible.We further focused on the respiratory flux from the TCA cycle. This flux can be reflected by the proportion of oxaloacetate (OAA) synthesized from either the TCA cycle or amphibolic pathway. The increased fraction of OAA derived through the TCA cycle in the AV3 culture without added ammonium indicates significantly increased flux through the respiratory TCA cycle. This data aligns with the notion that enhanced nitrogenase activity requires highly efficient respiratory metabolism to produce additional ATP. Interestingly, we also found that the relative respiratory activity of the TCA cycle increased as the growth rate decreased in cultures for different ammonium scenarios, with the most extreme negative relation in the case of diazotrophic growth of the AV3 mutant . The biochemical reactions and biomass equation adopted in our model come from Andres’s work16 and are listed in Supplemental Table A. vinelandii genome but showed marginal activity according to flux ratio analysis as presented in Fig. A. vinelandii.The estimated fluxes from the model fit are presented in Supplemental Table The global fluxomics datasets enable us to estimate the bioenergy and redox production from the stoichiometry of the metabolic network Fig. . We thus13C-tracer studies, we developed a fluxomic model to formulate the quantitative relationship between central carbon fluxes and nitrogen metabolism in A. vinelandii and elucidated the integrated responses of metabolic networks to nitrogen perturbation. Using this fluxomic model framework we were able to probe the bioenergetic basis of a diazotrophic phenotype in the ammonium-excreting AV3 mutant, which allows further refinement of the model to guide strategies toward metabolic engineering goals.Using A. vinelandii, which carries complete gene sets for two critical glycolytic options, the EMP and ED pathways. This work shows that A. vinelandii predominantly utilizes the ED pathway, while the EMP pathway has very low activity. Similar glycolytic flux partitioning is also observed in Pseudomonas18, a genus phylogenetically close to Azotobacter, in which the ED pathway accounts for over 90% of utilized glucose20. These two glycolytic pathways vary in reaction schemes and in how much ATP they produce for each glucose molecule metabolized21. Given the substantial energetic requirements for diazotrophic growth, why A. vinelandii prioritizes the ED pathway for glycolysis represents an intriguing open question. Compared with the EMP pathway, the most energetically efficient glycolytic pathway, the ED pathway oxidizes phosphorylated glucose to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is cleaved into one pyruvate and one glyceraldehyde-3-phosphate (G3P). Only the latter product supports substrate-level phosphorylation and can be used to produce ATP through subsequent glycolysis. Despite reduced ATP production, the ED pathway contains more exergonic reactions22 and is less thermodynamically constrained. This feature leads to tremendous benefit in enzyme kinetics as a stronger thermodynamic driving force will result in forward reactions being favored, which accordingly requires reduced amounts of catalytic enzymes to generate identical net flux. Indeed, the ED pathway could reduce by several-fold the enzymatic protein synthesis burden but still achieve the same glucose conversion rate as the EMP pathway21. It should be noted that in diazotrophic growth, the cost for protein synthesis can be considerable as it is largely based on the energetically expensive nitrogen-fixing process. From this perspective, natural selection of the ED pathway represents an energy balance consideration geared towards higher conversion rates of carbon substrates per N2 fixed and constrained by protein synthesis demands. The trade-off of this strategy is the decreased production of ATP from glycolysis, which must be replenished through another oxidative pathway as discussed below. Interestingly, our data also showed that the flux ratio of the ED pathway is positively correlated with the cell growth rate constraints by either ammonium availability or nitrogenase activity. The biological mechanism behind this correlation is still unknown. One hypothesis is that the activities of the ED pathway enzymes could be regulated by altered intracellular ammonium levels regardless of transcriptomic, proteomic, or metabolomic levels. Further research in systems biology is required to address this question comprehensively.Our study sheds light on the glycolytic strategy used by diazotroph 13C-fluxomic study also provides new evidence to better understand the role of respiratory metabolism in diazotrophs. Respiratory metabolism was proposed to protect the active nitrogenase against oxygen because the respiratory electron transport system bound to the peripheral cytoplasmic membrane can scavenge O2 and prevent the diffusion of O2 into the cells. This mechanism is believed to explain how nitrogenase can function when cells grow diazotrophically in the presence of O2. Although the respiration-protection hypothesis is widely accepted, it was recently challenged when it was reported that A. vinelandii had almost constant respiration rates and nitrogenase activity at O2 concentrations ranging from 30 to 100% air saturation24. These results hardly support the concept of respiratory protection. As a complementary hypothesis, Oelze suggested that nitrogenase protection also depends on the maintenance of a sufficiently low redox state25. This hypothesis assumes that oxidation of the substrate can warrant the proper supply of reducing equivalents and ATP to maintain nitrogenase in a reduced state. Indeed, our data strongly supports this hypothesis in that the increased fluxes through respiratory TCA cycle and OPP pathway positively correlated with enhanced nitrogenase activity. As shown in Fig. 2 fixation activity. It also supports the notion that respiratory protection arises from stringently coupled respiratory electron transport with the TCA metabolism. Although the way in which diazotrophs increase TCA cycle flux in response to enhanced nitrogenase expression is still a mystery, knowledge of this correlation could lead to lead to new strategies to maximize nitrogenase activity for engineering increased ammonium release by alleviating apparent metabolic bottlenecks. One solution could be the use of alternative carbon substrates (e.g. citrate and acetate) that can increase the activity of the TCA cycle locally, and accordingly increase the supply of energy and reducing equivalents. Previous studies on diazotrophically growing A. vinelandii showed that growth rates obtained on acetate achieved 0.35 h−1, higher than those grown on glucose (0.15 h−1)26. Therefore, addition of direct precursors of TCA cycle will not only supply ATP and reducing power for the highly active nitrogen fixation in the AV3 mutant, but also compensate for the carbon loss in TCA cycle which promises an enhanced biomass accumulation. It is noteworthy that the AV3 mutant showed a higher metabolic flux through the OPP pathway than wt in different ammonium availabilities which also supports the hypothesis in that extra reducing equivalents produced from an enhanced OPP pathway could be used to stabilize the enhanced nitrogenase activity in an AV3 mutant background. Overall, by deciphering the glucose metabolism of A. vinelandii, this work exemplifies novel insights into a general strategy that diazotrophs adopt for oxidizing carbon substrates under different conditions. It is our expectation that this quantitative knowledge of central carbon metabolism in terms of environmentally or genetically altered N-status can guide the reprogramming of diazotrophic metabolism geared toward increased technical feasibility of biologically produced ammonium fertilizers for industrial applications.In addition, our Azotobacter vinelandii DJ (wt) and AV3 (ΔNifL) strains were kindly provided by Dr. Leonardo Curatti27 and were maintained on 1.5% agar modified DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) Azotobacter medium plates without added nitrogen. Our modified version of DSMZ Azotobacter medium consisted of 0.9 g L−1 K2HPO4, 0.1 g L−1 KH2PO4, 0.1 g L−1 MgSO4·7H2O, 0.1 g L−1 CaCl2·2H2O, 5 mg L−1 Na2MoO4·2H2O, 20 mg L−1 FeSO4·7H2O, 20 g L−1 glucose, 50 mM MOPS buffer, and was adjusted with HCl to a starting pH of 7.6. For cultures grown with added ammonium, NH4Cl was added to a final concentration of 50 mM prior to adjusting the pH. The MOPS buffer was included to prevent a drop in pH that truncated cell growth in cultures with added ammonium as has been previously reported28. Media was filter sterilized, and cultures were grown in unbaffled shake flasks at 30 °C and 225 rpm. Larger cultures of 250 mL in 1 L shake flasks were grown in triplicate to measure multiple metabolites during cell growth. U-13C and 1-13C glucose  labeled cultures consisted of 12.5 mL of culture in 50 mL shake flasks and were grown in duplicate. Cell growth was monitored by measuring optical density at 600 nm using a spectrophotometer . Dry cell weight measurements were determined by centrifugation of a known volume of culture and subsequent lyophilization and recording of the pellet weight.29. Culture supernatants were filtered through 0.22 µm filters before being loaded on an Agilent 1100 series HPLC equipped with a refractive index detector (RID)  and an Aminex HPX-87H anion-exchange . The mobile phase consisted of 4 mM H2SO4 at a constant flow rate of 0.6 ml min−1. The column temperature was maintained at 55 °C and the optical unit temperature for RID was held at 45 °C.Glucose was analyzed using a method slightly modified from literature30. After incubating with reagent1 and reagent2 at 37 °C for 30 min, samples were analyzed with a microplate reader .Ammonium was quantified using an assay kit  based on a modified version of the Berthelot method31. Freeze-dried cells (about 3 mg) were weighed and added to 500 μL butanolysis solution (consisting of 90% (w/v) 1-butanol, 10% concentrated HCl and 2 mg mL−1 Benzoic acid as internal standard). The samples were incubated at 110 °C for 3 hrs followed by the addition of 750 μL Milli-Q water and vigorous voretxing for phase separation. An Agilent 7890B GC equipped with an Agilent HP-5 GC column was used to quantify the butyl ester of 3-hydroxybutyrate in the upper organic phase. Samples were analyzed at split ratio of 1:20 and overall flow rate of 1 mL min−1 with the following temperature profile: 90 °C, hold for 2 min; linear temperature gradient of 30 °C min−1 from 90 to 300 °C; 250 °C, hold for 2 min.Intracellular PHB was quantified using a modified esterification method+Cl−) method32. Briefly, culture supernatants were added to a PHMBH+Cl− solution (purchased from BOC Sciences) (0.3% (w/v) in 1% sodium acetate) at volume ratio of 1:2 to form an alginate-PHMBH+ precipitate. After centrifugation at 17000 g for 5 min, absorbance of the supernatant with residual PHMBH+Cl− was measured at 235 nm using NanoDrop One .Alginate was determined according to a polyhexamethylenebiguanidinium chloride (PHMBH15. The cells (OD600 * culture volume ≥5) were harvested at three time points  by centrifugation at 13000 g for 5 min. The specific growth rates (µ) calculated at these time points and used for the subsequent metabolic flux analysis was as follows: 0.022 ± 0.009, 0.016 ± 0.009, 0.043 ± 0.011, 0.025 ± 0.007 for wt and AV3 without added ammonium, and wt and AV3 with added ammonium, respectively. Cell pellets were washed with Milli-Q water and resuspended in 500 uL of 6 M HCl followed by incubation at 105 °C for 16 hrs. The hydrolysate was evaporated down under nitrogen gas flow at 40 °C, which can either be stored at −20 °C or used immediately for derivatization. Derivatization was carried out at 85 °C for 1 hr in a solution containing 80 uL pyridine and 20 uL N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide. The derivatized sample was analyzed by GC-MS. The GC-MS parameters used were the same as previously described33.Sample preparation for GC-MS measurement of labeled amino acids followed the standard protocol15:i represents the fraction of the mass isotopomer with i carbon atoms labeled, and n is the number of carbons in the fragment. MDVAA was then corrected for natural abundance of non-carbon atoms in fragment and carbon atom in the derivatization reagent. To prove that the cells were in a metabolic steady state, fractional labeling (FL) was calculated for U-13C glucose fed cells as15:Calculation of metabolic flux ratios was based on mass isotopomer distribution vector (MDV) of amino acid fragments asM) can be further estimated via a least square method due to the convolution reaction of amino acid biosynthesis15, and flux ratios at metabolic nodes were calculated as least square solutions. In this study, flux ratios of serine from glycine, glycine from serine, pyruvate from malate (upper and lower bound), erythrose-4-phosphate from pentose-5-phosphate, oxaloacetate from the glyoxylate shunt, oxaloacetate from the TCA cycle, phosphoenolpyruvate from oxaloacetate, phosphoenolpyruvate from the PP pathway and phosphoenolpyruvate from transketolase were calculated according to the principles proposed by Fischer et al.34. Additionally, the flux ratios of serine from glycolysis, and pyruvate from the ED pathway were obtained using 1-13C glucose. If all G3P molecules originate through the EMP pathway, half of the resulting serine will be unlabeled and the other half will be labeled at position C3, while all serine molecules will be unlabeled if carbon flows through the ED pathway or the OPP pathway (Supplemental Fig. Mass isotopomer distribution of the precursors of amino acids (MDVSimilarly, if all pyruvate originates from the ED pathway, alanine will be 50% unlabeled and 50% labeled at position C1 (Supplemental Fig. All calculations were implemented with Python3.6.A. vinelandii was reconstructed according to García et al.16 and provided in Supplemental Table 35. Determined MDVs and measured growth parameters were used as inputs for the following least-squares minimization problem36:sim and vsim are simulated mass isotopomer distribution and flux as a function of u, while MDVexp and vexp are experimental data; ∑exp is square matrix with measurement variances of MDV elements on the diagonal; varexp is the variance of determined reaction rate; N is the null space of stoichiometry matrix of mass balance. The problem can be solved through a gradient-based optimization algorithm. For flux estimation, the reaction rate of alginate formation was set to zero since alginate was not detected in any conditions. The flux between malate and pyruvate was also set to zero because the measured flux ratio of pyruvate from malate is negligible. The optimization was repeated 10 times for each condition to obtain a global solution, and 95% confidence intervals were calculated using a local linearization method, which is symmetric37. This method decreases computational burden compared to parameter continuation or Monte Carlo methods. All computation was implemented with Python3.6.Primary metabolic network of Fluxomics analysis of Azotobacter vinelandii supplemental information


Psammechinus miliaris and evaluates the transcriptional contribution to larval resilience. RNA-Seq was used to determine how the expression profiles of larvae spawned into low pH from pre-acclimated adults differed to those of larvae produced from adults cultured under ambient pH. The main findings demonstrated that adult conditioning to low pH critically pre-loads the embryonic transcriptional pool with antioxidants to prepare the larvae for the “new” conditions. In addition, the classic cellular stress response, measured via the production of heat shock proteins (the heat shock response (HSR)), was separately evaluated. None of the early stage larvae either spawned in low pH (produced from both ambient and pre-acclimated adults) or subjected to a separate heat shock experiment were able to activate the full HSR as measured in adults, but the capacity to mount an HSR increased as development proceeded. This compromised ability clearly contributes to the vulnerability of early stage larvae to acute environmental challenge.The pre-conditioning of adult marine invertebrates to altered conditions, such as low pH, can significantly impact offspring outcomes, a process which is often referred to as transgenerational plasticity (TGP). This study describes for the first time, the gene expression profiles associated with TGP in the green sea urchin  In the marine field, much of the early work manipulating pH and CO2 in marine systems, popularly known as Ocean Acidification (OA), has concentrated on early developmental stages (embryos and larvae), as these are potentially the most vulnerable part of the life cycle and key to continued population success2. However, many of these early experiments used adults from the wild and spawned them in present day pH sea water. The resulting larvae were then directly decanted into lowered pH sea water and their development monitored. Whilst these types of experimental manipulation mimic the effect of seasonal, episodic high pCO2 upwelling, such as that observed along the US western coast4, they do not mirror the real-time trajectory of climate change. These experiments exclude examination of the abilities of organisms to respond via phenotypic plasticity, developmental plasticity and transgenerational plasticity (TGP)6.To make accurate predictions about the resilience of biodiversity in the future, it is essential to understand the cellular mechanisms that underpin species’ responses to change. Evaluations at the molecular level provide 10. This is often called TGP, if both male and female parents are pre-acclimated and maternal conditioning or maternal provisioning if pre-acclimation is restricted to females only6. In a recent review of TGP in echinoderms and molluscs, the majority of studies reported beneficial effects, although neutral and negative effects were also recorded, with the authors acknowledging that such responses are complex to predict and often time-scale, taxa or species-specific5. For example, pre-acclimation of adult sea urchins to low pH, had deleterious effects on the reproductive success of the tropical Echinometra mathaei11. No effects were observed on the larvae of Echinometra species living on venting coral reefs from Papua New Guinea12. Mixed effects were discovered in the temperate Strongylocentrotus droebachiensis, which showed positive effects on egg size, no effect on female fecundity after prolonged pre-acclimation, but negative effects on F1 recruitment and juvenile survival7. In both the temperate Psammechinus miliaris and Antarctic Sterechinus neumayeri positive effects were observed on larval survival at low pH, especially if the whole reproductive cycle was included10. In a further study on sea urchins and pollution, maternal pre-conditioning of the temperate Evechinus chloroticus preloaded the resulting larvae with antioxidants to help mitigate against pollution-induced oxidative damage13. To emphasize the complexities of TGP responses, mortality was 100% before the blastula stage in embryos produced from low pH pre-acclimated adults of the cold water sea cucumber (Cucumaria frondosa) in the only study to date on lecithotrophic larvae14. The picture is even more complicated from a multi-stressor perspective, for example experiments (temperature and low pH) in the bivalves Mercenaria mercenaria and Agropecten irradians showed negative effects with pre-acclimated adults producing more vulnerable offspring15. Similarly studies using the Sydney rock oyster Saccostrea glomerata showed positive effects when low pH alone was investigated, but maladaptive responses to multiple stressors16. Additionally, the stage at which the embryos or larvae are introduced into low pH is also critical. Sea urchin larvae that were spawned and cultured up to the 4-arm stage in control conditions and then introduced to low pH were more robust than cleaving embryos introduced directly to low pH19. Thus predicting the outcomes from the experimental manipulation of environmental parameters in marine species is complex, with both parental conditioning and the stage at which the larvae are exposed to the new conditions both critical factors6.An increasing number of studies, many of them in sea urchin species, now show that adult acclimation to low pH prior to spawning can significantly impact larval success1 generation. For cellular mechanisms underpinning these physiological responses, the number of examples is limited. Recent data on larvae spawned from adult populations of Strongylocentrotus purpuratus from across a latitudinal pH gradient and also populations pre-acclimated in the laboratory to low pH have demonstrated the upregulation of metabolic pathways redirecting ATP production towards enhancing acid-base homeostasis and ion regulatory capacity, but with potential cellular trade-offs21. Hence, there is still a significant knowledge gap of not only the differential gene expression underpinning TGP, but also larval robustness through development to altered conditions5.In summary, most of the studies on TGP have described the morphological and developmental responses of the FPsammechinus miliaris raised under low pH (~1000 µatm). These larvae were produced from either adults raised under ambient conditions (~400 µatm) or pre-acclimated to low pH in IPCC year 2100 conditions (~1000 µatm) to evaluate the cellular level effect of parental conditioning. There was an expectation that larvae produced from adults cultured under ambient conditions and spawned directly into low pH would exhibit the classical stress response to new conditions, namely the heat shock response (HSR). This virtually ubiquitous cellular reaction involves the up-regulation of chaperone proteins, specifically members of the 70 kDa and 90k Da heat shock protein families (Hsp70 and Hsp90). These are highly conserved proteins that deal with the stress-induced denaturation of other cellular proteins22. The lack of such a response in the gene expression data was surprising. Therefore the ability of early stage larvae to mount an HSR was further investigated in a separate experiment where both adults and larvae were subjected to an acute heat shock and the resulting expression levels of nine hsp70 and hsp90 genes were evaluated by Q-PCR.In this study we analysed the gene expression profiles of early stage larvae from the green sea urchin Expression profiles were produced from cohorts of larvae at 2, 3 and 4 dpf (days post fertilisation) raised in low pH ~1000 µatm). One set of these larvae were produced from adults cultured at ambient pH (~400 µatm) to mimic rapid pH transfer experiments, whilst a second set was produced from low pH acclimated adults to study the effect of parental acclimation. In each case the expression profiles were compared to larvae raised under ambient conditions and produced from adults kept under ambient conditions (controls at ~400 µatm). The number of annotated transcripts which were up-regulated in the treated samples was highly variable  and inflammation (mep1B and clec4A) ) and also other transcription factors impacting cell differentiation and growth such as the map kinase mapk1 and protein kinase (prkcd)   with 48.5% identity and 72.4% similarity at the amino acid level in a 263 amino acid (aa) overlapping region. The first contig (52063) contained the methionine start codon, along with five motifs characteristic of the Hsp90 family (NKEIFLRELISNSSDALDKIR (at aa position 28–48); LGTIAKSGT (aa 95–103); IGQFGVGFYSAYLVAD (aa119–134); IKLYVRRVFI (aa 346–355) and GVVDSEDLPLNISRE (aa 372–386)), but was not full-length, missing the C-terminus with the cellular localisation motif  23. The second contig (51464) was shorter and only encompassed the central portion of the gene, containing only the last two motifs listed above. The remaining seven transcripts were putatively identified as belonging to the hsp70 family, both on Blast sequence similarity searching and motif searches  all had the signature for non-organelle family members  (Supplementary Information grp78 (Glucose-regulated protein 78 kDa), whilst the remaining three contigs  were particularly close in terms of sequence similarity. Analysis of the overlapping 265 amino acid region between these three sequences revealed that the more divergent member of this sub-group (37720) shared most sequence similarity to hsp70IV and 86.8–88.7% amino acid similarity to the other two contigs. Contigs 62830 and 62842 matched the same accession number (W4Y0E3: Hsp701A) sharing 84.2% amino acid identity (91.3% similarity). The level of differences between the three transcripts indicated that they were most likely separate isoforms, rather than allelic variants and these data were validated via the Q-PCR analyses.Nine contigs were initially identified in the backbone transcriptome, which shared sequence similarity with heat shock proteins (as defined by Blast sequence similarity searching) and also had sufficient overlap in sequence to enable them to be identified as distinct family members Table . Whilst es Table . Of theshsp transcripts under the different treatments and time points  and all were below 2 . The only exception was transcript 37720 (hsp70IV) in the CE3 (low pH cultured larvae produced from ambient adults) samples with an expression ratio of 18. Contig 13435 coding for a putative member of the hsp70 family was not expressed above the analysis threshold in any of the treated samples.Interrogation of the OA-generated transcriptome data revealed highly variable expression of the nine different ts Table . In geneP. miliaris experiment, adult and 2 dpf and 4 dpf larvae, all cultured and raised in ambient pH, were subjected to an acute heat shock of 25 °C for one hour to evaluate their HSR. All of the nine hsp70 and hsp90 gene family members identified from the larval transcriptome were successfully amplified in these samples by standard PCR and Q-PCR. 18s rRNA was a suitable reference gene as its expression was stable across all samples: Cq values were not significantly different between control and heat shock samples for 2 dpf larvae (t(3.84) = 1.87, p > 0.05), 4 dpf larvae (χ2(1) = 0.05, p > 0.05) and adults (t(17.473) = −0.010, p > 0.05). There was an increase in the number of significantly up-regulated hsp transcripts in response to heat shock through development: two transcripts were up-regulated in 2 dpf larvae, whilst five were up-regulated in 4 dpf larvae. Eight transcripts were up-regulated in adults  did not show significant differential expression between heat shock and control treatments. Developmental stage also had an effect on the magnitude of up-regulation: the combined log2 fold changes in gene expression across all transcripts were significantly different between stages . The effect was most evident between 2 dpf larvae and adults  translational activation of maternal mRNA, as has been shown for hsp90 or post-translational regulation of hsp7033.Despite the possession of an active immune system, gastrulation is an extremely sensitive stage of early life history development in sea urchinshsps were still developmentally limited with only two hsp transcripts activated in 2 dpf larvae, increasing to four transcripts in 4 dpf larvae. This is much lower when compared with the adults, in which eight hsps were expressed in response to an acute heat stress. Additionally the larval transcripts were also expressed at lower levels compared with the adults . These showed altered larval morphometrics with time in the low pH treatment. The larvae raised in low pH had significantly smaller body length-width ratios  than counterparts reared under ambient conditions. In addition, larvae raised under low pH had an increased percentage of abnormalities (25–40%) than those raised under ambient conditions. The severity of abnormalities in low pH larvae was more pronounced with missing arms, substantial length differences in arm length, asymmetric body shape etc. compared to ambient pH raised larvae, which had only minor length differences in arm pairs and minor imbalance to body shape , particularly if there are consequential trade-off effects20. A meta-analysis involving molecular profiling of sea urchin larvae cultured under low pH suggested that shape and size changes in low pH cultured larvae were due to metabolic depression with up-regulation of genes involved in metabolism, ion transport and biomineralization48. This meta-analysis did not show over-representation of gene ontologies related to development even though genes annotated to “developmental process” were up-regulated in certain studies48. Whilst the most striking finding about the transcriptional profiles described here was the delayed expression of key signalling genes, a number of ion channels and solute carriers were also up-regulated under the different treatments  and in general genes involved with skeletogenesis were down-regulated  of transcripts41. The data here show that the initial disruption to developmental signalling cascades occurs very early in development with the altered expression of the critical transcription factors   of early stage larvae to mount an effective “stress” response clearly contributes to the vulnerability of this early stage of life. Thus maternal conditioning and assessment of the vulnerability of larvae via the characterisation of particularly sensitive stages is important not only for experimental design, but also for modelling where an understanding of recruitment potential underpins future biodiversity predictions.In summary, investigations into larval sensitivities to environmental challenge are revealing the complex nature of the responses, particularly at the molecular level. The data here show that adult Psammechinus miliaris sea urchins were collected by scuba divers at 3–10 m in February 2010 from Rubha Garbh, Loch Creran, Scotland. The urchins were transported in aerated cool boxes and transferred into flow-through aquaria at Bangor University’s School of Ocean Sciences, Anglesey. They were then acclimated to ambient (control) pH aquarium conditions (~pH 8) for two months prior to the start of the experiment. Urchins were fed to satiation (approximately 5% mean body weight) twice a week on an artificial diet largely composed of mussels (Mytilus edulis) and macroalgae (Laminaria digitata). After the two months, animals with a test diameter over 25 mm were chosen as broodstock for the OA trial as this is the size generally considered as reproductively contributing adults in this species50. These animals were randomly assigned to experimental tanks in an OA flow-through mesocosm as described previously18. Treatments used in this study were derived from the IPCC predictions using the ‘RCP8.5 business-as-usual’ scenario51 with parameters calculated and reported following OA community guidelines52. Present day control sea water, imported directly from the Menai Straits, measured ~pHNIST 8.06 and pCO2 calculated at 412 ± 6 µatm (referred to from here on as 400 µatm or ambient), with the year 2100 pH level calculated at ~pHNIST 7.72 and a pCO2 of 1013 ± 12 µatm (referred to from here on as 1000 µatm or low pH)  Table . No dataP. miliaris from each treatment were induced to spawn at the beginning of July when all urchins, irrespective of treatment, were at stage IV maturity 18. Spawning of adults via injection of 0.5 M KCl and fertilisation of embryos was performed in the same conditions as the parents had been cultured in either ambient or low pH (400 µatm or 1000 µatm) as appropriate, with methodology as described previously18. Five females and two males were successfully spawned for each treatment with gametes pooled prior to fertilisation, with the production of three replicates. Embryos and larvae were either cultured under 400 µatm or 1000 µatm conditions, using 1 μm filtered and UV treated seawater obtained from the OA system. 48 hours after fertilisation larval stocking densities were adjusted to one larvae mL−1 and a daily feeding regime was started using Dunaliella tertiolecta (CCAP 19/6B) cultured in sterile conditions with a daily feeding rate of 1500 cells ml−1. 50 mls of larval culture were sampled 2, 3 and 4 days post fertilisation, at which point they were at the feeding pluteus (4 arm) stage. After brief centrifugation at 2,000 rpm to concentrate the larvae, the seawater was carefully removed and the larvae flash frozen in liquid nitrogen and stored at −80 °C until RNA extraction.Following two months exposure to 400 µatm or 1000 µatm, adult 2 concentration (TCO2) was measured twice weekly, in triplicate using a Carbon Dioxide Analyser . The TCO2 analyser was blanked to zero and calibrated with Reagecon 2 g L−1 CO2 standards before each set of readings. Silicate (SiCO3) and phosphate (PO4) nutrients were determined with a Lachat 8000 two flow injection nutrient analyser in the Scottish Association of Marine Science, Oban, UK. Values for the parameters of total alkalinity (TA), and Ω were calculated with CO2SYS with refitted constants following OA community guidelines52 (Table Daily measurements of temperature and pH were taken using a hand-held temperature compensated pH meter (HI98160 Hanna Instruments UK Ltd). Weekly salinity measurements were made using an ATC portable refractometer calibrated with distilled water prior to use. Total COsphate PO nutrientP. miliaris. The adults were taken from the control system (400 µatm) of the OA experiment described earlier. The control temperature used was 15.8 °C and the heat shock performed at 25 °C for one hour. This temperature was chosen as sufficiently high in relation to the normal temperature range experienced by these urchins to take them over a “stress” threshold and induce a heat shock response22. Larvae were produced from broodstock (test diameter (±SE) = 34.87 ± 0.82 mm, whole animal wet mass = 20.50 ± 1.10 g) which were reared under the control conditions described in Table Heat shock experiments were conducted on both larvae and adult l system 00 µatm oRNA was extracted from three replicates of pooled larval samples using TRI reagent (Bioline) and purified on RNeasy mini-columns (Qiagen) according to manufacturer’s instructions. RNA was quantified using either a NanoDrop 1000 photometer  or a Tape Station 2200 (Agilent Technologies). The OA experimental RNAs were subjected to RNA-Seq on an Illumina Hi-Seq (100 bp paired end reads) at The Earlham Institute, Norwich.56. The raw Illumina reads were assembled using SOAP denovo (soap.genomics.org.cn) . All kmer contigs greater than 200 bp were run through CD-HIT58, following which the remaining contigs were run through CAP359. All contigs and singletons greater than 500 bp were then screened for contamination from various sources, including the food  using BLAST and any reads identified as contaminants removed. The remaining sequences were used for both producing the backbone sequence database and also for mapping differential expression patterns. A proportion test was carried out for all comparisons in R60 with an adjustment made for multiple testing61 with an FDR cutoff set at 0.01. Only mappings where both paired-end reads mapped to the same contig were used to generate expression levels and calculate significance of expression with the following additional criteria for selection and designation of significant change in expression: a fold change greater than 2; an adjusted p value of <0.01; both sets of treatment reads had to have in excess of 10 reads per contig and at least one treatment had to have in excess of 50 reads per contig. Contigs were searched for sequence similarity using BLAST62 against the GenBank non-redundant database63 with a threshold score of <1e−10. Transcripts with sequence similarity to members of the 70 kDa and 90 kDa HSP family were identified from searches of the BLAST data, manually verified and the sequences manipulated, including translation into putative protein sequences, using Bio-Linux64. Sequences annotated through GenBank were further searched against the Swissprot databases of different model species. The Swissprot Homo sapiens database produced the highest number of matches below 1e−10 by at least a factor of 10 compared to the lower vertebrates and invertebrates when searched with the P. miliaris data and so it was chosen for further analyses. The Swissprot identifiers were used to input into Panther GO-Slim65 and the STRING program, a database of protein-protein interactions (http://string-db.org/), to provide an overview of putative gene networks.In total, 194,339,075 100 bp paired end Illumina reads  were clipped and trimmed for adapter sequence and quality using EA-UTILSRNA was extracted from three replicates of pooled larval samples using ReliaPrep™ RNA Miniprep Systems (Promega) according to manufacturer’s instructions. The protocol for non-fibrous tissue (tissue input ≤ 5 mg) was followed, with a centrifugation speed of 13,000 × g. RNA from the coelomic fluid of adults was extracted using RNeasy Mini Kit (Qiagen) following the protocol for animal cells, with centrifugation speed of 10,600 × g. Both protocols included a DNase digestion step. Larvae and coelomic fluid were lysed using a TissueRuptor rotor-stator homogeniser (Qiagen), and the final elution volume was 30 µL. RNA concentration and integrity were assessed using a Tape Station 2200 (Agilent Technologies). Complementary DNA (cDNA) was produced from 150 or 500 ng of RNA from adults or larvae respectively, using the QuantiTect® Reverse Transcription Kit (Qiagen) according to manufacturer’s instructions, with a further genomic DNA elimination step.66 or manually in the case of the three very closely related hsp70 genes  and 3.24 µM bovine serum albumin with 0.5 µL of cDNA.Q-PCR primers for each of the nine HSP transcripts were designed either using the Primer3plus programmax) with the following thermal cycling conditions: 3 min at 95 °C followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C. Amplification specificity was assessed through melting curve analysis, with an additional step immediately subsequent to the thermal cycling: the products were denatured for 15 s at 95 °C, allowed to re-anneal for 15 s at 55 °C and the temperature was then raised to 95 °C at a ramping rate of 0.25 °C/s. Samples were run in duplicate wells and all assays included no template controls and negative controls (nuclease free water). Amplification efficiencies for each assay were calculated by the EcoStudy software (Illumina) from a standard curve produced through serial dilutions of cDNA template pools . The number of dilutions was ≥4 starting from undiluted or 1:2 cDNA for all assays and the dilution factor was either 4 or 10, depending on the assay-specific amplification behaviour. Each q-PCR reaction comprised 1x Brilliant III SYBR® Green master mix (Agilent), 570 nM of each primer, 721 nM bovine serum albumin and 0.5 µL cDNA . Adult sample cDNA was diluted 1:10 for all transcripts except for contigs 46238 and 62830, for which a 1:4 dilution was used. Day 2 and day 4 larval cDNA samples were diluted 1:2 and 1:10 respectively. A 18 s rRNA transcript was used as the reference gene for normalisation, after confirming its expression was constant between control and heated samples.Q-PCR was performed using an Eco Real-Time PCR System  with automatic baseline correction and threshold adjustment  was used in the analysis. Stable expression of the reference gene used for normalisation (18s rRNA) was confirmed by comparing raw Cq values  were not normally distributed, a Kruskal-Wallis test and a Dunn’s post-hoc test with Holm’s correction for multiple comparisons were performed. All statistical analyses were performed in R60.Amplification efficiency and RSupplementary information and tables


Through variation of additives like CuI, KOH, and various amounts of zinc powder, orthogonal chemoselectivities in the deuteration process are observed. Mechanistic investigation indicates the presence of different, defined Ru‐complexes under the given specific conditions.A method for the selective deuteration of polyfunctional organic molecules using catalytic amounts of [RuCl C−H activation: A method for the selective deuteration of polyfunctional organic molecules using catalytic amounts of [RuCl2(PPh3)3] and D2O as a deuterium source is presented. Through variation of additives like CuI, KOH, and various amounts of zinc powder, orthogonal chemoselectivities in the deuteration process are observed (see scheme). Herein we report the first results of this study, in which we demonstrate the selective deuteration of different polyfunctional organic substrates. NMR spectroscopic investigations were used to identify the in situ formed Ru catalysts 2–4 3]  catalyze3)3] (2) is formed in the presence of an excess of zinc in H2O using CuI as an additive starting from precatalyst [RuCl2(PPh3)3] (1)  (3)  in the chemoselective reduction of olefins in the presence of carbonyl groups has been described in the literature.3 is one of the most active Ru catalysts in the reduction of carbonyl groups and quasi inert to olefin/alkyne reductions.The study presented here was based on in‐depth NMR spectroscopic studies on the influence of the additives KOH and CuI on the preacatalyst activation. The quintessence of this extensive work is summarized in Scheme (PPh3)3]  [Eq. 1)3)3] (2) 2O under the established conditions was studied by the reduction of diarylalkynes 5–7 and acetophenone derivatives 11–13 , Eq. (1), Scheme 11–13, upon addition of catalytic amounts of KOD, reacted cleanly to give the corresponding alcohols 14–16  1 was not necessary under these conditions. In the presence of CuI as an additive, however, no reduction of the carbonyl group and no deuteration at the α‐carbon atom was observed. Interestingly, under these conditions a selective deuteration of the two ortho‐carbon atoms in the aromatic moiety ketones 17–19 was observed  (1) and KOD in the absence of stoichiometric amounts of zinc (conditions (C)) an efficient ortho‐selective C(sp2)–H–D exchange was possible. The use of catalytic amounts of [RuCl2(PPh3)3] (1) and CuI (conditions (A)), on the other hand, provided the expected deuterated aromatics only when using zinc.Carbonyl groups are able to direct the ty Table . In factortho‐deuteration of C(sp2)−H bonds using catalytic amounts of KOD was highly selective, catalytic depletion of CuI led both to C(sp2)−H‐ and C (sp3)−H bond deuteration plus in some cases ring opening of the directing group, for example, oxazolidines. Since C(sp3)−H‐deuteration of benzoic acid propylamide 33 with the addition of both KOD and CuI led to almost identical results −H‐deuteration occurs prior to the opening of the oxazolidinone. Importantly, no dehalogenation reactions were observed under either reaction conditions.2 from D2O is likely.ortho‐selective H–D exchange occurs with addition of catalytic amounts of KOD even without zinc, a direct deuteration from D2O can be assumed under these conditions.Depending on the additive, significant differences in the deuteration were observed. While the p‐ and m‐alkynylpyridyl‐substituted aromatics 36 and 37 were tested under the established conditions –(4), Scheme Finally, we tested both methods on complex drugs such as piribedil Thus, in the presence of catalytic amounts of CuI (conditions (A), Scheme 46  1 is converted into different defined Ru complexes 2–4 by using either CuI (cat.)/Zn, or KOD (cat.) or KOD (cat.)/Zn. Each of these complexes shows a different selectivity in H–D exchange reactions. D2O is used as the common deuterium source in all cases. When using zinc as an additive, the formation of D2 gas could be experimentally proven. Future work will aim to improve the present protocols, for example, through systematic variation of ligands, in order to amplify the selectivity trends.Herein, we present a preparatively simple method for the deuteration of functional organic molecules under mild conditions. Depending on the additive, the precatalyst [RuClThe authors declare no conflict of interest.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.


To the Editor: The 1 h post-load plasma glucose (1hPG) measurement has the potential to serve as a sensitive screening tool for identifying people who, despite having normal glucose tolerance (NGT), are at high-risk of developing type 2 diabetes over the next few years [High 1hPG has been found to perform as well as the 2 h post-load plasma glucose (2hPG) measurement in predicting type 2 diabetes risk after median follow-up times of 9 and 13 years . Robust In a cross-sectional study, we previously described reduced euglycaemic clamp insulin sensitivity and impaired beta cell glucose sensitivity (BCGS) in people with NGT but with high 1hPG in 1205 healthy participants in the European Group for the Study of Insulin Resistance (EGIR) cohort: Relationship between Insulin Sensitivity and Cardiovascular Risk (RISC) [We now report 3 year longitudinal data from 797 participants with NGT at baseline, who had complete baseline and follow-up glucose data .In the current group of 797 participants we present, as medians (interquartile range) or %, the metabolic features of individuals with NGT whose glucose status progressed (‘progressors’) vs those who did not (‘non-progressors’) according to 1hPG < and ≥8.6 mmol/l) at baseline  vs 66 (8%) (with 26 of these progressing in relation to both), so the metabolic features described in the paragraph above for progressors reflect more the metabolic impairment of participants who developed isolated IFG rather than isolated IGT or combined IFG and IGT; indeed we observed a higher clamp insulin sensitivity for isolated IFG than for isolated IGT, in both 1hPG groups


Graphene based photocatalysts are highly suitable for the system, but their industrial scale use requires immobilization for improved separation and recovery of the photocatalyst. Therefore for practical purposes, design and fabrication of film type graphene photocatalyst with higher solar energy conversion efficiency is an absolute necessity. As a means to achieve this, we report herein the successful development of a new type of flexible graphene film photocatalyst that leads to >225% rise in visible light harvesting efficiency of the resultant photocatalyst-biocatalyst integrated artificial photosynthesis system for highly selective solar fuel production from CO2 compared to conventional spin coated graphene film photocatalyst. It is an important step towards the design of a new pool of graphene film based photocatalysts for artificial photosynthesis of solar fuels from CO2.Our growing energy demands must be met by a sustainable supply with reduced carbon intensity. One of the most exciting prospects to realize this goal is the photocatalyst-biocatalyst integrated artificial photosynthesis system which affords solar fuel/chemicals in high selectivity from CO This has triggered keen interest in the development of graphene based nanoparticle powder or nanocomposite photocatalysts7. However the industrial scale application of these photocatalysts is largely limited due to difficulty in their filtration and separation. Therefore for practical and economic purposes design and fabrication of film type graphene photocatalyst with higher solar energy conversion efficiency is an absolute necessity.Graphene is as an ideal material for light harvesting applications15. Although easy to formulate, uncontrolled capillary flow and dewetting processes during solvent evaporation result in non-uniform films with random orientation of graphene-chromophore units leading to increased charge recombination and consequently lower photocatalytic activity. Moreover, chromophore coupling to graphene sheet is usually carried out through functional groups such as –COOH present on graphene oxide (GO) or its reduced forms such as chemically converted graphene (CCG)16. They contain a large number of non-conjugated tetrahedral sp3 carbons which lie slightly above or below the graphene plane, further limiting electronic conductivity of the photocatalyst17. Keeping in view these crucial bottlenecks, it is imperative to develop a new design and fabrication strategy for graphene film photocatalysts.Graphene films have been previously fabricated by spin coating method2 compared to conventional spin coated graphene film photocatalyst. The monolayer graphene for this work was supported on a flexible polyimide sheet leading to a practical and easy to use photocatalyst. It also helped to solve the problem of insufficient mechanical strength of monolayer graphene film photocatalyst. The lack of functional groups on graphene monolayer for chromophore attachment presented another major challenge. This was ingeniously overcome by attaching chromophore units to sp2 carbon centers on graphene via 1,3-dipolar cycloaddition reaction. To the best of our knowledge, graphene film photocatalyst for solar fuel production using this approach has not been fabricated prior to this work.Here we report the successful development of a new type of flexible graphene film photocatalyst  collects incident photons as an electronic transition between localized orbital around it (HOMO to LUMO) and conducts via graphene (as a multi electron acceptor) to reduce the rhodium complex Rh ([Cp*Rh(bpy)H2O]2+;Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine). Upon reduction, the rhodium complex abstracts a proton from aqueous solution and transfers hydride ion to NAD+, which gets converted into NADH, thus completing the photocatalytic cycle. In this way, Rh behaves as an electron mediator between the graphene film photocatalyst and NAD+ leading to regeneration of NADH cofactor. Finally the enzymatic (Formate Dehydrogenase) conversion of CO2 substrate to formic acid consumes NADH. The NAD+ thus released again acts as a substrate for photocatalytic cycle, leading to photoregeneration of NADH. The photocatalytic-enzymatic cycles thus couple integrally, leading to exclusive formic acid production from CO23.Figure via 1,3-dipolar cycloaddition. More details are provided in the experimental section. The integration of GFPC 1 photocatalyst with biocatalyst (formate dehydrogenase enzyme) afforded the integrated artificial photosynthesis system for carrying out highly selective formic acid production from CO2  with 1,3-Dioxo-1H-dibenzoisoquinoline-2[3 H]-carbaldehyde (hereinafter DdIC chromophore) 18. This blue shift in absorption maxima can be attributed to the 1,3-dipolar cycloaddition of DdIC to GF. This observation indirectly suggested the covalent attachment of DdIC chromophores on the monolayer graphene.The existence of DdIC in GFPC 1 was confirmed by UV-vis spectroscopy. A strong soret band at 433 nm was observed in the absorption spectrum of DdIC chromophore in DMF Fig. . On the −1)19. Moreover in GFPC 1, the C=O stretching was observed at 1772 and 1707 cm−1 while C=C stretching was observed at 1670 and 1597 cm−1  data provided clear evidence for the coupling of chromophore to graphene film Fig. . The GF m−1 Fig. . In comp19. An increased thickness of 1.4681 nm in GFPC 1 indicated functionalization on the GF surface. Apart from increased thickness, the presence of DdIC on the GF surface was further supported by the bloated appearance and roughness of the sample. The difference in the high resolution transmission electron microscopy  and DdIC chromophore23.Raman studies further confirmed the coupling of DdIC chromophore to GF Fig. . The G a24 of DdIC a single new peak at 397 eV ascribed to N atoms of the C-N bond appeared  and GFPC 1 was also studied by thermogravimetric analysis (TGA) Fig. . TGA wasThe most critical part of the work was to evaluate the photocatalytic performance of the GFPC 1 in comparison to the DdIC chromophore, graphene-DdIC powder photocatalyst and its spin coated film sample . Therefore, visible light-driven 1,4-NADH photo-regeneration ability of all these were examined along with GF. While GF failed to carry out any NADH regeneration, DdIC chromophore afforded 21.8% NADH regeneration over a period of 120 minutes. On the other hand, graphene-DdIC powder photocatalyst and GFPC 2 afforded 48.9 and 40.1% of NADH regeneration over a period of 120 minutes, respectively. Since GFPC 2 was obtained by spin coating the graphene-DdIC powder, a lower NADH regeneration by GFPC 2 can be clearly attributed to irregular orientation of the photocatalyst species on the polyimide sheet. In comparison to this 91.8% NADH regeneration was observed when GFPC 1 was evaluated for the photocatalytic activity Fig. . In othe2  and another one without visible light irradiation were also carried out which also failed to show any detectable amount of formic acid. Additionally, no formic acid formation was detected in another set of control experiments carried out in the absence of rhodium complex Rh and/or NAD+ which confirmed their vital role in the functioning of the photocatalyst/biocatalyst integrated artificial photosynthesis system. To evaluate the possibility of rhodium complex Rh functioning as the CO2 reduction photocatalyst a control experiment consisting of rhodium complex and CO2 was performed. However, no formic acid was detected in this case also. This indicated that rhodium complex does not carry out photocatalytic CO2 reduction. Along with the high stability of GFPC 1 under visible light irradiation the various control experiments clearly point towards exclusive visible light driven photocatalytic NADH regeneration followed by enzymatic CO2 reduction as the sole route for formic acid production. This was further confirmed by C-13 isotope labelling experiment which clearly indicated that CO2 is the sole source of formic acid formation in our system H2O]2+; Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine) reduction was observed at −0.97 V . The vicinity and potential gradient between the light harvesting GFPC 1 photocatalyst and Rh center enable this efficient electron transfer from former to latter. The resultant reduced Rh species [Cp*Rh(bpy)] upon chemical protonation carries out catalytic regeneration of enzymatically active 1,4-NADH from NAD+ (E = −4.20 eV). This photocatalytic pathway is schematically shown in Fig. To understand the electron transfer pathway, cyclic voltammetry (CV) measurements were carried out. The reduction potential at cathodic peak current of GFPC 1 was observed at around −1.2 V Fig. . On mixi7 V Fig. . This isADH Fig. . From th−2 in response to the on/off simulated sunlight (1 sun) illumination  under identical conditions . The Fermi level of graphene was calculated to be 4.20 eV lower than vacuum level, which lies around the center of bandgap of the chromophore, about 0.6 eV lower than the lowest unoccupied molecular orbital (LUMO) level of chromophore. Although this “divide-and-conquer” method is simple and powerful to deal with a large system without too much computational burden, the two components (DdIC chromophore and graphene monolayer) of the photocatalyst are tightly bonded via strong covalent bond. Thus the electron can be transferred spontaneously to make the Fermi levels equal. So, electronic structure calculations of the whole system were performed, secondly. The optimized atomic geometry and electronic band structure are shown in Fig. To elucidate the origin of the photocatalytic activity and activity enhancement of GFPC 1 theoretically, a series of first principles calculations using plane-wave based density functional theory (DFT) code, VASP were carried out2. We believe that this work would serve as a benchmark example of a new pool of graphene based photocatalysts that can efficiently harvest and transfer solar light and thereby trigger renewed interest in the area of solar fuel/chemical production from CO2. Our strategy may also provide broad contours for application of flexible graphene films in other fields such as solar energy powered wearable electronic devices.In conclusion, the newly designed and fabricated flexible graphene film photocatalyst shows high photocatalytic activity attributed to a regular arrangement of light harvesting units with spatial orientation towards visible light as well as improved delocalization of π electrons and direct electron transfer from covalently attached chromophore to graphene monolayer. The graphene film based artificial photosynthesis system thus obtained shows remarkably higher formic acid production from COThe GFPC 1 photocatalyst for this research work was obtained by coupling CVD grown graphene film with 1,3-Dioxo-1H-dibenzoisoquinoline-2[3 H]-carbaldehyde (DdIC chromophore) via 1,3-dipolar cycloaddition. This was carried out by the following procedure.3. The film was then dried in the oven at 125 °C for 24 hours to obtain GFPC 1 photocatalyst with the loading of one DdIC group per 70 carbon atoms of the graphene film.Graphene film was suspended in 100 ml round-bottom flash via wire in 10 ml of ortho-dichlorobenzene (ODCB) along with sarcosine (2.5 mg), and DdIC (2.0 mg). The solution was stirred at 180 °C under argon atmosphere for 7 days. The GFPC 1 photocatalyst was then washed with ODCB, followed by water and CHCl16. The graphene thus obtained was coupled to DdIC chromophore by following the procedure as outlined for GFPC 1 except for the graphene powder used in this case. This sample is graphene-DdIC powder photocatalyst with the loading of one DdIC group per 68 carbon atoms of graphene.Graphene for this sample was prepared by following a literature method2 polyimide sheet and then dried in an oven at 80 °C for 3 days to obtain GFPC 2.A DMF suspension of the graphene-DdIC powder photocatalyst was spin coated  on 1 × 1 cm+ (1.24 μmol), rhodium complex Rh (0.62 μmol), Ascorbic acid (0.1 mmol) and photocatalyst  in 3.1 mL of sodium phosphate buffer . The regeneration of NADH was monitored by UV-vis spectrophotometer .The photochemical regeneration of NADH was performed within a quartz reactor under an inert atmosphere at room temperature, using a 450 W Xenon lamp (Newport 66921) with a 420 nm cut-off-filter as light source. The photocatalytic regeneration of NADH was carried out as follows. The reaction was performed in a quartz reactor. The reaction consisted of β–NAD2 was also performed within a quartz reactor at room temperature, using a 450 W Xenon lamp with a 420 nm cut-off-filter as light source. The reaction consisted of photocatalyst , β–NAD+ (1.24 µmol), rhodium complex Rh (0.62 μmol) and formate dehydrogenase enzyme (3 units) in 3.1 mL of sodium phosphate buffer  with Ascorbic acid (0.1 mmol) in the presence of CO2 (flow rate: 0.5 mL/min). The amount of formic acid was estimated by GC .The artificial photosynthesis of formic acid from CO2 film of GF, GFPC 1 and GFPC 2 contained 0.5 mg (approx.) of the photocatalyst.Note: The 1 × 1 cmk-points sampling was done with 3 × 3 × 1 grid including Г-point for the integration of the irreducible Brillouin zone. For the molecular system, only Г-point is included. The Kohn–Sham wave functions of the valence electrons were expanded using a plane wave basis set within a specified energy cutoff that was chosen as 400 eV. The positions of the nuclei in the initial structures were first relaxed by the conjugate-gradient algorithm, until the Hellman-Feynman forces on each nucleus were lower than 0.01 eV/Å. After the relaxation, spin-polarized total energy of the electronic structure was calculated using a self-consistent field method that terminated when a change in total energy between two subsequent steps was less than 10−6 eV.All density functional theory (DFT) calculations were performed using the plane-wave approach as implemented in the VASP code within the generalized gradient approximation (GGA) using the Perdew-Burke-Ernzerhof (PBE) exchange-correlation function. Frozen-core projector augmented wave pseudopotentials were used. For the simulation of periodic system (8 × 8 unit cell of graphene), the Monkhorst-Pack scheme Supplementary Information


Changes in external and internal postharvest quality parameters were assessed for up to 8 weeks for ‘Afourer’ mandarins and 10 weeks for Navel oranges. At all storage temperatures, high levels of ethylene were found to increase the level of calyx senescence, weight loss, loss of fruit firmness and respiration rates. Also, there were significant effects of ethylene and storage temperatures on total soluble solids (TSS) content, titratable acidity (TA), and ethanol accumulation in both citrus species. Continuous exposure to high ethylene also significantly reduced vitamin C and ferric reducing antioxidant power (FRAP) in ‘Afourer’ mandarins after 8 weeks of storage. Overall, ethylene treatments had a significant effect on both the external and internal qualities of the fruit during storage. The relationship between ethylene concentrations and storage temperatures demonstrate that lowering atmospheric ethylene levels at reduced storage temperatures maintain fruit quality during long term storage.Calyx browning and internal quality loss are major physiological causes for the loss of quality in citrus fruit during storage. While the symptoms of calyx senescence are only superficial, it can affect the appearance and consumer acceptability of citrus fruit. In this study, continuous ethylene exposure at different storage temperatures was investigated to assess their effect on calyx senescence and internal qualities in ‘Afourer’ mandarin and Navel orange fruit during storage. ‘Afourer’ mandarin fruit were stored at ≤0.001 , 0.01, 0.1 and 1 µL L Fruit appearance is an important aspect of consumer purchase and acceptability where the green fresh condition of the fruit calyx (button) is a desirable consumer attribute of citrus appearance ,2. ChangCitrus fruit can be stored for up to 8 weeks under optimal conditions, but storage life depends on pre-harvest conditions, cultivar and storage environment, where lower storage temperatures reduce fruit senescence . However−1 ethylene) has also been shown to protect fruit from stress related postharvest disorders such as CI and non-chilling peel pitting [−1) during degreening induced calyx senescence in ‘Valencia’ oranges and increased calyx abscission. However, studies by Li et al. [−1, the incidence of calyx senescence in ‘Afourer’ mandarins diminished. The development of undesirable characteristics in citrus as a consequence of ethylene exposure varies with citrus type and cultivar [−1 at 2 °C storage reduced calyx abscission in ‘Lane Late’ oranges, the same treatment conditions for ‘Navelate’ oranges resulted in high calyx abscission.Citrus fruit are considered non-climacteric, but are responsive to postharvest application of ethylene , which h pitting . Storage pitting . However pitting . Brown a pitting  found thi et al.  showed tcultivar . Lafuentcultivar  found th−1) are applied for a short period of time (a few days) [−1) of ethylene during storage [Many studies on the effects of ethylene on citrus fruit quality have been conducted examining the effects of commercial degreening treatments, where high levels of ethylene (up to 4 μL Lew days) ,13,14. T storage .−1 ethylene at 5 or 20 °C. Accumulation of volatiles such as ethanol is often associated with off-flavour sensory qualities, particularly in citrus juice products [Ethylene exposure at low concentrations during storage has been linked to compositional changes of citrus ,16. Li eproducts ,19 and mThere are few reports on assessing the effect of long-term ethylene exposure on calyx condition and internal quality attributes of mandarins and oranges held in long-term storage (cold or ambient temperature) ,10. The Citrus reticulata Blanca cv. ‘Afourer’) were obtained from a commercial packinghouse in Gayndah, Queensland, Australia . Fruit used for this experiment had their calyx intact, were waxed with commercial carnauba wax and were not treated with the growth regulator 2,4-dichlorophenoxyacetic acid . Fruit for the experiment were uniform in size and free from visible defects. Fruit were randomized and placed into 48 replicate net bags (n = 20 fruit per bag). The bags were then evenly distributed into 6.3 L plastic containers (n = 16) fitted with inlet and outlet airflow ports and stored at either 5, 10 or 20 °C. Individual containers within each temperature environment were then connected to one of four ethylene containing humidified air streams  operating at a flow rate of 50 mL min−1. The ≤0.001 µL L−1 stream was considered to be ethylene-free (air) as the ethylene concentration is below the limit of detection by gas chromatography. The experiment was monitored daily. Mandarin fruit stored at 20 °C were assessed as weekly removals for four weeks, while fruit stored at 10 and 5 °C were assessed every two weeks for eight weeks.Mature mandarin fruit (Citrus sinensis L. Osbeck) were harvested at commercial maturity from a New South Wales Department of Primary Industries (NSW DPI) research farm at Somersby on the NSW Central Coast, Australia . Fruit were transported to NSW DPI postharvest laboratories  (10 min), where the fruit was sanitised with commercial sodium hydrochlorite solution (50 ppm) before being washed and air dried. The fruit was then sized and sorted as previously described, randomized and placed in net bags as individual treatment units (n = 20 fruit). The treatment units were then placed in 60 L steel drums and stored in two different temperatures (1 and 10 °C). The individual drums within each temperature were connected to one of the four humidified air streams containing ethylene  operating at a flow rate of 400 mL min−1. The drums were inspected daily. There were four replicates  for each treatment with assessments after 1, 5 and 10 weeks at both storage temperatures and again with an additional shelf-life assessment following 5 days storage at 20 °C.In a parallel study, Navel oranges  from the final weights (W2) divided by the initial weights and multiplied by hundred percent (%).Weight loss of the fruit was assessed using an electronic balance , where fruit weight of each treatment unit was recorded each assessment day. Weight change was expressed as a percentage value determined by deducting the initial weights  was used to determine firmness of ten fruit per replicate after the fruit had conditioned to 20 °C. The maximum force (N) was measured by compressing the fruit in the equatorial zone between two flat surfaces closing together at the rate of 1 mm minrecorded .2 kg−1 h−1) was measured on five fruit from each replicate. Fruit were sealed into an airtight 2 L glass jars fitted with a septum for 3 h to accumulate respiratory gases. Carbon dioxide (CO2) concentration in the jar was determined by withdrawing a 1 mL gas sample from the headspace and injecting into a gas chromatograph  fitted with two stainless steel columns (60 cm × 1 mm i.d.) connected in series. Operating temperatures for the detector, injector, and column were 110 °C, 50 °C and 110 °C respectively. The carrier gas used was high purity argon  at a flow rate of 25 mL min−1. Respiration rate was calculated according to the method of Huque et al. [Respiration rate  and expressed as °Brix. TA was determined by titrating 5 mL of the juice with 0.1 M NaOH to pH 8.2 with an automatic titrator  and data were expressed as percentage citric acid. For ethanol accumulation, 10 mL of juice was transferred into a 20 mL glass vial with crimp-top caps sealed with silicone septa. The sealed sample was then incubated in a water bath of 30 °C for 10 min before analysis. Ethanol accumulation was determined by headspace analysis using a gas chromatograph  with a flame ionization detector and a column . The injector was set at 190 °C, the column at 68 °C, the detector (FID) at 190 °C with gas flow rates of 30, 30 and 300 mL min Ascorbic acid was determined by the indophenol titration method adapted from the method of Nielsen et al. . Brieflys-triazine (TPTZ) reagent in 40 mM HCl and 20 mM ferric chloride in the ratio of 10:1:1 and warmed to 37 °C in a water bath. 2.85 mL of the FRAP working solution was then added to a 150 mL aliquot of mandarin juice and the resulting solution allowed to react in the dark for 30 min at 20 °C. The solution absorbance (λ = 593 nm) was then recorded using a UV-Vis spectrophotometer  and the result referenced against a standard curve and expressed as µmol of Trolox equivalents (TE) per 100 mL fresh mandarin juice.The antioxidant activity of the ‘Afourer’ mandarin fruit were determined using ferric reducing antioxidant power (FRAP) assay as described by Thaipong et al. , with sop ≤ 0.05. The method used to discriminate among the means (Multiple Range Test) was Fisher’s Least Significance Difference (LSD) procedure at 95% confidence level.Data from each of the experiments were subjected to two-way analysis of variance (ANOVA) using SPSS Microsoft version 24.0 software package . Tests for significance difference of treatment means were at p < 0.001) on calyx browning during storage. However no significant interaction was observed between ethylene concentration and storage temperature. The data presented (−1) resulted in reduced calyx browning and maintained visible quality. Fruit at stored 5 and 1 °C under low atmospheric ethylene concentration (≤0.1 µL L−1) showed reduced calyx browning compared with the fruit stored at 20 and 10 °C. These results with ‘Afourer’ mandarins and Navel oranges are in agreement with the findings of Li et al. [External fruit appearance is an important characteristic that influences consumer purchasing decisions. The results of these experiments showed a significant effect of both ethylene concentration and storage temperature  at different temperatures resulted in significantly lower calyx abscission compared to the other ethylene concentrations. These results support previously published findings showing that lower ethylene levels (‘air’ or <0.1 µL L−1) and low storages temperatures (<10 °C) reduce calyx drop in Navelate orange fruit and ‘Afourer’ mandarins [There was a significant interaction between ethylene and storage temperature on the incidence of calyx abscission during storage. Both Navel oranges and ‘Afourer’ mandarins had higher levels of calyx abscission at increased ethylene levels and storage temperatures. The data presented in andarins ,10. The andarins  who repop < 0.001) and their interaction on the firmness of ‘Afourer’ mandarins and Navel oranges during storage. The results presented , while fruit exposed to an ethylene concentration of <0.1 µL L−1 at all storage temperatures recorded the highest firmness level. There was a significant interaction with ethylene concentration and storage temperature, where fruit firmness levels were lower in fruit stored at higher temperatures for both citrus types (There was a significant effect of both ethylene treatment and storage temperature (resented  show fruus types . This obus types . This isp < 0.001) and their interaction on chilling injury (CI) of Navel oranges. No CI symptoms were observed in ‘Afourer’ mandarins stored at 5 °C. Storage of Navel oranges at 1 °C produced CI symptoms in Navel oranges after 5 weeks of storage followed by 5 days at 20 °C (−1). It is known that ethylene treatment at low storage temperature plays a synergistic role in the maintenance of cell integrity, particularly in cell membranes under stress [There was significant effect of both ethylene and storage temperature  interaction of storage temperature and ethylene on the weight loss of both ‘Afourer’ mandarins of Navel oranges during storage (−1) with weight loss also increasing with increasing storage temperature. Conversely, lower levels of weight loss were observed in fruit stored in low ethylene concentrations at low temperatures, which can be attributed to reduced transpiration and metabolic activity.There was significant ( storage . The higp < 0.001) on the respiration rate of ‘Afourer’ mandarins and Navel oranges during storage but there was no significant interaction between ethylene concentration and storage temperature. The data presented (−1) at all the storage temperatures reduced respiration rate. In both citrus species, exposure to ethylene-free air (<0.001 µL L−1) resulted in the lowest respiration rate within each storage temperature during storage. However, a differential response to ethylene exposure and temperature was observed, with Navel oranges exhibiting a significantly lower respiration rate compared with ‘Afourer’ mandarins exposed to similar ethylene concentrations and storage temperature (10 °C) (There was significant effect of both ethylene concentrations and temperature (resented  shows th (10 °C) . Althougp < 0.001) on the ethanol accumulation in ‘Afourer’ mandarins and Navel oranges. The data presented  on total soluble solids (TSS) in both mandarins and oranges (There was a significant effect of both ethylene and storage temperature ( oranges . While dp < 0.001) on titratable acidity (TA), with no significant interaction between these factors. The TA of both ‘Afourer’ mandarins and Navel oranges decreased during storage, with ‘Afourer’ mandarins exhibiting lower TA levels than in Navel oranges. The data presented . The average vitamin C level at the beginning of the experiment was 7.1 mg 100 mL−1 (The concentrations of vitamin C (L-ascorbic acid) in ‘Afourer’ mandarins showed a significant interaction between storage temperature and ethylene treatment (100 mL−1 . As expe3+) and ferrous ion (Fe2+) by antioxidants present in the sample [p < 0.001) in relation to antioxidant activity in ‘Afourer’ mandarins during storage with results showing a decrease in FRAP levels across the storage period. There was also a significant decrease in activity observed in fruit exposed to 1 µL L−1 ethylene at all storage temperatures (Ferric reducing antioxidant power (FRAP) assay measures the antioxidant potential through the reduction of ferric ion  significantly reduced calyx senescence changes . Lower ethylene concentrations at all storage temperatures were also found to be beneficial to most postharvest quality factors. However the primary factor limiting the storage life of ‘Afourer’ mandarins and Navel oranges was storage temperature. For both citrus species, storage temperatures of 5 and 1 °C exhibited the slowest calyx browning and abscission. Storage at 5 °C under low ethylene concentration (≤0.1 µL L−1) was found to be more beneficial to calyx senescence than other storage temperatures. Ethanol accumulation in fruit during storage is known to contribute to the development of off-flavours. In this experiment, ethanol production was the higher in ‘Afourer’ mandarin than Navel oranges and storage with high atmospheric ethylene concentrations contributed to higher ethanol levels in the juice. In ‘Afourer’ mandarins, continuous exposure to low levels of ethylene also had a significant effect on both vitamin C concentration and ferric reducing antioxidant power (FRAP) after 8 weeks of storage. Chilling injury was associated with low ethylene concentrations, which only occurred on Navel oranges held at 1 °C. Further studies assessing the effects of low ethylene concentrations and storage temperature on calyx senescence and CI need to be conducted on other citrus species and cultivars to obtain a more comprehensive understanding of individual behavior.Effects on calyx senescence and postharvest quality of ‘Afourer’ mandarins and Navel oranges were investigated as a function of ethylene concentration and storage temperatures. The results showed that continuous exposure to lower ethylene concentrations (≤0.01 µL L


Currently, ribosome-binding protein 1 (RRBP1) is considered to be a novel oncogene that is overexpressed in colorectal cancer, lung cancer, mammary cancer, esophageal cancer and other carcinomas. However, the relationship between RRBP1 and endometrioid-type endometrial carcinoma (EC) remains unknown. Our purpose is to explore the function of RRBP1 in endometrioid-type endometrial carcinoma.n = 35) and normal endometrial tissues (n = 19) by western blotting.We investigated the expression of RRBP1 protein by immunohistochemistry on paraffin-embedded surgical specimens from one hundred thirty patients with endometrioid-type endometrial carcinoma. We also evaluated the differences in RRBP1 expression between endometrial cancer samples (P < 0.05). High levels of expression of RRBP1 were strongly correlated with pathological features, such as the Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, depth of myometrial invasion and lymph node metastasis (P < 0.05). Furthermore, RRBP1 expression was an independent prognostic factor for overall survival (OS) and disease-free survival (DFS) in patients with EC (both P < 0.05).RRBP1 was more highly expressed in endometrial cancer samples than in normal samples (This experiment identifies the utility of RRBP1 in predicting EC prognosis, revealing that it may be a potential target for therapeutics of EC. Endometrial cancer (EC) is the most common gynecological cancer in developed countries . Recent RRBP1 is an endoplasmic reticulum membrane protein that is critical for the transportation and secretion of nascent proteins in mammalian cells . RecentlThis study aimed to determine RRBP1 expression in endometrioid-type endometrial carcinoma and to reveal the connection between RRBP1 and the clinical significance of endometrial cancer.n = 35) and normal tissues (n = 19), were collected and stored at − 80 °C for the western blot experiment.Our research was approved by the Ethical Committee of the Harbin Medical University Cancer Hospital,and informed consent for tissue experiment was obtained from all patients.In this study, we examined one hundred thirty endometrial endometrioid adenocarcinoma patients from January 2010 to May 2013 who were treated at Harbin Medical University Cancer Hospital and underwent hysterectomy, bilateral salpingo-oophorectomy, pelvic and/or paraaortic lymphadenectomy, partial omentectomy, and peritoneal washing for cytology. None of them had received chemotherapy or radiation before the surgery. We assessed the stage of their endometrial cancer based on the International Federation of Obstetricians and Gynecologists (FIGO) . Normal All of the endometrial cancer patients were followed up for survival analysis until December 1, 2016. They were followed-up for a mean of 58.94 months .Fifty four samples were frozen and then homogenized in RIPA buffer , and the desired protein samples were collected. The appropriate amount of sample buffer was added to the collected protein samples and heated at 100 °C in a boiling water bath for 3–5 min to fully denature the protein. After cooling to room temperature, the protein sample was electrophoretically separated on a 10% sodium dodecyl sulfate polyacrylamide gel and then the proteins were transferred to a polypropylene fluoride membrane . After the completion of the transfer of the membrane, it was immediately placed in a previously prepared Western washing solution and rinsed for 1–2 min to wash the transferring solution off the membrane. The antibodies used in this study include anti-RRBP1 antibody  and anti-β-actin antibody .The embedded samples were cut into 4-μm sections and stained with hematoxylin. After dewaxing in xylene, the slides were dehydrated. The sections in 0.3% hydrogen peroxide were incubated in the dark for 10 min at room temperature to block endogenous peroxidase activity and then conducted antigen repair in 6 mmol/L sodium citrate buffer (pH 6.0)  at a temperature higher than 100 °C for 4 min. After washing with Phosphate-Buffered Saline(PBS) the slides were wiped dry. Then, they were placed in a humid chamber and incubated with blocking solution (BSA) for 20 min at room temperature. The blocking solution was blotted off and the sections were incubated with a 1:100 dilution of anti-RRBP1 antibody  overnight at 4 °C. The slides were washed with PBS, wiped and placed in a humid chamber and then incubated at room temperature with the secondary antibody for 20 min. The slide was washed again with PBS, wiped, and placed on a wooden board. Diaminobenzidine (DAB) staining was then performed.The staining was observed under a microscope, and then the staining was stopped by immersing it in PBS. Hematoxylin counterstaining was applied. According to the number of positive tumor cells, the staining was scored as follows: ‘0’ < 5%, ‘1’ 5–24%, ‘2’ 25–49%, and ‘3’ 50–100%. The intensity of the staining was scored as blank (0), weak (1), moderate (2), and strong (3). Based on the percentage of positively stained tumor cells and the staining intensity, a semiquantitative classification of RRBP1 protein expression levels was scored as < 4 indicates low expression and ≥ 4 indicates high expression .We invited two independent pathologists to calculate the immunohistochemistry scores in duplicate for each slide. They were experienced pathologists who were good at assessing immunohistochemistry and were blinded to any clinicopathological information about the slides.Chi-square tests were used to analyze the relationship between RRBP1 and the clinicopathological parameters. The Kaplan–Meier method and log-rank test were used for survival analysis. Cox proportional hazards regression was performed for the multivariate analysis of prognostic factors.P < 0.05, Fig. Western blot analysis showed that RRBP1 was highly expressed in EC tissues and weakly expressed in normal tissues (P = 0.003), deep muscular layer infiltration (P < 0.001), and high histological grades (P = 0.005) (Table P = 0.021) . Through multivariate analysis, we also found that high RRBP1 expression was an independent prognostic factor for both OS and DFS (Table P = 0.033 and P = 0.016).Through log-rank test analysis, we found that high expression of RRBP1 had a strong correlation with poor overall survival (OS) and disease-free survival (DFS) in EC patients . In addition, patients with RRBP1 high expression had a shorter duration of OS than patients with RRBP1 low expression. Thus, RRBP1 may be a valuable biomarker for predicting EC progression and patient prognosis. Our findings are in agreement with the previous studies on the roles of RRBP1 in tumor progression in various cancers, such as lung cancer , breast There is growing evidence that RRBP1 plays a multifaceted role in cancer progression. There is also evidence that RRBP1 is an important ingredient that enhances tumorigenicity both in vitro and in vivo. Knockdown of RRBP1 mRNA in an orthotopic lung model significantly reduced its tumorigenicity . Jen-ChiThis study also has several limitations. First of all, only a relative small sample size was available in our study. Secondly, it is a retrospective study without the mechanism research. The third disadvantage was that only patients with endometrioid-type endometrial endometrioid adenovarcinoma were included in our study. Therefore, a much larger study would needed to effectively test our conclusion, and most importantly, investigate the RRBP1 expression in any of the other histologic subtypes.In summary, this research suggested that overexpression of RRBP1 is closely correlated with a poor prognosis of EC patients. RRBP1 may become a useful target for treating endometrial cancer and a marker for identifying patients with poor prognoses. This conclusion needs additional experiments conducted to develop a better test for the biomarker and to validate the results.This experiment identifies the utility of RRBP1 in predicting EC prognosis, revealing that it may be a potential target for therapeutics of EC.