Minerals stabilize organic carbon (OC) in sediments, thereby directly affecting global climate at multiple scales, but how they do it is far from understood. Here we show that manganese oxide (Mn oxide) in a water treatment works filter bed traps dissolved OC as coatings build up in layers around clean sand grains at 3%w/wC. Using spectroscopic and thermogravimetric methods, we identify two main OC fractions. One is thermally refractory (>550 °C) and the other is thermally more labile (<550 °C). We postulate that the thermal stability of the trapped OC is due to carboxylate groups within it bonding to Mn oxide surfaces coupled with physical entrapment within the layers. We identify a significant difference in the nature of the surface-bound OC and bulk OC . We speculate that polymerization reactions may be occurring at depth within the layers. We also propose that these processes must be considered in future studies of OC in natural systems. Minerals are known to stabilize organic carbon in sediments, affecting biogeochemical cycles and global climate, but the mechanism is not understood. Here, the authors suggest that manganese oxides can trap organic carbon and may act as a ‘mineral pump', transforming carbon between labile and refractory forms. Recent estimates suggest that after rocks (70 million Gt of organic carbon), soils 2,500 Gt) and marine sediments 3,000 Gt) account for the largest reservoirs of organic carbon on Earth1,500 Gt a,000 Gt aet al.et al.Manganese is known to strongly interact with dissolved organic carbon (DOC)56To further add to the complexity of studying mineral–OC interactions in natural systems, most OC in sediments is molecularly uncharacterized carbon (MUC)Traditionally, OC in sediments has been analysed using acids/alkalis or solvents for extraction and then chromatography for characterization, but these methods are flawed due to the changes induced in the organic carbon during extraction11121516Geopolymerization describes the oxidative polymerization of low molecular weight monomers to humic substances that can be catalysed by mineral surfaces2021In contrast to the ‘black box' approach of studying natural systems, we decided to present experimental data from a more controlled non-natural system. Our data are from a clean water treatment works (WTW) where the main inputs and outputs of Mn and DOC are well constrained. The WTW filter bed consists of birnessite-coated sands (confirmed with extended X-ray absorption fine structure (EXAFS) spectroscopy, data not shown), which represent a far simpler and relatively homogenous mineralogy compared with natural marine sediments or terrestrial soils. Studying this simplified system has significant advantages over natural samples, as we are able to look at the specific chemical mechanisms involved in OC stabilization in concentrated mineral assemblages, largely without biotic interference (see below). This approach enables us to show that the birnessite, one of the most common forms of Mn oxide in marine202118−1) from the influent water (see Methods for further details). The influent water contains natural DOC ∼10 mg l−1 with a δ13C signature of ∼−27‰ ref. The birnessite-coated sand is from a clean water treatment works (WTW) gravity filter bed designed to remove soluble Mn (∼0.2 mg lThe surface of the intact birnessite coatings was washed repeatedly in de-ionized water (as a control) and in 1 M NaOH (in an attempt to remove surface bound OC) and analysed by micro-FTIR spectroscopy to observe visible Mn-OC see  to ident−1, which can be attributed to C-H groups−1 after washing in NaOH indicates that not all the C-H bonds are removed, confirming a strong association between this remaining OC and the birnessite.The micro-FTIR images show that OC distribution on the surface of the birnessite coating is not homogenous (−1 can be assigned to the stretching C=O (νC=O)−1 (ref. sym COO-), whereas the shoulder corresponding to the anti-symmetric stretching vibration (νasym COO-) is observed at 1,595 cm−1  such as in alkenes or aromatic rings−1 (mostly attributable to carboxylic groups) is not readily apparent after washing with NaOH, those at 1,565 and 1,400 cm−1 (attributable to carboxylate groups) and those at 1,650 cm−1  are all apparent in the most concentrated spot analyses of the birnessite washed in NaOH −1 ref.  can be am−1 ref.  in the svsym COO-) and asymmetric stretching (vasym COO-) of carboxylate groups and the difference in frequency between them (Δv)31323334v≈165–195 cm−1 suggests that the carboxylate groups could be forming either bridging or bidentate complexes with the Mn-OH functional groups on the birnessite surface, in a ligand exchange complexation reaction313233v≈220  and can be attributed to the presence of hydroxides, alcohols or, more likely, water. The bands observed around 2,550 cm−1  and Peak C (40 nm) fluorophoresDOC, which have a lower aromaticity than the original DOC for both Peak A and Peak CThere is a blue shift in the emission wavelength from original peaty water DOC to Mn-OCTaken together, the FTIR spectroscopy and fluorescence spectroscopy data suggest that the DOC is strongly adsorbed to the birnessite via carboxylate functional groups, and that, as a result of the adsorption reaction, the DOC is at least partially transformed into more aliphatic molecules.m/z 44 (CO2 and N2O—see Methods section) trace. Carbon dioxide (m/z 44) is detected at 200–375 °C  and 550–750 °C . The thermally labile fraction is represented by the pink peak components (labelled nos. 2–4) and represents ∼89% of the Mn-OC, which is available to 800 °C. The thermally refractory fraction is represented by the blue peak component (labelled no. 6) and represents ∼11% of the Mn-OC, which is available to 800 °C. Inorganic carbon (IC) as calcite, with no interference by other decomposition reactions, gives a weight loss at around 720 °C. The purple peak (labelled no. 7) present at 712 °C is assigned to IC breakdown. This is corroborated by an endothermic reaction at 712 °C, visible in the differential scanning calorimetry (DSC) curve  and inorganic  pools based on the TGA (up to 800 °C) of the bulk material is shown in urve see .m/z 18) is detected at 25–200 °C and 200–400 °C and constitutes the largest proportion of the overall weight loss. Water loss is associated with surface dehydration and mineralogical transformation of birnessite4042442  of the TGA data  reveals The peak at 600 °C representing thermally refractory OC has not previously been reported in the literature as soil organic matter or mineral-associated organic carbon12cf. MnO550 Bulk) and consist of only carboxylate compounds, since the carbonate fraction is decomposed at 712 °C  and the bulk birnessite coating thermally treated to 550 °C (MnO550 Bulk) was undertaken see . The Mn-2 °C see . This wa2 °C see . The XPS2 °C see .The bulk birnessite coating as received (MnOAR Bulk) has a C:N ratio of 15:1, which is much lower than the original DOC C:N ratio of 60:1 ref. . This maThe Mn-OC bound at the surface of the birnessite coatings has a significantly lower ratio of alkene/aromatic:aliphatic hydrocarbons (1:4) compared with the Mn-OC present throughout the bulk birnessite coatings (3:2) , which is statistically significantly less depleted in δ13C compared with the influent water (∼−27.0‰). Lalonde et al.13C signature in Fe-OC in marine sediments, which they attributed to selective sorption of an isotopically less depleted fraction of OC by mineral phases. Interestingly, the bulk birnessite coating that has been thermally treated to 550 °C  has a δ13C signature of ∼−27.6‰  that is statistically significantly different from the bulk material as received (P<0.01) but not statistically significantly different from the influent water signature (∼−27.0‰).The bulk birnessite coating as received (MnOAR Bulk) has a δOur data show for the first time that Mn-OC is adsorbed to the surface of birnessite  via a strong manganese carboxylate bonds , point 1Phenoxy radicals are then either converted to quinones, or encounters between phenoxy radicals result in radical coupling to produce dimers or polymeric productsWhen the next layer of birnessite precipitates, DOCx is then trapped and the uppermost surface of the DOCx molecule can adsorb to the new birnessite surface via the manganese carboxylate bonds . Further research is required to confirm whether OC is physically trapped in birnessite found in the natural environment.2 and H2O) and the formation of polymerized products in the bulk material where the concentration of phenoxy radical products can build upFurthermore, we propose that phenol groups within the DOC molecule are oxidatively transformed to phenoxy radicals, following the well-known birnessite-phenol reaction pathwayWe suggest that this initial mechanistic understanding of the role of birnessite in carbon stabilization may help in our understanding of the global carbon cycle.14C analysis was used to confirm that the bulk Mn-OC is 100% modern organic matter to ensure that there was no interference from the anthracite.The birnessite-coated sands come from Mosswood Water Treatment Works (WTW) in the UK (54°51°N-53°59°W). Mosswood is a Mn-removing WTW  that employs chemical oxidation and filtration to remove dissolved Mn(II) from potable water. Such WTWs are prevalent globally in temperate regions where influent water contains dissolved Mn and DOC, which are both problematic for the water industry for aesthetic reasons−1 present as both particulate organic carbon (POC or suspended solids) and dissolved organic carbon (DOC). Dissolved Mn is ∼0.2 mg l−1. Ferric sulphate is added as a coagulant to the influent water at the WTW to remove the majority of suspended solids (both organic and inorganic particulates) in the secondary treatment process before the water enters the Mn filter beds. A flocculant (a modified starch with cationic amine groups) is also added at very low dosage rates  in the secondary treatment process and most of this is removed as flocculated solids known as water treatment residual. Any flocculant remaining in solution could enter the Mn filter beds but the ratio of influent DOC to flocculant is likely to be high and we assume that the flocculant does not constitute a large part of the Mn-OC.Northumbrian Water Ltd. confirmed that these sands were in place for ∼40 years. Replacement of the filter media usually takes place due to drop in Mn removal efficiency either due to lack of porosity or an unknown cause. The influent water at Mosswood (132.5 Ml per day) originates from the Pennines peat uplands and influent water TOC is ∼10 mg l14C analysis was carried out at SUERC (Glasgow University) to confirm that Mn-OC was not anthracite.The birnessite-coated sand was analysed in two fractions, at the surface of the intact coating and at depth that was represented by the bulk material separated from the sand grain. The 63–149 μm fraction of the birnessite coating (captured from sieved intact sand grains by shaking) was (arbitrarily) used to represent the bulk material. All the other size fractions also contained ∼3%w/wC. TC, TIC, TOC and N were determined using a LECO Elemental analyser. Organic carbon (carbonate-free) isotopic abundances were analysed by IsoAnalytical Ltd , using a Europa Scientific 20:20 instrument . Results were recorded relative to the Vienna Peedee Belemnite standard (v-PDB). Samples were acid washed with 2 M hydrochloric acid and analysed by elemental analyser-isotope ratio mass spectrometry. Replication had a s.d. better than ±0.1‰ and values for reference materials were within two s.d. of the expected result. et al.m/z range 10–300). Individual powder samples  were placed within an alumina crucible, using an identical empty reference crucible, and were heated from ambient temperature to 990 °C under flowing He80-O20. Intensities for m/z 44 (CO2 +N2O) and m/z 18 (H2O) were recorded. No correction was made to m/z 44 for evolved N2O, which was assumed to be negligible compared with CO2 given failure to detect measurable m/z 30 (NO). Evolved gas evolution curves were interpreted using GRAMS/AI peak fitting software (www.AdeptScience.co.uk) that permits investigation of the contribution of several individual peaks to an overall trace. By defining initial conditions (including a presumed Gaussian peak shape), the range of potential values for each peak centre  and the number of iterations to perform (in this case 50), the software fits individual labile, recalcitrant and refractory carbon peaks2 trace.Thermal analysis was carried out to determine the relative proportions of different C components according to the method of Lopez-Capel Thermal treatment (to generate MnO500 and MnO1000 samples) was conducted by placing 2 g sample in a ceramic thimble then heated for 8 h in a 6 litre Carbolite ELF muffle furnace and allowed to cool to ambient temperature before being placed in 10 ml glass vials and sealed with Al foil between the screw caps for storage.in situ using a gas injection system. Several in situ polishing steps were performed on exposed cross-sections to produce a clean surface with minimal beam damage. High-resolution backscattered electron imaging was carried out in the same system, with images being captured using a low kV immersion lens.FIB cross-sections were performed using FEI Helios Nano Lab 600 Dual Beam system, equipped with focused Ga liquid metal ion source. Ion beam thinning carried out at 52° with respect to electron column. Initially a ‘rough cut' was carried out after depositing a protective platinum layer SEM imaging was performed on a Hitachi SU70 high-resolution analytical SEM, equipped with Oxford instruments INCA energy 450 and WDS 700. The JEOL 2100F transmission electron microscope was operated at 80 KeV.−1 wavenumber range, at a resolution of 4 cm−1, beam diameter 15 μm.Micro-FTIR analyses were carried out using a Perkin Elmer Spotlight FT-IR imaging system. Reflectance micro Fourier Transmitted Infrared spectra were collected over the 4,000 to 700 cm+ gas cluster ion beam (GCIB) gun at 4 kv for 30 s. The Ag+GCIB sputter etching was carried out in polyatomic mode to avoid degrading the chemistry during the etching process. The samples were then analysed using a Thermoscientific K-alpha X-ray photoelectron spectrometer (XPS) .XPS spectra were obtained at the National EPSRC XPS Users' Service (NEXUS) at Newcastle University. 0.5 grams of sample was immobilized onto a Si wafer using 3 M double-sided adhesive. The immobilized samples were decontaminated within the instrument by sputter etching, using a AgLow-resolution full spectra were acquired using a monochromatic Al Kα X-ray source with an output energy of 1,486.6 eV. An X-ray beam spot size of 50 μm, and a dwell time of 100 ms was used for both high resolution and survey spectra. The surface charge compensation was achieved using a low-energy electron flood gun, which was operated at 40 eV. Survey spectra were obtained between a binding energy of 0 and 1,350 eV with a 1.0 eV step size and pass energy of 200 eV.Analysis of the survey spectra was carried out using CasaXPS software , and major peaks were selected for element identification using the Handbook of X-ray Photoelectron Spectroscopy61The spectral data of the peaks, their components and their backgrounds, from each of the replicates, were acquired from the C1s and the N1s high-resolution scans. These were obtained from the surface fraction, the bulk fraction and the 550 and 1,000 °C thermally treated samples. The digital CasaXPS spectral data were transferred to Microsoft Excel 2010. The mean spectral intensity of replicates and their components were calculated and the mean Shirley backgrounds were deducted. The mean data were then normalized to the highest spectral intensity. The mean normalized spectra and their normalized mean components were then plotted against binding energy.http://dx.doi.org/10.15128/br86b364d.The data presented in this paper are available from How to cite this article: Johnson, K. et al. Towards a mechanistic understanding of carbon stabilization in manganese oxides. Nat. Commun. 6:7628 doi: 10.1038/ncomms8628 (2015).Supplementary Figures 1-2


Helicoverpa armigera”  in vitro synthesized dsRNAs for gene silencing in Helicoverpa armigera. These dsRNAs were specific against H. armigera enzymes/proteins such as proteases like trypsins , chymotrypsin (HaChy4) and cysteine proteases such as cathepsin (HaCATHL); glutathione S-transferases ; esterases ; catalase (HaCAT); super-oxide-dismutase (HaCu/ZnSOD); fatty acid binding protein (HaFabp) and chitin deacetylase (HaCda5b). These dsRNAs were fed to second instar larvae at an optimized dose (60 µg/day) for 3 days separately. Effects of dsRNA feeding were observed in terms of larval mass gain, percentage mortality and phenotypic abnormalities in later developmental stages of H. armigera. These findings might provide potential new candidates for designing sequence-specific dsRNA as pesticide in crop protection.The data presented in this article is related to the research article “RNAi of selected candidate genes interrupts growth and development of  Specialization TableValue of the data•This data will be useful to the other investigators to follow the same system for implementing in the field experiments by using nanoparticles for the stability of dsRNA.•Environmental parameters need to be integrated in these experiments hence environmental factors can be explored for its concrete use in the ground level.•The data support the development of further experiments on the use dsRNA in other devastating insect pests.•Findings from this work will be valuable for designing potent dsRNA as pesticide in crop protection.1viz. dsRNA stability, concentration and amplicon size were assessed based on ingestion by Helicoverpa armigera  , glutathione S-transferases (3), esterases (2), catalase (1)/super-oxide-dismutase (SOD) (1), fatty acid binding protein (1) and chitin deacetylase (1). Green fluorescent protein (GFP) was used as a negative control while glyceraldehydes phosphate dehydrogenase from H. armigera (GAPDH) was used as positive control. Sequence analyses for each of these target mRNA class was performed using CLUSTAL W and primers containing gene-specific sequence adapted to T7 promoter sequence in inverted orientation were designed manually . Once synthesized, dsRNAs were purified and assessed quality and quantity by agarose gel electrophoresis and Nanodrop 1000 spectrophotometer .cDNA synthesized from mRNA of AD and pigeon pea fed larvae and was used for amplification of target regions. These were cloned into pGEMT, confirmed and then linearlized plasmids were used as template for of dsRNA A and B. 2.3Artificial diet (AD) was prepared by the method of Nagarkatti and Prakash 2.3.1HaGAPDH and HaTry3 were used for this analyses. dsRNA (60 µg) was coated on surface of AD cube (~1 cm2) and allowed to diffuse dsRNA in the diet. These cubes were kept at control conditions for insect growth (24 °C temperature and 65% humidity). RNA was extracted from these cubes by using Trizol reagent  after 6, 12, 24 and 36 h of dsRNA coating. The quality, quantity and stability of RNA were determined by agarose gel electrophoresis and spectrophotometric analysis using Nanodrop (Thermo Scientific). It was found that dsRNA was stable up to 24 h . dsRNA was allowed to diffuse into diet and fed to insects. In this way, dsRNA (60 µg/cube of AD) feeding was carried out for selected 17 genes and AD coated with DEPC treated water was used as non-dsRNA control. dsRNA targeting GFP was used as negative control and one targeting GAPDH as a positive control. dsRNA applications were done thrice at 24 h intervals for all target genes, fresh AD cube with fresh 60 µg application was used each time. Following to this, larvae were reared on normal AD and observed for their growth and development till they became pupa. During dsRNA feeding experiments, 25 larvae from each of the treatment groups were harvested in liquid nitrogen at 2, 4 and 8 days post first dsRNA exposure (DPE). Silencing effects were observed in terms of larval mass gain, percent mortality, phenotypic abnormalities, egg laying and reduction of target transcript expression as well as enzyme activity studies. Larval growth was found to be significantly retarded in most of the candidate dsRNA fed insects. This was evident from the reduction in mass gain of the dsRNA fed larvae unlike the control group  and


Fragility fractures caused by osteoporosis affect millions of people worldwide every year with significant levels of associated morbidity, mortality and costs to the healthcare economy. The pathogenesis of declining bone mineral density is poorly understood but it is inherently related to increasing age. Growing evidence in recent years, especially that provided by mouse models, suggest that accumulating somatic mitochondrial DNA mutations may cause the phenotypic changes associated with the ageing process including osteoporosis. Methods to study mitochondrial abnormalities in individual osteoblasts, osteoclasts and osteocytes are limited and impair our ability to assess the changes seen with age and in animal models of ageing. To enable the assessment of mitochondrial protein levels, we have developed a quadruple immunofluorescence method to accurately quantify the presence of mitochondrial respiratory chain components within individual bone cells. We have applied this technique to a well-established mouse model of ageing and osteoporosis and show respiratory chain deficiency. The pathogenesis of declining bone mineral density, frequently culminating in a diagnosis of osteoporosis, is poorly understood. Following attainment of peak bone mass at approximately 30 years of age, a decline of bone mineral density ensues and continues throughout life unabated, universally affecting men and women1356Current theories on the underlying causes of declining bone mineral density include falling production of sex hormones810A growing body of evidence in recent years has suggested a causative link between mitochondrial dysfunction and the ageing process13141617181920−/−) mutator mouse has a mutation in the proof reading domain of the mitochondrial DNA polymerase which leads to the accumulation of mitochondrial DNA mutations at a much faster rate than in wild type counterparts. The prematurely aged phenotype includes a significantly reduced bone mineral density compared to age matched wild type mice24Mitochondrial mutator mouse models provide the strongest evidence of a causative link between mitochondrial dysfunction and an ageing phenotype, including osteoporosis. The polymerase gamma (PolgAc oxidase (COX) and succinate dehydrogenase (SDH) histochemistryAssessment of mitochondrial respiratory chain dysfunction in individual cells in other tissues such as skeletal muscle, myocardium, brain and intestine is well documented. The classical method for assessing the presence of complex IV defects in individual cells, established in 1989, is sequential cytochrome In our endeavours to characterise respiratory chain dysfunction at a cellular level in bone tissue, we found the application of COX/SDH histochemistry to frozen mouse bone to be a challenging method due to loss of tissue morphology and difficulty in identifying individual cell types. We have therefore adapted a method developed for the assessment of mitochondrial respiratory chain components in muscle tissue sections−/−) were generated that had a knock-in missense mutation (D257A) in the second endonuclease proofreading domain of the PolgA catalytic subunit of the mtDNA polymerase, using a C57BL/6 background mouse−/− mice (n = 9) and wild type littermates aged 4 (n = 5) and 11 months (n = 8) was compared. All animal experimental procedures were conducted in accordance with the UK Home Office guidelines and under its approval (licence 60/4540).Mitochondrial mutator mice  for 72 hours at room temperature. The bones were then decalcified for 21 days at 4 °C in 14% tetra-EDTA (Affymetrix), the pH of which was adjusted to 7.4 using glacial acetic acid. The decalcification solution was changed three times a week during this period.The tissue was then embedded in paraffin using a standardised automated process at our institution following which, 4 μm-thick longitudinal sections were cut and mounted on SuperFrost glass slides; these were allowed to air dry for a week.Frozen, calcified mouse femurs from the same animals were used for COX/SDH histochemistry. Whole femurs were embedded in OCT and 6 μm-thick sections were cut using a Leica Cryojane tape transfer system. The tissue sections were left to air-dry for one hour prior to performing COX/SDH histochemistry.c stock solution  with 800 μl of 3,3′diaminobenzidine tetrahydrochloride (DAB)  and few crystals of 20 μg ml−1 catalase. The SDH medium was prepared by combining 100 μl of sodium succinate stock solution (1.3 M sodium succinate in 0.2 M phosphate buffer pH 7.0), with 100 μl phenazine methosulphate (PMS) stock solution (2 mM PMS in 0.2 M phosphate buffer pH 7.0), 10 μl sodium azide stock solution (100 mM sodium azide in 0.2 M phosphate buffer pH 7.0) and 800 μl of NitroBlue tetrazolium (NBT) stock solution (1.5 mM NBT in 0.2 M phosphate buffer pH 7.0).The COX medium was prepared by combining 200 μl of cytochrome The air-dried sections were incubated in COX medium for 40 minutes at 37 °C, washed twice in PBS, then incubated in SDH medium for 30 minutes at 37 °C. Tissues were dehydrated through graded ethanol series and mounted using DPX. Brightfield images of sections were obtained using a Zeiss Axioimager M1.Formalin-fixed, paraffin-embedded sections (FFPE) were dewaxed at 60 °C for 30 minutes then deparaffinised using 2 changes of xylene (Histoclear) and rehydrated through graded ethanol series (100% to 70%). The sections were then immersed in 70% ethanol with 0.25% ammonium hydroxide for 1 hour to reduce autofluorescence, before rehydration was continued in 50% ethanol for a further 10 minutes.Antigen retrieval was performed in a 1 mM tetra-EDTA (Affymetrix) buffer, pH 8.0 at 80 °C for 30 minutes before immediate transfer to phosphate buffered saline (PBS). Sections were then incubated with 10% normal goat serum (in PBS) for 1 hour at room temperature (RT). Mitochondrial endogenous biotin activity was blocked using an avidin and biotin blocking kit (Vector laboratories) as per the manufacturer’s instructions prior to application of Vector’s mouse-on-mouse blocking kit.c oxidase (COX) subunit 1 (COXI). These were applied in combination with a monoclonal antibody against the outer mitochondrial membrane protein porin (VDAC1) . Porin is a nuclear-encoded, voltage gated ion channel present in abundance in the mitochondrial membrane, and its presence serves as a marker for mitochondrial massRespiratory chain complex subunits were detected using mouse monoclonal primary antibodies against NADH dehydrogenase [ubiquinone] I beta subcomplex subunit 8 (NDUFB8) of and cytochrome Following PBS washing, sections were incubated for 2 hours at 4 °C with the following secondary antibodies at a concentration of 1 in 200: 405 Dylight (Jackson) to porin, Alexa 488 (Life Technologies) to COX-I, biotinylated anti-IgG1 (Jackson) to NDUFB8 and goat anti-rabbit Alexa 546 (Life Technologies) to target the cell marker. Sections were then incubated for a further 2 hours at 4 °C in streptavidin-conjugated Alexa 647 (Life Technologies) at a concentration of 1 in 100, before wash steps and mounting using Prolong Gold (Life Technologies).No primary control sections were prepared concurrently using duplicates of each section, omitting application of primary antibodies (apart from the cell marker), in order to enable subsequent detection and subtraction of background signal.Imaging was performed using a Nikon A1 confocal microscope at 60× optical magnification with a 1.55× digital magnification applied, using 405, 488, 546 and 647 nm wavelengths. Sections from wild type mice aged 4 months with primary and secondary antibodies applied, were used to calibrate laser power settings in each of the 4 channels such that the pixel intensity was set with care taken to avoid any areas of under or over pixel saturation within a pixel intensity range of 0 to 4095.Imaris image analysis software  was used to automatically detect osteoblasts (546 nm) and areas within these that were positive for the mitochondrial mass marker porin (405 nm). For each of these areas of intracellular mitochondrial staining, the software provided average signal intensity values for porin (405 nm), COX-I/MTCO1 (488 nm) and Complex I/NDUFB8 (647 nm). An average signal intensity value for each of these components across the whole osteoblast cell surface was provided by the software.In order to calculate non-specific background fluorescence, corresponding no primary control sections were used to obtain average signal intensity levels for 405, 488, and 647 nm wavelengths, using the whole cell surface.The average COX-I, NDUFB8 and porin signal intensities detected within mitochondrial syncytium of each individual osteoblast was recorded. For each channel, the average non-specific background fluorescence measured in the corresponding no primary control section was subtracted. These background corrected values were log transformed, and normality was verified in the 4-month control mice using the Anderson-Darling test.−/−).Data from 4 month old wild type animals were used as the reference control. The following methodology has been previously reportedLinear regressions of COX-I vs porin and NDUFB8 vs porin were also performed on the same pooled control data to yield regression estimates. The linear regressions were used to generate Z-scores for the COX-I and NDUFB8 intensity in each osteoblast based on their expected intensity according to their porin intensity .Osteoblasts were classed as having normal levels of NDUFB8 and COX-I if Z-scores were no more than −3SD from the mean, intermediate (+) if Z-scores were between −3SD and −4.5SD, intermediate (−) if Z-scores were between −4.5SD and −6SD, and deficient if Z-scores were more than −6 SD below the mean. The level of intracellular porin detected was also classified according to Z-scores as “very low” if below −3SD,“low” if between −3SD and −2SD, “normal” if between −2SD and +2SD, “high” if between +2SD and +3SD, and “very high” if above +3SD.http://research.ncl.ac.uk/mitoresearch/).All statistical analysis was performed using RCOX/SDH histochemistry on bone tissue is shown in Different fixation times using 10% NBF in combination with different decalcification methods were trialled . FixatioImmersion of tissue sections in a 0.25% ammonium solution for one hour reduced the degree of autofluorescence to satisfactory levels and was particularly effective in the 405 and 488 nm emission wavelengths as per the findings of previous workApplication of the quadruple immunofluorescence protocol to FFPE mouse bone tissue sections allowed preservation of excellent morphology and accurate delineation of osteoblasts, osteoclasts and osteocytes as well as clear demarcation of mitochondrial syncytium within individual cells, and identification of COX-I and NDUFB8 activity  and 4. W−/− mice (n = 9). The relationship between mitochondrial mass (porin), COX-I and NDUFB8 protein abundance in the 4 month-old mice was linear. The relationship was also linear in 11 month wild type mice though with visibly higher variation, and notably more variable in 11 month PolgA−/− animals with many cells displaying low COX-I and NDUFB8 protein abundance (Wild type mice aged 4 months (n = 5) and 11 months (n = 8) were compared to 11 month PolgAbundance .−/− mice displayed mean COX-I levels 13.4SD lower (p < 0.0001) and NDUFB8 levels 8.2 SD lower (p < 0.0001) when compared to 4 month wild-type counterparts. When age-matched wild type and PolgA−/− mice were compared (both 11 months old), COX-I and NDUFB8 levels in the latter were found to be 5.6SD and 3.9SD lower respectively (p < 0.0001). Numerical data for individual mice is shown in −/− mice.Compared to the 4-month old wild type mice, 11-month wild type mice demonstrated COX-I levels 1.8SD lower on average (p = 0.136) and NDUFB8 levels 1.9SD lower on average (p = 0.014). The 11 month PolgAAs humans age, somatic mitochondrial DNA mutations accumulate13Processing bone for the purpose of immunofluorescence requires consideration of several technical factors. Adequate fixation of bone tissue is imperative to prevent autolysis and maintain morphology of the tissue during the prolonged process of EDTA decalcification. 10% NBF is the most commonly used fixative in laboratory practice, although there is no recommended standard in the literature regarding required fixation times for whole bone, the penetration of which by fixatives is hindered by the hard outer cortex, relying predominantly on Volkmans canals which provide communication between the periosteum and endosteum. Different fixation time of 24, 48 and 72 hours using mouse femurs were trialled and it was observed that with fixation times less than 72 hours, a loss of signal intensity occurs with a fall in signal to noise ratio in all 4 channels when decalcification time was increased by 7 days or decalcification temperature was increased from 4 °C to room temperature. This would suggest that mouse femur is not adequately fixed after immersion in 10% NBF for 24 or 48 hours, the assumption being that loss of signal following these lower fixation times is due to degradation of target epitopes.The method of decalcification is also of vital importance with the use of acid based decalcifying agents being detrimental, leading to increased tissue autofluorescence, increased precipitation of secondary fluorophores within the tissue compared to EDTA decalcification and inactivation of tartrate resistant alkaline phosphatase (TRAP) enzyme activity, rendering any requirement for TRAP staining of the tissue impossible. Although acid decalcification is much faster, our preference was for the use of 14% tetra-EDTA decalcification for 21 days at 4 °C which provided adequate decalcification, allowed easy microtomy and preserved morphology of target epitopes.Antigen retrieval can be particularly problematic with formalin fixed, paraffin embedded bone due to the fragile nature of the tissue and its tendency to separate from the slide. Adequate fixation and decalcification helps to counteract this, as does allowing sections adequate time to dry following microtomy. Pressure cooker or microwave heating mediated antigen retrieval is not suitable due to the tendency of bone tissue to separate from slides under these conditions. Immersion of sections in buffer solution at temperatures greater than 80 °C also caused tissue loss; limiting the temperature to 80 °C for 30 minutes using a water bath proved effective. 1 mM tetra-EDTA was the most effective buffer for this process. It should also be noted that the form of EDTA used for antigen retrieval is of great importance with mono EDTA and di-EDTA being comparatively poor, especially for NDUFB8 epitope retrieval. Enzyme antigen retrieval had a detrimental effect on tissue morphology and was not as efficient at unmasking the mitochondrial epitopes of interest compared to heat induced epitope retrieval methods. Overnight incubation of primary antibodies at 4 °C and subsequent 2 hour incubations with secondary antibodies at the same temperature provided a more homogenous signal throughout the entirety of the tissue section and increased signal to noise ratios.et al.33Bone is naturally a very autofluorescent substance particularly when illuminated with light emanating from the near visible ranges of the spectrum, primarily due to collagen. This leads to a reduction in the signal to noise ratio of images obtained and steps are required to counteract this. We have been unable to find any previous bone studies using four channel autofluorescence, possibly because of this difficulty. The use of an epi-fluorescent light source as opposed to a laser point scanning confocal microscope exacerbates the problem significantly, yet the use of the latter is mandatory for high quality imaging and accurate assessment. Immersing sections in 0.25% ammonia/70% ethanol was successful in reducing autofluorescence. This is a method initially introduced by Kardasewitsch in ref. We used a semi-automated method of quantification in which software identifies not just cells of interest but also mitochondrial syncytium within them, and provides signal densitometry values for expression of porin, COX-I and NDUFB8 in each cell. The values for COX-I and NDUFB8 are recorded relative to signal intensity of porin . The ability to incorporate an antibody to target porin is invaluable and provides a more accurate means of quantifying COX-I and NDUFB8 expression, removing problems of intra- and inter- observer variability seen with the method of COX/SDH histochemistry.−/− mice. It is also of note that data from wild type mice aged 11 months show evidence of respiratory chain deficiency compared to their younger counterparts. Both of these findings are of interest as they provide evidence for a potentially significant role in increasing mitochondrial dysfunction occurring with age at a cellular level and potentially diminishing bone mineral density, a universal process associated with ageing.Application of this novel quadruple immunofluorescence protocol to formalin fixed, paraffin embedded bone tissue allowed morphology preservation, clear delineation of cells, and quantification of respiratory chain subunits within. The method has shown for the first time that osteoblasts are a cell type that is vulnerable to mutations in a mitochondrial mutator mouse model, with significant COX-I and NDUFB8 deficiencies observed at 11 months in PolgA−/− mice. Diminishing respiratory chain function seen in osteoblasts may play a significant role in the pathogenesis of bone loss with age.In conclusion, our data shows that this quadruple immunofluorescence method is superior to standard COX/SDH histochemistry for use in bone as it eliminates much of the variability in result interpretation and can easily be applied to formalin fixed paraffin embedded sections of bone, allowing clear delineation and characterisation of the various cell types present. We have used the method to demonstrate respiratory chain deficiencies in the osteoblasts of aged and PolgAHow to cite this article: Dobson, P. F. et al. Unique quadruple immunofluorescence assay demonstrates mitochondrial respiratory chain dysfunction in osteoblasts of aged and PolgA−/− mice. Sci. Rep.6, 31907; doi: 10.1038/srep31907 (2016).


T. brucei cathepsin B from 80 in vivo grown crystals with an average volume of 9 µm3 obtained by serial synchrotron crystallography at a microfocus beamline is reported.The structure solution of  Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80  Since the 1990s, advanced third-generation synchrotrons have been used to produce micrometre-sized high-flux X-ray beams whose focus size matches the size of small crystals , ultimately reducing the crystalline order and corresponding to increasing levels of ‘radiation damage’. As a consequence, even under cryogenic conditions, the quality of the diffraction data, in particular those at high resolution, quickly deteriorates as a function of the X-ray dose applied to the crystal. Since the earliest days of macromolecular crystallography, this problem has been addressed by combining partial data sets collected from multiple crystals into a full data set to high resolution. In the 1990s cryogenic cooling became routine in macromolecular crystallography  and invisible to optical light because the lipidic mesophase in which the crystals grow turns opaque upon cryogenic cooling. Recently, an alternative procedure was proposed in which radiographs are taken of the entire sample mount to localize crystals . Cherezov  al. 2009 describe al. 2009 from poset al., 2000et al., 2012et al., 2012et al., 2011Cheetah, CrystFEL , have recently been shown to overcome the dose limitations mentioned above. Referred to as ‘diffraction before destruction’, the inertia of the atoms prevents significant atomic displacement during the short exposing pulse (Neutze Trypanosoma brucei procathepsin B (TbCatB) in complex with its native propeptide represents the first novel bioinformation obtained by applying the SFX approach at a free-electron laser . Prior to diffraction data collection, the suspension of needle-shaped crystals containing approximately 5 × 108 crystals per millilitre with average dimensions of 0.9 × 0.9 × 11 µm (approximately 9 µm3 in volume) was supplemented with 40%(v/v) glycerol as a cryoprotectant. A small volume of approximately 13 nl of the crystalline suspension was mounted after settling in a standard 20 µm thick nylon loop  containing approximately 5000 crystals.Spontaneous crystallization of TbCatB was obtained in baculovirus-infected Sf9 insect cells following our previously established protocol  and kept at 110 K using a gaseous nitrogen stream . Diffraction data were recorded on a PILATUS 6M-F detector .Diffraction experiments were conducted on the P14 microfocus beamline at the PETRA III storage ring  with a 4 × 5 µm (FWHM) microfocus beam, a total photon flux of 1.2 × 10viaRADDOSE . Using a series of short rotation exposures, by visual inspection of diffraction images and using the ability to index them as a criterion (see Supporting Information), we empirically determined a crystal lifetime of 0.5–1 s under the conditions at hand. This observed crystal lifetime corresponded well to the lifetime of 0.88 s calculated To collect a complete data set, we employed a data-collection strategy in which a region of interest of 600 × 600 µm was raster scanned with rotation exposures Fig. 2. 120 par2.2.CrystFEL software suite . These groups were further treated as regular rotation data for re-indexing and integration applying XDS  apparently reflects the variation in the crystal size in TbCatB preparations 〉 statistics and on the basis of the CC* criteria recently advocated as a single statistically valid guide for deciding the resolution cutoff of the obtained data  shares the papain-like fold which is characteristic of cathepsin B enzymes, including the propeptide residues 27–72 and 79–85 without defined electron density in between, as well as a carbohydrate chain consisting of two N-acetylglucosamine (NAG) monomers N-linked to Asn58 (in the propeptide) and another carbohydrate chain consisting of two NAG monomers and one β-mannose (BMA) molecule N-linked to Asn216 of the enzyme domain. Overall, the 3.0 Å resolution electron-density map is well defined by the TbCatB model. No electron density is observed for nine flexible amino-acid side chains mainly located within a loop region spanning residues His195–Asn209 or for ten atoms of the carbohydrate structures. In particular, as for the SFX structure determination, the expected features of the electron-density map that were not part of the search model are well defined by the propeptide and two carbohydrate chains after manual model building and refinement . A slight shrinking of the unit-cell parameters of the TbCatB in vivo crystals observed for the synchrotron data set . Slight differences were only observed for the second N-acetylglucosamine residue of the propeptide carbohydrate, which represents the most flexible carbohydrate within the model. This is further reflected by the almost identical number of amino-acid side chains/carbohydrate atoms not defined by electron density in both TbCatB structures (nine side chains and ten carbohydrate atoms in this structure versus 11 side chains and eight carbohydrate atoms in the SFX structure).For detailed comparison of the t Table 1 can be aBSG〉 = 57 Å2) is higher than that averaged over all atoms (〈BAll〉 = 41 Å2). In the SFX TbCatB structure refined using an identical protocol at 3.0 Å resolution, 〈BSG〉 (38 Å2) is lower than 〈BAll〉 (45 Å2). This observation is consistent with a significant diffraction contribution from reduced disulfide bonds in the synchrotron data but not in the SFX data.Despite the overall similarity in atomic coordinates, systematic differences were observed in the relative heights of the electron-density peaks at the 12 Cys SG atoms involved in disulfide bridges. Considering refined Debye–Waller factors as an (anticorrelated) measure of the height of electron-density maxima, we note that in the synchrotron structure the average Debye–Waller factor  than those of cypovirus polyhedra (CPV) studied by Metcalf and coworkers . In addition, the TbCatB crystals are embedded in a matrix giving rise to a high scattering background, similar to the situation of GPCR crystals mounted in LCP, where the crystals are commonly of larger volume  crystals. Parameters can be tuned to maximize accuracy  or to maximize resolution .The method presented here is conceptually simple and could be implemented at many microfocus synchrotron-radiation beamlines employing existing helical scan schemes. It lends itself to data collection on small crystals in suspension, such as those obtained from et al., 2011In addition to the promising application as a standalone approach, the combination of serial synchrotron and SFX data collected for a given crystallized protein further offers a new strategy for scaling and phasing of SFX data. In comparison to SFX data collection, serial synchrotron crystallography allows the extraction of accurate diffraction data, albeit to lower resolution owing to the finite rotation range and the onset of radiation damage during the exposure, from a small number of microcrystals by the systematic acquisition of structure-factor amplitudes followed by the application of well defined scaling models modelling a finely controlled experimental process. In contrast, at present, a three orders of magnitude larger number of microcrystals is required for the convergence of Monte Carlo intensity integration when arithmetic means of partially recorded intensities are used without scaling (Kirian cathepsin B, 4n4z PDB reference: 10.1107/S2052252513033939/jt5002sup1.pdfSupporting information. Includes Table S1 as well as Figs. S1, S2 and S3, which provide details of the quality measures of the diffraction data set, an exemplary diffraction pattern, the size distribution in the groups of adjacent indexed frames and details of the structural comparison of the T. brucei procathepsin B structure determined by SFX using free-electron laser radiation and by serial synchrotron crystallography in this study.. DOI:


Synechocystis sp. PCC 6803 ΔglgC strain incapable of glycogen storage. When deprived of nitrogen, the ΔglgC excretes the organic acids alpha-ketoglutarate and pyruvate for a number of days without increasing cell biomass. This study examines the relationship between the growth state and the photo-catalytic state, and characterizes the metabolic adaptability of the photo-catalytic state to increasing light intensity. It is found that the culture can transition naturally from the growth state into the photo-catalytic state when provided with limited nitrogen supply during the growth phase. Photosynthetic capacity and pigments are lost over time in the photo-catalytic state. Reversal to growth state is observed with re-addition of nitrogen nutrient, accompanied by restoration of photosynthetic capacity and pigment levels in the cells. While the overall productivity increased under high light conditions, the ratio of alpha-ketoglutarate/pyruvate is altered, suggesting that carbon partition between the two products is adaptable to environmental conditions.A key objective in microbial biofuels strain development is to maximize carbon flux to target products while minimizing cell biomass accumulation, such that ideally the algae and bacteria would operate in a photo-catalytic state. A brief period of such a physiological state has recently been demonstrated in the cyanobacterium  PCC 6803 (hereafter referred to as S6803) to make it incapable of glycogen storage by deleting the gene at locus slr1176 (glgC), which encodes a glucose-1-phosphate adenylyltransferase. When deprived of nitrogen, the wild-type (WT) strain increases cell biomass with glycogen accumulation, while the mutant instead produces and excretes alpha-ketoglutarate and pyruvate without biomass increase. The mutant continues to photosynthesize at rates similar to WT  and utilize newly fixed atmospheric carbon, instead of stored carbon, to produce the excreted keto acids . Figure Finally, we supposed that under certain conditions, light should be limiting for photo-catalytic production of organic acids and that higher light flux should result in faster generation of products. This result has been observed in many other cyanobacterial systems  and two additions: 20 mM sodium phosphate (pH 7.1) and 5 μg ml−1 gentamicin. The media were typically prepared without NaNO3, which was added separately to the indicated final concentrations as needed following preculture biomass accumulation in complete medium (17.6 mM NaNO3), harvesting by centrifugation (4000 × g) and washing with nitrogen free BG-11. For all experiments, cultures were maintained in 250 ml Erlenmeyer flasks in growth chambers in the presence of 5% CO2 enriched air atmosphere. Continuous (24 h) illumination was provided by cool white fluorescent bulbs with a light flux of ∼50 μE* m−2s−1 at the surface of the flasks or by cool white LEDs at light intensities as high as 300 μE* m−2s−1 at the surface of the flasks.Cultures of S6803 Δ−1 and detecting with UV detector at 210 nm. Peak retention times and signal integrals were compared against known standard solutions prepared in modified BG11 media with addition of purchased standards of sodium alpha-ketoglutarate and sodium pyruvate . No peaks were observed by high-performance liquid chromatography other than from alpha-ketoglutarate and pyruvate that were attributable to biologically produced compounds. All organic acid measurements were determined as the average of at least three independently grown cultures from identical conditions with error bars in figures representing ± 1 standard deviation.Aliquots (1 ml) of culture were harvested and centrifuged at 13 000 × g. Supernatants were filtered through 0.20 micron filters prior to injection (100 μl) in an Agilent Technology 1200 Series HPLC fitted with Bio-Rad Aminex HPX-87H column, and eluting with 5 mM sulfuric acid at a flow rate of 0.6 ml minCulture aliquots (1 ml) were measured in a Beckman Coulter DU 800 Spectrophotometer for whole-cell absorbance wavelength scans or scattering at 730 nm. All optical density measurements were determined as the average of at least three independently grown cultures from identical conditions with error bars in figures representing ± 1 standard deviation.D. Carrieri and T. Paddock are now employed at Matrix Genetics, LLC, a company that engineers cyanobacteria strains of commercial interest.


We programmed the device to 24 discrete resistance levels, and also demonstrated over a million (220) epochs of potentiation and depression, suggesting that our devices can be used for both multi-level non-volatile memory and neuromorphic computing applications. More importantly, we directly observed a sub-10 nm Ta-rich and O-deficient conduction channel within the HfO2 layer that is responsible for the switching. This work deepens our understanding of the resistance switching mechanism behind oxide-based memristive devices and paves the way for further device performance optimization for a broad spectrum of applications.Memristive devices are promising candidates for the next generation non-volatile memory and neuromorphic computing. It has been widely accepted that the motion of oxygen anions leads to the resistance changes for valence-change-memory (VCM) type of materials. Only very recently it was speculated that metal cations could also play an important role, but no direct physical characterizations have been reported yet. Here we report a Ta/HfO We also programmed the device to 24 resistance levels with long retention by controlling the compliance currents (CCs). Furthermore, we demonstrated over a million (220) potentiation and depression epochs with this device using electrical pulse trains. Using scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS), we directly identified a Ta-rich and O-deficient conduction channel that is responsible for the resistance switching. Finally, we proposed a physical model and attributed the switching behavior to the composition modulation of a sub-10 nm conduction channel implemented through the motion of both cations and anions in the oxide layer driven by electric field and thermal effect.We herein report a Ta/HfO2 and a 50 nm thick Ta top electrode (TE) (see Methods for fabrication details). HfO2 was chosen as the switching layer because it is widely used as the gate dielectric in metal oxide semiconductor field effect transistors. Our cross point device consisted of a 20 nm thick Pt bottom electrode (BE), a 5 nm thick HfO2 device can be reversibly switched within 5 ns. It needs to be pointed out that the switching speed measurement was limited by the equipment rather than the device itself. A faster speed could potentially be achieved with an increased pulse amplitude if a shorter pulse were available and a special device structure was designed to allow for the transmission of higher frequency signal to the junction area. For endurance measurement, we used 100 ns pulses , suggesting even better endurance is possible if a verify-write algorithm were adoptedThe device can be switched reliably between the HRS and LRS states using 5 ns electrical pulses of 2.2 and −4 V pulse amplitude for SET and RESET, respectively. The resistance read at 0.1 V DC voltage after each switching event is plotted in 2/Pt device also showed excellent retention properties (6 s) without noticeable degradation at room temperature. To further evaluate the retention properties, temperature dependent measurements were carried out for the devices. 5, 7.5 × 104, 1.4 × 104, 2.7 × 103, and 1.3 × 103  s at 250, 275, 300, 325, and 350 °C, respectively  are relatively poor2324a from 2O5−x/TaO2−x/Pt2/TiNx devices26The impressive retention performance is fairly surprising given that previously reported retention results for HfO4 s, confirming the nonvolatile behavior and good retention properties. Precise tuning of the resistance into even more states could be implemented through a circuit with one transistor-one memristor (1T1M) configurationMultiple resistance states have been achieved for the device by using different compliance currents and stop voltages during programming. As shown in 2/Pt device, and over 220 (>1 million) potentiation/depression epochs have been demonstrated  under electrical and thermal effects. As shown in x based devices30−4/K for LRS and −4.37 × 10−4/K for HRS. The different signs of the TCR at LRS and HRS suggest the switching was caused by the modulation of the conduction channel composition while not the channel size (TCR is independent of geometry)2/M2 stack is a leading candidate for memory applications and thus one of the most extensively used stack, where M1 is a relatively inert metallic layer, such as Pt or TiN, and M2 is a reactive metal layer, such as Hf, Ta and Ti232 devices when mechanisms and performances are discussed. However, the impressive retention observed in our device that is comparable to that for TaOx devices instead of HfOx devices26x device and the reactive metal electrode plays a more important role than expected in determining the device properties. Specifically, in our device stack, it is highly likely that Tantalum cations have migrated into the HfO2 layer and directly contributed to the formation of conduction channel(s).We believe the switching of our Ta/HfO2/Pt device with a 3 μm diameter Ta via as the top electrode. A thicker (10 nm) HfO2 was used to provide sufficient material volume for a better TEM characterization. The device was repeatedly switched for several cycles and left at LRS before being cut using a focus ion beam (FIB) microscope. During the resistance switching cycles, a small part of the device was deformed because of Joule heating and the evolution of compressed oxygen bubble, consistent with previous results331.7 (Z is the atomic number of the species) and the atomic density. Typical core-loss EELS spectrum taken at the unchanged HfO2 layer has a dominant Hf-M edge peak at (1716 eV) while that from Ta electrode has a typical Ta-M edge peak at (1797 eV)  and electron energy loss spectroscopy (EELS) analyses were conducted for a Ta/HfO1797 eV) . The EEL1797 eV) . Similar1797 eV) . Core-lo1797 eV) . The con2/Pt device can be explained as follows. During the forming step, Ta is oxidized into Tax+ when a positive voltage is applied on it. Due to the strong electric field and concentration gradient, the mobile Tax+ cations migrate into the HfO2 layer and serve as dopants. In the meantime, O2− anions are attracted towards the Ta TE, which introduces O vacancies (VOs) as dopants in the oxide layer. This is similar to the well-studied Ta anodizing process, in which case the tantalum oxide growth on the surface is attributed to both the inward migration of O anions to the metal/oxide interface and the outward migration of Ta cations to the oxide/solution interface193536235x+ into and O2− out of the HfO2 layer increases the doping levels of Ta and VO to the HfO2 layer and finally form localized conduction channel(s), bringing the device to LRS. In the first RESET process, a negative bias is applied at the Ta TE. In the vertical direction (perpendicular to the electrode/oxide interfaces), electric field drives O anions toward Pt BE while pulls Ta cations back toward Ta TE. The motion of both ions leads to lower Ta but higher oxygen concentrations in the conduction channel, and hence a more resistive device that is at HRS. On the other hand, a positive voltage during the first SET process reverses the process and turns the device back to LRS. It is worth noting that although it is easier to understand the channel composition modulation through the electric field induced vertical drift, thermally enhanced lateral diffusion also plays an important role for the device operation2/Pt device to the growth and reoxidation of Ta-rich and O-deficient conduction channel(s) through motions of Ta cations and O anions. This theory is very different from previously popular switching mechanism for HfO2 based devices, in which only O anions at the electrode/oxide interface contributes to the switching while the electrodes  only serve as the oxygen gettering layer2 device leads to similar switching behaviors (such as long retention and high endurance) as TaOx-based memristive devices10In-situ TEM studies on nanoscale vertical devices are desired in the future to uncover the whole dynamic switching process .Based on the electrical measurements and physical characterization, the switching mechanism of the Ta/HfO2O5: −760 kJ mol−1 and Ag2O: −60 kJ mol−1)x) while that between Ta and O is very small. As a result, motion of Ag or Cu ions is more preferable than O anions in ECM devices. The transition from VCM- to ECM- type switching behaviors in typical VCM material system (Ta/TaOx/Pt) has been achieved by inserting a thin amorphous carbon layer at Ta/TaOx interfacex to suppress the role of O anionsFinally, although we directly observed Ta cations migration, the co-existence of O anion movement makes our device fundamentally different from traditional ECM devices based on motion of Ag or Cu ions. The difference is a result from a number of reasons. First, Ta is more easily oxidized than Ag due to the much stronger negative Gibbs energy for the formation of oxides (Ta2/Pt memristive device with fast switching speed (≤5 ns), record high endurance (1.2 × 1011 cycles), and reliable retention (extrapolated to be ≫10 years at 85 °C). We also achieved 24 non-volatile long-retention resistance states by controlling compliance current during DC programming, and over a million (220) potentiation and depression epochs using electrical pulse trains. The device performance suggests that our devices can be used for both memory and computing applications. More importantly, we studied the switching mechanism and attributed the switching to the composition modulation of a sub-10 nm Ta-rich O-deficient conduction channel by both anion and cation migration under electric field and thermal effect. The results strongly suggest that the reactive metal electrode in the oxide based memristors plays a much more important role than previously expected in determining the switching mechanism and device performance. We further built a model that successfully explains the excellent behavior of our device. This work broadens our understanding of the resistance switching mechanism behind oxide-based memristive devices and paves the way for further device performance optimization and applications.We have developed a Ta/HfO2 on top as the substrates. For the 10 × 10 μm2 micro-devices, the bottom electrodes were patterned by ultraviolet photolithography. After that, 1.5 nm Ti/20 nm Pt were deposited sequentially by electron beam evaporator, followed by a lift-off process in acetone. A 5 nm HfO2 blanket layer was prepared by atomic layer deposition (ALD) using water and tetrakis(dimethylamido)hafnium as precursors at 250 °C. The 50 nm thick Ta top electrodes were defined by a second photolithography step and a 15 s O2 descum, metallization using DC sputtering and liftoff. The devices for FIB cutting were prepared in the similar fashion, except that a blank 20 nm Pt/1.5 nm Ti layer was deposited as the BE, followed by the ALD of 10 nm HfO2. A 40 nm thick SiO2 was sputtered on as isolation layer and a 3 μm wide via hole was opened using photolithography and reactive ion etching before the 50 nm thick 75 μm wide Ta pad was deposited as the TE.We used Si wafers that have 100 nm thermally grown SiOThe DC electrical characterizations were carried out with an Agilent 4156B semiconductor parameter analyzer in a voltage-sweep mode. Pulse measurements for switching speed, cycling endurance and analog switching were conducted with an Agilent 81160a pulse generator. The devices were programmed to ON or OFF states and the resistance was read at 100  mV DC voltage between switching events. The retention tests at 250, 275 and 300 °C were performed on a Cascade Summit 11000 probe system equipped with a thermal chuck . The retention performances at 325 and 350 °C were measured in a variable temperature micro probe system (MMR Technology) . The device resistances were periodically monitored by Agilent B1500 at different temperatures in every 60 s with a low read voltage  to avoid disturbance of the device states. For all the electrical measurements, the bottom electrodes were grounded while the top electrodes were biased.The HAADF-STEM images, and EELS analysis were acquired in an aberration-corrected Hitachi HD2700C Scanning Transmission Electron Microscope operated at 200 keV. The FIBed TEM cross-section samples were prepared with FEI Helios 600 nanoLab.How to cite this article: Jiang, H. et al. Sub-10 nm Ta Channel Responsible for Superior Performance of a HfO2 Memristor. Sci. Rep.6, 28525; doi: 10.1038/srep28525 (2016).


This article contains the data related to the research article “in vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay”   Specifications TableValue of the data•The image data provide various phenotypes of the GFRα1-positive stem/progenitor spermatogonia near GDNF-soaked beads which were transplanted into the interstitium of mouse testes in vivo.•These image data are useful for the estimation of the active state of GFRα1-positive spermatogonia by comparing their phenotypic similarities to those in other testes under various conditions.•These data allow researchers to elucidate the effects of various growth factors in mammalian spermatogenesis in vivo.•This bead transplantation technique will be helpful for the preservation and renewal in sub- and infertile males of various mammal species.1A novel bead transplantation assay into mouse testicular interstitium  [1] prov22.1Wild-type male mice  were used. All animal experiments were performed in strict accordance with the Guidelines for Animal Use and Experimentation at the University of Tokyo .2.2Affi-Gel blue beads  were soaked in a solution of recombinant GDNF  for 1 h at room temperature. To mark the tubular wall adjacent to the transplanted beads, the beads were immersed in DiI  solution for 15 min.For transplantation, the adult testes were gently extracted from the abdominal cavity under anesthesia, and then the GDNF- or BSA (negative control)-soaked beads were transplanted into the testicular interstitium  via vitrified micro-capillary under a dissecting microscope.2.3For whole-mount immunostaining, seminiferous tubule fragments around the transplanted beads were isolated, fixed in 4% PFA-PBS for 8 h at 4 °C, and washed with PBST, as described previously


During another set of experiments, nitrification rates exhibited a diel periodicity throughout much of the photic zone, with the highest rates occurring at night when competition with phytoplankton is lowest. Together, the results of our experiments indicate that nitrification rates in the photic zone are more strongly regulated by competition with phytoplankton for ammonium than they are by light itself. This finding advances our ability to model the impact of nitrification on estimates of new primary production, and emphasizes the need to more strongly consider the effects of organismal interactions on nutrient standing stocks and biogeochemical cycling in the surface of the ocean.Nitrification, the microbial oxidation of ammonium to nitrate, is a central part of the nitrogen cycle. In the ocean’s surface layer, the process alters the distribution of inorganic nitrogen species available to phytoplankton and produces nitrous oxide. A widely held idea among oceanographers is that nitrification is inhibited by light in the ocean. However, recent evidence that the primary organisms involved in nitrification, the ammonia-oxidizing archaea (AOA), are present and active throughout the surface ocean has challenged this idea. Here we show, through field experiments coupling molecular genetic and biogeochemical approaches, that competition for ammonium with phytoplankton is the strongest regulator of nitrification in the photic zone. During multiday experiments at high irradiance a single ecotype of AOA remained active in the presence of rapidly growing phytoplankton. Over the course of this three day experiment, variability in the intensity of competition with phytoplankton caused nitrification rates to decline from those typical of the lower photic zone (60 nmol L Accounting for this process is therefore needed in order to make accurate estimates of new primary production The quantity of nitrogen (N) supplied to the sunlit layers of the ocean regulates levels of primary production and phytoplankton community composition −3 in the photic zone is regenerated within it by nitrification, causing model-based estimates of oceanic new production to be 1.5 to 3-fold higher than actual Despite decades of observations of nitrification in the photic zone amoA mRNA transcripts) required to carry out this process It has long been believed that nitrification is regulated by light in the photic zone of the ocean. Primary support for this hypothesis comes from repeated reports of nitrification rates being low in the surface mixed layer and then increasing exponentially with depth, as irradiance intensity decreases, to a maximum near the photosynthetic light compensation point (1% blue light) +4) +4 plays a larger role than light itself in determining the distribution of nitrification rates and activity of the AOA in the photic zone of the ocean.As the ultimate driver of photosynthesis, light intensity in a given ocean layer influences rates of primary production as well as the rate that N, a widely limiting and essential macronutrient, is withdrawn from seawater by phytoplankton +4. In upwelling systems, disphotic waters, high in nutrients and free of growing phytoplankton, are transported to the surface layer where they are first exposed to light and then, a few days later, rapidly growing phytoplankton Isolating the influence of light or competition on nitrification remains difficult, because their intensities co-vary in stratified surface waters amoA) genes and mRNA transcripts associated with three major groups of marine ammonia oxidizing microorganisms (AOM), ‘Water Column A’ (WCA) and ‘Water Column B’ (WCB) ecotypes of the AOA and betaproteobacterial ammonia oxidizing bacteria (β-AOB), confirmed that putatively light-sensitive AOM were present and active at the start of our experiments (amoA gene (79%±20%) and transcript (88%±5%) pools in all samples  and photoperiod (12 h) of the surface layer , comprised mainly of centric diatoms (42% of biomass) ; low chlce layer .−1 of NO−3 in ‘surface’, ‘deep’ and ‘bloom’ treatments, respectively  by Day 1 in ‘surface’, after which no activity was detected. The ‘deep’ and ‘bloom’ had remarkably similar initial rates of 57 and 58 nmol L−1 d−1. By the end, rates declined to 29 nmol L−1 d−1  in ‘deep’ and to 0.3 nmol L−1 d−1  in ‘bloom’  for the shared substrate and the half-saturation value (Km), or the substrate concentration supporting half the maximum rate of nutrient uptake max or Km, was most important in determining the outcome of our experiments can be addressed by considering the results of the multiday enclosures , isolated from aquarium sediments +4 concentration in both ‘surface’ and ‘bloom’ treatments , 15% (0.057 of 0.39 µmol L−1) and 17% (0.058 of 0.34 µmol L−1) of the substrate pool was oxidized during the first day of the experiment in ‘surface’, ‘deep’ and ‘bloom’  +4 from remineralization, that influences nitrification rates in the photic zone.Interpreted in the context of the Michaelis-Menten model, the linearity of the rate-substrate relationship in our dataset  shows no+4 concentrations and phytoplankton abundances varied, meaning the data are not the result of a classic kinetics experiment +4 concentrations <10 nmol L−1. If the AOA have complete command of their substrate pool, the basic tenets of the kinetic model still hold true, whether phytoplankton are present or not. In our results, this would be indicated by nitrification rates increasing with substrate concentrations to asymptotically approach a maximum rate, as has been observed at much lower substrate concentrations in dark kinetics experiments −3 depletion were lowest . Once the primary sink is saturated, the residual NH+4 becomes available for nitrification.The substrate-rate relationship  was detee lowest , nitrifie lowest , and thee lowest . Yet, conscripts  and ratenscripts  decrease2 = 0.93, P<0.01) between rates of nitrification and N depletion by phytoplankton, calculated as changes in concentrations of NH+4+NO−3 between days , leaving incrementally less NH+4 available for nitrification  (−1 d−1) to those of well-lit layers (<1 nmol L−1 d−1) The results presented here are further evidence activity  and rateactivity  during od (≤17%) . Surprisd (≤17%) , when nod (≤17%) . Within +4 by maintaining a lower Km and higher Vmax for NH+4+4 under these circumstances, consistent with decreasing rates of nitrification in the photic zone between coast and gyre Compared to the coastal ocean, phytoplankton in the oligotrophic ocean have adapted to the extremely low availability of NHWestern Flyer during October of 2011 at stations M1 , M2  and 67–70  within and offshore of Monterey Bay (Northeast Pacific Ocean), as part of MBARI’s CANON initiative (cruise: CANON11). Seawater samples were collected using a previously described −3, SiO4−4, PO3−4 and NO−2 concentrations were frozen at sea and transported to shore, where macronutrient concentrations were determined following previously described colorimetric methods +4 concentrations were measured in unfiltered seawater samples immediately after collection by fluorescence, as described previously Sample collection and experimentation were done aboard the R/V −3 and NH+4 concentrations, using the same methodologies described above. Due to the water constraints during the multiday experiments, chlorophyll concentrations were measured at sea on Days 0 and 3 and estimated for Days 1 and 2 assuming 1 µg L−1 chlorophyll accumulated for every 1 µmol L−1 of NO−3 depleted Waters used for multiday (competition) experiments were collected at station M1  from 2 m and 60 m. Upon return to deck, 2 m (‘surface’) and 60 m (‘deep’) waters were drained directly from the rosette into triplicate, acid rinsed 4 L polycarbonate bottles (3 per depth). To a third set of bottles, 0.4 L of 2 m water (10% of volume) were added to 3.6 L of 60 m water (90% of volume), to mimic mixing following an upwelling event (‘bloom’). The resultant 9 bottles (3 treatments×3 replicates) were subsampled for determination of chlorophyll, NOFollowing the initial subsampling, bottles from all treatments were placed into an on deck incubator cooled with ambient surface waters (12.5–16°C). The incubator was covered with neutral density screening, which attenuated incident irradiance by 50% . The intensity of irradiance reaching the deck incubator was monitored throughout our experiments using a photosynthetically active radiation (PAR) sensor  affixed to the top of the incubator. The irradiance intensities during the experimental period, including start and end times, are shown in 15NH+4 tracer was added to each of the 280 ml incubation bottles , after which the samples were mixed by inversion and placed in the on deck incubator for 24 hours. Subsamples (50 ml) for determination of the 15N enrichment of the NO−3+NO−2 pool, taken at 0 and 24 h, were passed through a 0.2 µm pore size syringe filter and stored frozen (−80°C) until analysis. At the end of the experiment, on Day 3, the water remaining in the 4 L incubation vessels (1 L) was filtered to harvest bacterioplankton biomass for later molecular genetic analyses (described below).Subsamples for determination of macronutrient and chlorophyll concentrations were taken on each day of the experiment, according to the methods described above. Additionally, an aliquot for water from each of the 4 L enclosures was transferred to clear 280 ml polycarbonate bottles for determination of nitrification rates. 15NH+4 tracer to a final concentration of 0.5 µmol L−1, after which bottles were held in the on deck incubator described above for 24 hours. Samples to determination the rate of 15N accumulation in the NO−3+NO−2 pool were taken at 0 and 24 hours, passed through a 0.2 µm pore size filter and frozen at −80°C.Waters from 30 and 60 m at M1 and 30, 60 and 200 m at M2 were used to study the effects of light on nitrification in the absence of actively growing phytoplankton. For these experiments, water from each depth of study was drawn from the sampling rosette into either duplicate 280 ml polycarbonate (light) or 280 ml brown HDPE (dark bottles). Incubation experiments were initiated by addition of 15NH+4 tracer to a final concentration of 0.2 µmol L−1, seawater samples were incubated at estimated in situ levels of light  were collected before dawn at station 67–70 and drawn into clear 280 ml polycarbonate bottles (N = 2 per depth). Following addition of 24 hours , passed 15N of NO−3+NO−2 was determined at the University of California Davis Stable Isotope Facility (http://stableisotopefacility.ucdavis.edu/no3.html) using the denitrifier method PLUS. δ 15N values were calibrated against nitrate isotope reference materials USGS32, USGS34 and USGS35 analyzed in parallel. Precision of the N isotope analysis, determined by repeat analysis of the reference materials, was found to be ±0.4 ‰ for the entire data set.The δ15N-ammonia oxidation rates 15N in the NO−3+NO−2 pool measured at time  through 25 mm filters housed in Swinnex filter holders ; each sample was first passed through a 10 µm pore size polyester prefilter  and then a 0.2 µm filter . Filters were then flash frozen in liquid nitrogen in gasketed 2 ml bead tubes containing a mixture of 0.1 and 0.5 mm glass beads.−1 of seawater.Total nucleic acids were extracted from a single filter sample using a two-step co-extraction protocol, as described previously −1 of seawater. An aliquot of the purified RNA was immediately subjected to removal of contaminating DNA using the Turbo DNA-free kit following manufacturer’s protocol. cDNA was synthesized using random hexamers and the SuperScript III First-Strand Synthesis System for RT-PCR  according to manufacturer’s protocol, except for increasing the reverse transcription incubation step to 5 hours at 50°C. Negative reverse transcription control reactions were performed for each sample, replacing the reverse transcriptase enzyme with water.Following passage through the DNeasy column, the eluent was immediately processed for total RNA purification using the mirVana miRNA isolation kit  following the manufacturer’s protocol. RNA was eluted from the capture column with 75 µl of 95°C nuclease-free water. RNA yields averaged (±S.D.) 1.8±0.5 µg RNA LamoA genes related to WCA and WCB Thaumarchaeal ecotypes was estimated with two independent, non-overlapping qPCR assays amoA gene and transcript abundances were determined using commonly used oligonucleotides The abundance of 8 gene copies per reaction were generated from purified, linearized plasmids obtained from clone libraries constructed with each primer set. The limit of detection of was 1 copy per reaction for WCA and WCB AOA amoA, and 10 copies per reaction for β-AOB amoA genes and transcripts. In the event that the coefficient of variation for a set of triplicate reactions exceeded 10%, one of the replicates was omitted or the sample was reanalyzed. Both assays had efficiencies of 94–99% across all samples; all results were consistent and reproducible. To facilitate comparison between samples, estimates of gene and transcript abundances obtained by qPCR were normalized to the volume of seawater filtered and reported with the units of genes L−1 or transcripts L−1 throughout the manuscript.All qPCR assays were run in triplicate using a StepOnePlus Real-Time PCR System (Applied Biosystems). Standard curves ranging from 1 to 1×10All statistical analyses were performed on untransformed data in SPSS version 20 (IBM). Fit analysis for the predictive relationships was done with Igor Pro version 6.04 (Wavemetrics).Figure S1Flux of photosynthetically active radiation (PAR) to the deck incubator where all experiments were conducted. The yellow box highlights the incubation period for the light-dark experiments with waters from Station M1; the orange box denotes the incubation period for the same experiments conducted with waters from station M2 –2. The b(TIF)Click here for additional data file.Table S1Phytoplankton community structure assessed by epifluorescence microscopy and expressed as percent of total phytoplankton biomass as carbon, in surface waters (2 m) at the time of sampling stations M1 and M2.(PDF)Click here for additional data file.Table S2amoA and Thaumarchaeal 16S ribosomal RNA genes and transcripts at the start and end of multiday experiments. †Values represent the average of replicate samples  for end point samples (± S.D). Initial time point samples were not replicated. B.D., below detection limit of assay . All gene and transcript copies were normalized by the volume of seawater filtered for each sample, and expressed as copies per L−1 of seawater.Abundance of AOA and β-AOB (PDF)Click here for additional data file.


Defining responders to glucose lowering therapy can be important for both clinical care and for the development of a stratified approach to diabetes management. Response is commonly defined by either HbA1c change after treatment or whether a target HbA1c is achieved. We aimed to determine the extent to which the individuals identified as responders and non-responders to glucose lowering therapy, and their characteristics, depend on the response definition chosen.We prospectively studied 230 participants commencing GLP-1 agonist therapy. We assessed participant characteristics at baseline and repeated HbA1c after 3 months treatment. We defined responders (best quartile of response) based on HbA1c change or HbA1c achieved. We assessed the extent to which these methods identified the same individuals and how this affected the baseline characteristics associated with treatment response.Different definitions of response identified different participants. Only 39% of responders by one definition were also good responders by the other. Characteristics associated with good response depend on the response definition chosen: good response by HbA1c achieved was associated with low baseline HbA1c (p<0.001), high C-peptide (p<0.001) and shorter diabetes duration (p = 0.01) whereas response defined by HbA1c change was associated with high HbA1c (p<0.001) only. We describe a simple novel method of defining treatment response based on a combination of HbA1c change and HbA1c achieved that defines response groups with similar baseline glycaemia.The outcome of studies aiming to identify predictors of treatment response to glucose lowering therapy may depend on how response is defined. Alternative definitions of response should be considered which minimise influence of baseline glycaemia. In clinical care identifying when a therapy has been ineffective and can be stopped may benefit both the patient (e.g. avoiding side effects from ineffective therapy) and care provider (reduced cost); this approach has been incorporated into clinical guidance in the UK advising discontinuation of many more expensive glucose lowering treatments if response criteria are not achieved Being able to identify patients who respond particularly well or poorly to a therapy is important for both the study and application of a stratified  approach to the management of diabetes. Studies aiming to determine predictors and mechanisms of altered treatment response to a therapy may select patients with extremes of response for intensive phenotyping to maximise power . We measured HbA1c at baseline and 3 months (10–14 weeks) and assessed the following baseline clinical characteristics: HbA1c, BMI, duration of diabetes, age of diabetes diagnosis, glucose, C-peptide, triglycerides, creatinine. Blood tests were performed fasting.We prospectively studied 230 non-insulin treated participants with HbA1c ≥58 mmol/mol (7.5%) commencing GLP-1A therapy as part of their usual diabetes care recruited to Predicting Response to Incretin Based Agents (PRIBA) study  identified similar individuals and associated characteristics we defined good response to GLP-1A therapy (‘responders’) as follows:HbA1c change (from baseline to 3 months)<−30 mmol/mol (−2.7%) (n = 38)HbA1c achieved (HbA1c at 3 months on therapy) <56 mmol/mol (7.3%) (n = 38)Definitions were chosen based on the quartile of best responders for each method, using the closest thresholds which allowed equal numbers of responders. ‘Non responders’ were defined as those not achieving these criteria within each category. We assessed agreement in classification of responders between different response definitions by calculating percentage agreement and Kappa statistic and assessed differences in baseline characteristics using Mann-Whitney U (continuous variables) or Chi Squared (dichotomous variables).st centile of response for each definition):We developed a novel alternative method to define good responders to therapy, combining HbA1c achieved and HbA1c change using cut-offs based on the same centile so that only those meeting both criteria are designated responders. For comparison to the above methods the following definition was chosen to give 38 responders again (equating to the 41HbA1c change<−21 mmol/mol (−1.9%)ANDHbA1c achieved <62 mmol/mol (7.8%)We also defined response groups using HbA1c change, adjusted for baseline, using linear regression, with responders defined as the 38 participants with the greatest baseline adjusted HbA1c change .We examined the characteristics of responders and ‘non-responders’ (remaining participants) for both methods and assessed the extent to which these two methods identify the same responders and associated characteristics.All statistical analysis was performed using PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc.HbA1c, creatinine, triglycerides and glucose were measured in recruitment centres’ local laboratories . HbA1c measurement was standardised to the IFCC reference method procedure, all repeated measurements within the same individual were analysed within the same laboratory. C-peptide was measured in the Biochemistry Department at the Royal Devon and Exeter Hospital, Exeter, UK, using the routine automated E170 immuno-analyser from Roche Diagnostics .The study was approved by the South West Research Ethics Committee (UK). Written informed consent was obtained from all participants.Baseline characteristics of participants are shown in .Agreement between the two conventional definitions of response was poor . Other baseline variables are not associated with response group using either definition .Where treatment response is based on HbA1c achieved, low HbA1c and fasting glucose, high C-peptide and shorter duration of diabetes are associated with good glycaemic response to therapy. However when using HbA1c change to define response, the trend for these variables is in the opposite direction  . The best 38 responders using this method have similar baseline HbA1c to non-responders (median 86.0 vs 84.0 mmol/mol (10.0 vs 9.8%) responders/non responders, p = 0.60, Figure S1). Characteristics of responders and non responders defined using this method are also similar ,  similar Figure 3This study demonstrates that defining responders to glucose lowering therapy by HbA1c change or HbA1c achieved will identify different individuals, with different baseline glycaemia and associated characteristics. We suggest a simple practical alternative combining HbA1c change and HbA1c achieved that may be used to identify response groups that are independent of baseline HbA1c.In our study fewer than 40% of the top quartile of responders by one definition of glycaemic response were also responders by the alternative definition. This means studies categorising response to treatment may compare very different response groups should they choose alternate definitions of glycaemic response. Baseline HbA1c, a major source of potential confounding, is markedly different in responders defined by different definitions. This is consistent with previous research that has shown that baseline HbA1c strongly influences response to glucose lowering therapies, with high baseline associated with greater HbA1c fall but less likelihood of achieving glycaemic targets Our findings are also important in defining response in clinical care. Almost all participants (89%) with baseline HbA1c ≥90 mmol/mol (10.4%) in this study achieved an HbA1c fall of ≥11 mmol/mol (1%), a criteria for continuing therapy beyond 6 months in the UK Failure to compare patients with similar baseline HbA1c in studies of treatment stratification may lead to the finding of associations between a variable and treatment response that are in fact simply due to an association between the ‘predictive variable’ and baseline glycaemia. This will result in opposite directions of effect if response is defined by target HbA1c achieved rather than absolute HbA1c change. Where a baseline characteristic is a true predictor of response but has the opposite relationships with baseline HbA1c the association with treatment response may be missed due to negative confounding. This is seen with C-peptide in our study, which is more strongly associated with response when comparing participants with similar baseline HbA1c. While it is possible to adjust results for baseline HbA1c when comparing ‘responders’ and ‘non responders’ to glucose lowering therapy, this has the potential to increase error We have demonstrated a very simple and practical way to define response groups to glucose lowering therapy that do not differ on baseline HbA1c, by combining HbA1c change and HbA1c achieved. This method identifies similar participants and characteristics to using response groups based on HbA1c adjusted response . However unlike regression this method can be used to create clear clinical criteria and a mathematical calculation is not needed to define the responder group for each participant. This is a major advantage for studies recruiting participants into response groups for phenotyping. This method can also be used as a non-parametric method for the examination of existing datasets, which could have potential advantages where regression assumptions are not met.th centile of HbA1c change (−16 mmol/mol) and HbA1c achieved (69 mmol/mol) will define a comparison group of 38 non responders in this cohort.While this study is limited to one glucose lowering treatment class it is likely our findings will apply to all other glucose lowering medicines; baseline HbA1c is associated with response to all glucose lowering interventions including placebo A potential disadvantage of this method is that in studies including a wide range of baseline HbA1c it may preferentially select those with average baseline HbA1c as responders: a participant with a very high baseline will be unlikely to achieve a low on treatment HbA1c, a participant with a low baseline may be unlikely to have a large HbA1c fall. There may be advantages to restricting phenotyping to individuals with similar baseline glycaemia when studying physiology.Table S2). Repeating our analysis including those who discontinued other therapy does not substantially change our results. Although this study suggests C-peptide but not other examined participant characteristics are associated with glycaemia response to GLP-1 therapy these results are based on a small cohort examining extremes of response over a short (3 month) timescale to address a methodological question, further research will be needed to determine if these associations are of relevance to clinical practice.A potential limitation of this study is that differences in baseline HbA1c observed between different response groups in this study may have been exaggerated by the study design. We have excluded participants with other treatment change from this analysis as it is not possible to know what the response to GLP-1 therapy would have been had other therapy remained stable. Those with low baseline glycaemia and good response may have been preferentially excluded as they will be more likely to have hypoglycaemia and stop an adjacent therapy than those with high baseline. However this appears to have little effect: including participants who have reduced concurrent therapies does not substantially change the association between baseline HbA1c and glycaemic response  and by the combined method.(PPTX)Click here for additional data file.Table S1A: Comparison of ‘responders’ and ‘non responders’ to GLP-1A defined by HbA1c achieved B: Comparison of ‘responders’ and ‘non responders’ to GLP-1A defined by HbA1c change(DOCX)Click here for additional data file.Table S2The effect of excluding participants with co-treatment reductions on baseline HbA1c:HbA1c change association (linear regression HbA1c change on baseline hbA1c).(DOCX)Click here for additional data file.