Ectothermic organisms are thought to be severely affected by global warming since their physiological performance is directly dependent on temperature. Latitudinal and temporal variations in mean temperatures force ectotherms to adapt to these complex environmental conditions. Studies investigating current patterns of thermal adaptation among populations of different latitudes allow a prediction of the potential impact of prospective increases in environmental temperatures on their fitness.Paramecium caudatum. These different clones have been isolated from 12 freshwater habitats along a latitudinal transect in Europe and from 3 tropical habitats (Indonesia). The sensitivity to increasing temperatures was estimated through the analysis of clone specific thermal tolerances and by relating those to current and predicted temperature data of their natural habitats.In this study, temperature reaction norms were ascertained among 18 genetically defined, natural clones of the microbial eukaryote thermal generalists with a broad thermal tolerance and similar optimum temperatures. The weak or missing co-variation of thermal tolerance with latitude does not imply local adaptation to thermal gradients; it rather suggests adaptive phenotypic plasticity among the whole European subpopulation. The tested Indonesian clones appear to be locally adapted to the less variable, tropical temperature regime and show higher tolerance limits, but lower tolerance breadths.All investigated European clones seem to be P. caudatum genotypes at the most southern edge of their geographic range seem to suffer from the predicted increase in magnitude and frequency of summer heat waves caused by climate change.Due to the lack of local temperature adaptation within the European subpopulation,  Temperature is one of the most important environmental factors determining a variety of ecosystem elements, e.g. species ecophysiology, abundance and distribution, as well as species diversity and population dynamics To make predictions of organisms' and population responses to global warming, studies on genetic and phenotypic diversity over a species' geographic range are important. Such investigations can unveil patterns of evolutionary temperature adaptation to the current thermal heterogeneity on Earth by determining which ectotherms have a high acclimatisation capacity and which only occur at specific temperatures. Adaptive phenotypic plasticity, for instance, may cause a higher tolerance to changing thermal conditions thermal generalists. In contrast, tropical ectotherms, experiencing less variation in temperature, should be selected for narrow thermal niches resulting in thermal specialistsSeveral studies could show a co-variation of latitude and thermal tolerance e.g.  suggestithermal generalist pattern for ubiquitous species, populations at the ‘warm edge’ (such as the tropics or subtropics) might be most at risk due to global warming  as well as the optimum temperature (Topt) and the maximum performance  served as a genetic and phenotypic outgroup. This globally distributed ciliate species inhabits the mud-water interface of littoral freshwater environments, which are considerably affected by atmospheric temperature changes P. caudatum has to cope with large temporal and spatial variations in temperature. It therefore constitutes a suitable ectotherm to test hypotheses in thermal adaptation as well as the consequences of climate change on such ubiquitous protists. Here, we performed temperature dependent growth experiments to (i) test for a hypothesized local temperature adaptation of different P. caudatum genotypes; (ii) investigate thermal constraints resulting from evolutionary temperature adaptation; and (iii) understand the sensitivity of P. caudatum to predicted future temperatures.In the present study, the microbial eukaryote Paramecium caudatum cells were isolated from freshwater samples of 12 different natural habitats along a north-south transect in Europe as well as from three tropical habitats in Indonesia, Sulawesi (see Paramecium does not require specific permission and samples were not taken from water bodies where private property was indicated or from nature reserves where sampling is prohibited. The field studies did not involve endangered or protected species.Enterobacter aerogenes were obtained from the American Type Culture Collection (ATCC 35028) and the kanamycin-resistant strain Pseudomonas fluorescens SBW25 EeZY-6KX The food bacteria P. caudatum stock cultures were maintained in a 0.25% Cerophyl infusion, prepared according to the methods of Sonneborn erophyl infusion inoculated with Enterobacter aerogenes to establish mass cultures. Later, cultures were kept at 10°C, lowering the growth and ageing of P. caudatum. Previous to the start of the experiments, monoxenic P. caudatum cultures were established at 22°C in a Cerophyl infusion with Pseudomonas fluorescens serving as the only food bacteria  during the acclimation period by doubling the culture volume with Pseudomonas fluorescens inoculated Cerophyl infusion  as appropriate (1–5 ml per day). Due to the different acclimation phases from 22°C up to 35.5°C or down to 7°C, respectively, temperature-dependent experiments were conducted time-delayed. All experiments were performed in microprocessor-controlled, cooled incubators obtained from BINDER GmbH (Type KB 53).Cells from exponentially growing, monoxenic P. caudatum pre-cultures were adjusted to ∼250 cells ml−1 with CMP. Two millilitres of these starting cultures were added to each microcosm containing 18 ml CMP and resulting in an initial abundance of ∼25 cells ml−1. Growth experiments were run in triplicate in 60-ml tissue culture flasks with filter lids (TPP® AG) over two to eight days depending on the experimental growth temperature. The bacterial start density was regulated to a saturating prey level of about 2•108 cells ml−1. If the P. caudatum pre-culture densities were below 250 cells ml−1 because of growth-limiting temperatures (e.g. 7°C or ≥34°C), initial cell abundance was adjusted to the highest possible cell number (≥10 cells ml−1).Before the experimental start, acclimatised Paramecium cell abundance was estimated by sampling 1 ml every nine to 41 hours depending on the experimental growth temperature. This resulted in five to eight samples per replicate. For precise counting, cells were fixed by the addition of Bouin's solution μ, d−1) for each replicate and at each experimental temperature was calculated over the period of exponential increase using the slope of the linear regression of loge-transformed cell densities versus time (t).P. caudatum genotypes from different geographic regions, the mitochondrial cytochrome c oxidase subunit I (COI) gene was sequenced following the protocol of Barth et al.−1) at 56°C. Afterwards, the mixture was boiled for 20 min and frozen at −20°C; the supernatant was used for subsequent PCR reactions. Each PCR reaction mix contained 10 µl of Chelex® extracted genomic DNA, 10 pmol of each primer, 1 U Taq-polymerase , 1×PCR buffer with 2 mM MgCl2 and 200 µM dNTPs in a total volume of 50 µl. PCR conditions were as follows: 5 min initial denaturation (95°C); 35 cycles of 1 min at 95°C, 1 min at 50°C and 45 s at 72°C; and a final extension step of 5 min (72°C). Using the primers CoxL11058 and CoxH10176  to a thermal optimum (Topt) where the investigated biological function reaches its maximum. With a further increase in temperature above Topt the TPCs show a rapid decline towards a critical temperature maximum  was performed to describe the temperature dependent growth rate data of the individual  CTmax; . It was nm0a and nm0b, cf. nm2, cf. nm4, cf. Lactin-2 function  were used as fixed effects. In model nm0a, all four parameters were also used as random effects while for models nm0b, nm2, and nm4 only Tmax, Δ and λ were used because of the high correlation between the parameters ρ and λ resulting in a low model convergence. The decision which of the two parameters had to be omitted for nm2 and nm4 was made by comparing the respective AIC values (not shown). Model nm0b is shown for comparison only . The maximum growth rate  was calculated analytically as the growth rate (μ) at Topt using the Lactin-2 function (Eq.1), while Topt was calculated using its first derivative (Eq.2) as follows:ρ is a constant influencing μmax and the slope of the low-temperature branch, Tmax is the maximum temperature, and Δ defines the temperature range of the thermal inhibition above Topt. Parameter λ is an intercept parameter that forces the curve to intersect the abscissa at low temperatures and allows the estimation of CTmin.Then, the ecophysiological characteristics Lactin-2 function parameters  as well as the mean maximal surface air temperature , both for the warmest three months of the specific habitat. The WorldClim database is a set of interpolated global climate layers considering monthly precipitation as well as mean, minimum and maximum temperatures of the years ∼1950–2000. The database also provides 19 derived bioclimatic variables. The 2.5 arc-minutes resolution database was used to obtain the habitat mean temperature of the warmest quarter  and to calculate the habitat mean maximum temperature of the warmest three months . Furthermore, we used climate change data (2.5 arc-minutes) provided by DIVA-GIS to calculate future conditions for the respective habitats of the investigated P. caudatum clones. These data were derived from high-resolution simulations of global warming 2 doubling until 2100  and the latitude of the respective natural habitats were performed to compare these thermal adaptation indicators with the geographical origin of the different clonal P. caudatum cultures. We used a subset of data containing all investigated European clones and a second dataset including also the Indonesian paramecia. Additionally, the latitudes of the European habitats were corrected for the altitude by assuming that 100 m elevation translates into a ∼100 km latitudinal increment within the temperate zone p-values were estimated by Pearson correlation on ranks.Correlation analyses between each key ecophysiological characteristic  as well as the warming tolerances of maximum temperatures  were calculated to correlate the clonal specific thermal adaptation indicators and the local climate conditions with the altitude-corrected latitudinal gradient  and Topt values as well as all μmax, cal data and thermal tolerance breadths (TTB = CTmax−CTmin) was used to test for the so-called warmer is better hypothesis Jack-of-all-temperatures is a master of none hypothesis A Spearman rank correlation analysis between all calculated maximum growth rates (CV) of the mean growth rate of all investigated clonal P. caudatum cultures were analysed to estimate the intraspecific variation among all tested European clones as well as to assess intra-populational divergence. The calculation for each experimental temperature was as follows:iμ) at the investigated temperatures (iT) and its arithmetic mean , while the clone from Greece (GRL-1) showed the highest (CTmax = 35.12±0.06°C) among all investigated European clones. Conversely, the Spanish clone (ESH-2) possessed a comparatively low heat tolerance (CTmax = 32.96±0.06°C) compared to another Swedish clone . Not only the CTmax values showed high differences among the tested European clones, the calculated CTmin values were also considerably different  at the same experimental temperature of 28°C, while the calculated optimum temperatures (Topt) of the fitted TPCs ranged from 27.94±0.23°C (ESH-2) to 29.49±0.15°C (GRL-1).Using the wed TPCs . These cTopt. More precisely, large relative variations (CV) were obtained at low temperatures [CV(μ7°C) = 21.14%] and especially at temperatures above Topt [CV(μ32.5°C) = 91.41%], compared to the low relative variation at Topt [CV(μ28°C) = 7.50%]. While clones from the same habitat  belonging to the same as well as to different COI haplotypes (see CV(μ7°C) = 14.41%, CV(μ28°C) = 7.59%, CV(μ32.5°C) = 17.91%]. Performing one-way ANOVAs of clone specific growth rates at each experimental temperature suggests significant differences for the lowest and the highest temperatures tested . This result indicates the existence of an intra-populational variation even though we found no significant differences for the intermediate temperatures.The comparison between relative differences of intrinsic growth rate data of all European clones showed considerably larger differences with increasing distance from ypes see  showed sP. caudatum clones . In addition, an AIC based comparison of nonlinear mixed-effects models with different levels of spatial aggregation (nm2) as the second best model. While the null model nm0a with common fixed effects for all 18 individual clones was the most parsimonious and significantly superior model, the model with two regions was significantly better than the four regions model  and CTmax  with latitude using the complete dataset. When the correlation analyses were restricted to the European subset, we could only detect a significant correlation for CTmin  distances. Here, analyses on the complete dataset revealed highly significant correlations for all tests , while Mantel tests on the European subset showed non-significant relationships at all .Our results further showed that thermal safety margins increased with latitude as well as altitude . All lowCTmax with increasing latitude by analysing the European dataset. Therefore, the decrease in maximum air temperature with increasing latitude was considerably higher than the decrease in CTmax of the respective P. caudatum clones. This resulted in a steep increase in maximum warming tolerance with increasing latitude for the European P. caudatum clones  would show negative maximum warming tolerances  and calculated maximum growth rates  revealed a significant positive correlation of Topt and μmax, cal  and thermal tolerance breadth was detected using the complete dataset and performing Spearman's rank correlation  and genetic distances. Excluding the Indonesian clones from the analyses resulted in non-significant relationships  as well as high genetic distances (0.073–0.087 substitutions per site) compared to the European P. caudatum clones, which exhibit comparatively low genetic distances among each other (0–0.02 substitutions per site). Performing Mantel tests for the genetic and geographic distances resulted in significant correlations for both datasets  to which the southern European clones were barely exposed to, while southern European clones should possess higher critical maximum temperatures (CTmax). Our data partly support this general assumption of a co-variation of the critical thermal limits  and the optimum temperature (Topt) with the latitude when analysing the European dataset. Performing correlation analyses on this dataset revealed a significant dependence of CTmin with altitude-corrected latitudes, although we partly obtained high standard errors based on the used fitting procedure  as significantly superior compared to the four regions model nm4 . These results imply a stronger impact of latitude on the thermal performance of the eukaryotic microbe P. caudatum when comparing data on a large inter-continental geographic scale, in contrast to the analyses on the intra-continental European scale. Further, significant positive correlations of all ecophysiological distances with genetic distances could be identified only when using the complete dataset, but not among the European subset  compared to the temperatures they experienced in their natural habitats  have generally higher maximum growth rates (μmax) giving evidence for warmer is better concerning the growth performance as an important component of overall fitness. Nevertheless, we could not observe a significant correlation of Topt and μmax for the European subset, which indicates that the scale at which investigations are performed is of importance. Further studies on geographically well described organisms are necessary to understand if this finding is of general relevance for a variety of ectotherm species, for microbial eukaryotes only, or just for Paramecium caudatum.In this study, a significant correlation between optimum temperature and maximum population growth rates could be shown , which sJack-of-all-temperatures is a master of none hypothesis μmax) and thermal tolerance breadths Click here for additional data file.Table S2Geographical and meteorological details for natural habitats of investigated Paramecium caudatum clones.(DOC)Click here for additional data file.Table S3Mantel test for the correlation between geographic (x matrix) and ecophysiological distances (y matrix).(DOC)Click here for additional data file.Table S4Mantel test for the correlation between genetic (x matrix) and geographic distances (y matrix).(DOC)Click here for additional data file.


The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors are widely used in solid organ transplantation, but their effect on kidney disease progression is controversial. mTOR has emerged as one of the main pathways regulating cell growth, proliferation, differentiation, migration, and survival.The aim of this study was to analyze the effects of delayed inhibition of mTOR pathway with low dose of everolimus on progression of renal disease and TGFβ expression in the 5/6 nephrectomy model in Wistar rats.This study evaluated the effects of everolimus (0.3 mg/k/day) introduced 15 days after surgical procedure on renal function, proteinuria, renal histology and mechanisms of fibrosis and proliferation.Everolimus treated group (EveG) showed significantly less proteinuria and albuminuria, less glomerular and tubulointerstitial damage and fibrosis, fibroblast activation cell proliferation, when compared with control group (CG), even though the EveG remained with high blood pressure. Treatment with everolimus also diminished glomerular hypertrophy.Everolimus effectively inhibited the increase of mTOR developed in 5/6 nephrectomy animals, without changes in AKT mRNA or protein abundance, but with an increase in the pAKT/AKT ratio. Associated with this inhibition, everolimus blunted the increased expression of TGFβ observed in the remnant kidney model.Delayed mTOR inhibition with low dose of everolimus significantly prevented progressive renal damage and protected the remnant kidney. mTOR and TGFβ mRNA reduction can partially explain this anti fibrotic effect. mTOR can be a new target to attenuate the progression of chronic kidney disease even in those nephropathies of non-immunologic origin. In the last few years, mTOR inhibitors such as rapamycin or its derivative everolimus are increasingly used as potent immunosuppressants in renal and cardiac transplant therapy Chronic allograft nephropathy (CAN) is the main cause of renal allograft loss after one year of transplantation. Despite the impact of modern immunosuppression on reducing acute graft rejection, there has been little impact in long term graft survival Although the pathogenesis of chronic damage responsible for CAN is still largely unclear both immune and non-immune mechanisms may participate and they are characterized by an inflammatory response and the subsequent release of profibrotic cytokines and growth factor within the kidney mTOR is a major downstream component in the phosphoinositide 3-kinase pathway (PI3K), and has emerged as one of the main signalling routes utilized by cells to control their growth, proliferation, differentiation, migration, organization and survival In addition to lymphocytes, mTOR inhibitors act as anti proliferative for several other cell types such as vascular smooth muscle cells, mesangial, tubular and endothelial cells. Massive urinary protein excretion has been observed in renal transplant recipients with CAN after conversion from calcineurin inhibitors to mTOR inhibitors, especially sirolimus Controversy exists about the beneficial effects of mTOR inhibition in experimental nephropathies with some reports showing that it may be useful to diminish progression The model of mass reduction with right nephrectomy plus ligation of two branches of the left renal artery (5/6 nephrectomy) has been extensively used to study renal disease progression. Rats with 5/6 nephrectomy develop severe hypertension, proteinuria and progression to end stage renal disease The effect of mTOR inhibitors on disease progression in this model also is controversial. Diekmann et al The aim of this study was to analyze the effects of delayed mTOR inhibition on progression of renal disease in the 5/6 nephrectomy model in Wistar rats, using a low dose of everolimus introduced 2 weeks after nephrectomy and to evaluate its effects on fibrosis mediators as TGFβ.Blood pressure, BUN, plasma creatinine, plasma bicarbonate and proteinuria were significantly lower and creatinine clearance was significantly higher in sham group (SG) when compared with control group (CG) and everolimus-treated group (EveG) . There wHistological analysis showed less glomerulosclerosis (GS) in EveG compared with CG when evaluated with PAS stain , and with Masson's stain . There was also less tubulointerstitial (TI) damage in EveG compared with CG  in PAS stain and in Masson's stain  .Proteinuria is related with the magnitude of tubulointerstitial (TI) damage. When we considered all the animals together, the relationship between proteinuria (mg/day) and TI score, evaluated by Masson's stain, showed a regression coefficient of 0.86, (R2 0.73 p<0.05).Renal fibrosis was also assessed by morphometric analysis of renal tissue stained with Sirius red. Data from morphometric analysis are given as mean ± SD and as frequency distribution, as fibrosis develops in a focal and segmental pattern and its distribution does not show a gaussian pattern. 2, p<0.05) whereas treatment with everolimus significantly diminished glomerular hypertrophy (18132±5056 µm2) although it remained significantly higher than the SG (p<0.05) (The CG showed a higher glomerular area than the SG (21405±6612 vs 10553±1985 µm(p<0.05) .2. Statistical analysis reveals that the CG showed more mesangial fibrosis than the SG (1153.7±566.2 vs 185.6±104.8 µm2 p<0.05). Treatment with everolimus significantly diminished mesangial fibrosis when compared with CG .2, p<0.05), and the group treated with everolimus showed lower interstitial fibrosis than CG  although it was significantly higher than the SG (p<0.05) ((p<0.05) .We evaluated the effect of everolimus on glomerular expression and TI expression of αsmooth muscle actin. The EveG showed less staining at glomerular level  and TI level  when compared with CG .Glomerular and TI PCNA staining was significantly reduced in the EveG compared with CG  and  respectively .There were no differences in macrophage infiltration measured by CD68 staining .mTOR mRNA is over expressed more than 4-fold in CG when compared with SG . This increase is blunted in the EveG  . There wTGFβ mRNA significantly increased in the CG when compared with SG . TGFβ in the EveG returned to the SG levels of expression  .There were no differences in pERK/ERK ratio among the different groups.Treatment with everolimus 0.3 mg/kg/day for 8 weeks starting two weeks after 5/6 nephrectomy significantly reduced proteinuria, albuminuria, glomerular and tubulo-interstitial fibrosis, fibroblast proliferation and/or activation, and cellular proliferation.It also significantly diminished glomerular hypertrophy. Rovira et al showed that sirolimus treatment before 5/6 nephrectomy diminished glomerular hypertrophy but the treatment after nephrectomy was unable to reduce hypertrophy As the fibrosis process develops in a focal and segmental pattern we decided to show the frequency distribution of fibrosis areas in the tissue  and inhibition of the eukaryotic initiation factor 4E–binding protein1 (4E-BP1), enhancing protein production and cell proliferation The well known increase in mRNA TGFβexpression observed in the remnant kidney model Recent reports indicate that one of the most relevant noncanonical TGFβ¿ pathways is mTORC1 We found a significant increase in pAkt/tAkt(308) ratio in the CG, consistent with the stimulation off mTOR signaling pathway and also mTORC2 in this model. Recently Sarbassov showed that prolonged exposition to rapamycin is able to inhibit mTORC2 On the other hand, Vogelbacher et al We used a dose of everolimus more than 8 folds lower than Vogelbacher et al de novoWe believe that the earlier introduction of high dose everolimus can not only aggravate a pre existent nephropathy but can also produce a new and more aggressive experimental model of nephropathy. Letavernier et al., reported that high sirolimus levels may induce focal and segmental glomerulosclerosis Beneficial effects of mTOR inhibitors were also reported in anti-thy1 nephritis in vivo evidence that everolimus significantly prevented progressive renal fibrosis and protected the remnant kidney in an experimental model of reduced renal mass. In order to obtain these beneficial effects everolimus should be administrated after the acute effects of renal ablation and reparation have taken effect and in a lower dose than previously described.Our findings provide The mTOR and TGFβ inhibition can partially explain the anti fibrotic effects produced by everolimus.This study supports that mTOR can be a possible new target to attenuate the progression of chronic kidney disease All the animals were handled according to the Principles of the Laboratory Animal Care . The experimental protocol was approved by the University Animal Experimental Committee and by the local ethic committee. The animals were housed in individual cages in a constant-temperature room with 12:12-h dark-light and free access to water and food with a normal protein diet (24%). Rats were anesthetized with sodium thiopental 35 mg/kg i/p for all surgical procedures.Male Wistar rats with an initial body weight of 300±30 g were used for this study. Initially, simultaneous systolic blood pressure and pulse in the awake animal were registered by the tail cuff method using CVMS-20 software .Rats were randomly assigned to 3 groups. Two of these groups underwent 5/6 nephrectomy by selective ligation of renal artery branches followed by contra lateral nephrectomy. SG underwent abdominal incision and manipulation of both kidneys without excision.Two weeks after nephrectomy the EveG received everolimus 0.3 mg/kg/day or vehicle for CG, administered by daily gavages during 8 weeks. Dose election was based in a preliminary study using different doses . The animals treated with higher doses showed higher mortality rate and severe adverse effects: diarrhea, weight loss, arrest in wounds healing, and encapsulated abdominal abscess that were not observed with 0.3 mg/kg/day. Meanwhile, recent reports have described the use of a lower dose of everolimus in order to reduce mortality Everolimus for oral administration was provided by Novartis Pharmaceuticals, Inc.The rats were assigned to 3 groups: SG (n = 7), CG (n = 11) and EveG (n = 8). After 8 weeks of treatment 24 hours urine collection was performed and simultaneous systolic blood pressure and pulse were registered. The remnant left kidney was immediately perfused with phosphate-buffered saline at 4°C and 4% paraformaldehyde, and stored for renal morphology and immunohistochemistry studies.Serum creatinine, BUN, plasma bicarbonate, urinary protein, albumin, low weight molecular protein excretion and creatinine clearance were determined.We performed the same experimental procedure with three new groups of animals: SG (n = 7), CG (n = 5) and EveG (n = 7) to obtain renal tissue for real time RT PCR and Western blot. At week 8 remnant kidney was perfussed with cold PBS. The kidney was removed and renal cortex excised, snap-frozen in liquid nitrogen and stored at −80°C until processing.Serum and urinary creatinine were measured by the buffered kinetic Jaffe reaction. BUN was determined by spectrophotometric method, plasma bicarbonate with a blood analyzer  and urinary protein excretion was assayed by the turbidimetric method with sulfosalicylic acid. Albuminuria and low weight proteinuria were assessed by High Performance Liquid Cromatography according to Turpeinen Coronal sections of the kidney were immersion-fixed in paraformaldehyde solution and embedded in paraffin. Light microscopy was performed on 4-µm sections of tissue stained with periodic acid-Schiff's (PAS) and Masson trichrome reagent (MT) to assess GS and TI fibrosis. GS was assessed by semi quantitative analysis of 20 consecutive glomeruli (400×) according to El Nahas et al TI damage was evaluated using a semi quantitative analysis of 20 cortical fields (200×) according to Veniant el al The score index in each rat was expressed as a mean value of all scores obtained.All the histological analysis were performed by an observer unaware of the treatment received by each group.Morphometric studies were performed in sections 5 µm thick and Sirius Red stained. In brief, images were captured at 20-fold magnification using a green optical filter (IF 550) and a high-resolution videocamera (SONY CCD-iris) connected to a light microscope . The evaluation and image analysis procedures were performed with specific software A total of 25 glomerular images and 10 interstitial images random fields of renal slides  were captured and processed. The values obtained for each image were: a) glomerular fibrotic area or percentage of Sirius red-stained area which is contained in Bowman's capsule, b) renal corpuscular area and c) interstitial fibrotic area, or percentage of tubulointerstitial area occupied by Sirius red staining (excluding glomeruli and big arteries). These values, saved in data ASCII files conveniently labeled, can be exported to any statistical analyses program and calculate several parameters that can express the degree of renal fibrosis and glomerular sclerosis in an objective and quantitative way.Sections of paraformaldehyde fixed kidney tissues were processed by indirect immune detection technique with Biotin streptavidin amplified detection system  using three primary antibodies: (1) proliferating nuclear cell antigen (PCNA)  (dilution 1∶200), as a marker of cell proliferation, (2) α smooth muscle actin as a marker of myofibroblast transformation  (dilution 1∶100) and (3) CD68 as a marker of macrophages  (dilution 1∶100). Next, samples were incubated with secondary antibody conjugated with peroxidase, EnVision®  for 30 minutes and stained with DAB . Tissues were counter stained with hematoxilin.Normal rabbit IgG  was used as a negative control. Mean score per biopsy was calculated as follows: glomerular PCNA and CD68 as the mean number of positive cells in 20 glomeruli (cells/glomerular cross section) (400×), CD68 and PCNA tubular/interstitial score was obtained as the mean number of stained cells in 20 fields (cells/fields) (200×).For the evaluation of α-smooth muscle actin each glomerulus and tubulointerstitial field was graded semi quantitatively according to the extent of the staining from 0 (absent) to 4 more than 75% in the glomerular tuft or more than 75% of tubulointerstitial field. Mean scores of 20 glomeruli and 20 fields were calculated. Immunohistochemistry images were acquired  and digital analysis was performed .Tissue protein extracts were homogenized in lysis buffer  and centrifuged at 14,000 g for 30 min. Supernatant was recovered and proteins were quantified. Western blot analysis was performed as previously described 1 (Rn-00572010_m1). It is directed to locus gene NM 021578 that codifies for the translated protein NP_067589, precursor of total TGFβ1 , and the ABI Prism 7700 Sequence Detector. The level of target gene expression was determined using the ΔCt method as described Total RNA was obtained following the Trizol manufacturer's instructions . The RNA used for the study had a 28 s/18 s ratio between 1.8 and 2.0. Total RNA was reverse-transcribed as follows: 2 µg of RNA were incubated with 1 µl of 50 µM random hexamers followed by RNA denaturalization. Then, 5× reaction buffer, 0.4 µl of 100 mM dNTP mix, and MMLV retrotranscriptase 200 u/µl  were added in a final volume to 20 µl and the reaction was incubated during 5 min at 25°C, followed by 30 min at 42°C and 5 min at 85°C to stop the reaction. qRT-PCR was performed using 5 µl of cDNA, primers and the TaqMan probe for Akt (Rn-00583646_m1), mTOR (Rn-00571541) and TGFβAll data were expressed as Mean ± SD. One way analysis of variance and post hoc with Student-Newman–Keuls test was used to determine the statistical significance. Statistical significance was defined as P value less than 0.05.


Small parallelepiped beam-shaped human cortical bone specimens were subjected to cyclic four-point bending fatigue in several steps. The specimens were prepared to control damage localization during four-point bending fatigue cycling and to unambiguously identify resonant modes for NRUS measurements. NRUS measurements were achieved to follow the evolution of the nonlinear hysteretic elastic behavior during fatigue-induced damage. After each fatigue step, a small number of specimens was removed from the protocol and set apart to quantitatively assess the microcrack number density and length using synchrotron radiation micro-computed tomography (SR-µCT). The results showed a significant effect of damage steps on the nonlinear hysteretic elastic behavior. No significant change in the overall length of microcracks was observed in damaged regions compared to the load-free control regions. Only an increased number of shortest microcracks, those in the lowest quartile, was noticed. This was suggestive of newly formed microcracks during the early phases of damage accumulation. The variation of nonlinear hysteretic elastic behavior was significantly correlated to the variation of the density of short microcracks. Our results suggest that the nonlinear hysteretic elastic behavior is sensitive to early bone microdamage. Therefore NRUS technique can be used to monitor fatigue microdamage progression in  Bone microdamage is a natural phenomenon caused by daily loading in vitroAmong various bone quality factor, bone microdamage is certainly the less understood, as in vivo.Recently, synchrotron radiation micro-computed tomography (SR-µCT) enabled the 3-D assessment of microcracks at a micro-scale resolution Several measurement modalities, including positron emission tomography (PET) in vivoQuantitative ultrasound is widely used to assess skeletal status In previous NRUS studies, our group has reported that, under resonance conditions, the resonance frequency of damaged femoral diaphysis was down shifted with increasing vibration amplitude. We found a correlation of progressive fatigue of human bone samples to their nonlinear dynamical response Small parallelepiped beam-shaped human cortical bone specimens were subjected to cyclic four-point bending fatigue in several steps. The specimens were prepared to control damage localization during four-point bending fatigue cycling and to unambiguously identify resonant modes for NRUS measurements.Fourteen human cortical bone specimens were prepared from the femoral mid-diaphysis of four female donors . Ethical approval for the collection of samples was granted by the Human Ethics Committee of the Centre du don des Corps at the University Paris Descartes . The tissue donors or their legal guardians provided informed written consent to give their tissue for investigation, in accord with legal clauses stated in the French Code of Public Health. The specimens were wet machined  as parallelepiped beams (50*4*2 mm), defatted dryρ) was evaluated by measuring the specimen volume and weight. Bone specimens were dried at 37°C during one night in a climate chamber  at relative humidity 15% in the presence of desiccators. Drying and rewetting procedure does not affect bone properties as the collagen molecular structure remains intact Apparent dry density :The principles of NRUS measurements have been extensively described elsewhere k is the wave number and L is the specimen length. The resonance peak frequency f of the first compression mode was derived for each voltage drive level from the strain amplitude measured as a function of frequency at the corresponding excitation level. From the resonance peak data, the nonlinear parameter fα can be calculated using the following equation f is the resonance frequency at increased strain level and 0f its corresponding value at the lowest drive amplitude f is proportional to the peak strain amplitude Δε via the nonlinear elastic fα parameter. This parameter, so-called nonlinear hysteretic elastic parameter, is typical of nonlinearities that appear for strains above approximately 10−50f used to compute fα was repeated before each excitation level. In doing so, the measurements become less affected by changes in environmental conditions such as temperature and yields more precise and stable fα estimates The widely used NRUS measurement protocol During the NRUS measurements, specimens were kept at fixed temperature (37°C ±0.1°C) and relative humidity (15%±5%) into a climate chamber The piezoceramic emitter attached to the specimen for NRUS measurements was removed and the specimen was rehydrated during 48 hours before each mechanical testing. All specimens were progressively damaged by cyclic four-point bending at 2 Hz under load control in a saline solution at 37°C (±1°C) using a hydraulic testing machine  with a 1 kN loading cell (accuracy 0.5%) and the internal displacement transducer (accuracy 1%). The specific four-point bending assembly composed of 6.35 mm diameter roller-bearings with a 40 mm outer span and a pivoting 20 mm inner span minimizes the formation of grooves under the rollers Fmax) corresponding to 5000µε  at the mid-span was computed for all specimens Fmax. During the cycling session, the load and displacement curves were recorded to extract the linear elastic beam theory (LEBT) modulus (LEBTE) as defined by Landrigan LEBTE is a combination of elastic (secant modulus) and plastic  biomechanical parameters. After each damage step, the LEBTE modulus is normalized by the initial value measured for the first loading cycle of the first damage step. LEBTE has been shown to decrease as bone microdamage accumulates LEBTE or pre-determined number of cycles ( = 6000) or anomalous LEBTE decreasing speed. This multi-criteria definition of each step was chosen to achieve progressive damage accumulation and to avoid specimen failure before the end of the fourth step.From the initial Young’s modulus, the load  were investigated. VOI1, located in the load-free region at one distal end of the sample, outside of the roller-support, was assumed to be free of damage . VOI2, located in the central portion of the beam, is the region where most microdamage was assumed to accumulate during mechanical fatigue. The photon energy was 25 KeV and the size of the VOI was 2.8×2.8×1.96mmMicrodamage is generally characterized on transverse or longitudinal 2-D sections by conventional or epifluorescence microscopy 2]) and microcrack length (Cr.Le [µm]) were measured using the software ImageJ  with the plugin NeuronJ The surface microcrack density  as well as after removing the outlier (N = 13). P-values less than 0.05 were considered significant.Matlab 7.8 with statistics toolbox 7.6  was used for statistical analyses. A non-parametric one-way analysis of variance (ANOVA) dedicated to repeated measurements (Friedman Test) followed by post hoc multiple comparison (Nemenyi test) was applied to test whether the levels of nonlinear elasticity achieved at each steps of the fatigue protocol were statistically significantly different. This analysis was performed on two subsets of the total set of specimens (N = 14). Group 1 (N = 8) includes all specimens having undergone the first three damage steps and Group 2 (N =  4) includes all specimens having undergone all the four damage steps. The effect of fatigue loading on microdamage characteristics (Cr.Dn and Cr.Le) in the control region and the damaged region was investigated with a non-parametric Wilcoxon signed rank test for all specimens (N = 14). The relationship of the nonlinear elastic parameter Individual results for mechanical and NRUS testing are presented in LEBTE has been assessed on five dedicated specimens that were not included in the protocol. They went through 20 cycles (after system stabilization) of four-point bending test. The process was repeated 6 times with repositioning. The coefficient of variation (standard deviation/mean) was found to be 6.3% for LEBTE.The variability of LEBTE modulus was 15.1±3.0 GPa the average apparent dry density dryρ was 1792±155 g.mm−3 and the average porosity was 12.0±5.2.The mechanical characteristics  were fouNo significant trends could be observed between stopping criteria and the other measured variables (nonlinearity or microcracks characteristics).fα. The initial nonlinear values of fα ranged between −4.3 and −6.2, except for one highly nonlinear specimen (fα  = −15.5). The average initial fα value was −5.8±2.9. After the last damage step (step 1 to 4 depending on the specimen), fα values ranged between −4.7 and −16.2.The measurement precision of NRUS, assessed by the coefficient of variation of three measurements with intermediate debonding of the piezo-electric source and repositioning, was found to be 8.5% for fα increased with the number of fatigue steps. However, a disparity could be observed between the specimens.On average, the nonlinear parameter fα was measured for both groups . Group 1 including the eight specimens having undergone the first three stages of damage is represented in fα between damage steps for both groups except between the initial and first step and between the third and last step for specimens of the Group 2.A significant effect of fatigue on fα or its variation Δfα/fα and the initial elastic modulus LEBTE, the total number of cycles, the apparent density dryρ nor the sample porosity.Finally, note that no correlation was found between the initial nonlinear elastic parameter The total number of microcracks found by pooling the data of all the specimens and both VOIs was 4106 with 1380 microcracks leading to the specimen surface and 2726 microcracks fully embedded within the bone volume.2 and VOI2: Cr.Le = 69.6±25.7µm; Cr.Dn = 3.33±1.58#/mm2) nor after excluding from the analysis, microcracks leading to the specimen surface . Damage characteristics of microcracks fully embedded in bone are summarized in 2 to 3.88#/mm2 (VOI1) and between 0.25#/mm2 to 4.59#/mm2 (VOI2).The difference in microdamage characteristics between VOI1 (control region)  and VOI22 and VOI2: Cr.Dn.Q1 = 1.06±0.60#/mm2; p<0.05) or when microcracks leading to the surface were excluded  , whereas the quantity of longer microcracks remained unchanged. The density Cr.Dn.Q1 of the shortest microcracks was significantly different between VOI1 and VOI2, both when all microcracks were included (VOI1: Cr.Dn.Q1 = 0.77±0.38#/mm p<0.01) . The mea p<0.01) .f/αfα in fα variation) was excluded, a significant correlation of r = 0.6 (p<0.05) was found between both quantities.The relative variation between VOI2 and VOI1 of density of short microcracks embedded within the bone matrix ΔCr.Dn.Q1/Cr.Dn.Q1 is plotted against the relative variation of the nonlinear parameter Δfα) and microdamage characteristics derived from SR-µCT, concurrently assessed in calibrated human cortical bone specimens during a four point-bending fatigue cycling protocol. By repeating the NRUS measurement protocol after each damage step, we were able to monitor the evolution of the nonlinear behavior during progressively induced mechanical damage, each specimen being its own control. We observed that the damage steps had a statistically significant effect on the nonlinear hysteretic parameter fα measured by NRUS. This suggests that the parameter fα is a sensitive marker to bone microdamage induced by ex vivo mechanical fatigue test. These results confirm the seminal observations made in previous studies by our group This is the first study reporting the nonlinear elastic hysteretic behavior (assessed by the nonlinear elastic parameter fα) of human cortical bone samples was measured in this study, allowing the comparison with other materials. Here, the nonlinear hysteretic elasticity typical of damaged materials is measured, as opposed to the classical elastic non linear response which exists in most materials, including undamaged solids, due to intrinsic anharmonicity of, for instance, the crystalline lattice-atomic level vibration.The nonlinear hysteretic elasticity (fα = −5.8±2.9) is consistent with the value previously reported for undamaged cortical bovine bone (fα = −5.0±2.5) The initial (pre-fatigue) nonlinear value  nonlinear behavior of cortical bone. It may have its origin in damage, microcrack or diffuse type, native or induced by sample preparation. Such damage is observed by measuring the control regions located at the ends of the specimens. As for other potential sources of damage , it would be necessary to assess their magnitude and explore them by dedicated experiments. However, as each sample is its own control, we can attribute unambiguously the increase in the nonlinear hysteretic behavior after fatigue to damage accumulation.Besides, water saturation, capillarity effects and fluid flow pressure may also play a role as in rocks 2 in VOI1) is one order of magnitude larger compared to the values found in most studies on human cortical femur (0.21±0.16#/mm22222 to 5#/mm2 for women older than 45 years old and higher than 1.5#/mm2 for human femur older than 70 years old The density of microcracks (Cr.Dn) embedded within undamaged bone volumes  When we considered all the detected microcracks, leading to the surface of the specimens and embedded within the bone matrix, we found no significant change of microcrack density and length. This is at odds with several previous studies, although once again, the difference in technologies used in different studies to quantitatively assess microdamage should be emphasized, namely, X-ray micro-tomography in our study versus optical–based microscopy in previous reports. Several studies report that three or four-point bending fatigue tests on calibrated cortical bone specimens induced the progression of microdamage by increasing microcrack density and length in human When the microcracks leading to the specimen surface were analyzed separately, the density of short microcracks was not statistically significantly different between VOI1 (control region) and VOI2 (damage region). This is in contrast with the results obtained when we considered fully embedded microcracks only. The reason for this discrepancy remains unclear. One possibility is that pre-existing and/or preparation microcracks leading to the specimen surface release stress concentration, thus preventing significant growth of microdamage at the periphery of the specimens. On the contrary, microcracking process, as an essential mechanism inside bone specimen to release stress concentration, may explain why small microcracks density increases. Further experiments are required to elucidate this issue.Microcracking process is well known in fiber-reinforced composite materials as the so-called three-phase modulus degradation curve Our results seem to be in line with the progressive growing of microdamage described in fα and the variation of Cr.Dn.Q1 is suggestive of the sensibility of the nonlinear hysteretic elastic parameter to newly formed microcracks in early phases of bone damage. However we cannot reject an effect of early diffuse or nanodamage to hysteretic nonlinearity as this damage was not investigated, neither the initiation and growing of a single macrocrack (i.e. known to produce large nonlinear elastic behavior) that was not contained by the microcracking process.The significant correlation found between the variation of Altogether our results evidence:  an increased number of short microcracks in damaged   regions compared to the load-free (control) regions. These   shortest microcracks, with length in the first quartile, are   suspected to be newly formed microcracks as a result of   fatigue cycling fα.  a significant effect of damage steps on the nonlinear   hysteretic elastic parameter  a significant relationship between the relative variation of  nonlinear elasticity and the relative variation of the density  of newly formed microcracks.fα) is sensitive to early bone microdamage. Our results suggest that NRUS can be used to monitor fatigue microdamage in in vitro experiments. The ability to non invasively quantitatively assess microdamage accumulation in living bones would represent an important step to improve our understanding of skeletal status. However, several scientific and technical problems have to be solved first, such as the adaptation of the nonlinear ultrasound techniques to in vivo measurement requirements.The hysteretic nonlinear parameter (


A systematic review showed that patients convey important informations in spontaneous co-speech hand gestures (HG) when presenting their complaints and describing their pain experiences. Only a minority of practitioners and therapists were reported to have actively analyzed patients’ hand gestures during case taking. Sensation method (SM) homeopaths were one of the reported exceptions. This survey was designed to gain a better understanding of the perspective, usage, appraisal and general relevance of manual co-speech gestures by SM homeopaths.Ninety-four out of 306 seminar attendants  at two seminars on SM homeopathy with varying degrees of expertise, answered a standardized 54-item questionnaire. For 20 items on “perspective, utilization and relevance of gestures in patients’ symptom description” a factor analysis was performed, by means of principal components analysis and varimax rotation to arrive at a solution that demonstrates both the best simple structure and the most coherence. Nine items were excluded after reliability analysis due to low item total correlations.Eleven remaining items formed a set of three factors explaining 66.6 % of variance. The first factor with five items describes “Hand gestures in relation to verbal expressions” (α = 0.81). The second factor includes four items regarding “Hand Gestures describing the experience of bodily and mental symptoms” (α = 0.74). The third factor is regarding the “practitioners’ behaviour and active attitude in observing hand gestures” (α = 0.86).This survey shows how SM homeopaths actively observe HG and judge them to help patients in expressing their symptoms qualities and illness experience. Whether this view is shared by other physicians or medical professions should be investigated in the future.


B14 shares 95 % amino acid identity with the 183 protein encoded by modified virus Ankara (MVA), an attenuated VACV strain being developed as a vaccine vector. To evaluate whether the immunogenicity of MVA might be increased by manipulation of MVA immunomodulatory proteins, the MVA counterpart of B14, protein 183, was characterized. Unlike B14, protein 183 was unstable in eukaryotic cells unless proteasome-mediated protein degradation was inhibited. Furthermore, 183 did not inhibit NF-κB activation in response to cytokine stimulation, and did not restore the virulence of VACV strain Western Reserve lacking gene B14R. The instability and non-functionality of 183 are probably explained by a deletion of 6 aa within α-helix 6 of the B14 crystal structure.Vaccinia virus (VACV) encodes multiple proteins to evade host innate immunity, including B14, a virulence factor that binds to the inhibitor of  Vaccinia virus (VACV) is the best-studied member of the family Poxviridae; it is a double-stranded DNA virus that replicates in the cytoplasm of host cells (st cells . VACV wast cells . In the st cells . MVA lacB14R deletion mutant (vΔB14) was attenuated in a mouse intradermal (i.d.) infection model and affected the inflammatory response to infection (κB (NF-κB) activation by binding to the inhibitor of κB kinase (IKK) complex via the IKKβ subunit  in MVA. In different VACV strains and other orthopoxviruses, alanine, valine or threonine is found at this position  downstream of a human cytomegalovirus  immediate-early promoter. HEK 293T cells  for 8 h. NF-κB activation was assessed by firefly luciferase activity and normalized by Renilla luciferase activity. Triplicate samples were collected and data are expressed as the mean fold induction relative to pCI. Whilst B14 inhibited NF-κB activation as expected, MVA 183 had no such activity  mAbs  that were infected with vΔB14. A recombinant virus containing MVA 183, vΔB14-MVA183, was selected by transient dominant selection . vΔB14-MVA183 was purified in parallel with viruses vB14  and vΔB14 (B14R into vΔB14 restored lesion size, as reported previously (183R into vΔB14 did not alter lesion size (Fig. κB activation in cell culture.MVA does not replicate in many mammalian cell lines and is avirulent in mice. Therefore, to investigate the role of MVA 183 in virus virulence, the R, vΔB14 . A pSJH7election  and PCR ize Fig. . Therefoα-tubulin (Fig. 183R, as the nucleotide sequence upstream of MVA 183R was the same in WR B14R, and so the difference probably reflected protein instability as noted above (Fig. To examine 183 expression in infected cells, lysates from CEFs infected with MVA or vΔB14-MVA183 were analysed by immunoblotting using the anti-B14 polyclonal antibody  and α-tulin Fig. . As befoove Fig. . A majorove Fig. . This shAnalysis of VACV protein D8 expression levels in these cells showed a substantial decrease in the presence of MG132. This observation was in accord with the report that MG132 blocks a post-entry step in VACV replication (Escherichia coli under conditions that yielded stable, soluble B14 protein that was used for protein crystallography (α6 helix. This deletion is found only in MVA and in no other VACV strain, including the parental virus CVA, and so arose during MVA passage in CEFs. The observation that MVA 183 does not inhibit NK-κB activation may explain, at least in part, why MVA infection activates NF-κB in contrast to other VACV strains (α. Possible explanations for this are alterations to the proteasome-mediated degradation of proteins after TNF-α stimulation, or that a component of the TNF-α-induced signalling pathway leading to NF-κB activation can somehow stabilize the 183 protein once the pathway is activated. However, if the latter were true, it is notable that, even when 183 is present, at the levels tested it did not inhibit NF-κB activation. It is also formally possible, but less likely, that TNF-α treatment is affecting 183 mRNA levels.Several lines of evidence indicate that MVA 183 is a labile protein. First, following transfection of mammalian cells with plasmids encoding 183, the protein was not detected, whereas B14 was. Second, after infection of avian cells by MVA or a VACV WR virus encoding MVA 183, the 183 protein was not detectable unless the proteasome was inhibited by MG132. Third, attempts to express protein 183 in  strains . InteresκB activation and contribution to virus virulence. Other MVA genes encoding immunomodulatory proteins with small deletions or mutations relative to counterparts in other VACV strains are as yet uncharacterized and these changes may have important functional consequences. Attempts to increase the immunogenicity of MVA by engineering or deletion of genes encoding immunomodulators should be informed by knowledge of whether the encoded protein is functional, and priority should be given to engineering genes known to encode functional proteins.The original bioinformatic analysis of the MVA genome recorded open reading frames that were retained, deleted or disrupted by mutation compared with other VACV strains (κB activation following addition of TNF-α, nor contributes to VACV strain WR virulence. The loss of function is probably attributable to a 6 aa deletion within the α6 helix of the Bcl-2 fold and it is unlikely that removal of this gene from the MVA genome will enhance MVA immunogenicity.In summary, MVA protein 183 is a non-functional counterpart of VACV WR protein B14 and neither inhibits NF-


Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined “fuzziness”, often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs. Understanding how proteins recognize each other is central to deciphering the inner workings of living things and for biomedical research. It has long been known that the sequence of a protein, which is a string of different amino acids, can dictate how a protein molecule folds into a well-defined shape required for biological tasks. Many folded proteins recognize and bind with each other by a tight geometric fit similar to that between a lock and its key. Recently, however, it has become clear that some proteins function as a flexible string, in constant motion, without forming a stable shape. Understanding how such “disordered” proteins work is challenging. To gain insight, we studied a disordered protein region that causes a large family of human cancers. Employing an innovative combination of experimental and theoretical techniques, we describe a new mode of protein interaction based on multiple simple contacts between one type of amino acid (aromatic) in the disordered protein and another type (positively charged) on the partner protein. Because this mechanism also underlies the ability of the disordered protein to cause cancer, further investigation of this unprecedented mode of protein-protein interaction may open up new avenues for cancer therapy. Understanding the sequence-function relationship of a protein and how it might malfunction is central to biomedical research. While many proteins function in their folded states, recently it became clear that intrinsically disordered proteins (IDPs) also play key functional roles To gain insight into “fuzzy” interactions, we have studied the Ewing's Sarcoma (EWS)-activation domain (EAD) in the TET family of RNA-binding proteins Functional studies of EFPs have provided a foundation for understanding sequence-function relationship of EAD. Most notably, the transcriptional and oncogenic activity of EAD is conferred by multiple tyrosine (Y) residues due to their aromaticity but not hydrophobicity In light of the versatile roles of cation-π interactions in protein folding and protein-protein interactions In view of the abundance of aromatic residues in EAD (38 Ys in the native EAD) and the significant strength of cation-π interactions The intact EAD spans ∼280 highly repetitive residues but such a long sequence is not particularly amenable to mutagenesis and is also quite impractical for computational studies. This hurdle can be overcome by exploiting small EAD regions (∼40 residues) that faithfully mimic the salient features of the intact EAD and whose transcriptional activity (transactivation) can be readily tested using a multisite reporter  density . Transac density . As for To assess the polycation-π idea, we constructed a coarse-grained chain simulation model that embodies the hypothesis. The EAD is represented by a flexible ribution  resembliribution , which wribution , the intribution  of whichribution  by usingPbs rationalize the functional data regarding the effect of Y (aromatic) number. A similar agreement with model simulation was also observed for the activities of a set of previously studied EAD sequences . Binding increases with aromatic density C  is comparable but weaker than the average pairwise cation-Y energy ≈−3.3 kcal/mol we determined from our simulation using a cation-Y potential energy well depth of ≈3.6 kcal/mol  residues . Adding The relative simulated riations . Quantitriations . Simulatriations ,F,  of 1k values). The data in k. This trend is consistent with the analytical model results for these sequences  of the Ys while maintaining overall density and total Y number? In ractions . Taken tThe significance of protein disorder in the bound state or “fuzziness” has only recently emerged Our hypothesis is intuitive given that cation-π interactions have wide and versatile biological roles, the interaction is strong We have assumed a globular target because the biophysical aspects of the proposed model strongly predict that a large number of real globular proteins interact with EAD. Nonetheless, a disordered (IDP) target that enables favorable cation-π contacts with the EAD is also possible  althoughPolyelectrostatic (Sic1/Cdc4) and polycation-π interactions share some similarities. Each may well reflect a general mode of interaction for polyvalent IDPs. In contrast to Sic1-Cdc4, however, the properties of the EAD studied herein are related to the diseased state The molecular recognition events studied here are related to pathological EAD function and, accordingly, are not obviously shaped by evolution In this regard, knowledge of EAD-target interface might provide therapeutic avenues How may polyaromatic molecular recognition by EAD relate to normal EWS protein function? This is a challenging question given the strong evolutionary conservation of EWS To conclude, the proposed model for molecular recognition by EAD expands the seemingly endless modalities for IDP function and malfunction. The hitherto unrecognized polycation-π mode of IDP-target binding can be versatile. It offers a highly plausible biophysical basis for EAD and perhaps other scaffold/networking proteins to interact with many distinct target proteins Plasmids: pZΔE Proteins: pZΔE expresses a protein lacking EAD sequences and containing only the ATF1 region and zta bZIP domain Transactivation assays and Western blotting: Transfections, trans-activation assays and quantitation of transactivation under linear assay conditions were performed as previously described α chain. Pairwise interactions between amino acid residues depend on whether they are aromatic, hydrophobic, charged, or polar  contain the experimental sequences related to the N-terminal 66 residues of EAD1-66 (box with purple Ys), the region of ATF1 protein (ΔATF1) present in the EWS/ATF1 oncogene and the DNA-binding domain of zta protein (ztaDBD). In (A)–(C), amino acid residues are denoted by the standard one-letter code. Sequences for (JPG)Click here for additional data file.Figure S2The chain simulation model. (A) The generic EAD binding target (partner) is a sphere of radius r here corresponds to S1) or S2). (E) Model cation-π interaction potentials in the form of S1) and (S2) respectively  whereas the dashed curve is for (PDF)Click here for additional data file.Figure S3Evidence for the polycation-π hypothesis from a re-analysis of early experiments on 33-residue EAD sequences. Sequences and experimental data were taken from ref. 3 simulation box. (A) The sequences are defined in the above reference. The experimental relative activities and the simulated relative binding probabilities are represented by the black and grey bars respectively. (B) The sequences in (A) are grouped according to their Y number (PDF)Click here for additional data file.Figure S4Simulated binding probabilities of monomer and dimer EAD sequences follow similar trends. Similar dependences on Lee ref.  studied (PDF)Click here for additional data file.Figure S5Components of the analytical model. (A) Schematic of cation-π contacts along the IDP. Here we only consider IDP chains with evenly spaced aromatics that are k residues apart; thus the contour length between two cation-contacting aromatics is always in the form of A = 0.5365, B = 0.53139, ω = 0.02786, and σ = 0.33604 for A = 0.40915, B = 1.12627, ω = 0.05373, and σ = 0.39353 for ithm see . As can (PDF)Click here for additional data file.Figure S6Conformational entropy loss upon loop formation. The quantity n  that have one chain end (bead number 1) touching an excluded-volume plane at a given point Click here for additional data file.Figure S7Applying the lattice conformational entropy estimates to the analytical model. (A–C) The fitting parameters A = 0.13748, B = 7.04181, and C = 0.52115; (B) A = 0.97499, B = 0.93564, and C = 0.97495; and (C) A = −5.19530, B = 2.98286, C = 0.31975, and D = 2.79004. These expressions were used to estimate l>16 by extrapolation. (D) The extrapolated l = 60. (E) Two methods for estimating the entropic cost of loop formation in the analytical model are compared. Plotted are the binding free energies of the model EAD chains in l≤16 and extrapolated estimates for l>16, whereas the red data points (triangles) were obtained by using entropy estimates from exact enumeration for l≤16 but random-flight estimates for l>16. The plot here shows that the predicted (PDF)Click here for additional data file.Figure S8Exploring other EAD-target binding scenarios. The EAD sequences are the same as those in 3 (black symbols) or (600 Å)3 (blue symbols). (B) A snapshot of an 3. During the binding simulations, both the EAD and the hypothetical IDP target were allowed to sample all accessible conformations while the center of mass of the IDP target was kept at a fixed position in the center of the simulation box. We considered a class of such targets, each of which is a chain consisting of 64 alternating cations and anions (32 cations and 32 anions). The adjacent cation and anion are connected by a 5 Å virtual bond with a stiff bond-angle force constant equal to 10.0 cations  that we GG refs. . Here th(PDF)Click here for additional data file.Table S1Numbers of conformations, or self-avoiding flights, on the simple cubic lattice. Conformational counts as functions of chain length (number of beads) n are obtained by exact enumeration. A chain with n beads has n−1 bonds. Here, n is even, n is odd] making a contact with an impenetrable plane Click here for additional data file.Table S2Loop probabilities determined by exact lattice conformational enumeration. Tabulated here are examples (not a complete list) of conformational counts x,y) coordinates. In the present treatment of our analytical model, x,y (where x<y) that have nonzero n≤17 were used to estimate the conformational entropic cost of loop formation (ormation  and S7.(PDF)Click here for additional data file.Table S3Exact lattice enumeration data for loop formation probability. Tabulated here as examples are the exact l = 16 and n = 17. The horizontal and vertical labels correspond, respectively, to the x and y coordinates of the positions on the impenetrable plane. One end of the chain (first bead) is always anchored at the origin . In this table, the entry at a given position  is the number of conformations that have the chain's last Click here for additional data file.Text S1Experimental and Computational Details and Rationale.(PDF)Click here for additional data file.


DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of visual processing in a contingent negative variation (CNV) task.Dopamine plays an important role in orienting and the regulation of selective attention to relevant stimulus characteristics. Thus, we examined the influences of functional variants related to dopamine inactivation in the dopamine transporter . Early and late CNV as well as preceding visual evoked potential components were assessed.DAT1 and COMT on the occipito-temporal early CNV were observed. In addition, there was a trend towards an interaction between the two polymorphisms. Source analysis showed early CNV generators in the ventral visual stream and in frontal regions. There was a strong negative correlation between occipito-temporal visual post-processing and the frontal early CNV component. The early CNV time interval 500–1000 ms after the visual cue was specifically affected while the preceding visual perception stages were not influenced.Significant additive main effects of DAT1 and COMT during motor post-processing but not motor preparation. We propose the hypothesis that similar dopaminergic mechanisms modulate working memory encoding in both the visual and motor and perhaps other systems.Late visual potentials allow the genomic imaging of dopamine inactivation effects on visual post-processing. The same specific time-interval has been found to be affected by  Event-related potentials offer the possibility to examine the influence of genes on cognitive processes with a high time resolution. In visual processing, various stages can be described during perception, ranging from the spatial analysis of a visual stimulus during the P1 and N1 COMT and DAT1 genes on time resolved visual processing. The analyses ranged from initial stimulus perception  to stimulus post-processing after the offset of the visual stimulus (early CNV).Dopamine modulates the neuronal signal-to-noise ratio in order to focus prefrontal cortical resources COMT polymorphism, characterized by the substitution of valine for methionine at codon 158 DAT1 and the 6-repeat allele of a VNTR in intron 8 have been found to lead to greater DAT1 expression DAT1 genotype on EEG or neuroimaging data, there is a wide agreement that this DAT1 polymorphism is functional. Thus, here we examined the DAT1 haplotype and its interaction with the COMT Val158Met polymorphism in relation to visual early CNV in adolescents:We examined a functional A continuous performance test (CPT) in the A-X version was employed. In this task, the cue ‘A’ was follwed with a probability of 50% by the target stimulus ‘X’ which required a speeded motor response. We analyzed different visual ERP components evoked by the cue ‘A’ from initial visual perception , to early and late visual CNV. Movement-related potentials have been addressed in a previous paper The study was approved by the institutional review board of the Medical Faculty of the University of Heidelberg/Mannheim. Written informed consent was obtained from all participants and their parents according to the Declaration of Helsinki.The exact sampling procedure has been described before Hand dominance was assessed by the Edinburgh Handedness Inventory Continuous 64-channel DC EEG was recorded by Neuroscan Synamps amplifiers . Sintered silver/silver chloride electrodes were positioned by an equidistant electrode cap . Electrode impedances were kept below 10 kOhm. Vertical electrooculogram (VEOG) was recorded by electrodes 1 cm below and above the left eye. Horizontal electrooculogram (VEOG) was calculated by leads F9' and F10' next to the outer canthi. Small deviations of electrode positions from the international 10-10 system are indicated by apostrophes. The recording reference was placed near the left mastoid. Offline, data were transformed to average reference. The sampling rate was 500 Hz. An anti-aliasing low-pass filter with a cut-off frequency of 100 Hz was employed. The visual stimulation was presented by Gentask of the Neuroscan Stim software package. Reaction times were collected from response triggers from the response pad.Subjects performed a computerized A-X version of the continuous performance test  were employed as distractors. The distractor ‘H’ occurred 160 times (frequent distractor). The distractors ‘B’, ‘C’, ‘D’, ‘E’, ‘F’, ‘G’, ‘J’, and ‘L’ appeared 40 times each.Average reference was calculated offline. Data were 30 Hz low-pass filtered by a zero-phase shift Butterworth filter with a slope of 48 dB/octave. Continuous recordings were segmented into epochs which comprised the time interval from 400 ms prior to the distractor before the warning stimulus ‘A’ to 1600 ms after the imperative stimulus ‘X’ . Data were corrected for eye movements and blinks by the algorithm of Gratton and Coles . Potentials exceeding 150 µV amplitude were rejected automatically as artifacts; remaining smaller artifacts were removed by an experienced EEG technician who was blind to the study hypotheses.Visual post-processing . As the main target parameter, we analyzed visual post-processing after the warning stimulus ‘A’  during the interval 600–900 ms after stimulus onset, i.e. 450–750 ms after stimulus offset at the pooled leads P7', P8', P9', P10' Visual perception and visual stimulus anticipation during late CNV. In order to exclude that increased stimulus post-processing could be a by-product of increased anticipation or increased attention during the initial stimulus perception, we examined the same genetic effects on preceding visual potential components:Stimulus anticipation durin late CNV: The parieto-occipital late contingent negative variation was determined over posterior parietal/dorsal occipital areas  during the 200 ms preceding the target stimulus Perception/attentio: P1 was calculated as the mean amplitude in a time window ±10 ms around the positive peak 70–140 ms after the warning stimulus at the pooled leads O1', O2', P7', P8' Supramodal orienting response. Finally, in order to complete the stimulus-locked analysis, genetic effects on orienting  were examined. Frontal early CNV amplitude was measured at its topographical maximum at Fz during the time interval 600–900 ms DAT1 6R–10R/6R–10R+COMT Met/Met group  and applied to other groups after refitting the dipole orientations.Source analysis was carried out on the group grand averages which provide the best signal-to-noise ratio on time intervals which yielded significant genetic effects in the surface potential analysis In a second step sLORETA was performed in BESA research 5.3  on the same early CNV time interval in order to describe the extension of the distributed cortical sources of visual early CNV as exact as possible. Default regularization parameters were employed assuring that neither over-regularization would lead to a low number of widespread midline sources nor an under-regularization to numerous superficial sources.EDTA anticoagulated venous blood samples were collected. Leukocyte genomic deoxyribonucleic acid (DNA) was isolated with the Qiamp DNA extraction kit .COMT single nucleotide polymorphism (SNP) was completed using TaqMan (SNP) Genotyping Assays . Amplification conditions for COMT rs4680 were: 3.0 µl TaqMan® Mastermix, 0.3 µl/0.15 µl TaqMan® oligonucleotide mix (20×/40×), 1.70 µl dH2O and 1 µl DNA solution (∼30 ng) in 96-well format in a 6 µl reaction. Amplification was performed by initial heating of 10 min–95°C, 40 cycles of 15 sec–95°C/1 min–60°C and final 10 min–4°C. TaqMan® assay-on-demand ID C_25746809_50 detected the alleles of rs4680 (hCV25746809) in the sequence context of CCAGCGGATGGTGGATTTCGCTGGC[A/G]TGAAGGACAAGGTGTGCATGCC.Genotyping of the DAT1 was genotyped with the primers and reaction conditions of Sano et al. DAT1 VNTR in intron 8  and the 40 bp DAT1 VNTR at the 3′UTR . No deviations from Hardy Weinberg equilibrium were detected .The 40-bp VNTR polymorphism in 3′-untranslated-region (UTR) of DAT1 VNTRs were analyzed combined as haplotype. In accordance with previous literature and to avoid small groups containing only a low number of subjects, with respect to the DAT1 haplotype, subjects were dichotomized into homozygous carriers of the 6R–10R haplotype, which have previously been demonstrated to increase the risk of psychiatric disorders Both DAT1 haplotype: 6R–10R/6R–10R (N = 90) versus at least one non-6R–10R haplotype (N = 105); and (2) COMT: Val/Val (N = 43) versus Val/Met (N = 107) versus Met/Met (N = 45).In detail, the following genotype groups were formed: (1) DAT1 haplotype  and COMT  on visual early CNV amplitude, linear regression analyses were performed. Gender was entered into the model as a covariate. In order to test for a significant epistasis between DAT1 and COMT, regression models with and without an interaction term were compared. Significant interactions were further examined by univariate regression analyses with COMT alleles as predictor separately for each DAT1 haplotype level, again controlling for gender effects.To examine the effect of the We also tested whether the results would be confirmed when the visual N700 following the distractor and the visual N700 following the target stimulus ‘X’ were used as dependent variables, as we hypothesized that the visual N700 following any single visual stimulus could be the equivalent to early CNV following the cue ‘A’.Finally, possible genetic effects on earlier stimulus processing and supramodal working memory updating/orienting were assessed by t-tests to examine the possibility that increased post-processing was only a by-product of increased earlier processing. No correction for multiple testing was applied in order to detect any confounding effects. Pearson correlation coefficients were computed to assess associations between occipito-temporal and frontal early CNV components as well as between initial visual perception-related components  and subsequent visual post-processing .The task was well accomplished by the subjects, with on average 2.5±2.7 omission and 2.4±3.0 commission errors. Further details on mean reaction time, reaction time variability, omission and commission errors have been reported previously DAT1 haplotype or COMT genotype on P1, N2, or occipito-parietal late CNV amplitude . For any qualitative differences in P1, N2 or CNV between the genotype groups, see COMT Met/Met (−6.2±4.0 µV) versus the Val/Val group . Interactions between the DAT1 haplotype and COMT genotype with respect to event-related potential components that preceded early visual CNV were far from being statistically significant .We obtained no significant effects of DAT1 and COMT polymorphisms had a significant effect on occipito-temporal early CNV amplitude .Both DAT1 and COMT specifically affected the visual post-processing window following the distracter stimuli, the warning stimulus ‘A’ and the target stimulus ‘X’. The visual early CNV topography with the characteristic maximum over occipito-temporal visual areas is also presented. COMT effects tended to be stronger in the presence of the homozygous 6R–10R DAT1 haplotype. There were no changes in visual early CNV topography or duration, but occipito-temporal early CNV amplitude was affected.DAT1-haplotype-defined groups indicated that the COMT genotype only had a significant effect on visual early CNV amplitude in homozygous carriers of the 6R–10R DAT1 haplotype  but not otherwise . The largest early visual CNV amplitudes occurred in those subjects carrying both the Met/Met COMT polymorphism and the homozygous 6R–10R DAT1 haplotype . DAT1 regression slope was −0.67±0.40 µV  and COMT regression slope was −0.59±0.29 µV . Again, there was a trend towards an interaction of DAT1 and COMT  in the model with an interaction term .The results were also roughly confirmed for the occipito-temporal early CNV followed by non-target stimuli in an additive model  and DAT1  and the trend towards an interaction between DAT1 and COMT  were confirmed.Accordingly, when all trials involving the cue ‘A’ (both followed by a target and by a non-target stimulus) were analyzed together, the additive effects of COMT genotype and early frontal CNV amplitude , there was no significant association of DAT1  or an interaction between COMT and DAT1  with respect to early frontal CNV. However, there was a strong negative correlation between early occipito-temporal and early frontal CNV , i.e. larger negativity over visual areas was associated with less frontal negativity  and commission errors  but not reaction time , indicating that larger negativity during visual post-processing was associated with a higher number of both types of errors.In contrast, there was no significant correlation between P1  or N2 amplitude  and early occipito-temporal CNV amplitude despite the large sample size.Source analysis: Residual variance during the visual early CNV time interval could be reduced to 7.7% by the 4-dipole model for the 6R–10R/6R–10R+Met/Met – group revealing the most pronounced visual early CNV. DAT1 other+COMT Val/Val group. For all groups, the dipole moments returned to baseline after the end of the visual early CNV component. sLORETA results pointed towards activation around Brodman areas 19 and 37. There were no hints towards topographic differences between the genetic groups. DAT1 and COMT polymorphisms influenced the extent to which secondary visual areas became transiently and selectively active during visual post-processing.DAT1 and COMT effects  and the interaction thereof  failed to reach trend or significance level in the linear regression model.Despite qualitative results for the visual N700 following the target stimulus ‘X’ which pointed in the same direction as for the visual N700 following the cue  and 2, tDAT1 and COMT effects on the visual N700 following non-target stimuli which had been preceded by the cue ‘A’ showed a trend for DAT1  but no effect of COMT , while the interaction between DAT1 and COMT reached trend level in this case .The corresponding additive DAT1  or COMT  alone, but a trend towards an interaction between the two . Separate linear regression analyses for the two DAT1-haplotype-defined groups indicated that the COMT genotype had a significant effect on visual N700 amplitude in homozygous carriers of the 6R–10R DAT1 haplotype only  but not otherwise . Again, the largest visual N700 amplitudes occurred in the subjects with the Met/Met COMT polymorphism in the presence of the homozygous 6R–10R DAT1 haplotype.With respect to the visual N700 following the distracter which preceded the cue, there were also no significant effects of DAT1 and COMT influence the amount of cortical activity during visual post-processing in the same specific time window about 500–1000 ms after the stimulus which we previously described for the motor system during movement post-processing COMT exhibited intermediate occipito-temporal early CNV amplitudes. Individuals with the homozygous 6R–10R DAT1 haplotype showed higher visual early CNV amplitudes than subjects with at least one non-6R–10R DAT1 allele. In agreement with Alexander's model DAT1 and COMT with respect to visual and motor post-processing/working memory encoding.The present investigation reports data on genomic imaging of the time course of visual processing. The findings outlined above provide first evidence that genetic variation in There was a strong negative correlation between visual post-processing  and the orienting reaction as reflected by frontal early CNV, and both source analyses confirmed that these two negativities reflect distinct sources. Like in the motor system, where increased motor post-processing was associated with longer reaction times as an indicator for less attention and response preparation, again, visual post-processing was associated with a higher number of both omission and commission errors. I.e. worse task performance and less controlled attention were associated with increased post-processing. This could indicate that post-processing is used to compensate for deficits during initial stimulus perception.DAT1 and COMT polymorphisms as did the occipito-temporal early CNV.However, frontal early CNV did not show the same or an inverse modulation by There was no significant correlation between ERP components during initial visual perception of the cue  and subsequent occipito-temporal early CNV. Taking into account the large sample size, this means that if initial visual perception would directly influence the amount of visual post-processing, any effect would have to be extremely small.DAT1 and COMT on visual post-processing are not limited to paired stimulus paradigms (early CNV), but that a single visual stimulus is sufficient to evoke the same visual post-processing DAT1 and COMT was not as clear as in the motor system DAT1 and COMT have been described previously for the visual system in a working memory task There was a trend towards an interaction between DAT1 and COMT for the visual early CNV following the cue ‘A’, the N700 following the preceding distractors and the non-X stimuli following the cue ‘A’ which required response inhibition. With respect to the N700 following the target stimuli, a ceiling effect may have been present, as overall higher amplitudes were obtained than during early CNV or N700 following a distractor. We suggest that the effects of DAT1 and COMT and not N700 duration. This suggests that it was not the direct effect of DAT1 or COMT on dopamine inactivation by reuptake or metabolism that was operative, but rather the effects of DAT1 and COMT on tonic dopamine levels mediated the influences on visual post-processing DAT1 haplotype could also point towards visual stimuli eliciting larger phasic dopaminergic responses in the presence of lower tonic dopamine levels, taking into account that the 6R–10R DAT1 haplotype has been associated with a higher dopamine turnover in children and adolescents As already observed in the motor system, the amplitude of visual early CNV/N700 was affected by Preceding visual event-related potential components during visual perception:COMT Met/Met genotype among homozygous carriers of the 6R–10R DAT1 haplotype, we obtained rather a combination of (non-significantly) lower initial processing followed by higher post-processing. Thus, no tentative influence of COMT and DAT genotypes pointed into the same direction as the influences of COMT and DAT on visual early CNV amplitude. There was no hint that the effects of DAT1 and COMT on increased post-processing could have been a consequence of increased initial visual processing. COMT and DAT1 genotype did not affect reaction time variability in this sample without subjects with attention deficit hyperactivity disorder Overall, the results of the control conditions did not uphold the correction for multiple testing and must be examined in further studies. However, for the Our results, thus, suggest several phases of dopaminergic effects on visual processing in visual event-related potentials and early CNV:DAT1 and COMT polymorphisms did not affect early visual processing in our study. It remains unclear whether the association of reduced P100 in schizophrenia and working memory deficits may be an indirect effect due to a common source which affects both parameters.Early visual processing: Dopamine may exert influences on early visual perception in the retina, though it is not an important neurotransmitter for the central pathways as shown in patients with Parkinsons's disease who have decreased visual P100 amplitudes Early CNV and working memory encoding: Early CNV shows pronounced age effects during development with an amplitude decrease over frontal sites For the first time, dopaminergic genetic influences were demonstrated on the time course of visual processing in 15-year-old adolescents. We found that the same specific time interval as for the motor system was affected. We propose that modality-dependent memory traces in perceiving  or executing (motor) systems are modulated during memory encoding by a dopaminergic system which affects the time interval of about 500–1000 ms after the stimulus or movement. Earlier perceptual stages were unaffected. Future studies will have to reveal the extent to which an inefficient resource allocation during the orienting response  affects performance, and contributes to an increased number of errors especially in adolescents.


Glycerol is a by-product of the manufacture of biodiesel and its supply as a sustainable raw material is anticipated to grow steadily due to a shift towards “greener” fuels. Since glycerol provides a precursor to various industrially valuable products, such as glyceric acid, tartronic acid and hydroxyacetone, this increasing glycerol supply represents an industrially important feedstock for synthesizing such chemicals by oxidative reaction. Heterogeneous catalysts play a key role in the promotion and control of such reactions, and can also lead to “greener” synthesis routes when compared with alternatives, such as the addition of stoichiometric reagents during synthesis.Nanoparticulate gold dispersed on a variety of oxide supports has been studied for a wide range of catalytic redox reactions,113 products (glyceric and tartronic acids) can be achieved at ambient temperatures without employing a base. We further report microstructural characterization of these bimetallic catalysts using aberration-corrected scanning transmission electron microscopy (STEM), high-angle annular dark-field (HAADF) imaging, and energy-dispersive X-ray spectroscopy (XEDS).While these previous studies have focused on monometallic nanoparticulate gold, we have since demonstrated that alloying gold with palladium can lead to a 25-fold enhancement in the activity of alcohol oxidation.2 due to hydrolysis during the immobilization process, which is carried out in aqueous solution.Bimetallic catalysts were synthesized by immobilizing colloidal metal particles on an MgO support. X-ray diffraction (XRD) analysis . In contrast, Au–Pd catalyst prepared with a 1:3 mol fraction and similar metal loading demonstrated significantly lower activity under these conditions, and the selectivity to glyceric acid was lower than that displayed for the Au–Pt catalyst, despite the lower conversion observed. These results suggest that at similar conversion levels, the Au–Pd bimetallic catalysts are significantly less selective to the desired C3 products and data at iso-conversion shows this is the case . Indeed, the [Mg2+] is 2–3 orders of magnitude lower in amount than the products observed. Furthermore addition of this minimal level of base did not significantly affect the conversion and there was no reaction in the presence of Mg2+.It is possible that MgO may be acting as a sacrificial base during the reaction and hence we investigated possible leaching of Mgα lines. These results suggest that the individual metal particles do contain both elements. However, this analysis is complicated by the inherently low XEDS signal collected from such small particles, pathological overlaps between the Au-Mα and Pt-Mα peaks, and the degradation of the underlying Mg(OH2) support under the electron beam. Whether the metal particles are homogeneous or phase-separated within each particle (e.g. a core–shell morphology similar to that observed for Au–Pd) could not be resolved due to the extremely small size of the particles and the difficulty in obtaining spatially resolved XEDS data from such small particles at near atomic resolution.Transmission electron microscopy (TEM) measurements demonstrate that the Au–Pd catalysts have a broader particle-size distribution than the Au–Pt catalysts . The micZ) between Pd (Z=46) and Au (Z=79), the Au–Pd particles were determined to be homogeneous. If these particles experienced substantial core–shell segregation, it would have been clearly visible because the HAADF cross-sections of the two elements differ by more than a factor of 2.A representative selection of STEM-HAADF images of the Au–Pd and Au–Pt (both 1:3 mole fraction) particles supported on MgO are presented in Figure 2 catalyst both for reactions at ambient temperature and at 60 °C at 60 °C . After r60 °C