N-glycomes in mammals. This means that cellular differentiation is accompanied by changes in the N-glycan profile. Yet when the N-glycomes of cell types with differing fates diverge is unclear. We have investigated the N-glycan profiles of two different clonal populations of mesenchymal stromal cells (MSCs). One clone (Y101), when differentiated into osteoblasts, showed a marked shift in the glycan profile toward a higher abundance of complex N-glycans and more core fucosylation. Yet chemical inhibition of complex glycan formation during osteogenic differentiation did not prevent the formation of functional osteoblasts. However, the N-glycan profile of another MSC clone (Y202), which cannot differentiate into osteoblasts, was not significantly different from that of the clone that can. Interestingly, incubation of Y202 cells in osteogenic medium caused a similar reduction of oligomannose glycan content in this non-differentiating cell line. Our analysis implies that the N-glycome changes seen upon differentiation do not have direct functional links to the differentiation process. Thus N-glycans may instead be important for self-renewal rather than for cell fate determination.Different cell types have different  Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells that contains both adult multipotent and immunomodulatory cell types  despite their differing phenotypes.While glycosylation differences are a well-accepted feature of different cell types, the functional significance of these variations is less clear. Moreover, it is unknown whether MSC sub-types with different properties, such as differentiation-competent MSCs vs. primarily immunomodulatory MSCs, have differences in their glycosylation profiles. We therefore asked if differences in the glycosylation state between the various MSC sub-populations in our model cultures could be observed, and could potentially contribute to phenotypic differences between the MSC subtypes within a primary mixture. In accordance with previous findings  supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% GlutaMax-I. To stimulate osteogenesis, cells were seeded at a density of 20,000 cells/cmN-glycan separation (FANGS) method was carried out as described  and 2 μL of 20 mg/mL 2,5-dihydroxybenzoic acid (in 70% methanol). Two microliters of this mix was spotted onto a ground steel MALDI target plate (Bruker) and allowed to air dry. Immediately afterwards, 0.2 μL of ethanol was added to the spot and left to air dry for re-crystallization. Samples were analyzed using an ultraflex III MALDI-TOF mass spectrometer (Bruker). The spectra were acquired over the m/z, using the Savitzky-Golay algorithm. Glycan peaks were identified from their mono-isotopic peak m/z values on comparison with those for established N-glycan structures. For a glycan to be included in the analysis at least two of its isotopic peaks had to have intensities >3x the noise, and the associated peaks had to fall into an isotopic pattern as predicted based on the glycan's atomic composition (Bruker Compass's Isotope Pattern function). The peak intensities of all isotope signals >3x noise for a given glycan were summed to provide that glycan's total peak intensity. To compare different spectra, the total peak intensity values were normalized to the total peak intensities of all glycans identified in that spectrum. Normalized peak intensities could then be averaged across different spectra of the same sample type.Spectra were analyzed using flexAnalysis 3.3 (Bruker), first processed using the centroid peak detection algorithm, using a signal to noise limit of 3, and smoothed for 1 cycle at 0.1 Cells were washed twice with PBS, and incubated with washing buffer  at 37°C until cells detached. Cells were centrifuged for 5 min at 450 g, resuspended in PBS and 150,000 cells pelleted and resuspended in 120 μL ice-cold PBS. Following 15 min incubation on ice, 120 μL of 10 μg/mL FITC-ConA (Vector Labs) was added to each sample. After two 15 min incubations interspersed with flicking of the tubes, 1 mL of washing buffer was added and the samples were centrifuged for 5 min at 450 g. The cell pellet was resuspended in 100 μL of washing buffer containing 1 μg/mL DAPI and incubated on ice in the dark for 5 min, followed by the addition of 1 mL washing buffer, and centrifugation for 5 min at 450 g. The pellet was resuspended in 400 μL PBS for flow cytometry. Data were gated for DAPI-negative FITC-positive cells. Median fluorescence intensity was determined for all gated cells in the sample.Analysis of cell surface markers was performed as previously described  and a test of equal variance was performed. For comparing two groups if data passed, a Student's N-glycans of one of these lines, Y101, were profiled using the FANGS method . Among the complex glycans, the singly sialylated, fucosylated biantennary species (m/z 2605.24), followed closely by the same glycan without sialic acid (m/z 2244.08) and then the bis-sialylated version (m/z 2966.42) are most abundant. It is worth noting that the majority of the large complex glycans are fucosylated , which most likely indicates the presence of core fucose.To study the sub-type-specific properties of cells from a primary human MSC mixture, previously generated telomerase immortalized lines derived from primary human MSCs, termed hTERT-MSCs , followed, at about half the intensity, by the same complex glycan without sialic acid (m/z 2244.08) and the fully sialylated species (m/z 2966.39). The most abundant oligomannose glycan in osteoblasts, the Man6 species (m/z 1783.87), is only ranked fourth in relative intensity in the N-glycome of osteoblasts (Figure Changes in the S Figure . The res Figures . In the s Figure , Table 1N-glycomes of osteoblasts, although these are among the most abundant glycans in those of the MSCs (Figure m/z 2600.22 Figures Averaging the glycan peak intensities from several biological replicates showed that the most abundant complex glycans were indeed reproducibly much more abundant in osteoblasts than undifferentiated MSCs Figure , Table 1s Figure , Table 1N-glycan profile, we wondered whether MSCs with diverse differentiation abilities present variations in N-glycan profiles. We compared two hTERT-MSC lines: Y101 and Y202. Y101 cells differentiate well into osteoblasts. Y202 cells in contrast, which do show the cell surface markers characteristic of MSCs (Supplementary Figure Given that differentiation considerably alters the N-glycan repertoire, we used FANGS followed by MALDI-TOF-MS to profile their glycans. Both MSC lines were cultured for 24 h in basal medium post passaging, prior to N-glycan sample harvest and analysis. The overall N-glycan profiles of these lines look very similar to each other (Figure N-glycan profile is between the MSCs and the differentiated progeny, rather than within the different lines of MSCs (Figure To assess if the differences in differentiation capacity between the different clonal MSC lines are associated with a variation in their r Figure . Profilir Figure . This lar Figure . A heat s Figure .It is clear that differentiation alters the glycan profile, yet the glycan profile of the stem cells does not provide clues to their differentiation potential. We therefore wondered if the glycosylation changes are restricted to cells undergoing differentiation, or whether non-differentiating cells, when incubated under differentiation-promoting conditions, alter their glycan profiles. The reduction of oligomannose glycan content was investigated for MSCs incubated in osteogenic medium for 8 days. As expected, Y101 cells showed a clear reduction in oligomannose content, as evidenced by reduced ConA-FITC staining measured using flow cytometry when compared with cells cultured in basal medium (Figure As a second test we wondered if glycosylation changes are necessary for differentiation into osteoblasts to take place. We used the glycan-processing inhibitor swainsonine, which inhibits mannosidase II. As expected, this caused a major shift in the glycan profile, in which complex glycans almost completely disappeared, and were replaced by hybrid ones (Supplementary Figure per se as in our study. Nevertheless, the shift in oligomannose glycan abundance from 70 to 80% in the stem cells to 30% in the fibroblasts (An et al., A previous glycomic study concluded that oligomannose glycans are more abundant in parental stem cells than in their differentiated osteoblasts (Heiskanen et al., Another change within the glycan profile is increased overall fucosylation intensity in osteoblasts. Single fucosylation could be present either on the core or the antennae, but glycans with multiple fucoses have to contain fucose on the antennae. From MSCs we observed several glycans with multiple fucoses as opposed to osteoblasts where single fucosylation predominates. Thus while the overall intensity of fucosylation is elevated in osteoblasts, the level of fucosylation on antennae is likely higher in the stem cells, leading us to speculate that fucosylation on the antennae could be a property of stem cells rather than osteoblasts Figures . Indeed,N-glycan profiles, despite a marked difference in their differentiation capabilities. The profiles of the stem cell lines we report are also similar to the previously reported profiles of primary MSCs (Heiskanen et al., N-glycan profiles are a good indicator of overall MSC identity. Importantly, changes to the glycan profile following prolonged culturing of the stem cells are also small, enforcing the usefulness of glycans as an MSC marker. The main difference between the glycan profiles of MSCs before and after prolonged culturing is a significantly higher abundance of degradation products (Table N-glycans that cannot be made during biosynthetic processing in mammals is most likely a consequence of intermediates accumulating in the lysosome during glycan degradation (Uematsu et al., N-glycans. The amount of these glycan species in the MSCs and osteoblasts is in line with the levels of paucimannose glycans found in other glycan profiling studies of cultured cells (Abdul Rahman et al., Comparison of the two MSC lines shows a remarkable similarity in their ts Table . The appN-glycans in MSCs is neither required nor sufficient alone to drive osteogenesis. Inhibition of complex N-glycan formation by swainsonine does not inhibit osteogenesis. In contrast, the shift to a lower proportion of oligomannose glycans in Y202 cells is not accompanied by osteoblast mediated mineralization. Rather, it could more likely be the stem-like properties of MSCs that may benefit from certain glycan features.A major question in glycobiology is the function of glycans during cellular processes. While it is well documented that there are changes in glycan profiles between cell types, the functional consequence of these changes is unclear. Our results suggest that the shift to complex N-glycan profile as the differentiating Y101 MSC line, it is unlikely that glycans directly determine the differentiation potential of these cells. However, continued proliferation and self-renewal are also very important properties of stem cells. Indeed terminal fucosylation has been suggested to be an important factor in stem cell maintenance (Li et al., Our detailed analysis of two MSC cell lines allows us to speculate about the requirements for the various glycan subtypes of different stem cell functions. Given that the Y202 line, which is unable to differentiate effectively, has the same KW, PG, and DU designed the study; KW performed the experiments; KW, JTO, and DU analyzed the data; KW, JTO, PG, and DU wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Non-infectious chronic uveitis is a serious and disabling sight-threatening disease accounting for up to 10% of pathologies leading to blindness. Currently, a step-by step escalating immunosuppressive therapy is generally used, in children as well as in adults, and anti-TNFa biologic therapies have markedly increased the treatment options for sight-threatening uveitis refractory to conventional immune-modulatory therapy (DMARD) in addition to topical and/or systemic corticosteroids. However, a subset of patients fails to respond to TNFa blockers or is unable to tolerate these therapies and may therefore benefit from switching to another drug. In this clinical setting, the large availability of several different molecules, mostly off-label, poses the clinical question if it can be useful and safe to administer another class of biologic drugs, such as Abatacept or Rituximab, for patients with refractory auto-immune uveitis.To summarize the evidence regarding the effectiveness and the safety of switching to a Non anti-TNF biologic modifier immunosuppressant treatment (NTT) currently available in clinical practiceAcomprehensive systematic review was undertaken involving a literature search between January 2000 and April 2013 was conducted using EMBASE, Ovid MEDLINE, Evidence Based Medicine Reviews–ACP Journal Club, Cochrane libraries, and EBM Reviews. Studies investigating the efficacy of NTT as biologic modifier immunosuppressant medication for autoimmune chronic uveitis, refractory to topical and/or systemic steroid therapy, were eligible for inclusion. The primary outcome measure was the improvement of intraocular inflammation, as defined by the SUN working group criteria. We determined a combined estimate of the proportion of subjects responding to NTT.We initially identified 526 articles, of which 89 were potentially eligible. From the selection process, a total of 10 retrospective chart reviews and 1 randomized single-blind controlled study, providing a total of 12 children and 34 adults, were deemed eligible. The studies were related to Rituximab (n=3), Abatacept (n=3), Tocilizumab (n=3) and single studies on Alemtuzumab and Anakinra. Before the NTT treatment, all the eligible subjects received several combinations of one or more DMARD and at least one anti-TNF strategy. Considering the observational studies, thus excluding 7 adults enrolled in the RCT, 8 children out 12, and 18 adults out of 27 responded to NTT treatment: 0.66 was the combined estimate of the proportion of subjects improving on NTT treatment for children (95% CI: 0.58-0.81) and adults (95% CI: 0.64-0.79). Further statistical comparison between different NTT strategies was not possible due to the small sample sizeThe only RCT reported a success rate of 2 out of 7 adult Behçet’s disease patients with a 6-month exposure to RituximabAlthough randomized controlled trials are needed, the available evidence suggests that a NTT strategy may be useful in selected categories of autoimmune chronic uveitis in adults as well in childhood, refractory to a previous course of immunosuppressive treatment, both with DMARDs and anti-TNFa.None declared.


Standard flow territory maps were subdivided into proximal, intermediate and distal flow territories, based on the measured ATT. The coefficient of variation (CV) and physiological correlations with age and gender were compared between the three perfusion parameters.Two pseudo-continuous arterial spin labeling (ASL) scans with  but the CV of ATT (4–9%) was much smaller. The total gray matter correlations with age and gender were most significant with ATT (p = .016 and p<.001 respectively), in between for CBFcrushed (p = .206 and p = .019) and least significant for CBFnon-crushed (p = .236 and p = .100).There was no difference in CV between CBFThese data show the feasibility and added value of combined measurements of both crushed CBF and ATT for group analyses in elderly with hypertension. The obtained flow territories provide knowledge on vascular anatomy of elderly with hypertension and can be used in future studies to investigate regional vascular effects. Paralathology –5.crushed) and CBF of the macro- and micro-vascular compartments together (CBFnon-crushed)  by the flow-encoding arterial spin tagging (FEAST) method. FEAST is based upon the subdivision of an imaging voxel into macro- and micro-vascular compartments based on differences in blood flow velocity . By perfcrushed) . The ratcrushed may be preferable to CBFnon-crushed, since micro-vascular CBF changes are generally assumed to reflect (patho-)physiological changes in neuronal activity and energy demand whereas macro-vascular CBF is believed to be dominated by cardiovascular fluctuation [Several advantages exist for the application of a vascular crusher and the estimation of ATT using FEAST. CBFctuation . ATT hasctuation ,9. Compactuation , relativctuation . Howeverctuation . Therefoctuation . Howevercrushed and ATT for large clinical imaging studies in elderly with hypertension. To investigate the spatial distribution of these parameters, we used the estimated ATTs to divide standard flow territory maps into proximal, intermediate and distal flow territories. We study the reliability of these perfusion parameters in terms of their population variation and physiological correlations with age and gender.The primary purpose of this study was to assess the feasibility and value of the combined measurement of CBF195 community-dwelling elderly  with hypertension (systolic blood pressure higher than 140 mmHg) participating in the Pre-DIVA study were eligible for inclusion . ExclusiAll patients provided written informed consent and the study was approved by the institutional review board of the Academic Medical Center, Amsterdam.crushed, CBFnon-crushed and ATT within clinical scanning time, was added to a routine clinical dementia protocol. Two ASL scans were performed with (CBFcrushed) and without (CBFnon-crushed) flow-crushing gradients in three directions (velocity encoding 50 mm/s). Identical imaging parameters of the two consecutive gradient-echo single-shot EPI pseudo-continuous ASL (pCASL) sequences were: matrix = 64x64, FOV = 240 x 240 mm, 17 axial slices, slice thickness 7 mm, no gap, echo time/repetition time = 17/4000 ms, flip angle = 90 degrees, SENSE = 2.5, initial post-label delay (PLD) = 1525 ms; slice readout time = 34.9 ms; resulting PLD range for 17 slices = 1525–2080 ms, labeling duration = 1650 ms and two background suppression pulses at 1710 and 2860 ms after a pre-labeling saturation pulse. 20 label-control pairs were acquired ‒ resulting in a duration of 2:40 minutes for each scan. The labeling plane was positioned parallel and 8.3 cm inferior to the center of the imaging volume [3 3D T1-weighted scan was included in the imaging protocol for segmentation and registration purposes.All imaging was performed on a 3T system  equipped with an 8-channel head coil. Foam padding was used to restrict head motion. A slightly adapted version of the original FEAST acquisition, which enables the simultaneous acquisition and quantification of CBFg volume . An isot2*a is the transverse relaxation time of arterial blood (50 ms) [a is the equilibrium magnetization of arterial blood, for which an average scanner value was used [inv is the correction for label loss due to background suppression pulses (0.83) [1a is the T1 relaxation time of arterial blood (1650 ms) [crushed, which is replaced by the PLD (1525 ms + 34.9 ms/slice) for CBFnon-crushed.The raw EPI control and label images were 3D motion corrected using SPM8 . After pair-wise subtraction, these raw maps were converted to CBF with a single compartment model, assuming that the label decays with the T1 of blood ,14:CBF[05 g/mL) , ΔM is t (50 ms) , M0a is was used , calculawas used , α is thwas used , αinv iss (0.83) , T1a is 1650 ms) , PLD = 1ATT was calculated based on the following two FEAST equations :ΔM=A algorithm and the resulting normalization fields were applied to the CBF and ATT maps [The 3D T1-weighted anatomical scans were segmented using SPM8 into gray matter tissue probability maps. After pair-wise subtraction, the CBFATT maps .The total cerebral gray matter was defined as tissue probabilities >70%. Standard flow territory templates (left and right combined) were used to investigate vascular territories supplied by the bilateral anterior, middle and posterior cerebral arteries  . Within crushed and CBFnon-crushed, the sign test and Brown-Forsythe test were used . To provide insight in the distribution of CBF and ATT values in the ACA, MCA and PCA, median group-level histograms were generated from the histograms of individual maps . To investigate physiological correlations, robust linear regression analyses were performed to model cross-sectional correlations between the predictors age or gender and the dependent variables CBFnon-crushed, CBFcrushed or ATT, adjusted for total brain volume (defined as the combined volume of gray and white matter segmentations).Prior to all analyses, the distributions of investigated values were tested for normality using the Shapiro-Wilk test. Because most distributions of perfusion parameters deviated from normal, distributions were summarized by the median and mean absolute deviation (instead of mean and standard deviation). To compare variation between the perfusion parameters, the coefficient of variation (CV) was used, calculated as mean absolute deviation divided by the median. To test whether respectively the median or CV differed between CBFThe ATT-based flow territories showed an almost entirely continuous sequence from anterior-inferior to posterior-superior (ACA), inferior to superior (MCA) and anterior-inferior to posterior-superior (PCA) .crushed and CBFnon-crushed maps had a similar appearance. Whereas the median CBFcrushed differed in nearly all flow territories from the median CBFnon-crushed, there was no CV difference between CBFcrushed and CBFnon-crushed , both ranging from 15% to 20% , 12–23% (MCA) and 7–14% (PCA) .crushed and FEAST-based ATT measurements within acceptable scanning time in a population of elderly with hypertension. These perfusion parameters showed stronger correlations with age and gender compared to conventional CBF measurements, demonstrating the potential value of CBFcrushed and ATT for group analyses. The observation that the variation of perfusion parameters within flow territories was comparable with the variation of the total gray matter, suggests that the reliability of crushing and FEAST within flow territories is sufficient on a group level, and no further spatial averaging is required. For large clinical imaging studies in elderly with hypertension, therefore, there appears to be potential to study more perfusion parameters than CBF alone.The results of this study prove the feasibility of ASL-based CBFThe ATT maps comply with the trajectory of the cerebral vessels, with shortest ATTs where the vessels enter the cerebrum and longest at the superior-posterior watershed area . The resThe observed correlations of the perfusion parameters with age lie within the range of previously reported values, whereas the differences with gender were relatively small compared to previous values –30. Thescrushed) and ΔM (CBFnon-crushed), and will thus not propagate into the FEAST-based ATT measurement, which is derived from the ratio of the two perfusion scans , but short enough to image before all the labeled blood enters the microvasculature (i.e. PLD<micro-vascular ATT). When the PLD is shorter than the macro-vascular ATT, there will be no labeled signal in either the crushed or non-crushed acquisition, and the division of noise with noise will lead to a Cauchy distribution around a mean ratio of 1, corresponding to an ATT that is equal to the PLD where in reality the ATT is longer. On the other hand, in the case that the PLD is longer than the micro-vascular ATT, all label will already have arrived in the micro-vascular compartment and the signal in both crushed and non-crushed acquisitions will be similar. This again leads to a ratio of 1, corresponding to an ATT equal to the PLD where in reality the ATT is shorter. Both situations would lead to narrow ATT histograms with minimum values coinciding with the PLD.To which extent the skewness in the ATT histograms reflects the physiological distribution of ATT within flow territories, or is due to too long macro-vascular ATT or too short micro-vascular ATT relative to the PLD, cannot be differentiated with these data. According to our comparison of the FEAST-based ATT values with PLD-values , we estiNevertheless, it is remarkable that the PCA exhibited the lowest proportion of artifactual ATT-values  but alsocrushed compared to CBFnon-crushed is, at least partly, due to our shorter PLD [To satisfy PLD criteria for both CBF and ATT measurements, we applied a PLD that is a trade-off between conventional CBF measurements and FEAST-based ATT measurements. Therefore, our mean PLD (1800 ms) and labeling duration 1650 ms) were shorter than advised for CBF measurements in this population (2000 ms and 1800 ms respectively) in the recent ASL consensus paper . It can 50 ms werrter PLD .It should be noted that because of the abovementioned velocity cutoff and PLD-limitations, the ATT-values presented in this study should not be referred to as absolute values. Because of logistic reasons and time constraints we were unable to perform multi-TI measurements in a small subset of subjects, which would have allowed us to calibrate the FEAST-based ATT-values ,10. Neve3. However, acquisition-related partial volume effects are expected to be equal for CBFcrushed, CBFnon-crushed and ATT maps, as these are all acquired with the same resolution and orientation. Hence, comparisons between these acquisitions are not expected to be significantly influenced by partial volume effects. In the current study, the labeling plane was fixed 8.3 cm inferior to the AC-PC line. Although this location has been shown to provide optimal labeling efficiency in young healthy volunteers [Partial volume effects form another potential limitation, considering the relatively large voxel size of 3.75x3.75x7 = 98.4 mmlunteers , the inclunteers .In conclusion, we have shown the feasibility and value of combined measurements of CBF and FEAST-based ATT for large imaging studies in elderly with hypertension. The obtained flow territories can be used in future studies to identify regional vascular effects. On a group level, crushing can improve correlations with physiological parameters such as age and gender. The high physiological correlations of ATT suggest that this perfusion parameter can be more relevant than conventional CBF measurements. However, the PLD should be carefully selected and one should account for the possible under- and overestimation of ATT. These data encourage future clinical imaging studies in elderly with hypertension to investigate multiple MRI perfusion parameters, instead of focusing at CBF only.


Non-allelic homologous recombination (NAHR) is a common mechanism for generating genome rearrangements and is implicated in numerous genetic disorders, but its detection in high-throughput sequencing data poses a serious challenge. We present a probabilistic model of NAHR and demonstrate its ability to find NAHR in low-coverage sequencing data from 44 individuals. We identify NAHR-mediated deletions or duplications in 109 of 324 potential NAHR loci in at least one of the individuals. These calls segregate by ancestry, are more common in closely spaced repeats, often result in duplicated genes or pseudogenes, and affect highly studied genes such as GBA and CYP2E1.The online version of this article (doi:10.1186/s13059-015-0633-1) contains supplementary material, which is available to authorized users. Non-allelic homologous recombination (NAHR) is a biological mechanism for repairing broken chromosomes, which results in gross genome rearrangements. A significant portion (approximately 10% to 22%) of all genome rearrangements in humans, also called structural variations, is thought to be the result of NAHR . UnderstDespite its importance and prevalence, NAHR is challenging to detect with either computational or experimental techniques. The difficulty stems from four crucial properties: (1) NAHR is mediated by highly homologous repeats, (2) there are millions of repeats across the human genome , 3) the the3,11,a , then all of the reads agree with the reference at both v1 and v2. This is very strong evidence in favor of the hybrid over either mediating LCR; indeed, the log-odds ratio   on LCR A is identical to the corresponding sequence on LCR B. Hence, all NAHR duplications whose breakpoint lies somewhere in  will have identical resulting hybrid LCRs.


Pigl, Tiam1, and Rffl, among others. These genes are implicated in glycosylphosphatidylinositol biosynthesis, NADPH oxidase function, and inflammation. These mutants: (1) are resistant to two different oxidative stressors, paraquat and the omission of 2-mercaptoethanol, (2) have reduced levels of endogenous reactive oxygen species (ROS), (3) are capable of generating live mice, and (4) transmit the stress resistance phenotype to the mice. This strategy offers an efficient way to select for new mutants expressing a stress resistance phenotype, to rapidly identify the causative genes, and to develop mice for in vivo studies.Longevity is correlated with stress resistance in many animal models. However, previous efforts through the boosting of the antioxidant defense system did not extend life span, suggesting that longevity related stress resistance is mediated by other uncharacterized pathways. We have developed a high-throughput platform for screening and rapid identification of novel genetic mutants in the mouse that are stress resistant. Selection for resistance to stressors occurs in mutagenized mouse embryonic stem (ES) cells, which are carefully treated so as to maintain pluripotency for mouse production. Initial characterization of these mutant ES cells revealed mutations in  Stress resistance is a trait intimately linked to increased life span and health span in multiple species, including yeast, fruit flies, worms, and mice   as a mutagen to facilitate gene identification. We report here the recovery of 7 PB-mediated, stress-resistant mutants identified via mass-selection for paraquat (PQ) resistance. These stress-resistant ES cells retained germline competence and are being converted into living mice for further in-depth investigations. The genes responsible for stress resistance are implicated in diverse functions including NADPH oxidase (Nox) activities, inflammation, and protein anchoring. These new mutants will be valuable animal models to investigate how those mutations affect stress resistance and their potential roles in extension of both life and health-span.In our previous studies we demonstrated that mice derived from stress-resistant ES cells retained the stress-resistance Phenotype in tail skin fibroblast and were able to transmit increased stress resistance to their offspring  genes could be turned off by doxycycline , designated as Bcl2 gene, bovine growth hormone polyA signal (bGHpA), phosphoglycerate kinase (PGK) promoter, neomycin resistant cassette (neo), internal ribosomal entry site (IRES), and splice-donor (SD) from the Hprt gene. This UPA trap was then subcloned into pCyL50 (a gift from the Wellcome Trust Sanger Institute) at the XhoI site which is flanked by the 3′ and 5′ long terminal repeat (LTR) of PB. The resulting piggyBac-unbiased polyA-trap vector was designated as PB-UPA  trap vector was cloned by routine restriction digestion, or PCR, and ligation so that genetic elements were put in the order of splice-acceptor (SA) from the A Figure .6) were electroporated with 1 μg of PB-UPA and 20 μg of mPB transposase (a gift from the Wellcome Trust Sanger Institute)  were plated onto a 150-mm gelatinized culture dish  and exposed to 10 μM PQ for 7 days. Colonies of surviving cells typically arose 7 days after reinstating normal culture medium; these colonies were isolated, expanded, and analyzed.Prior to stress selection, gene-trapped ES cells from each sub-library were expanded on a 100-mm culture plate in the presence of doxycyline (1 μg/ml) for 12–14 days, to generate PB transposon, were cloned  and sequenced. The DNA sequence flanking the transposon thus obtained was aligned with the mouse genome using BLAST to identify the integration site and the gene being disrupted.Genomic DNA of stress-resistant ES cells was isolated using Qiagen's GenePure kit and splinkerette PCR was performed as described  of PB were employed for PCR  for 12–14 days, during which time the cells were passaged every 48 h. Next, 1 × 10PB transposons could be remobilized by transient expression of PB transposase. Excision of the PB element causes reversion to the original wild-type DNA sequence and the associated loss of PQR would be tested. Gene-trapped ES cells (5 × 106) were electroporated with 20 μg of an expression plasmid containing PB transposase and a puromycin-resistance cassette (mPB-Puro). Transfected ES cells were selected with puromycin (1 μg/ml) for 2 days to enrich for clones with reversion. Individual ES cell colonies were picked and analyzed for G418 sensitivity (conferred by the loss of PB-gene-trap cassette) and genotyped by PCR.Inserted Total RNA from ES cells were isolated by Qiagen RNeasy kit. The first strand cDNA was then synthesized from the total RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem). Gene specific primers . The expression level of the genes in these cells was assessed by reverse transcription (RT)-qPCR, using a probe set that spans two exons between which the PB had been inserted  were plated onto a 60-mm gelatinized culture dish with culture medium containing PQ (10 μM) or omission of 2-mercaptoethanol  and cultured for 48 h. After that, the total number of viable cells (those that excluded trypan blue) was counted. PQ at 10 μM kills about half of the wild-type ES cells in 48 h and is an optimal concentration for measuring stress resistance. The percentage of cells surviving stress treatment was calculated based on the total viable cells recovered divided by the total cells seeded. The parental clone C9 and a known stress-resistant clone, 4C11   and incubated for 15 min with shaking followed by plating onto a 96-well plate  for an additional 15 min incubation. The fluorescence signal was then measured using the well-scanning mode by a BioTek H1 fluorescence plate reader. Afterward, Hoechst 33342 dye  was added to each well (1 μg/ml) and incubated at room temperature for 1 h to stain the total DNA, as a normalization factor for cell number. The ROS content was expressed as the ratio of relative fluorescent unit (RFU) of DCFDA to that of Hoechst.Cultured cells were trypsinized and resuspended in Hank's balanced salt solution (HBSS) containing 8 μM of 5-(and 6-)chloromethyl-2′,7′-dichlorodihydroflurorescein diacetate, acetyl ester . Male chimera (indicated by the ES cells specific agouti coat color) recovered from the litter were mated with several young virgin C57BL/6J females to test for germ-line transmission. All animal work was approved by the IACUC.PiglPB/+ fibroblast lines were thawed at the same time, and cultured on 60-mm plates for 24 h. After that, they were trypsinized, counted, and plated onto a 96-well plate at 15,000 cells per well (6 wells per line), and allowed to attach overnight. Cells were then exposed to 4 mM PQ for 6 h, after which normal media were reinstated for an overnight recovery. Next morning, cell viability was measured by MTT assay, with untreated cells serving as controls. Viability after PQ treatment was expressed as percentage of live cells with respect to the untreated cells.Tail skin fibroblasts were isolated from 3-month old mice and cultured as described  and mPB transposase (20 μg) by electroporation. Approximately 0.03% of ES cells were stably integrated with PB-UPA, trapping a gene. Transfected ES cells were distributed evenly into ten 96-well plates and exposed to G418 to select gene-trapped clones. After 4 days of G418 selection, most of the non-trapped ES cells were killed while the surviving clones, on average about 9 clones per well, were proliferating. Each plate contained a unique set of gene-trapped clones, designated as a sub-library (C9PA-01 to C9PA10). Each was handled separately in downstream processing. The combined library contained approximately 9000 gene-trapped clones in total  were cultured in the presence of PQ to isolate resistance mutants. PQ was chosen as our primary stressor because it kills cells effectively and cleanly, which facilitated the unambiguous isolation of surviving clones. Moreover, PQ was used in a similar strategy to identify longevity mutants in invertebrate species. We found that both the omission of the feeder layer and a low density of cells were crucial for the selection, otherwise non-resistant ES cells would not be killed by the stressors. After a 7-day exposure to PQ, we observed that surviving ES cell colonies, which varied in number, appeared in nine of the culture dishes. We picked at least 10 colonies from each of those dishes and a total of 96 putative stress-resistant colonies were isolated. Colonies isolated from the same dish frequently harbored the same gene-trap mutations indicating that they arose from a single clone. The observation of multiple PQ resistant (PQR) sibling colonies within a given dish strongly suggests a prevalent stress-resistance phenotype associated with that clone. Since colonies isolated from the same sub-library usually contained identical mutations, we analyzed only 2 colonies from each sub-library. These PQR colonies were analyzed by Splinkerette PCR to identify the trapped gene. For two putative PQR clones isolated from two separate sub-libraries, we failed to locate the PB insertion site, presumably due to difficulty in PCR amplification, and they were excluded from further testing. Clones with successfully identified gene-trapped mutation were further characterized and reported here. Overall, by screening a total of 6 × 107 cells (9000 unique gene trapped clones), we identified 7 mutant clones that were resistant to PQ  to generate clones of homozygous mutants. Dox-treated cells . All three clones have a single PB insertion, as determined by qPCR with the TaqMan probe binding to the neo, and are heterozygous with respect to the gene-trap mutations. The use of C9 ES cells containing the BLM deletion allowed us to easily generate homozygous clones in cell culture to analyze how this affects the stress-resistance phenotype. In addition, PB provided a molecular tag for us to easily genotype the cells by PCR using a 3-primer setup to differentiate wild-type, heterozygote, and homozygote  provided by PB-UPA, leading to a truncation in the encoded mRNA  at the N-terminal   Figure . The PHn) Figure  were losRfflPB/+ mutant was analyzed by RT-qPCR, a 7-fold increase of expression was observed when compared to the wild-type cells. Homozygous PB/PBRffl mutant cells showed a 12-fold increase in expression  media without 2- mercaptoethanol, and (2) media with 10 μM PQ. Cells were cultured under stress for 2 days, after which the number of viable cells was counted. Compared to the wild-type ES cells (C9),  Figures , confirm Figures . The obsR phenotype may correlate with ROS level in these cells. When measuring basal cellular ROS level by CM-H2DCFDA, we observed all three resistant clones exhibited a lower level of ROS , from which no live homozygous PB/PBPigl pups were identified, suggesting that homozygous Pigl gene-trap mutations are embryonic lethal. We have isolated tail snips from both the PiglPB/+ heterozgyotes and the wild-type littermates at 3-months of age to culture fibroblasts for assessing cellular resistance to stressors. PiglPB/+ fibroblasts have lower ROS content than wild-type and are more resistant to PQ-induced cell death . We envision that a full coverage can readily be achieved by accumulating mutants over repeated independent mutagenesis experiments.The use of a selectable mutant phenotype in ES cells, followed by conversion of these cells into living mice with the same phenotype, offers a novel and efficient method for performing a large-scale, high-throughput forward genetic screen to produce mouse mutants for any phenotype that can be studied in cells. A major advantage of such a screen is that it is unbiased, allowing the discovery of unexpected mutants and novel mutational events. Based on the large body of evidence that stress resistance is closely linked to life extension, both across and within species, we selected for stress-resistant ES cells as a surrogate, at the cellular level, for longevity mutants. Here we report on this novel strategy to create ES cells that are resistant to oxidative stress. Using a The diploid nature of mammalian cells has been a limiting factor in conducting genetic studies because inactivation of one allele usually is insufficient to yield an observable phenotype due to the compensatory effect of the unaffected allele. Recessive mutations must exist in the homozygous state for them to be detected. To allow the discovery of recessive mutations causing stress resistance, we adopted the method of knocking out the Bloom syndrome helicase like protein (Blm) , both for randomness and for transposition efficiency  knock out was first shown to reduce skin tumorigenesis in mice  , and in turn mediates ROS generation. The Nox family of enzymes is one of the major sources of ROS in a variety of cell types, and has recently been shown to have strong implications in inducing oxidative stress and damage. Many studies suggested that Nox activities could contribute to oxidative stress in various tissues, including the microglia . This gene encodes for an enzyme catalyzing the second step of the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. PIGL catalyzes the deacetylation of N-acetylglucosaminyl-phosphatidylinositol at the cytosolic side of endoplasmic reticulum (ER). The deacetylated glucosaminyl-phosphatidylinositol is flipped into the lumen of ER for further maturation to a full GPI anchor and transferred to proteins that have a GPI attachment signal at the C terminus by GPI transamidase. GPI anchored proteins are then transported to plasma membranes and membranes of cellular organelles. PIGL mutations, causing GPI deficiency, lead to the CHIME syndrome  staining using flow cytometry (data not shown). The stress-resistance phenotype in PiglPB/+ ES cells may be mediated by a modification of the composition of GPI-anchored proteins on the plasma membrane or the secretion of a subset of proteins without proper attachment of the GPI anchor . We also observed ROS reduction in PiglPB/+ ES cells but how the Pigl mutation mediate such an effect is currently unknown.Another hit was Rffl)  encodes a RING domain-containing ubiquitin protein ligase (E3), a potential negative regulator of TNF-induced NF-kB activation. Rffl targets receptor interacting protein (RIP) for ubiquitination and degradation resulting in a downregulation of NF-kB transcriptional activity so as to prevent sustained inflammatory reaction . These newly identified genes offers considerable promise as novel longevity genes in the mouse. Although we have only demonstrated the use of PQ for selection, we believe this strategy is useful in identifying novel genetic factors that confer resistance to virtually any stressor such as ER stress, heat shock, as well as cytotoxic stress such as heavy-metal exposure. Thus, this forward genetic screen has broad implication in a many field of study. Current efforts in our lab focus on generating additional PB-UPA trap libraries covering the whole genome and to exhaustively screen for additional stress-resistant mutants. We are also generating mice from these stress-resistant ES cells for whole-animal studies.Wallace S. Chick, Michael Ludwig, David Kitzenberg, and Thomas E. Johnson conceived of the experiments. Wallace S. Chick, Michael Ludwig, David Kitzenbery, Xiaoyun Zhao, and Kristina Williams performed the experiments. Wallace S. Chick and Thomas E. Johnson wrote the manuscript with input from Michael Ludwig, David Kitzenberg, and Kristina Williams.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Based on classical two-cell, two-gonadotropin theory, in the follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) put on their main effects on the granulosa and theca cells. LH is essential for androgens production. Androgens are used for estradiol production by granulosa cells. Profound suppression of LH concentrations in some normogonadotropic patients can cause several adverse effects. Objective: The main clinical purpose of this study was that normoresponder women treated with controlled ovarian super ovulation for IVF or ICSI may benefit from co-administration of rLH.Materials and Methods: 40 patients who were candidates for assisted reproductive technology (ART) were randomly selected. In all patients long luteal protocol was used for ovulation induction. Patients were randomly divided into two groups: Group 1 (n=20) with standard long protocol (GnRH agonist) and r-FSH alone, Group 2 (n=20) with standard long protocol (GnRH agonist) and r-FSH with r-LH. Results were statistically analyzed and compared in two groups. Results: The number of retrieved oocytes, mature oocytes, cleaved embryos, transferred embryos, estradiol levels in Human chorionic gonadotropin (hCG) administration day, implantation rate and clinical pregnancy rate in group 2 were higher but not significantly different. Conclusion: Administration of rLH in late follicular phase had no beneficial effect on outcomes in young women with mean age of 31 years. Maybe a greater sample size should be used to see the effects more accurately; also it is possible that rLH will be useful in older patients.Registration ID in IRCT: IRCT201304302575N4 Development of follicles is a complicated process affected by a variety of hormones, peptides and cytokines produced both in situ and at remote areas. Based on classical two-cell, two-gonadotropin theory, in the follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) put on their main effects on the granulosa and theca cells , 2. LH iet al reported higher pregnancy rates in infertile patients treated with combined rFSH and rLH in comparison to control group. Another study showed that patients with high endogenous LH had higher implantation rates  analogues suppression, which in some patients degree of suppression is like hypo-hypo patients, so during ovarian stimulation some, not every patient requires exogenous LH replacement, because low levels of endogenous LH is insufficient for thecal cell function . The main clinical purpose of this study is to investigate the benefits from co-administration of rLH in normoresponder patients undergoing controlled ovarian hyper-stimulation for IVF or ICSI.This prospective study was done with financial support of Yazd Research and Clinical Center for Infertility as a randomized single center clinical trial in 2012. The study was approved by the Ethics Committee of Infertility Center. Written informed consent was obtained from all patients prior to participate in the study. A total of 40 normoresponder patients were included in this study. The inclusion criteria were infertile women younger than 35 years old, day 3 FSH serum levels <10 U/L, male infertility or unexplained infertility, and body mass index (BMI) less than 30. Exclusion criteria were: azoospermia, uterine myoma, mild endometriosis, hydrosalpinx, history of previous IVF (successful or unsuccessful), history of endocrine diseases such as diabetes or thyroid disorders, and patients who had hysteroscopic surgery due to intrauterine lesions such as uterine sub-mucosal myoma or intrauterine adhesions . ProtocolPatients underwent pituitary down-regulation with Buserelin , using a daily dose of 500 mg, s.c., according to the long agonist protocol, starting on day 21 of the cycle preceding gonadotropine treatment and continued 250 mg/daily with start of menstruation until the day of hCG administration. Then treatment with r-hFSH alone  (75 IU per ampoule) was started on day 2 of menstruation with 2-3 ampoule/day based on ovarian response as assessed by sequential vaginal ultra sonography and serum estradiol measurement until the leading follicle had reached a diameter of 14 mm. Then patients were randomized to two groups of 20 patients, one group received r-hLH , 75 IU s.c., for a maximum of 10 days (group 2) and the other group continued r-hFSH without r-hLH (group 1). Randomization was done with random numbers table. Ovulation was induced by administration of HCG , 10,000 IU i.m., when at least two or three follicles had reached a diameter of >17 mm. Endometrial thickness and estradiol levels were measured on HCG injection day. Transvaginal ultrasound-guided needle aspiration of oocytes was performed 34-36 h after HCG injection. IVF or ICSI were performed depending on the semen parameters. In both groups embryo transfer was done 2-3 days after follicular aspiration. The maturational status of oocytes and embryo grading (Hills 1998) was also performed. The embryos of highest morphological grade were transferred into the uterine cavity. Number of transferred embryos was determined based on patient's age, number and quality of embryos and up to 3 embryos per patients were transferred. For luteal phase support 100 mg/daily intramuscular injections of progesterone was administrated.14 days after embryo transfer serum β-hCG was checked. If it was positive, pregnancy would be approved. 3 weeks after positive βhCG, transvaginal sonography was done to confirm the presence of fetal sac and heart beat . The results were compared in two groups. Luteal phase support was continued for 10 weeks of pregnancy. Statistical analysist-test, Mann-Whitney U-test and χ2-test was used as appropriate. P-values below 0.05 were considered significant.All statistical analysis was performed using SPSS software. Student’s 40 women fulfilling the criteria defined in materials and methods participated in this controlled trial study. Patient characteristics are shown in 2 biosynthesis as explained by two-cell, two-gonadotropin model  in 79 patients after down-regulation with triptorelin 0.1 mg, caused an increased pregnancy rate (In this study the total number of obtained oocytes, mature oocytes, resulted embryos, the number of FSH ampoules, endometrial thickness, fertilization rate, embryo quality, implantation rate, chemical and clinical pregnancy rate were higher in r-hLH supplementation group, although they were not significantly different in two groups. It was indicated in a study by Lisi ncy rate . Such diet al the addition of r-hLH had no significant effect on the number of MII oocytes and implantation rate in the study population. In women over 35 years implantation rate was significantly higher in rLH group, which suggests that older patients may benefit from LH supplementation (In present study stradiol levels on the day of hCG administration were higher in r-hLH supplementation group but it was not statistically significant. It is not clearly understood how stradiol effects follicle and oocyte development, although its role as growth factor is shown by different studies and evaluation of estradiol patterns is one of important markers of ART success -28. Alsoentation . It shouet al it was demonstrated that in a group of patients who received r-hFSH alone implantation and pregnancy rates were higher in comparison to the other group who received r-hFSH and supplementary r-hLH, although the differences were not statistically significant (Some recent studies have shown that FSH efficacy when co-administrated with rLH, is decreased which is reflected in increasing FSH vials needed for ovarian stimulation , 30, 31.nificant . In contet al the effect of rLH in late stimulation phase was evaluated after down regulation with long protocol. They found that rLH supplementation caused a higher E2 level on hCG administration day but it had no beneficial effect on ART outcome in young patients population (In a study by Tarlatzis pulation . In summThe authors declare no conflict of interest regarding the relevant research and the present article.


Two previous studies concluded that proenkephalin A (PENK-A) had predictive capabilities for stroke severity, recurrent myocardial infarction, heart failure and mortality in patients with stroke and myocardial infarction.This study aimed to investigate the value of PENK-A as a biomarker for predicting mortality in patients with type 2 diabetes mellitus.Patients with type 2 diabetes mellitus were included from the prospective observational ZODIAC (Zwolle Outpatient Diabetes project Integrating Available Care) study. The present analysis incorporated two ZODIAC cohorts (1998 and 2001). Since blood was drawn for 1204 out of 1688 patients (71%), and information on relevant confounders was missing in 47 patients, the final sample comprised 1157 patients. Cox proportional hazard models were used for evaluating the relationship between PENK-A and (cardiovascular) mortality. Risk prediction capabilities were assessed with Harrell’s C statistics and the integrated discrimination improvement (IDI).After a follow-up period of 14 years, 525 (45%) out of 1157 patients had died, of which 224 (43%) were attributable to cardiovascular factors. Higher Log PENK-A levels were not independently associated with increased (cardiovascular) mortality. Patients with PENK-A values in the highest tertile had a 49% (95%CI 1%-121%) higher risk of cardiovascular mortality compared to patients in the reference category (lowest tertile). C-values were not different after removing PENK-A from the Cox models and there were no significant differences in IDI values.The associations between PENK-A and mortality were strongly attenuated after accounting for all traditional risk factors. Furthermore, PENK-A did not seem to have additional value beyond conventional risk factors when predicting all-cause and cardiovascular mortality. The opioid peptide enkephalin is found in brain and endocrine tissues and has effects on neuroendocrine and nociceptive responses 2]. Enke. Enke2].As enkephalins have a very short half-life, a stable precursor fragment of enkephalin ) has been developed and investigated in clinical studies . Given tNo previous studies with either long-term follow-up, nor studies specifically in patients with type 2 diabetes mellitus (T2DM) have been performed. We hypothesized that increased PENK-A levels are associated with cardiovascular and all-cause mortality in patients with T2DMThis prospective observational study of primary care treated patients is part of the ZODIAC (Zwolle Outpatient Diabetes project Integrating Available Care) study; the design and details of which have been presented elsewhere . This prBaseline data consisting of a full medical history were collected in 1998 and 2001. Patients were considered to have macrovascular complications when they had a previous history of angina pectoris, myocardial infarction, percutaneous transluminal coronary angioplasty, coronary artery bypass grafting, stroke, or transient ischaemic attack. Physical and laboratory assessment data, such as blood pressure, body mass index, lipid profile, creatinine levels, HbA1c and urinary albumin-creatinine ratio, were collected annually. Blood pressure was measured twice with a Welch Allyn Sphygmomanometer in supine position after at least five minutes of rest. The mean blood pressure of two recordings was calculated for each visit.Since mature enkephalins that derive from the PENK-A precursor peptide, are unstable bioactive peptides, an immunoassay has been developed that measures a stable precursor fragment of PENK-A, termed PENK-A 119–159 . The abbThere were two clinical endpoints: all-cause and cardiovascular mortality. The vital status and cause of death were retrieved from records maintained by the hospital and the general practitioners up to the end of 2012. Causes of death were coded according to The International Classification of Diseases, 9th revision (ICD-9). Cardiovascular mortality was defined as any death in which the principal cause of death was cardiovascular in nature (ICD-9 codes 390–459).Analyses were performed with SPSS version 18.0  and STATA version 12 . Continuous variables are represented as mean (± standard deviation) for normally distributed values and as median [interquartile range] for the non-normally distributed variables. Normality of the variables was examined by inspecting Q-Q plots. Univariate linear regression analyses were used to investigate whether PENK-A concentrations were associated with several clinical parameters. Due to a skewed distribution, PENK-A and serum creatinine were logarithmically transformed.Multivariate linear regression models were used to assess the association between PENK-A and baseline characteristics. Cox proportional hazards models were used to investigate the relationship between the serum concentration PENK-A, as a continuous and a categorical (PENK-A divided into tertiles) variable, and all-cause and cardiovascular mortality with and without adjustment for selected confounders. We used 1. an unadjusted model, 2. an age- and gender-adjusted model, and 3. a model in which we additionally adjusted for the following variables: duration of diabetes, smoking (dichotomous), macrovascular complications (dichotomous), body mass index (BMI), systolic blood pressure, HbA1c, serum creatinine, cholesterol-HDL ratio and albuminuria (dichotomous). Because of missing information regarding albuminuria, BMI, smoking and duration of diabetes in 33, 2, 6 and 6 patients respectively, all analyses were performed in 1157 out of 1204 patients (96%) whose PENK-A values were determined. STATA’s ph-tests were used to test the assumption of proportional hazards for baseline predictors. Survival curves were used to illustrate the relationship between PENK-A and mortality.For assessing the prognostic capabilities of PENK-A, we performed the following analyses stepwise. First, calibration was investigated using the Grønnesby and Borgan test assessing the goodness of fit; a nonsignificant result means an acceptable calibration . CalibraIn addition to investigate whether the relationship between PENK-A and mortality was affected by other clinical variables, interaction terms were added to model 3. Interaction was tested when variables were associated with PENK-A in the multivariate linear regression analyses. Interaction was evaluated as significant at a p-value of 0.10 .Both cohorts of the ZODIAC study (1998 and 2001) and the informed consent procedure were approved by the local medical ethics committee of the Isala hospital, Zwolle, The Netherlands. Verbal informed consent was obtained for all patients by the participating diabetes specialist nurses and the consent was documented in the patients records. According to Dutch law, written informed consent was not necessary for this type of study in 1998 and 2001. All data were analysed anonymously.The baseline characteristics are presented in After a follow-up period of 14 years, 525 (45%) patients had died of which 224 (43%) were attributed to cardiovascular causes. A total of 13 patients were lost to follow-up (1%) and cause of death was unknown for 25 patients (5%). The median serum concentration PENK-A in the survivors was 103 [84–132] pmol/L, compared with 126 [97–159] pmol/L in the deceased patients. Results of the Cox regression analyses are presented in After adding an interaction between serum creatinine and PENK-A to model 3, this interaction was only significant in the model with all-cause mortality (p = 0.019) . Also, tThe Grønessby and Borgan test indicated that predicted probabilities corresponded well with observed risks and all models were well calibrated (data not shown). So, the number of deceased patients as estimated by the model is in line with the number of patients that actually died. According to the Harrell C statistic , model 3After adjustment for all traditional cardiovascular risk factors, we observed no independent association between PENK-A, as a continuous variable, and mortality. Although patients with the highest PENK-A concentrations had a 49% higher risk of cardiovascular mortality compared to patients with the lowest concentrations, the association was strongly attenuated after adjusting for all traditional risk factors. These results put conclusions from previous studies in a different perspective .Notably, the 2 previous studies that both claimed that PENK-A was promising as a biomarker, did not systematically adjust for the same important risk factors as we did in our study 10]. Alt. Alt10].Although the IDI showed a significant improvement in discrimination for cardiovascular mortality prediction for the age- and gender-adjusted PENK-A levels, care must be taken to rely on these results because measures like IDI are not developed in the context of censored data. In addition, serious criticism has been published against the use of IDI and NRI indices 19]. Sim. Sim19].Strengths of this study were the prospective design, the high event rate (cause of death was available for 501 patients out of 525 deceased patients) and the long follow-up period. This study also had several important limitations. Firstly, the observational design prevented us from drawing conclusions on causality. Secondly, the serum concentration of PENK-A was assessed in 71% of the total study population, which could have led to selection bias. However, no relevant differences were observed in baseline variables between the final study group and those subjects without PENK-A measurements (n = 285). Furthermore, missing of PENK-A data was related to mortality and this could have led to an underestimation of the effects of PENK-A on mortality ). Furthermore, PENK-A was measured only once, therefore potential daily variability in concentration or measurement errors could not be accounted for.Serum PENK-A levels were not independently associated with all-cause mortality in patients with type 2 diabetes mellitus, and its association with cardiovascular mortality was strongly attenuated after accounting for all traditional risk factors. Furthermore, PENK-A did not seem to have additional value beyond conventional risk factors when predicting all-cause and cardiovascular mortality. The results of this study show that is plausible that the results from two previous studies could have been influenced by residual confounding. Future studies assessing the prognostic capabilities of PENK-A should systematically account for all important cardiovascular risk factors.S1 Statistical Analyses(PDF)Click here for additional data file.S2 Statistical Analyses(PDF)Click here for additional data file.S3 Statistical Analyses(PDF)Click here for additional data file.S4 Statistical Analyses(PDF)Click here for additional data file.


Transpulmonary bubble transit (TPBT) detected with contrast echocardiography is reported as a sign of intrapulmonary shunt during cirrhosis or exercise in healthy humans. However, its physiological meaning is not clear during acute respiratory distress syndrome (ARDS). Our aim was to determine the prevalence, significance, and prognosis of TPBT detection during ARDS.This was a prospective observational study in an academic medical intensive care unit in France. Two hundred and sixteen consecutive patients with moderate-to-severe ARDS underwent transesophageal echocardiography with modified gelatine contrast. Moderate-to-large TPBT was defined as right-to-left passage of at least ten bubbles through a pulmonary vein more than three cardiac cycles after complete opacification of the right atrium. Patients with intra-cardiac shunt through patent foramen ovale were excluded.2/FIO2 ratio between groups, and TPBT was not influenced by end-expiratory positive pressure level in 93% of tested patients. Prevalence of septic shock was higher in the group with moderate-to-large TPBT. Patients with moderate-to-large TPBT had fewer ventilator-free days and intensive care unit-free days within the first 28 days, and higher in-hospital mortality as compared to others.The prevalence of moderate-to-large TPBT was 26% (including 42 patients with moderate and 15 with large TPBT). Patients with moderate-to-large TPBT had higher values of cardiac index and heart rate as compared to those without TPBT. There was no significant difference in PaOModerate-to-large TPBT was detected with contrast echocardiography in 26% of patients with ARDS. This finding was associated with a hyperdynamic and septic state, but did not influence oxygenation. Continuous data were expressed as mean ± standard deviation, unless otherwise specified and were compared using the Mann-Whitney test for two groups comparison. For subgroups analysis, continuous data were compared using the Kruskal-Walis test followed by pairwise Mann-Whitney test with Benjamini-Hochberg correction. Categorical variables, expressed as percentages, were evaluated using the chi-square test or Fisher exact test. Two-tailed n = 7) or patent foramen ovale (n = 42). Thus, the present study includes 216 patients (150 men and 66 women), with a median age of 63 (50 to 76) years. Moderate-to-large TPBT was detected in 57 patients . Among the 159 patients without significant TPBT, 120 had no TPBT and 39 had a minor TPBT.A total of 265 ARDS patients underwent contrast TEE. Forty-nine patients were excluded because of inconclusive contrast study (p = 0.30). Respiratory settings and arterial blood gases at TEE day were not different between groups except for a lower tidal volume. Prevalence of septic shock was higher in the group with moderate-to-large TPBT  in 80 patients. TPBT was similar with lower and higher PEEP in the majority  of patients . TPBT was moderate at lower PEEP but minor at higher PEEP in one patient; conversely, TPBT was moderate at lower PEEP but large at higher PEEP in one patient and minor at lower PEEP but moderate at higher PEEP in four patients.We studied the effect of PEEP-level changes , leadinSeptic shock was more frequent in patients with moderate-to-large TPBT in our study and probably explains the association with higher values of heart rate, cardiac index, and features of hypovolemia (collapsibility of superior vena cava and lower E/A ratio). These latest features were not associated with lower cardiac index, probably because heart rate was also higher. Tachycardia may increase TPBT via a decrease in pulmonary capillary transit time . Previou2/FiO2 ratio did not differ between groups with or without TPBT. Numerous factors influence oxygenation during ARDS, including intrapulmonary shunt, but also effect of low PvO2 on PaO2 [2, and the net effect on PaO2 may vary from one patient to another. Moreover, PaO2/FiO2 ratio depends on FiO2 in a non-linear relationship which is influenced by the severity of shunt [Contrary to our expectations, PaO on PaO2 , intra-c on PaO2 . Higher of shunt . Increasof shunt . In addiof shunt , leadingTPBT was associated with longer duration of mechanical ventilation and ICU stay. No significant difference in ICU mortality was found, but hospital mortality was higher in the group of patients with moderate-to-large TPBT. The latter finding could be explained by a poorer condition after longer mechanical ventilation and ICU stay. Septic shock, which was more frequent in patients with moderate-to-large TPBT in our study, could also explain these findings.Limitations of this study include its mono-centric design and the seven-year period inclusion. However, our mechanical ventilation strategy did not vary during the study period. Second, we only report on a single TEE within three days of ARDS start. Third, we did not measure cardiac output and lung recruitment during PEEP changes. Fourth, we did not compare contrast TEE with other methods of measurement of shunt, such as measurement of venous admixture using a pulmonary arterial catheter, and as previously stated, detection of TPBT cannot be used as a direct surrogate of intrapulmonary shunt. Fifth, we did not explore TPBT in other ICU patients without ARDS and could not report on its general prevalence in critically ill patients and during mechanical ventilation or sepsis. In physiological studies performed in healthy humans, TPBT could be detected during exercise but not at rest ,10.In conclusion, we report the first evaluation of contrast echocardiography to detect TPBT in the setting of ARDS. Although moderate-to-large TPBT was identified in 26% of patients, we did not detect any massive TPBT in this setting. TPBT did not influence oxygenation, and may not be used as a direct surrogate of intrapulmonary shunt during ARDS. TPBT was mainly associated with a hyperdynamic hemodynamic status and septic shock. Whether TPBT is present in ventilated patients with septic shock but not ARDS requires further studies.


Evidence from randomized controlled trials (RCTs) has shown a significant survival advantage of trastuzumab. Although extant work in developed countries examined economic evaluation of trastuzumab in adjuvant treatment for early breast cancer based on the 1-year treatment, there is uncertainty about cost-effectiveness of trastuzumab in the Adjuvant Treatment of early breast cancer in developing countries. This study aimed to estimate cost-effectiveness of adjuvant trastuzumab therapy compared to AC-T regimen in early breast cancer in Iran.A cost-effectiveness analysis was performed using a Markov model to estimate outcomes and costs over a 20-year time period using a cohort of women with HER2 positive early breast cancer, treated with or without 12 months trastuzumab adjuvant chemotherapy. Transition probabilities were derived mainly from the BCIRG006 trial. Costs were estimated from the perspective of the Iranian health care system. Both costs and outcomes were discounted by 3%. One-way sensitivity analysis was undertaken to assess the associated uncertainties in the expected output measures.On the basis of BCIRG006 trial, our model showed that adjuvant trastuzumab treatment in early breast cancer, yield 0.87 quality-adjusted life-years (QALY) compared with AC-T regimen. Adjuvant trastuzumab treatment yielded an incremental cost-effectiveness ratio (ICER) of US$ 51302 per QALY.By using threshold of 3 times GDP per capita, as per World Health Organization (WHO) recommendation, 12 months trastuzumab adjuvant chemotherapy is not a cost-effective therapy for patients with HER2-positive breast cancer in Iran. Breast cancer (BC) is the most prevalent cancer among Iranian women ; 2001.Trastuzumab (Herceptin®) is a synthetic and recombinant humanized monoclonal antibody directed against the extracellular domain of the (HER2). The HER2 proteins stimulate cell proliferation and trastuzumab inhibits cell proliferation in HER2-dependent tumors  have shown a significant survival advantage of trastuzumab, with a reduction in the rate of recurrence and improvement in the rate of survival for early breast cancer, when added to conventional chemotherapy, for 1-year. The aveIn developed countries, several studies conducted to determine economic evaluation of trastuzumab in adjuvant treatment for early breast cancer based on a 1-year treatment . There iWe implemented a Markov model based on breast cancer disease states. A Markov model with four health states was designed to estimate outcomes and costs for a hypothetical cohort of women with positive early breast cancer.As shown in We constructed a decision tree in TreeAge Pro 2011®,  to estimate the costs and utility effects for a hypothetical cohort of women with HER2 positive early breast cancer in two treatment strategies. According to BCIRG 006 trial, two treatment strategies were selected: adjuvant chemotherapy plus trastuzumab and adjuant chemotherapy alone. This model applied for comparing 20 years costs and effectiveness of adjuvant chemotherapy plus trastuzumab and adjuant chemotherapy alone for patients with HER2-positive early breast cancer. The average age of the population in the model was 50 years old, similar to the BCIRG 006 trials. In the first strategy, randomly assigned women with HER2-positive early-stage breast cancer received 100 mg/m2 docetaxel, 60 mg/m2 doxorubicin, 600mg/m2 cyclophosphamide intravenously (AC-T arm) in each session, six times every 3 weeks that prolonged approximately 4 months. In second strategy, the same regimen plus 52 weeks of trastuzumab (AC-T plus H) is considered. We simulated a hypothetical cohort of women with an average age of 50 year with the same entry criteria as in the BCIRG 006 trial. The incidence of adverse effects associated with the adjuvant therapy was also taken into account.Analyses were projected to 20-year horizons. The cycle length was one year. The outcome was measured as life-year gained (LYG) and quality-adjusted life-year (QALY). Costs and QALYs were both discounted by 3%.The following assumptions were made in the analysis:Recurrence of contralateral breast cancer was combined with local and regional recurrence.There were no breast cancer recurrences beyond year 20 of the model .The benefit of trastuzumab treatment last for 5 years In BCIRG 006 study, CHF related deaths were not reported, thus mortality due to CHF was assumed to be zero The underlying clinical data and the treatment protocol, follows the BCIRG 006 trial . Table 1In BCIRG 006 trial at a median follow-up of 65 months, DFS rate at 5 years was 75% among the patients receiving AC-T and this rate was 84% among those receiving AC-T plus trastuzumab. Based on a same trial, the hazard ratio for recurrence in the control group, compared with the observation group, was 0.64  .We adjusted survival with quality of life using utility weights for years that every patient spent in each state. These weights were derived from the existent literature see .Direct medical costs were measured from the perspective of the Iranian health care system only. Direct non-medical and indirect costs such as transportation cost, out-of-pocket payments, time spend for seeking care and loss of productivity were not measured in our study. To calculate the dosage of drugs, treatment expense for an assumed average 70kg woman was used , quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICER). The results of the analysis are summarized in In the base case analysis (i.e. over 20-year time horizon) the QALYs gained with AC-T regimen (strategy 1) and with 52 weeks adjuant trastuzumab treatment for HER2-positive patients (strategy 2) were 11.11 and 11.98, respectively. Therefore, the new intervention produced an extra 0.87 QALYs. The total costs for AC-T adjuant and AC-T adjuant treatments were 12,388 USD and 56,984 USD, respectively. The base case analysis indicated that treatment with a 12-month adjuvant trastuzumab regimen generated an ICER of 51,302 USD per QALY.One-way sensitivity analyses were undertaken to assess robustness of the study results. Result of this analysis presented in Sensitivity analyses of variables in the model showed that model is sensitive to changes in the cost of trastuzumab, discount rate for outcomes and hazard ratio in AC-TH group. In order to assess model validation, the results of the model for two treatment strategies were compared with overall survival at 5 years in BCRG006 trial. Overall survival at 5 years in BCRG006 trial reported 92% and 87% in AC-T and AC-TH group. In our Markov model, overall survival in same period is 93% and 85%.In developed countries, 1-year adjuvant trastuzumab treatment is cost-effective in early breast cancer treatment, particularly in a long-term perspective .In base case analysis, our model yielded an ICER with 51302 USD per QALY after 20 years for the BCIRG-006 trial. High cost–effectiveness ratio yielded in our study is comparable with similar studies  estimated that adjuvant trastuzumab treatment in the United Kingdom has an incremental cost per QALY gained of £18,000, which is less than the general £30,000 threshold . MethodoWillingness-to-pay and acceptable threshold in developing countries such as Iran and Colombia has not been determined yet. Therefore, we used the WHO cost-effectiveness threshold  for relative cost-effectiveness in our study . This thSince 2007, the Iranian Ministry of Health and Medical Education provides some financial supports for patients but trasutuzumab is not covered with current insurances schemes. The high price of this drug can impose catastrophic expenditure to the patients and their households.One of the strengths of our study was that our clinical data were derived from 5-year analyses of BCIRG006 clinical trials, while most of the cost-effectiveness analyses done on trastuzumab, are based on a one-year HERA trials. Our analysis, nevertheless, has few limitations need to be acknowledged. First, we estimated only the direct medical costs from the health care system perspective and direct non-medical and indirect costs such as transportation cost, out-of-pocket payments, time spend for seeking care and loss of productivity were not measured in our study. Indirect costs and productivity gains are possibly substantial and adding these costs to the model would have increased lifetime costs. Second, some of the cost components such as the costs of managing congestive heart failure and cost of follow up for disease-free patients, were estimated based on international guidelines and may not reflect the real costs. Finally, due to the lack of utility weights for the breast cancer patients in Iran, utility weights were obtained from other settings than Iran. Geographical and cultural difference between countries may affect utility weights.Using WHO cost-effectiveness threshold base on GDP per capita, 12 months trastuzumab adjuvant chemotherapy in early breast cancer is not a cost-effective therapy for the patients with HER2-positive in Iran.This study was approved by the Ethics Committee of Iran University of Medical Sciences.


FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling. On the other hand, prolonged elevation of intracellular Ca2+ concentration is a cause of excitotoxicity which leads to neurodegeneration .i.e., FRET efficiency per se. However, in the context of the spatiotemporal aspect of signal transduction, anchoring of a sensor is equally important to consider.Over the past decades, Förster resonance energy transfer (FRET)—based biosensors have proved one of the most valuable and powerful tools for assessing the signal transduction within living cells. For instance, they have been widely used for assaying activity of enzymes, conformational changes in proteins, protein-protein interactions, fluctuations in cytoplasmic ion concentration and pH (reviewed by ). Accordper se for the performance of FRET sensors has not gained proper attention. Here, we directly compare the properties of two VEIDase sensors which differ only by the presence or absence of the microtubule-associated protein tau. Our results demonstrate that anchoring of FRET sensors is essential for high spatiotemporal resolution.We have previously reported detection of caspase-6-like (VEIDase) activity at subcellular levels using a tau-anchored FRET sensor preferentially processed by this caspase . In partPlasmid encoding tau-anchored VEIDase sensor (taFS-VEID) based on enhanced cyan fluorescent protein (ECFP)—enhanced yellow fluorescent protein (EYFP) FRET was previously designed and constructed in our laboratory . Plasmid2 atmosphere at 37 °C.Human neuroblastoma SH-SY5Y and SK-N-AS cells  were cultured in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1% non-essential amino acids (NEAA), 100 U/mL penicillin and 100 μg/mL streptomycin, from here on referred to as complete medium. The cells were maintained in a humidified 5% CO5 cells/dish, and allowed to attach overnight. The following day the medium was replaced with fresh complete medium and the cells were transfected with plasmids encoding taFS-VEID or naFS-VEID using X-treme Gene HP DNA Transfection Reagent  according to the manufacturer’s instructions. SK-N-AS cells were imaged the following day. SH-SY5Y cells were differentiated with retinoic acid , diluted to a final concentration of 10 µM in the complete medium, for 3 days following transfection. Prior to the imaging session, the medium was buffered with 10 mM HEPES. When applicable, staurosporine was added to the buffered medium to a final concentration of 1 μM prior to the start of the imaging session. For western blot analysis, SK-N-AS cells were seeded into 6-well plates  at a density of 3 × 105 cells/well and allowed to attach overnight. Transfection was performed as described above. Next day, the cells were treated for 3 h with 1 μM staurosporine. All the reagents were purchased from Life Technologies  if not otherwise indicated.For live cell imaging experiments, the cells were seeded into glass bottom Petri dishes , 3–5 × 10Z-axis and images linearly adjusted to 0.1% saturation.Localization of sensors was determined using an imaging system with a Nipcov spinning disk (CSU-22) on a Zeiss Axiovert 200 using solidstate 488 nm laser illumination  and an Orca-Flash 4.0 sCMOS camera . Z-stacks were acquired using a piezo objective actuator  to cover the focal planes of the cells and, for the SH-SY5Y cell protrusions, these focal planes were summed over the Z-axis, and intensity in the FRET channel was divided by the intensity in the ECFP channel.Time lapse imaging was performed on a Leica TCS SPI laser scanning confocal microscope  as previously described . ECFP waAnalysis of FRET images was performed by segmenting them into cells and analyzing each cell individually. The pixels of the cells were sorted into percentiles and the ratio value at the 10th percentile and the mean of the 10th–90th percentile was further analyzed, representing the fraction of pixels of the cell retaining the highest FRET and the average FRET, respectively. To correct for the asynchronous apoptosis induction by staurosporine, the data was temporally aligned to the initiation of FRET loss in each cell. The data was temporally interpolated to achieve optimal temporal alignment and subsequently fitted to a mono exponential decay curve using GraphPad Prism version 6.07 for Windows .w/v) and 0.1% Tween, followed by an overnight incubation at 4 °C with rabbit polyclonal anti-GFP antibody (1:2000) (Life Technologies). Finally, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-coupled donkey anti-rabbit (1:5000)  and the proteins were visualized by chemiluminescence using SuperSignal™ West Dura Extended Duration Substrate .Cells were harvested as previously described . 15 μg pIn order to evaluate how anchoring of FRET sensors affects their properties, we employed a tau-anchored sensor for monitoring VEIDase activity , here deTo compare the intracellular distribution of anchored and soluble FRET sensors, the constructs were transfected into human neuroblastoma SH-SY5Y cells. The cells were subsequently differentiated towards a neuronal phenotype with 10 µM retinoic acid for 3 days, which stimulated outgrowth of neurite-like processes. Analysis was performed by confocal laser scanning microscopy (CLSM). Cells expressing taFS-VEID displayed intense and defined fluorescence throughout the cytoplasm, readily detectable even within thin processes B, top. BThe fluorescent signal from taFS-VEID was lacking in the nuclei of SH-SY5Y cells, as compared to the other compartments, and was clearly absent from the nuclei of SK-N-A-S cells. By contrast, the signal from naFS-VEID was more concentrated in the nuclei of these cells B,C. ThisNext, we proceeded to investigate the properties of anchored and soluble FRET sensors in terms of detecting caspase activation at subcellular level. SK-N-AS cells overexpressing taFS-VEID or naFS-VEID were treated with 1 µM staurosporine and imaged by CLSM with 5 min intervals. FRET was approximated as the ratio between the FRET and the ECFP channels.In the cells expressing taFS-VEID, the rates of decline in FRET varied substantially among different regions of the cell, including adjacent areas within the same protrusion . By contThe differences in the local distribution of FRET signal between the anchored and non-anchored sensors were further demonstrated by a dynamic unbiased quantitative analysis of the time-lapse images . In partThus, anchoring of FRET sensors enables detection of subtle differences in caspase activation at subcellular level, while the spatiotemporal resolution is lost when using soluble FRET probes.To investigate fragmentation of the FRET sensors, we analyzed total lysates of SK-N-AS cells expressing taFS-VEID or naFS-VEID by western blot using anti-GFP antibodies. Intact taFS-VEID and naFS-VEID sensors migrated as a ~130 kDa and a ~55 kDa band, respectively, matching their theoretical sizes C. Stauroper se on their spatiotemporal resolution has not been considered.Over the past decades, FRET biosensors have proved to be useful tools for assessing the activity of a variety of enzymes, and have allowed new insights into different signaling pathways. However, FRET probes of conventional type have limited capacity for determining the important spatiotemporal aspect of enzyme activation. Although subcellular targeting of FRET sensors has been widely reported ,7,8,9,10Here, we present a case study using tau-anchored (taFS-VEID) and non-anchored (naFS-VEID) caspase (VEIDase) activity reporters. We provide evidence for the presence of an anchor as a necessary condition for sensitive detection of subcellular variations in enzyme activation. We show that anchoring of FRET sensors leads to their enrichment in the targeted location. In our case, tau mediates binding of the VEIDase sensor to the microtubules of neurite-like processes while retaining the availability of the sensor to the whole pool of cytosolic enzymes. Thus, anchoring is expected to enable detection of FRET at the locations of interest with higher signal-to-noise ratio. Furthermore, taFS-VEID, in contrast to naFS-VEID, was undetectable in the nuclei of neuroblastoma cells, implying that possible nuclear VEIDase activity can be neglected when using taFS. Thereby, anchoring of FRET sensors minimizes the impact of the activity of the enzyme in off-target compartments on the readout.Further, we show that taFS-VEID, but not naFS-VEID, allows for detection of active caspases at subcellular level. In particular, in neuroblastoma cells undergoing apoptosis, taFS-VEID enabled monitoring of local differences in FRET not only within the same cell, but even within the same protrusion. By contrast, no such differences could be observed using naFS-VEID. Accordingly, the rate of change in the cellular regions retaining highest FRET was markedly delayed in case of taFS-VEID. This demonstrates that anchoring enables detection of subcellular variations in the amount of active enzymes. We suggest that restricting the diffusion of FRET sensors by anchoring is advantageous for assessing the propagation of an enzyme activation within a cell, as in this case it is the diffusion of an active enzyme and/or its activating signal that affects the FRET. Conversely, free diffusion of non-anchored FRET sensors makes them uniformly available for an active enzyme throughout the cell, leading to a rapid and global “all-or-nothing” response, which might not reflect the actual pattern of the enzyme activation. Thus, anchoring can be expected to be advantageous not only for cleavable, but also for conformation dependent FRET sensors (e.g., cameleons  and RangIn conclusion, our results illustrate the advantage of anchored FRET sensors for assessing activation of enzymes at a subcellular level. The localization and duration of signaling is increasingly recognized as an essential factor determining the functional outcome. Thus, obtaining the spatiotemporal context is important for understanding the mechanisms of cell signaling pathways both in health and in disease, as well as for drug screening. In view of this, it is advisable to consider subcellular anchoring when designing FRET biosensors.


On average, the human hippocampus shows structural differences between meditators and non-meditators as well as between men and women. However, there is a lack of research exploring possible sex effects on hippocampal anatomy in the framework of meditation. Thus, we obtained high-resolution magnetic resonance imaging data from 30 long-term meditation practitioners (15 men/15 women) and 30 well-matched control subjects (15 men/15 women) to assess if hippocampus-specific effects manifest differently in male and female brains. Hippocampal dimensions were enlarged both in male and in female meditators when compared to sex- and age-matched controls. However, meditation effects differed between men and women in magnitude, laterality, and location on the hippocampal surface. Such sex-divergent findings may be due to genetic (innate) or acquired differences between male and female brains in the areas involved in meditation and/or suggest that male and female hippocampi are differently receptive to mindfulness practices. Meditation-specific features of the hippocampus, including its connecting fiber tracts, have been examined using different imaging modalities, such as magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). Outcomes of these imaging studies point to greater hippocampal dimensions in meditators, such as bigger hippocampal volumes, larger hippocampal distances, more hippocampal gray matter (GM), as well as a higher fractional anisotropy in fibers connecting to the hippocampus . MoreoveThis solid body of literature on hippocampus-specific meditation effects is matched by an at least equally significant body of literature on hippocampus-specific differences between male and female brains. For example, there are reports of sex differences in hippocampal anatomy , hippocaThe aforementioned meditation effects and sex differences within the hippocampus raise the question of whether hippocampus-specific meditation effects manifest differently in male and female brains. The lack of research addressing that question might be due to the rather small number of subjects included in many meditation studies and/or the unequal distribution of male and female subjects. We therefore conducted the present study leveraging an existing data set consisting of 15 male and 15 female meditators as well as 15 male and 15 female control subjects. Specifically, we set out to investigate the modulating effects of biological sex on hippocampal anatomy in the framework of meditation. For this purpose, we combined a traditional volumetric approach  with a modern surface-based mapping technique  and tested for significant group-by-sex interactions followed by mapping meditation effects in men and women separately.1 aiming at the closest pair-wise match with respect to sex, handedness, and age. The maximum allowed age difference within a sex-matched pair was 2 years. Criteria for eligibility and procedures to screen the control subjects are detailed elsewhere  each contained 15 males and 15 females and consisted of 28 right-handers and two left-handers . Handedness was determined based on preferences for selected activities, such as writing, throwing, holding, opening, etc., using a modified version of the Edinburgh Inventory . Age ranAll subjects (meditators/controls) were scanned on the same site, using the same scanner, and following the same scanning protocol. Specifically, magnetic resonance images were acquired on a 1.5 Tesla Siemens Sonata scanner  using an 8-channel head coil and a T1-weighted MPRAGE sequence . The obtained structural brain images were then corrected for intensity inhomogeneities and linearly transferred into a standard space using six-parameter (rigid-body) normalizations, as previously detailed .2 and the VBM8 toolbox3, as described elsewhere  differ in total brain volume. For this purpose, all image volumes were tissue-classified into GM, white matter (WM), and cerebrospinal fluid (CSF) using SPM8lsewhere . Tissue Ir = 0.95. In addition, the hippocampus was labeled five times, by the same rater, within one randomly selected brain revealing a volumetric overlap of 85% for all labels. The overlap was defined as the volume of the intersection of the five labels, divided by the mean volume of these labels, multiplied by 100.The hippocampus was labeled manually in contiguous coronal brain sections, as previously described . To dete3) based on the dimensions and number of voxels constituting the hippocampal labels. Left and right global hippocampus measures were statistically compared between groups defined by meditation status (meditators/controls) and biological sex (men/women). More specifically, we used a general linear model with the left and right hippocampal values as dependent variables, group as fixed factor, and sex as covariate. As a safeguard against type I error, Bonferroni corrections were applied using a threshold of p ≤ 0.025 to account for the two (left/right) dependent variables. A significant group-by-sex interaction was followed by post hoc comparisons within men and women, separately.Global left and right hippocampal volumes were established  were converted into three-dimensional shape representations of the left and right hippocampus. Then, parametric surface meshes ,b were gp = 0.002) but not for the right hippocampus (p = 0.46). Conducting post hoc comparisons separately within males and females, left hippocampal volumes were significantly larger in male meditators than male controls (p = 0.02) as well as in female meditators than female controls (p = 0.046). Significant meditation effects with respect to right hippocampal volumes were not detectable in males (p = 0.722) or in females (p = 0.291).Descriptively, left and right hippocampal volumes were larger, on average, in male meditators compared to male controls; they were also larger in female meditators compared to female controls  and 30 controls (15 men/15 women), where meditators had, on average, more than 20 years of experience (with a minimum of 5 years), thus constituting true long-term practitioners. In accordance with the outcomes of our previous study of meditation effects on hippocampal anatomy by pooling male and female brains together , we obseThe observed group-by-sex interactions and sex-divergent effects are intriguing and perhaps reflective of differential (innate) conditions in male and female brains, as also implied by the large number of reports on sex differences within the hippocampus (see Introduction). At the same time, it is possible that male and female meditators may require (or employ) different amounts or elements of practice to experience desired effects. Both possibilities, independently or interacting with each other, might be accompanied by a sex-specific engagement of certain hippocampal subsections during meditation. Such a sex-specific hippocampal engagement might result in a sex-specific impact on hippocampal anatomy, where additional effects may manifest if male and female hippocampi are differently susceptive to the practice. Future research is clearly necessary, not only to uncover the rather complex underlying mechanisms but also answer the age-old question if meditation induces (sex-specific) brain changes or if a (sex-specific) unique brain anatomy preceded the meditation practice, as further discussed elsewhere , 2013b. Our current observations are in line with prior reports of sex differences in hippocampal function, development, atrophy and pathology , which iAlthough existing mindfulness research seems to lack sex-specific analyses—at least with respect to addressing brain anatomy—the observed group-by-sex interactions seem to be in accordance with a recent study reporting sex-divergent outcomes when assessing the impact of a mindfulness intervention on behavioral measures/psychological constructs . More spImportantly, the hippocampus is not a single homogeneous structure but regionally segregated by architecture, connectivity, and functional relevance . Our refThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.