There are an estimated 18 million Alzheimer's disease (AD) sufferers worldwide and with no disease modifying treatment currently available, development of new therapies represents an enormous unmet clinical need. AD is characterized by episodic memory loss followed by severe cognitive decline and is associated with many neuropathological changes. AD is characterized by deposits of amyloid beta (Aβ), neurofibrillary tangles, and neuroinflammation. Active immunization or passive immunization against Aβ leads to the clearance of deposits in transgenic mice expressing human Aβ. This clearance is associated with reversal of associated cognitive deficits, but these results have not translated to humans, with both active and passive immunotherapy failing to improve memory loss. One explanation for these observations is that certain anti-Aβ antibodies mediate damage to the cerebral vasculature limiting the top dose and potentially reducing efficacy. Fc gamma receptors (FcγR) are a family of immunoglobulin-like receptors which bind to the Fc portion of IgG, and mediate the response of effector cells to immune complexes. Data from both mouse and human studies suggest that cross-linking FcγR by therapeutic antibodies and the subsequent pro-inflammatory response mediates the vascular side effects seen following immunotherapy. Increasing evidence is emerging that FcγR expression on CNS resident cells, including microglia and neurons, is increased during aging and functionally involved in the pathogenesis of age-related neurodegenerative diseases. Therefore, we propose that increased expression and ligation of FcγR in the CNS, either by endogenous IgG or therapeutic antibodies, has the potential to induce vascular damage and exacerbate neurodegeneration. To produce safe and effective immunotherapies for AD and other neurodegenerative diseases it will be vital to understand the role of FcγR in the healthy and diseased brain. Here we review the literature on FcγR expression, function and proposed roles in multiple age-related neurological diseases. Lessons can be learnt from therapeutic antibodies used for the treatment of cancer where antibodies have been engineered for optimal efficacy. Alzheimer's disease (AD) is a devastating illness with a hugely detrimental effect on the quality of life of patients and their families. An estimated 18 million people worldwide suffer from AD and with an ever ageing population; the number of cases will increase. The currently approved treatments for AD are: inhibitors of the enzyme acetyl-cholinesterase, and Memantine which is a blocker of the neurotransmitter channel for glutamate (NMDA). Both these therapies are able to transiently improve cognition , a 39–43 amino acid peptide cleaved from the amyloid precursor protein (APP)  and Solanezumab (EXPEDITION 1 and EXPEDITION 2) failed to meet primary clinical endpoints of improvement in cognition  bind to the constant domain of IgG, and are expressed on a wide variety of cell types including CNS macrophages- microglia. Activation of FcγR can result in a pro-inflammatory response including the release of cytokines and other mediators , inhibitory (FcγRIIb) or gpi linked decoy (FcγRIIIb). There are a number of subclasses of human IgG , each with varying affinity for the different FcγRs  are associated with and signal through a separate Fc gamma chain (Fcγ chain) which contains immune-tyrosine activation motifs (ITAMs). A common model used to study the roles of activating FcγRs −/− mice which have an exacerbated response to autoantibodies inducing more tissue damage , AD, and Multiple sclerosis 2 fragments of PD IgG prevented these responses , but binds with high affinity to Aβ (KD = 5.67 × 10−8 M). Aβ is a potent inducer of neuronal apoptosis, and this effect is ameliorated in FcγRIIb deficient neurons , reactive oxygen species synthesis, and the initiation of cell death in co-cultured neurons. The damage to neurons was shown to be dependent on NO production, as iNOS inhibitors but not cytokine blocking antibodies could inhibit neuronal death. This effect was shown to be Fcγ chain-dependent as microglia from Fcγ chain deficient mice fail to induce cytokine/ROS production or neuronal damage (Le et al., Neuronal function is tightly regulated and a small change in homeostasis can be detrimental depending on the levels of cytokines produced. At physiological levels TNFα has important roles in regulating normal brain activity including regulation of synaptic scaling (Stellwagen and Malenka, We propose that FcγRs may contribute to the pathology of neurodegenerative diseases through a number of mechanisms (summarized in Figure The development of anti Aβ immunotherapy has already been reviewed elsewhere Schenk, , this seNeuro-inflammation in AD is characterized by an upregulation of immune regulatory receptors expressed on: microglia, astrocytes and perivascular cells as well as increased cytokine concentration in the brain (Peress et al., The clinical trials to date have shown that immunotherapy against Aβ is able to clear plaques from the brains of AD patients. However they have also highlighted the danger of immune activation within the CNS, which can result in damage to brain tissue. It appears that ligation of activating FcγRs by therapeutic antibodies causes damage to the vasculature of the CNS; we propose that immunotherapy could also impair the function of neurons through mechanisms outlined in Figure Jeff B. Stavenhagne is a full time employee of H lundbeck A/S. Jessica L. Teeling and James P. Fuller have received funding from H Lundbeck A/S


Prior to the “onset of hearing,” developing cochlear inner hair cells (IHCs) and primary auditory neurons undergo experience-independent activity, which is thought to be important in retaining and refining neural connections in the absence of sound. One of the major hypotheses regarding the origin of such activity involves a group of columnar epithelial supporting cells forming Kölliker's organ, which is only present during this critical period of auditory development. There is strong evidence for a purinergic signalling mechanism underlying such activity. ATP released through connexin hemichannels may activate P2 purinergic receptors in both Kölliker's organ and the adjacent IHCs, leading to generation of electrical activity throughout the auditory system. However, recent work has suggested an alternative origin, by demonstrating the ability of IHCs to generate this spontaneous activity without activation by ATP. Regardless, developmental abnormalities of Kölliker's organ may lead to congenital hearing loss, considering that mutations in ion channels  involved in Kölliker's organ activity share strong links with such types of deafness. As first described in 1863 by a Swiss anatomist and physiologist Albert von Kölliker, Kölliker's organ is an epithelial structure present in the developing auditory sensory organ in a wide variety of mammals, including cattle, rabbits, cats, dogs, and humans . It is oThe cochlea develops from its base to apex in a time-dependent manner, both structurally and functionally. In the mouse, the putative sensory epithelium becomes visible around day 14 of gestation (embryonic day 14), when the endolymphatic duct is composed of tall columnar epithelial cells of ectodermal origin surrounded by mesenchymal tissue . This maAs the sensory structures mature in the cochlea, the epithelium forms two domains starting from around embryonic day 16 (mouse): the greater epithelial ridge (GER) containing Kölliker's organ lying on its medial aspect and the lesser epithelial ridge (LER) in the lateral portion. The epithelial cells that separate these two regions become the inner and outer pillar cells of the organ of Corti . While Iμm in height 3-4 μm in width, and have microvilli covering their apical or luminal surface. An occasional kinocilium has been observed on the surface of the epithelial cells  from internal stores and inward currents through P2X receptors-channel complexes [2+ waves in Kölliker's organ are comparable to those found between groups of astrocytes connected via gap junctions [2+ waves are also observed within the outer sulcus cells following ATP release or mechanical stimulation [2+ waves lead to further ATP release, resulting in a regenerative wave that allows the synchronisation of nearby cells [2+ waves can be observed from very early stages of development, with their frequency increasing dramatically (by 5-fold), along with an increase (1.7-fold) of the area of activation [2+ waves throughout Kölliker's organ at very early stages (P0-P1), naturally occurring Ca2+ waves are rare at that age, possibly due to lower levels of ATP release. The ATP-induced currents are also of smaller amplitudes at that age [Calcium is a major intracellular messenger in the cochlea, being involved in a number of signalling pathways. Within Kölliker's organ, a strong correlation was initially observed between spontaneously generated currents, morphological changes, and Caomplexes , 35. Theunctions . Each wamulation . These Cby cells , 35. ATPtivation . Althougthat age , 41.2+ action potentials in IHCs during development could lead to hearing impairment, particularly due to defects in the Ca2+ channel Cav1.3, as its dysfunction is detrimental to cochlear functioning [L-type channel is the predominant Ca2+ channel in IHCs of developing cochlea and is responsible for exocytosis of glutamate and potentially of other neurotrophic factors [2+ is also involved in rhythmic morphological changes within supporting cells of Kölliker's organ. One intriguing possibility is that the increase in intracellular Ca2+ within supporting cells can activate Ca2+-activated Cl− channels, leading to efflux of Cl−. A recent study has demonstrated a strong expression of Ca2+-activated Cl− channels (Anoctamin-1) in Kölliker's organ, particularly in supporting cells immediately adjacent to IHCs [− efflux could in turn cause the movement of water out of these cells, resulting in morphological changes within Kölliker's organ [− channel inhibitor 4,4′diisothiocyano-2,2′stilbene disulfonic acid on spontaneous morphological activity in Kölliker's organ [Clinically, lack of regular Cactioning . This pa factors . It is l to IHCs . While A to IHCs , its fun's organ , 41. Thi GJB2 gene, which encodes Cx26 and accounts for about 50% of prelingual childhood deafness [2+ transients within Kölliker's organ, and the Cx30-null mice also show an elevation of auditory thresholds [v1.3 channels are essential in creating and maintaining synapses during the critical spontaneous activity driven period of development, and adverse effects are seen as early as the first postnatal week in their absence, starting with OHC degeneration [The role of Kölliker's organ is quickly evolving from simply providing support for adjacent IHCs, to potentially initiating activity that is necessary for the development of a fully functional auditory system. While no direct link between Kölliker's organ dysfunction and deafness has yet been established, critical components of its organisation appear to be linked to major forms of deafness. One of the strongest potential links lies within mutations of thedeafness . Both Cxdeafness , 65) areresholds , 67. Furneration .In addition, the function as well as the transformation of Kölliker's organ to the inner sulcus is crucial for the maturation of the cochlear structure and function. Thyroid gland deficiencies have been shown to affect this process by maintaining Kölliker's organ past the onset of hearing, and in humans, impairment of thyroid hormone signalling is associated with hearing loss –70. This2+ or ectonucleotidase activity may act as a feedback for ATP release and thus control the rhythmicity of ATP release in the developing cochlea. Interestingly, both types of ion channels (connexins and P2 receptors) have been linked to hearing loss. Following sound detection by the cochlea (the “onset of hearing”), Kölliker's organ disappears, transforming into the adult inner sulcus region which contains cuboidal epithelial cells. From a physiological perspective, this is an important step in cochlear adaptation to external environment. From a developmental point of view, purinergic signalling in Kölliker's organ may provide a sophisticated mechanism central to the tonotopic organisation of the cochlea which starts to develop in the absence of sound.First discovered over a century ago, Kölliker's organ is present transiently in the developing cochlea, until the cochlea becomes sensitive to external sound. It is an epithelial structure composed of long columnar cells and lies immediately adjacent to IHCs. From this close proximity, epithelial cells of Kölliker's organ have been suggested to initiate spontaneous electrical activity in the IHCs, driving action potentials in the primary auditory neurons, and auditory nuclei in the brainstem. Although the exact mechanism remains unclear, rhythmic ATP release from connexin hemichannels and activation of purinergic P2 receptors may contribute to rhythmic current/morphological oscillations of Kölliker's organ. The rise in intracellular Ca


Fashion is an essential part of human experience and an industry worth over $1.7 trillion. Important choices such as hiring or dating someone are often based on the clothing people wear, and yet we understand almost nothing about the objective features that make an outfit fashionable. In this study, we provide an empirical approach to this key aesthetic domain, examining the link between color coordination and fashionableness. Studies reveal a robust quadratic effect, such that that maximum fashionableness is attained when outfits are neither too coordinated nor too different. In other words, fashionable outfits are those that are moderately matched, not those that are ultra-matched (“matchy-matchy”) or zero-matched (“clashing”). This balance of extremes supports a broader hypothesis regarding aesthetic preferences–the Goldilocks principle–that seeks to balance simplicity and complexity. Every day, people ask themselves the question: “What to wear?” People want outfits that are maximally fashionable, and this isn’t mere vanity: clothing influences perceived and signaled social identity The Goldilocks Principle represents a tradition of philosophical thought stretching back millennia: Aristotle’s Golden mean, Buddha’s middle way, and Confucius’ Doctrine of the Mean all represent a balance between two extremes. The Goldilocks Principle has psychological support in a variety of domains, as infants prefer looking at visual sequences that are neither too complex, nor too simple In terms of fashion, there are two popular approaches to style that represent “extremes.” On one hand, we often speak as if the most fashionable outfits are those that fully coordinate or “match” In this paper, we investigate whether the Goldilocks Principle predicts fashionableness across diverse color combinations in both men’s and women’s outfits. Support for the principle would be illustrated by a “peak” in ratings, such that any linear trends between coordination and fashionableness should be qualified by a quadratic effect such that maximum fashionableness is achieved by moderate color coordination.ageM = 35.4, SDage = 12.9) each saw 30 different color combinations, in one of four color palettes. Palettes 1 and 2 were in women’s clothing, and palettes 3 and 4 were in men’s clothing. Each palette included 4 colors, illustrated in This study was approved by the Institutional Review Board at The University of North Carolina at Chapel Hill. All participants provided written informed consent prior to completing the study and were recruited through Amazon’s Mechanical Turk (mTurk). A total of 239 mTurk participants , which were Z-scored within each palette.Rather than solicit global ratings of coordination, participants rated the coordination of all possible pairs of color swatches within each palette through 3 items–coordinated, matching and similar–answered on five point Likert scales (α’s >.81). These pairwise judgments were aggregated for each outfit, and Z-scored within each palette, to create an overall coordination score. Thus, combinations with all similar or highly coordinated colors  received high scores whereas those with very different colors  received low scores  = 13.04, p = .001, such that more coordination was linked to more fashionableness, consistent with the general importance of matching. Importantly, however, this linear trend was qualified by the predicted quadratic effect, R2 = .44, F  = 22.23, p<.001, such that peak fashionableness was achieved by moderately coordinated combinations. This quadratic effect accounted for twice as much variance as the linear effect.Analyses revealed a significant linear trend, F<1, but did reveal the predicted quadratic trend, R2 = .28, F  = 11.18, p<.001, such that peak fashionableness was again achieved by moderately coordinated combinations.Analyses did not reveal a significant linear trend, These data suggest a simple answer to the question “what to wear?” Select a color combination that is neither completely uniform, nor completely different. Certainly, moderate matching is not the only key to fashion, which varies across time and culture and depends upon many factors including cut, design, and trendiness. However, these studies reveal that, with all other factors held constant, the Goldilocks principle predicted judgments across four different color palettes in both men’s and women’s clothing. To examine the external validity of these findings, future research should test this idea in naturalistic settings, such as in magazines and runway shows.These results are consistent with both centuries of philosophical thought and more recent psychological studies on the importance of “the middle way.” The Goldilocks principle may also explain aesthetic judgments beyond fashion, reflecting a basic principle of human preference that seeks to balance simplicity and complexity, order and disorder. Indeed, people prefer music that balances melodic simplicity and complexity Figure S1Rating a sample outfit combination.(DOCX)Click here for additional data file.Figure S2Rating a sample color pair.(DOCX)Click here for additional data file.Table S1Clothing combinations by color palette.(DOCX)Click here for additional data file.Table S2Cronbach’s Alphas for scales used.(DOCX)Click here for additional data file.Materials and Methods S1Extended materials & methods.(DOCX)Click here for additional data file.Results S1Extended results.(DOCX)Click here for additional data file.


Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver. Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643.q value < 0.05). Many genes induced by PPARα activation were involved in lipid metabolism , xenobiotic metabolism  or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARα activation were several chemokines , interferon γ-induced genes  and numerous other immune-related genes . Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARα is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARα activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARα were identified that were commonly induced by PPARα activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L.Quantitative PCR indicated that PPARα is well expressed in human liver and human liver slices and that the classical PPARα targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARα activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARα activation  contains supplementary material, which is available to authorized users. The Peroxisome Proliferator Activated Receptors (PPARs) represent an important group of receptors involved in mediating the pleiotropic effects of various environmental contaminants, food components, and drugs . PPARs aPPARα is best known for its role in the liver, where it acts as the master regulator of lipid metabolism, especially during fasting –10. FastSimilar to other members of the PPAR family, PPARα is activated by a range of different fatty acids and eicosanoids –16. In aMost of our insights into the physiological, toxicological and pharmacological role of PPARα has been derived from experiments in animals and in particular from rodent studies. These studies have revealed that PPARα is not only involved in the adaptive response to fasting but also mediates the hepatocarcinogenic effects of peroxisome proliferators . WhetherNevertheless, the absence of more complex human model systems has hampered our ability to gain insight into the molecular function of PPARα in human liver, in particular with respect to target gene regulation. To overcome this limitation, we collected precision cut liver slices (PCLS) from human subjects and studied the effect of PPARα activation on gene expression using whole genome expression profiling. In contrast to primary hepatocytes, PCLS mimic the multi-cellularity and structural organization of whole liver and thus represent a superior ex-vivo model system for human liver . So far n = 15) were immediately frozen in liquid nitrogen and stored at −80 °C. The liver biopsies targeted for slicing (n = 4) were immediately placed in ice-cold oxygenated Belzer UW Cold Storage Solution  and quickly transferred to our laboratory for further processing of PCLS. Only macroscopically healthy livers were used for slicing. Biopsies were provided by the biobank of the Rijnstate hospital. Collection of biopsies for research purposes into the biobank was approved by the local institutional review board on behalf of the medical ethics review committee of the Radboud University Medical Centre. All patients signed informed consent for collection of the biopsy prior to surgery. Donor characteristics of the livers used for slicing are shown in Table In all patients a laparoscopic Roux-en-Y Gastric Bypass was performed under general anesthesia as treatment for their morbid obesity. Patients were instructed to fasten for solid foods and liquids starting at the night before surgery. During surgery a biopsy of the liver was obtained with the help of ultrasound dissection (UltraCision®). The biopsy was collected from the liver edge. The majority of the liver biopsies collected n = 5 were im2/80 % O2 under continuous shaking (70 rpm). Duplicate wells were used per donor with 3 liver slices per well. After 1 hour the media was replaced with fresh William’s E Medium in the presence or absence of Wy14643 (100 μM) dissolved in dimethyl sulfoxide . This concentration was chosen based on the affinity of human PPARα for Wy14643 [PCLS were prepared and cultured as described previously . 5 mm cy Wy14643 . After 2The treatment of primary human hepatocytes with Wy14643 has been previously described . BrieflyTotal RNA was isolated using TRIzol reagent (Invitrogen). RNA integrity number was found to be above 7.1 for all samples suggesting that the human liver slices were of good quality. The RNA integrity number is based on a digital electropherogram generated using a Agilent bioanalyzer. It describes the degree of degradation of RNA with level 10 representing completely intact RNA. RNA was reverse transcribed using a iScript cDNA Synthesis Kit . Real-time PCR was carried out using SensiMiX (Bioline) on a CFX 384 Bio-Rad thermal cycler (Bio-Rad). 36B4 was used as housekeeping gene. Primer sequences used are shown in Table P-value for the effect of Wy14643 treatment were calculated using an Intensity-Based Moderated T-statistic (IBMT) [q-value was calculated as measure of significance for false discovery rate [Total RNA was purified with RNeasy Minikit columns (Qiagen) and RNA quality was assessed using RNA 6000 Nano chips on the Agilent 2100 Bioanalyzer . Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Human Gene 1.1 ST array plate . Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform according to the instruction by the manufacturer. Arrays were normalized using the Robust Multiarray Average method , 34. Proc (IBMT) . The q-very rate . The micGene set enrichment analysis (GSEA) was used to find enriched gene sets in the induced or suppressed genes . Genes wFirst, we determined the relative expression level of PPARα in PCLS in comparison with human liver. After correcting for the housekeeping gene 36B4, mRNA levels of PPARα in human PCLS after 24 h incubation were about ten-fold lower as compared to snap-frozen human liver biopsies Fig. . To veriq-value of 0.05 as cut-off (IBMT regularised paired t-test), the expression of 1282 genes  was found to be significantly altered by Wy14643 treatment, of which 617 genes were upregulated and 665 genes were downregulated. The top 25 of most significantly induced genes, ranked according to statistical significance, are shown in Fig. To study the effect of PPARα activation on whole genome gene expression, we performed microarray analysis. Wy14643-induced changes in expression of the selected PPARα target genes were very similar between the microarray and qPCR analysis Fig. . Using aTo gain better insight into the biological function of genes regulated by PPARα activation in human liver slices, we performed gene set enrichment analysis (GSEA). Pathways related to lipid metabolism or directly involving PPARα featured prominently among the gene sets induced by Wy14643  combined with a fold-change cut-off of 1.2, we found 347 genes to be upregulated in human liver slices, as compared to 121 genes in the hepatocytes  and studied the response to Wy14643 of the same genes in the primary hepatocytes. Whereas the majority of genes induced by Wy14643 in the liver slices were also induced by Wy14643 in the hepatocytes , interferon γ-induced genes  and numerous other immune-related genes . Downregulation of gene expression is unlikely to be mediated by PPARα present in non-parenchymal cells, as PPARα expression in these cells is very low , 39. InsDespite the clear clinical efficacy of fibrates towards lowering of circulating triglycerides, the lack of peroxisome proliferation in human primary hepatocytes following PPARα activation has fed the idea that humans are largely insensitive to peroxisome-proliferator-induced hepatic effects and that the functional role of PPARα in human liver may be limited –47. SubsThe majority of gene sets enriched among the upregulated genes were related to lipid and xenobiotic metabolism, which are well-established target pathways of PPARα. Intriguingly, several highly enriched gene sets were part of the unfolded protein response and IRE1α-XBP1 signaling, two key factors involved in governing UPR. Currently, there are no published data linking PPARα to IRE1α-XBP1 signaling and regulation of UPR, though it has been observed that PPARα is involved in regulating proteome maintenance by inducing numerous heat shock proteins . SurprisOur analysis reveals differential regulation of a number of PPARα targets between liver slices and primary hepatocytes. For instance, VNN1 was significantly upregulated by PPARα activation in primary hepatocytes but showed no change in expression in liver slices. Conversely, expression of FADS2 was significantly increased in human liver slices but showed no change in primary hepatocytes. The differential regulation of specific PPARα target genes by Wy14643 between primary hepatocytes and liver slices may be a reflection of the different cellular context in the two models systems, with non-hepatocytes potentially exerting a stimulatory or inhibitory influence on PPARα-dependent gene induction. However, it should be realized that for a number of genes the seemingly differential regulation may reflect a quantitative difference rather than a true qualitative difference. For example, SLC25A20 was induced significantly by 1.58-fold in primary hepatocytes as compared to a non-significant 1.34-fold induction in liver slices, barely missing the statistical significance cut-off.Our analysis yielded a number of relatively poorly characterized genes that showed a pronounced and consistent upregulation upon PPARα activation in the two human liver model systems. These include TSKU, RHOF, CA12 and VSIG10L. Interestingly, many genes that were found to be induced by PPARα in early microarray analyses and which did not have an assigned function at the time were later shown to be involved in some aspect of lipid metabolism. Accordingly, it can be hypothesized that the above mentioned genes as well as other poorly characterized genes that are commonly induced by Wy14643 in various liver model systems may be directly or indirectly connected to lipid metabolism.The comparative microarray analysis of the effect of PPARα activation in primary human hepatocytes and human liver slices is somewhat hampered by a number of different factors, including the use of different types of Affymetrix gene chips, different human donors, and an unequal number of biological replicates per group. However, treatments of liver slices and hepatocytes were carried out for the same duration and with the same PPARα agonist. Furthermore, RNA was isolated and labeled via the same technique, hybridizations were performed on the same platform by the same technician, and the microarray data were processed in parallel using the same analysis methods.http://en.wikipedia.org/wiki/Peroxisome_proliferator-activated_receptor_alpha), which was generated largely by using published transcriptome datasets.On a final note, the data collected in this paper were added to a publicly available overview map of known (lipid) metabolic genes upregulated by PPARα in human liver (accessible via: In conclusion, our paper demonstrates the suitability and superiority of PCLS over primary human hepatocytes for studying the functional role of PPARα in human liver. Our data underscore the major role of PPARα in regulation of hepatic lipid and xenobiotic metabolism and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARα in human liver that may be therapeutically relevant for NAFLD. The data add to our growing understanding of the critical role of PPARα in gene regulation in human liver.


To evaluate the diagnostic performances of two training programs for residents with no prior ultrasound experience to reach competences in extended basic critical care transthoracic echocardiography (CCE) including Doppler capabilities.This is a prospective observational study in two intensive care units of teaching hospitals. Group I (five residents) completed a short training program ; group II (six residents) completed a longer training program . The residents and an expert examined all patients who required a transthoracic echocardiography. Their agreement studied by Cohen’s κ coefficient, concordance coefficient correlation (CCC) and Bland–Altman plots was used as an indicator of program effectiveness.Group I performed 136 CCEs  in 115 patients . Group II performed 158 CCEs  in 108 patients . Both groups adequately assessed left ventricular (LV) systolic function  and pericardial effusion . Group II appraised severe right ventricular dilatation and significant left-sided valve disease with good to very good agreement . Regarding left ventricular ejection fraction, E/A ratio, E/e′ ratio and aortic peak velocity assessed by group II, CCCs were all >0.70 and the bias (mean difference) ±SD on Bland–Altman analysis was 1.3 ± 8.8 %, 0 ± 0.3, 0.4 ± 2.2 and 0.1 ± 0.4 m/s, respectively. Detection of paradoxical septum , of heterogeneous LV contraction  and of respiratory variation of the inferior vena cava , as well as stroke volume measurement , was appraised by group II with moderate agreement requiring probably more comprehensive training.Although a training program blending 6-h theory and 12-h practical may be adapted to achieve some essential competences, it seems to be insufficiently to perform a complete extended basic critical care transthoracic echocardiography including Doppler capabilities.The online version of this article (doi:10.1186/s13613-016-0150-8) contains supplementary material, which is available to authorized users. Critical care transthoracic echocardiography (CCE) with Doppler is increasingly used at the bedside of intensive care unit (ICU) patients to establish diagnoses and guide the management of those with cardiopulmonary compromise . Though The ability to master spectral Doppler and tissue Doppler imaging (TDI) indices after a limited training program has not yet been tested . In factSo, this study was designed to evaluate the feasibilities and diagnostic performances of two different focused training programs (a short and a long program) for residents without any previous knowledge in ultrasound for performing extended basic CCE with Doppler capabilities.We performed a prospective, observational, educational study comparing two focused training programs to acquire extended basic CCE skills. Our study was considered to be part of routine clinical practice by the appropriate ethics committee , so no informed consent was required from the patients or their relatives. If patients were awake or if their relatives were present, information was given on the training of the residents.Two distinct training programs were conducted with non-cardiologist residents with no prior experience in ultrasound. Both training programs were conducted by V.L., a cardiologist expert with board certification in echocardiography and level 3 competence according to the American Society of Echocardiography Standards .Five residents (group I) participated in training program I, which was conducted within the ICU of the University teaching hospital of Colombes (France) between November 2010 and November 2011. It involved 2 months of theoretical and practical appraisal. The theoretical program  participated in training program II conducted within the ICU of the University Teaching Hospital of Tenon  between November 2012 and November 2013. This training program differed from program I as follows: (1) a 3-month theoretical and practical appraisal; (2) an extra hour of interactive clinical cases; (3) at least 25 “offline reviewed” extended basic CCEs were required; and (4) the ability to recognize a paradoxical septum was taught ; right ventricular (RV) dilatation ; left-sided valvular regurgitation or stenosis ; pericardial fluid ; and the respiratory variation of the inferior vena cava (IVC) Answer the following qualitative clinical questions: LV contraction pattern ; paradoxical interventricular septum Answer the following quantitative clinical questions: global LV function [visual evaluation of the LV ejection fraction (LVEF)]; stroke volume ; LV filling pressure parameters; LV diastolic function ; and aortic valve peak velocity.The extended basic CCEs were performed using two recent ultrasound systems equipped with full Doppler capability and a tissue Doppler imaging program . The extended basic CCEs aimed to:Additional details on the methods for answering these clinical questions are provided in Additional file p value of <0.05 was statistically significant.Results are expressed as mean ± standard deviation (SD), or number (percentage), unless otherwise stated. The durations of the CCEs were compared between investigators using a paired Wilcoxon test. Conditions and indications of “paired” CCEs were compared between group I and group II using a Chi-square test. The proportions of “optimal” vs “not optimal” imaging quality and unanswered clinical questions  were compared between residents and the expert using the Chi-square McNemar test. In patients for whom the qualitative and semiquantitative clinical questions could be addressed by both resident and expert, the agreement between responses provided by the two investigators was assessed using the Cohen’s κ coefficient and 95 % confidence intervals (CIs) . CCC canDetails of total training hours of the two programs are shown in Table Group I residents performed 136 “paired” extended basic CCEs  in 115 patients . Group II residents performed 158 “paired” extended basic CCEs  in 108 patients . Indications, conditions and global imaging qualities (graded by the expert) of CCEs were similar between both groups  with less optimal acoustic windows . Group I residents recorded at least one optimal acoustic window in 105 of 127 (83 %) extended basic CCEs versus 124 of 127 (98 %) extended basic CCEs by the expert (p < 0.0001). Group II residents recorded at least one optimal acoustic window in 112 of 123 (91 %) extended basic CCEs versus 116 of 123 (94 %) extended basic CCEs by the expert (p < 0.33).When compared to the expert, group I and group II residents performed longer extended basic CCEs , the number of unanswered clinical questions was not statistically different between residents in group II and the expert   and the detection of a significant left-sided valve disease . Group II residents detected a paradoxical septum with a moderate agreement . Lastly, both groups detected a heterogeneous LV contraction and a significant respiratory variation of IVC diameter with a fair to moderate agreement.Table p = 0.01), E/A ratio  and E/e′ ratio . The Bland–Altman plots demonstrating the bias (95 % LoA) of quantitative Doppler measurements between residents in group II and the expert are shown in Fig. Table This is the first study that aimed to validate whether a focused training program designed to achieve essential competences in extended basic CCE including Doppler capabilities for residents with no prior ultrasound experience was adequate. Basic critical care echocardiography by physicians inexperienced in ultrasound has been previously investigated, using training programs of 6–15 h –7. Of noThe shorter training program blending approximately 4-h theory and 3-h practical allowed residents to acquire only two important skills using 2D imaging including global systolic LV function and pericardial effusion assessment. However, residents failed to achieve others skills using 2D imaging and Doppler capabilities. The longer training program blending approximately 4 h theory and 12 h practical allowed residents to acquire to a good or very good degree of some essential competences in extended basic CCE, using 2D imaging  and Doppler capabilities . As expected, LV filling pressure and diastolic function were more adequately assessed by residents trained by the long program than by those trained with the short program, with a good reproducibility but a moderate agreement. Lastly, some skills such as the measurement of the stroke volume, the detection of heterogeneous LV contraction and of paradoxical septum and the assessment of respiratory variation of IVC diameter probably require more in-depth training.For the first time, our study shows that a focused training program in ICU setting allows to reach competences using Doppler capabilities. Using color and continuous Doppler, the residents trained by the long program adequately detected significant left-sided valve disease, as only 6 % of the evaluated patients were misclassified. Consistent with our results, Mjølstad et al.  found thSome essential basic skills using only 2D imaging seem easier to achieve since, in keeping with recent series –7, both Compared to residents trained by the short program, the agreement between responses provided by residents trained by the long program and the expert was generally higher. This may be related to the longer training curriculum, which included more interactive clinical cases, but may also be related to a learning curve effect, since residents trained by the long program performed a mean of 27 examinations, compared with 10 in the short training program. Our learning rate estimates are consistent with the “European Society of Intensive Care Medicine” recommendations of approximately 30 TTEs in ICU patients to reach competence in basic critical care echocardiography . HoweverOur study has some limitations. First, the training programs were conducted in two different places, at different times. However, indications, conditions and global imaging qualities of “paired” extended basic CCEs were mainly similar between both study centers, and both trainings were conducted with non-cardiologist residents with no prior experience in ultrasound. Second, we have included a limited number of trainees because of our small number of residents, their 6-month rotation period and the exclusion of those who had experience in ultrasound. However, each recently formed resident performed a large number of CCE during the evaluation period. Third, the mean duration of extended basic CCE examination was very long, even after a larger number of “offline reviewed” extended basic CCEs. We consider this point to be crucial because hemodynamic evaluation has to be performed quickly in order to rapidly optimize treatment. Fourth, because of the workload in the ICU, the residents were often hard-pressed to find time to perform the CCEs. A rotation specifically dedicated to learning echocardiography might be one way to improve results substantially. Fifth, our study wasn’t designed to assess the learning curve of residents. Finally, because the simulation system was unavailable in our units, this very efficient learning modality was not been tested in the study.A focused training program appears satisfactory to teach residents to adequately address some important clinical questions for a comprehensive hemodynamic assessment in critical care patients. However, a complete extended basic critical care transthoracic echocardiography including common parameters derived from spectral Doppler and tissue Doppler imaging requires a larger training program. Future research should aim to assess the ability of these training programs to result in adequate therapeutic proposals.


This article examines the impact of Cattaneo-Christov heat flux in flows of viscoelastic fluids. Flow is generated by a linear stretching sheet. Influence of thermal relaxation time in the considered heat flux is seen. Mathematical formulation is presented for the boundary layer approach. Suitable transformations lead to a nonlinear differential system. Convergent series solutions of velocity and temperature are achieved. Impacts of various influential parameters on the velocity and temperature are sketched and discussed. Numerical computations are also performed for the skin friction coefficient and heat transfer rate. Our findings reveal that the temperature profile has an inverse relationship with the thermal relaxation parameter and the Prandtl number. Further the temperature profile and thermal boundary layer thickness are lower for Cattaneo-Christov heat flux model in comparison to the classical Fourier’s law of heat conduction. Mechanism of heat transfer occurs when there is a difference of temperature between the bodies or between the various parts of the same body. Such mechanism has widespread industrial and technological applications like cooling of nuclear reactors, cooling of electronic devices, energy production, power generation and many others –3. FouriThe analysis of boundary-layer flow past a stretching surface is significant in various industrial and technological processes. Examples of such practical applications are wire drawing, extrusion of plastic sheets, hot rolling, paper production, glass fiber etc. There are many fluids in our daily life usage like shampoos, certain oils, sugar solution, tomato paste, mud, apple sauce, chyme, personal care products and several others which do not satisfy the classical Newton's law of viscosity. Such fluids fall in the class of non-Newtonian fluids. All the non-Newtonian fluids through their distinct features cannot be explained by using a single constitutive relationship. This fact of non-Newtonian fluids are quite distinct than that of viscous fluids. In the past various models have been developed to characterize the properties of non-Newtonian fluids. Amongst these the simplest subclasses of differential type fluids are the elastico-viscous and second grade –20. FurtThis communication presents a comparative study for Cattaneo-Christov heat flux model in boundary layer flow by considering the two classes of viscoelastic fluids. Constitutive relations for second grade and elastico-viscous fluids are considered. Most of the studies in the literature are explained through the classical Fourier’s law of heat conduction. There is not a single study in the literature that present the characteristics of Cattaneo-Christov heat flux in boundary layer flow of viscoelastic fluids. Hence the purpose here is to employ the Cattaneo-Christov heat flux model in the boundary layer flow of viscoelastic fluids. To the best of the author's knowledge, no such consideration has been discussed in the literature yet. Similarity approach is adopted to convert the partial differential system into the set of nonlinear ordinary differential system. The governing nonlinear system is solved through the homotopy analysis method (HAM) –32. Impax—axis is taken along the stretching surface and y—axis is orthogonal to it. Let Uw(x) = ax denotes the surface stretching velocity along the x—direction. The heat transfer process is studied through the Cattaneo-Christov heat flux theory. The governing two-dimensional (2D) boundary-layer flows in two cases of fluids are under considerationWe consider the steady two-dimensional (2D) flows of viscoelastic fluids over a linear stretching surface. Flow models for second grade and elastico-viscous fluids are considered. The Cartesian coordinate system is adopted in such a way that the u,v) are the fluid velocities in horizontal and vertical directions respectively, v = μ / ρ the kinematic viscosity, μ the dynamic viscosity, ρ the density, cp the specific heat, k0 = −α1 / ρ the elastic parameter, T the temperature and q the heat flux. Here k0 > 0 is for elastico-viscous fluid, k0 < 0 corresponds to a second grade fluid and k0 = 0 is for Newtonian fluid. According to the Cattaneo-Christov heat flux model  is the embedding parameter, ℏ the Eqs  and 25)f and ℏθ f and ℏθ. Such non-zero auxiliary parameters are important in accelerating the convergence of obtained homotopic solutions. The proper values of such parameters are important to get the convergent approximate homotopic solutions. To get the appropriate values of ℏf and ℏθ, the ℏ—curves are plotted at 14th order of homotopic deformations. Figs f ≤ −0.40 and −1.70 ≤ ℏθ ≤ −0.40 for elastico-viscous fluid f ≤ −0.20 and −1.50 ≤ ℏθ ≤ −0.50 for second grade fluid No doubt the approximate homotopic solutions contain the non-zero auxiliary parameters ℏγ and Prandtl number Pr on the non-dimensional velocity distribution f′(η) and temperature distribution θ(η). Here the elastico-viscous f′(η) for both fluids is sketched in f′(η) is reduced for larger values of elastico-viscous parameter f′(η) is higher for larger values of second grade parameter θ(η) for both fluids. Temperature distribution θ(η) and thermal boundary layer thickness are enhanced for elastico-viscous fluid while opposite behavior is observed in case of second grade fluid. γ on the temperature distribution θ(η). Temperature distribution θ(η) and thermal boundary layer thickness are decreasing functions of thermal relaxation parameter. Here the thermal relaxation parameter γ = 0 shows that the heat flux expression is reduced to the classical Fourier's law. Effect of Prandtl number Pr on the temperature distribution θ(η) for both fluids is displayed in θ(η) and thermal boundary layer thickness are reduced when Prandtl number Pr increases. Prandtl number has an inverse relationship with the thermal diffusivity. High Prandtl fluid possess weaker thermal diffusivity and the low Prandtl fluid has stronger thermal diffusivity. When we enhance the Prandtl number, then weaker thermal diffusivity appears. Such weaker thermal diffusivity creates a reduction in the temperature distribution and thermal boundary layer thickness for both fluids. θ′(0) for various values of thermal relaxation parameter γ for elastico-viscous θ′(0) is enhanced for larger values of thermal relaxation parameter γ in both fluids. It is also analyzed that the values of heat transfer rate at the surface −θ′(0) in second grade fluid The present section has been arranged to examine the impacts of viscoelastic parameter Boundary layer flows of two viscoelastic fluids over a linear stretching surface satisfying Cattaneo-Christov heat flux is analyzed. The main observations of this study are summarized below:f′(η) and momentum boundary layer thickness are reduced when we enhance the positive values of Velocity profile θ(η) and thermal boundary layer thickness are decreased when Pr enhances.Both the temperature distribution γ show a reduction in the temperature distribution θ(η) and thermal boundary layer thickness.Increasing values of thermal relaxation parameter Skin friction coefficient is reduced when we enhance the positive values of γ for both fluids.Heat transfer rate at the surface is more for increasing values of thermal relaxation parameter


The images directly reflect the spatially resolved phosphorus content of mineral tissue in dentin and enamel; they show a lack of signal from pulp tissue and reduced signal from de-mineralized carious lesions. We demonstrate for the first time that the signal in 31P MR images of mineralized tissue is enhanced by a 1H-31P nuclear Overhauser effect (NOE). Using teeth as a model for imaging mineralized human tissue, graded differences in signal enhancement are observed that correlate well with known mineral content. From solid-state NMR experiments we conclude that the NOE is facilitated by spin diffusion and that the NOE difference can be assigned to a higher water content and a different micro-structure of dentin. Thus, a novel method for imaging mineral content without ionizing radiation is proposed. This method has potential use in the assessment of de-mineralization states in humans, such as caries of teeth and osteoporosis of bones.Three-dimensional phosphorus MR images ( Clinically relevant MRI sequences that accommodate minimal dead times are UTE (Ultra-short echo time)691031P spins compared to 1H spins are additional challenges to perform 31P MRI of bones and teeth. One way to enhance the 31P signal is by generating a 1H–31P nuclear Overhauser effect (NOE)A major challenge in 31P MRI with 1H-31P NOE to obtain anatomical and structural information of mineral tissue of human teeth as a model system with known phosphorous content in distinct anatomical regions. We adapted SWIFT and ZTE pulse sequences and the MR system hardware for NOE enhanced 31P MRI at a minimized ‘dead time’, and determined the structural origin of the NOE by solid-state NMR of mineral samples from teeth.In this study we performed 3-dimensional 1H MR imaging. In the images several anatomical features, such as dentin, enamel and pulp tissues can be distinguished  T2* of 31P was ≈100 μs and the global T1 was ≈130 seconds for this human maxillary left second molar tooth. For other teeth samples, similar T2* values were obtained, and T1 values were all within the range of 100–200 seconds. In 31P MR images obtained in 100 seconds by SWIFT and ZTE (512 spokes), the contour of the molar tooth is already visible 31P signal of enamel is about 50% higher than that of dentin.In order to optimize the pulse sequence for  visible . In the 31P MR images obtained with ZTE offer better SNR than those obtained with the gapped SWIFT sequence , and dentin is visible as a hyperintense region . This enhancement is not the same for all regions: subtraction of the SWIFT MR images with and without NOE clearly reflects the shape of dentin, while enamel is less intense in the subtraction image  NMR spectra of both enamel and dentin, the water resonance at ≈5 ppm is the most pronounced signal  experiments are presented in 1H → 31P CP spectra of dentin at short and long contact times shows that the 31P line shape of dentin observed at long CP contact times is narrower 6(OH)2)31P in human mineralized tissue is challenging, owing to its low MR sensitivity and very long T1 and short T2* relaxations. To deal with the short signal persistence after excitation we implemented hardware for rapid switching between excitation and receive mode and ZTE and SWIFT sequences for immediate signal acquisition. The SNR of SWIFT images is expected to be lower compared to images obtained by ZTE due to gapped acquisition and is proportional to the square root receiver duty cycle drec(=0.5 in this study). This is in agreement with experimental findings: in vivo imaging. A recent modification of SWIFT, multi-band SWIFT, allows for a highly increased receiver duty cycle31P MRI of human bone and teeth. Various 31P MRI methods have been used to assess mineralization of bone, including a solid-state method similar to ZTE for human teeth1920It is attractive to use 31P NOE enhancement by 1H irradiation has been widely reported for phosphate compounds in solutions231H-31P NOE enhancement of the signal intensity in 31P MR images is observed. More importantly, the enhancement for enamel and dentin is different. Steady state NOE experiments show that the enhancement reaches its maximum value with 1H RF irradiation lasting about 5 × T1 of 31P spins, in agreement with our knowledge of heteronuclear NOE in the solid stateThe NOE is the fractional change in the integrated NMR absorption intensity of a nuclear spin system upon saturation of neighbor-coupled spins1H content in enamel compared to dentin. The change in line-width in the 31P CP spectra at longer contact times indicates that the materials consist of various components showing different dynamics. In dentin, the broader line-width at short contact times is attributed to a more heterogeneous, amorphous environment2627NOE and CP both rely on the dipolar interaction. As the phosphate structures in enamel and dentin are chemically very similar no big changes in the static dipolar interactions are expected. However, molecular motions will modulate the dipolar interactions and therefore affect CP and NOE efficiency. Different microstructures of chemically related moieties can therefore still give different CP and NOE response. The difference in microstructure of enamel and dentin is clearly reflected in the CP dynamics. CP is a coherent polarization transfer process and is very sensitive to motions in the kHz regime of the involved spins. The slower build-up in enamel most likely results from much lower relative 31P spectra were deconvoluted according to the number of components used in literature1431P spin diffusion (neighboring spins exchange polarization via dipolar couplings) taking place during the build-up process over hundreds of seconds. The NOE effect is a cross-relaxation effect, which requires a modulation of the dipolar interaction at the nuclear Larmor-frequency to be efficient. Therefore fast motions with correlation times in the ns regime must be present. The OH− concentration in enamel is much higher than in dentin, but the strongest enhancement is observed in dentin. This indicates that the hydroxyl resonance is not the main contributor to the NOE mechanism. As the water resonance around 5 ppm is a common feature and remains even after extensive dryingTo identify differences in NOE build-up for the various components of enamel and dentin, the NOE enhanced 1H resonance involved in the NOE by 1H saturation at selective frequencies, but no significant differences in the enhancement were observed (data not shown). It is virtually impossible to selectively saturate a single proton resonance as during the long NOE build-up period (hundreds of seconds), irradiating at a single frequency, spin diffusion will effectively equilibrate all polarization. This is in agreement with experiments on synthetic apatite, where all proton resonances show spin exchange within milliseconds31P spectra of dentin resemble it’s CP spectra at long polarization times , it is most probable that the more pronounced and faster NOE build-up in dentin arises in the amorphous phase and is then transferred by spin diffusion to the crystalline phase.We tried to identify the 1H-31P dipolar interactions are most likely to occur in these phases.A difference in dentin and enamel microstructure most likely explains the faster and more pronounced NOE in dentin: enamel consists mainly of highly crystalline apatite mineral (96 wt%), while dentin, besides being partly amorphous, also contains an organic matrix and water 31P MRI uses no ionizing radiation and it can be combined with 1H MRI for the visualization of calcified dental tissue. Additionally 31P MRI has an advantage of being a true volumetric method. Although the current results suggest some favorable prospects for biomedical applications, the realization of 31P MRI for human bone imaging to address clinical questions faces a number of challenges1H irradiation in the current NOE experiments was less than 5%, while much higher duty cycles for 1H-31P NOE of the human brain have been safely applied at 7T field strengthCompared to 3D imaging with cone-beam computed tomography, 31P MR images of teeth can be obtained by SWIFT and ZTE pulse sequences with minimum transmit/receive deadtime, in which dentin and enamel are spatially resolved. We showed that irradiation at the 1H frequency creates NOE enhancement of the 31P signal in these images. The signal of dentin is more enhanced than that of enamel which is caused by micro-structural differences. Thus the NOE provides an option for additional structural imaging of teeth. This new approach may also have potential in the assessment of other mineralized tissues in the human body, such as monitoring mineral changes due to osteoporosis.In conclusion, we demonstrate that 3D ex-vivo imaging. No data that could identify patients were recorded and therefore was Institutional Review Board exempt. Teeth were stored in an isotonic saline solution containing 0.1% of sodium azide as an antimicrobial agent. Before the MRI experiments, water on the surface of the teeth was removed by blotting with paper towels, and teeth were then wrapped with Teflon tape to reduce evaporation of water and isolate the tissue from equipment. A maxillary left second molar and a mandibular right second molar were chosen to test the feasibility of 31P MRI to access their overall anatomy and mineral content. In order to further test whether 31P MRI can detect mineral loss, two teeth with carious defects were selected: a mandibular right first premolar with buccal pit lesion and distolingual lesion in the crown; a maxillary left canine with restoration at the mesiolingual surface.Extracted teeth harvested as waste tissue were selected for 31P and 1H) probe. The phosphorus RF coil consisted of two parallel copper loops of 1.5 cm in diameter at 1 cm distance. The proton RF coil was a copper single loop surface coil with 1.5 cm in diameter positioned in between the phosphate coil loops and oriented perpendicularly to them. The RF hardware allowed minimum “dead” times between excitation and acquisition and was 2 μs for the 1H frequency and 3 μs for the 31P frequency.MRI was performed on a 9.4T MR system (Agilent-Varian) with a home-built double resonance  or 14 minutes for a higher resolution image , and receiver duty cycle (drec) of 0.5 in all the experiments. Almost all other parameters are similar, except for the short hard pulse excitation (5 μs length) applied in ZTE. Images were reconstructed to a matrix of 64 × 64 × 64 pixels. The global T1 and T2* relaxation times of the 31P spins were estimated (for the whole tooth) by inversion recovery and measuring line widths, respectively. Two regions of interests (ROIs) localized on dentin and enamel were manually drawn on the cross-sectional slices of the 31P images, to compare their difference in SNR. The SNR is evaluated by calculating the average signal divided by the standard deviation of the background signal.Proton SWIFT images were acquired with an excitation bandwidth of 125 kHz, 256 gaps for sampling, an oversampling factor of 4, and 15° flip angle (FA). The field of view (FOV) was 3 × 3 × 3 cm. In total 32,000 radial spokes were acquired with a repetition time (TR) of 2.5 ms which includes a 2 ms total pulse length. The total acquisition time (TA) was 100 seconds. Images were reconstructed to a matrix of 256 × 256 × 256 pixels. Phosphorus SWIFT and ZTE images were both acquired with an excitation bandwidth of 62.5 kHz, 64 gaps for sampling, FA of 3.7°, oversampling factor of 4, TR of 200 ms, FOV of 3 × 3 × 3 cm1H-31P NOE enhanced experiments, the radiofrequency saturation of proton spins was implemented in the steady-state imaging scheme. The proton spins were saturated for 1.282 s during TR (1.482 s) by a hard pulse train , with a 3.2 mm rotor and a H-X-Y triple resonance probe head using a relaxation delay of 750 s at 10 kHz spinning rate. The 31P excitation pulse length was 5.5 μs. The 1H nuclei were saturated by a train of 1 ms soft pulses (ν1 = 450 Hz) separated by a delay of 1 ms, with a total saturation time ranging from 50 to 500 seconds. 1H-31P CP spectra at different contact times (100 μs − 15 ms) were recorded for dentin and enamel at 9.4T with a recycle delay of 10 s, an 1H excitation pulse length of 5.4 μs and 10 kHz spinning.To quantify the NOE build-up and to identify the source of the enhancement, a series of solid-state NMR spectroscopy experiments were performed. Dentin and enamel were separated and crushed into powder. Single pulse How to cite this article: Sun, Y. et al. Imaging human teeth by phosphorus magnetic resonance with nuclear Overhauser enhancement. Sci. Rep.6, 30756; doi: 10.1038/srep30756 (2016).


Gas hydrates stored on continental shelves are susceptible to dissociation triggered by environmental changes. Knowledge of the timescales of gas hydrate dissociation and subsequent methane release are critical in understanding the impact of marine gas hydrates on the ocean–atmosphere system. Here we report a methane efflux chronology from five sites, at depths of 220–400 m, in the southwest Barents and Norwegian seas where grounded ice sheets led to thickening of the gas hydrate stability zone during the last glaciation. The onset of methane release was coincident with deglaciation-induced pressure release and thinning of the hydrate stability zone. Methane efflux continued for 7–10 kyr, tracking hydrate stability changes controlled by relative sea-level rise, bottom water warming and fluid pathway evolution in response to changing stress fields. The protracted nature of seafloor methane emissions probably attenuated the impact of hydrate dissociation on the climate system. Understanding the timescales of gas hydrate dissociation and methane release are critical to gauge the potential climate impact. Here, the authors report a methane efflux chronology from five sites in Barents and Norwegian seas and show methane release coincident with the release of ice sheet-induced pressure. Large quantities of methane exist as free gas, dissolved in pore fluids and solid gas hydrates within marine sediments234467891113C excursions in the geological record6414Methane released by the dissociation of marine gas hydrates has been inferred to be the cause of a number of negative δMethane is consumed at a variety of stages during transport through the sediment and overlying water column. On a global scale, >80% of methane migrating within the sedimentary package can be microbially consumed at the sedimentary sulphate–methane transition zone by the anaerobic oxidation of methane (AOM)11192–103 years (ice sheet collapse), whereas significant sea-level and bottom water temperature variations operate over 103 years212082–103 years8The processes that influence gas hydrate stability in continental margins act on timescales ranging from near instantaneous  to 102–103 years272629Direct information on the timing of past methane release events can be obtained through U-Th dating of methane-derived authigenic carbonates25The shelf areas of the Norwegian and Barents Seas offer ideal settings in which to assess the processes governing gas hydrate stability on glaciated continental margins with grounded ice and hence can serve as a palaeo-analogue of the Antarctic gas hydrate reservoir313The southwest Barents and Norwegian Sea are active provinces for oil and gas exploration, with extensively documented, focused, hydrocarbon-rich fluid migration through regional fault networks3323435338343133140313An autonomous underwater vehicle was used to map the seabed at high spatial resolution (<10 cm) in some pockmark areas and areas of known seepage33) is also present  (13C-depleted (δ13C <−45‰ VPDB) carbonates in the studied crusts further suggests that the principal source of methane is thermogenic, which is typically less 13C depleted than microbial methane13C-CH4 of −48‰ and δD-CH4 of −186‰ Vienna Standard Mean Ocean Water (VSMOW); Carbon and oxygen isotope compositions of the sampled carbonate crusts were measured on powders hand-drilled on a millimetre scale, to spatially resolve distinctive carbonate generations. Carbonate δd δ13C <−‰ VPDB ca18O values of the authigenic carbonates provide information about the temperature and oxygen isotope composition of the ambient porewater in which carbonates precipitate. The oxygen isotope composition of carbonates precipitating in equilibrium with the bottom water after deglaciation, that is, at 15 ka  and give dates with a precision on the order of ±0.5–1% (2σ). Early-stage cements prove to be more difficult to date effectively, owing to the presence of a high proportion of detrital material .Gas hydrate stability was modelled at steady state based on the presence of an ∼1,100-m-thick grounded ice sheet−1 ref.  and a ga1 ref. 37. Followients Sea . Based oents Sea  and the ents Sea , we esti14C dating of foraminifera associated with glaciomarine sediments3ca. 10 kyr. Although the carbonate crust U-Th data are limited in coverage, the clustering of U-Th dates . In addition, the rate of gas hydrate dissociation, position within the sedimentary column and the fluid flow velocity would potentially affect the time over which the fluids reach the seafloor. As carbonate crusts are forming at fluid flow velocities between 20 and 60 cm per yearThe retreat of the SIS from the shelf margin began at 19 ka and comprised several episodes of ice retreat and advance with grounding zone wedge formation35ca. 90 m uplift of the seafloor between 16 and 6 ka , it is likely that significant amounts of methane were stored in such shelf settings during the LGM. However, the impact of oceanic gas hydrate dissociation on the climate system is limited by the atmospheric transfer rate of methane, which is a function of timescales of dissociation and release rates at the seafloor, the efficiency of methane oxidizers and bubble dissolution in the water column, and the thickness of the water column. Modelling estimates show that under normal oceanographic conditions, methane released at water depths >200 m would be almost entirely (>80%) consumed during transit towards the sea surfaceWe estimate that 10 × 1014C characteristics of methane trapped in ice cores suggest insignificant emissions from marine gas hydrates during times of high atmospheric methane after deglaciation and stability of gas hydrates142 years from deglaciated shelf areas with grounded ice appears unlikely given the protracted nature of hydrate-derived methane efflux after the LGM, as our findings from the southwest Barents Sea indicate, as well as the asynchronous deglaciation of different shelf areas.The main episode of carbonate crust formation in the Barents Sea after the collapse of the SIS broadly overlaps with elevated atmospheric methane concentrations as recorded by ice cores from Greenland and Antarctica4ca. 10 kyr interval implying that flux rates were modulated by the second-order processes controlling both the rate of dissociation (isostatic rebound and bottom water warming), fluid transport (changes in stress fields leading to fault reactivation under the control of isostatic rebound) and consumption . Overall, the protracted nature of methane release on the Norwegian margin and its minimal impact on atmospheric methane concentrations highlight the complexity of the gas hydrate system and the importance of mechanisms mediating gas hydrate dissociation and fluid advection in response to abrupt climatic change.The analysis of methane-derived authigenic carbonate through the integration of U-Th geochronology and geochemical proxies, combined with gas hydrate modelling provides a means for evaluating past methane release from glaciated continental margins where gas hydrate dynamics are governed by glacial–interglacial cycles. Modelling results demonstrate that gas hydrate accumulation beneath grounded ice-sheets on the Norwegian margin, in a setting analogous to the present day Antarctic shelfα radiation in 2–70° 2θ range) on bulk carbonate powder. Identification of the main carbonate phases was done using EVA software  units relative to the VPDB reference for carbon and oxygen, and relative to the VSMOW reference for hydrogen isotopes.Stable carbon and oxygen isotopes of hand-drilled carbonate were analysed with a GasBench II preparation line connected to a Thermo Scientific Delta V Advantage IRMS (Thermo Fisher Scientific). Reproducibility is better than ±0.2‰ for both δ methane  were anaet al.3 for 1 h and centrifuged to separate soluble carbonate-rich fractions from less-soluble detrital material. Detritus fractions were dissolved over 1–7 days in a mixture of HClO4:HF:HNO3 (1:2:2.5), using ∼50 μl HF per estimated mg of material, dried down, re-dissolved in 8 M HCl and dried down again, to ensure the conversion of residual fluorides to chlorides. Detritus fractions were then dissolved in 8 M HNO3, recombined with their respective carbonate fractions, spiked with a mixed 229Th-236U tracer, left to equilibrate overnight and dried down. All samples went through two overnight oxidation steps in a mixture of 2 ml 16 M HNO3 and 0.2 ml 30% H2O2 followed by evaporation to dryness. Pre-concentration of U and Th through Fe co-precipitation and initial separation on 0.6 ml columns using AG-1 × 8 anion exchange resin were done following the procedure of Edwards et al.3 and 0.2 ml 30% H2O2, and dissolved in 1 ml 0.1 M HCl and 0.035 M HF. Before mass spectrometry analyses, all samples were filtered to remove particles originating from the perfluoroalkoxy alkane (FEP) beakers used for sample preparation.U-Th dating was carried out on 76 microdrilled carbonate samples weighing between 2–80 mg . A furth−1 uptake rate. Typical operating conditions included the addition of 4–8 ml min−1 high-purity N2 to the sample-carrying Ar stream, to minimize U and Th oxide formation in the plasma.Isotope ratio measurements were made on a Thermo Neptune Plus multi-collector ICP-MS, with samples introduced via an Aridus II desolvating nebulizer using an ESI PFA nebulizer tip with an ∼50 μl min234U measured on an axial secondary electron multiplier, and 233U, 235U, 236U and 238U measured on Faraday cups equipped with 1011 Ω resistors. Blocks of five samples were bracketed by analyses of CRM 112a U and CRM 112a U+IRMM 3636 spike. Exponential mass bias corrections were based on the measured values of the 233U/236U ratio of the IRMM 3636 spike normalized to a value of 1.01906. Secondary electron multiplier/Faraday gain corrections were based on the 234U/235U ratio of bracketing unspiked CRM 112a analyses. Hydride formation and tailing were monitored at the beginning of each analytical session, with measurements made at mass 237 and 239, while aspirating an unspiked CRM 112a solution, and were corrected during offline data reduction.U measurements were made using a normal sample cone and X-skimmer cone, and a static multicollector data collection protocol with 230Th was measured on the axial secondary electron multiplier, with 229Th and 232Th measured on Faraday cups equipped with 1011 Ω resistors. Blocks of ten samples were bracketed by blocks of five analyses of an in-house 229Th-230Th-232Th reference solution calibrated against CRM 112a, which was used to monitor mass bias and secondary electron multiplier/Faraday gain.Owing to the young age of the samples, Th measurements required better sensitivity and were predominantly made using a Jet sample cone and X-skimmer cone. 232Th/238U activity ratio variability on the geological interpretation of U-Th dates within the scope of this study was found to be negligible for samples with (230U/232Th) activity ratios above ∼2 (see 230Th/232Th)<2 (n=16) were either negative or statistically equivalent to zero (at the 2σ level) and were consequently excluded from the data set  and World Ocean Database 2005 bottom water temperature (http://www.nodc.noaa.gov/OC5/indprod.html) from NOAA and a feeding thermogenic gas composition of 96% methane, 3% ethane and 1% propane−1, but is mostly confined around 31±6 °C km−1 (2σ)5156The gas hydrate stability modelling was carried out assuming steady state using CSMHYD software4 km3) in the southwest Barents Sea shelf during the LGM. Considering a sediment porosity−3 at standard conditions for temperature and pressure (0 °C and 1 bar).The model of gas hydrate stability was used to estimate the volume of the GHSZ .Supplementary Figures 1-3, Supplementary Tables 1-3, Supplementary Note 1 and Supplementary References.U-Th isotopic dataU-Th detritus-corrected age


Our method enables the efficient and seamless assembly of short DNA blocks with dinucleotide overhangs through a simple ligation process. Next generation sequencing analysis of the assembled DNA library revealed that ligation was accurate, directional and unbiased. This straightforward DNA assembly method should fulfill the versatile needs of both  Most of these are due to the biased representation of codons as well as the formation of unexpected restriction sites. Traditional site saturation approaches using random poly-NNK (K: G or T) libraries avoids two out of the three stop codons while covering all twenty amino acid codons, but the length of the randomized region is short and a fixed lengthin vitro and in vivo analyses  and continuing up to 198 bp (11 blocks) in length, reaching the upper limit of the Miseq sequencing (14 DNA blocks (~320 pmol) for ligation, the number of non-redundant sequences for concatemers longer than 4 blocks can reach between 108 to 1013 depending on their length ; thus, we concluded this was a result of unintended misplacement during the ligation step. A heat map of 167 by 167 DNA block combination classified by their coding protein secondary structure showed clear overrepresentation of DNA block corresponding to helices, but not limited to helix-helix ligation. Moreover, approximately 98% (163/167) of all DNA blocks were represented within one order of magnitude between 2000–20,000 times . These rde novo proteins consist of limiting amino acid variation, etc.). As a proof of concept, we used low-priced standard oligonucleotides ($0.18/nt), which can be obtained through any commercial oligo synthesis company. The total cost for the 168 degenerate DNA blocks was approximately $1000, which is cost efficient given the flexibility of mix-and-matching DNA blocks to create a variety of combinatorial libraries. This method will benefit from the progress in the next generation oligonucleotide synthesis to further lower the initial cost of oligonucleotides. The simplicity of this method is advantageous for maintaining a high quality DNA library, since the error-producing steps are limited to annealing, ligation and PCR, resulting in 75.3% overall accuracy  with standard purification at 200 μM concentration in salt-free di-water. A total of 168 degenerate oligonucleotide pairs, two adapter sequences and PCR primers used in this work are listed in Each degenerate oligonucleotide pair was mixed in equal amount in a tube buffered with 10× H buffer  to a final concentration of 80 μM oligo in 0.4× H buffer . Oligonucleotides were denatured by heating at 94°C for 2 min on a Echotherm heat block  followed by gradual cooling and annealing at room temperature by moving the heat block from the heater to the bench top. Once the oligo mix reached room temperature, the tube was placed on ice to finalize the annealing step to generate DNA block with overhangs. Alternatively, a thermal cycler can be used to obtain programmed denaturation and stepwise cooling with a constant decrease of 10°C every 5 min. Annealed products (dsDNA with overhangs) were phosphorylated with T4 Polynucleotide Kinase  in a 10× ligase buffer at 37°C for 1 h. Then, T4 DNA ligase (New England Biolabs) was directly added to the phosphorylation reaction mixture and incubated at room temperature for 2 h to yield the randomly ligated DNA block concatemers .de novo dsDNA or a plasmid using type II restriction enzymes , will generate long 5′ and 3′ adapter sequences with dinucleotide overhangs (Two adapter dsDNA sequences (5′ and 3′) were prepared in a same way as DNA blocks, but only one side of the adapter dsDNA sequence contained phosphorylated overhangs in order to avoid circularization and self-ligation . Adapterverhangs  and 1c. The ligation product was purified using MinElute Reaction Cleanup Kit , and a small portion (10 ng) was PCR amplified with Platinum PCR Su perMix  with initial denaturation at 94°C for 30 seconds for melting, annealing at 52°C for 30 seconds and elongation at 72°C for 1 min, repeating 30 cycles . PCR products were cleaned up using Wizard SV Gel and PCR Clean-Up System (Promega). TruSeq DNA kit  was applied to a total of 200 ng of PCR product to add Illumina-compatible adapters, including end-repair, A-tailing and ligation, to the library. 10 cycles of adapter-complement PCR was performed to enrich the sequence-able fragments in the libraries. Size selection was performed with standard ampure magnetic beads at 0.8× to remove small fragments including adapter dimers. The prepared DNA sample was processed with 150 bp paired-end sequencing using Miseq v3 chemistry.https://usegalaxy.org) for trimming, checking quality and converting file to a multi-FASTA format. First, FASTQ Quality Trimmer was used to remove low quality ends until a sliding window of 50 bp exceeded QC15. FastQC package was then used to obtain average Phred quality score per base (http://www.broadinstitute.org/cancer/software/genepattern/).FASTQ output file was parsed using the online Galaxy platform15per base  and per per base . To extrper base . A heat Supplementary materials


Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine’s range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine’s mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy. Drosophila melanogaster and – being an immunosuppressant - its actual use for humans remains doubtful The understanding of the mechanisms underlying the aging process is of general interest; not least because it opens doors to modulate it and eventually postpone or prevent age-related pathologies and thus improve our health span. Aging research in the last three decades has elucidated a series of mutations in single genes that increase life span in many organisms Saccharomyces cerevisiae, the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and that of cultured human immune cells. A high-polyamine diet has also been reported to increase the life span and reduce the age-related pathology of a short-lived mouse strain Spermidine is a natural polyamine involved in many important molecular processes such as DNA stability, transcription, translation, apoptosis, cell proliferation, differentiation, and survival In yeast, worms, flies and Hela cells, spermidine directly induces autophagy Metabolic regulation has long been connected to life span modulation: many mutations known to increase life span also alter metabolism and nutrient sensing, for instance mutations in the IIS  pathway D. melanogaster. Importantly, we additionally uncover an impact of spermidine on lipid composition, which involves an increase in weight and TAG levels as well as alterations in fatty acid and lipid profiles, most of which seem to be autophagy-dependent. These results are the first showing spermidine’s potential to modulate lipid metabolism and suggest a possible involvement of this capability in the substance’s mechanisms to exert beneficial effects during aging.In this work, we expand spermidine’s scope of potential beneficial effects during aging by showing that it improves age-dependent locomotor activity in −/−)atg7, a gene essential for autophagy. These animals showed a much faster decline in activity compared with wild type flies and no improvement with spermidine .atg7 It should be noted that these mutant flies showed no significant differences on overall lipid levels  EPCd34 in male flies appears most interesting. CPE is the main sphingolipid in invertebrates According to our results, the above delineated qualitative and quantitative impact of spermidine on lipid levels and composition partly seems to depend on autophagy. Autophagy deficiency eliminates most of the effects of spermidine on lipid profiles or even reverses them as with the saturation ratio of EPCd34. On the other hand, lack of autophagy exacerbates spermidine-mediated TAG-accumulation, which as discussed above rather suggests that this effect is not associated to spermidine-mediated longevity, which is mainly autophagy-dependent It will be interesting to see whether the herein described modulation of lipid level and composition interacts with some of the previously described mechanisms of spermidine-mediated beneficial effects, including general hypoacetylation and phospho-regulatory pathways In summary, we here uncover the far-reaching impact of spermidine treatment on lipid levels and composition, which at least in part seems to be autophagy-dependent. It is tempting to theorize that these spermidine-induced changes might be involved in the plethora of beneficial effects that spermidine exhibits during stress and aging. Future studies will have to clarify the causality of the herein described phenomenon in order to establish a further branch of action in spermidine’s extensive impact on cellular metabolism.−/− flies were kindly provided by T. Neufeld (University of Minnesota) The atg7http://rsbweb.nih.gov/ij/). Lard was run on each plate as a control. Three independent replicates were performed, each with four independent samples for each group. Triglycerides content was also measured using a commercial colorimetric kit . In each vial, 20 flies were kept and given spermidine or not for one week. Three independent samples were prepared for each group consisting of 10 flies (fresh tissue), which were put in a Eppendorf tube with 200 µl PBS and 750 µl of a 1∶2 chloroform:methanol solution. The flies were homogenized and then transferred into glass vials. The samples were then mixed for 15 minutes on a vortex. A further 250 µl of chloroform was added and the samples vortexed, followed by 250 µl of water and vortexed again. The mixture was made biphasic by centrifugation for 5 min at room temperature at 2000 g. The lower phase was transferred into new vials, dried under nitrogen and re-suspended into 600 µl of a 1∶2 chloroform:methanol solution. A portion (200 µl) was transferred into a new vial and all vials were dried under nitrogen. The lipids in the vials in which 200 µl were transferred were re-suspended in 250 µl of the standard diluent assay reagent and triglycerides measured following the manufacturer’s instructions. Three independent replicates were performed.The protocol for measurement of triglycerides content by thin layer chromatography is as described in Al-Anzi et al. Lipid extracts from the vials in which 400 µl were transferred, were dissolved in 15 µl of choloroform:methanol (1∶2) and 15 µl of acetonitrile:iso-propanol:water (6∶7∶2) and analyzed with a Absceix 4000 QTrap, a triple quadrupole mass spectrometer equipped with a nanoelectrospray source.m/z 153 in negative ion mode. MS/MS daughter ion scanning was performed with collision energies between 35–90 V.Samples were delivered using either thin-wall nanoflow capillary tips or a Nanomate interface in direct infusion mode (∼125 nl/min). The lipid extracts were analyzed in both positive and negative ion modes using a capillary voltage of 1.25 kV. MS/MS scanning  were performed using nitrogen as the collision gas with collision energies between 35–90 V. Each spectrum encompasses at least 50 repetitive scans. Tandem mass spectra (MS/MS) were obtained with collision energies as follows: 35–45 V, PC/SM in positive ion mode, parent-ion scanning of m/z 184; 35–55 V, PI/IPC in negative ion mode, parent-ion scanning of m/z 241; 35–65 V, PE in negative ion mode, parent-ion scanning of m/z 196; 20–35 V, PS in negative ion mode, neutral loss scanning of m/z 87; and 40–90 V, for all glycerophospholipids  detected by precursor scanning for www.lipidmaps.org).Assignment of phospholipid species is based upon a combination of survey, daughter, precursor and neutral loss scans, as well previous assignments Full characterization and quantification of the fatty acids was conducted by conversion to the corresponding fatty acid methyl esters (FAME) followed by GC-MS analysis. Triplicate lipid extract aliquots were transferred to 2 ml glass vessels and spiked with an internal standard fatty acid C17∶0 (20 µl of 1 mM) and dried under nitrogen. Fatty acids were released by base hydrolysis using 500 µl of concentrated ammonia and 50% propan-1-ol (1∶1), followed by incubation for 5 h at 50°C.After cooling the samples were evaporated to dryness with nitrogen and dried twice more from 200 µl of methanol:water (1∶1) to remove all traces of ammonia. The protonated fatty acids were now extracted by partitioning between 500 µl of 20 mM HCl and 500 µl of ether, the aqueous phase was re-extracted with fresh ether (500 µl) and the combined ether phases dried under nitrogen in a glass tube. The fatty acids were converted to methyl esters (FAME), by adding diazomethane  to the dried residue, while on ice. After 30 min the samples were allowed to warm to RT and left to evaporate to dryness in a fume hood. The FAME products were dissolved in 10–20 µl dichloromethane and 1–2 µl analyzed by GC-MS on a Agilent Technologies  with a ZB-5 column , with a temperature program of at 70°C for 10 min followed by a gradient to 220°C at 5°C/min and held at 220°C for a further 15 min. Mass spectra were acquired from 50–500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern with a FAME standards (Larodan) and bacterial FAME standards (Supelco).Glycogen content was measured as described in Van Handel For protein content, in each vial 20 flies were kept and given spermidine or not for one week after which flies were then frozen in liquid nitrogen. Twenty five flies were homogenized in 100 µl PBS and incubate at 70°C for 5 min. The samples were then diluted 1∶10 in PBS ad centrifuge at maximum speed for 3 min. Standards (0.5–2 mg/ml) were prepared with albumin . Five µl of homogenates, standards or blank were added to 250 µl of Bradford reagent  into a 96-well plate and mix for 30 s. The absorbance was read at 570 nm after leaving the plates 30 min at room temperature. Three independent replicates were performed, each with 4 or 5 independent samples for each group.Food intake was measured as described in Libert et al. Flies were kept in individual vials in groups of ten. Once a week, the percentage of flies in each vial able to reach 8 cm in 10 s was measured until no fly could reach the threshold. Three independent replicates were performed. The experiment was performed in both normal and autophagy-deficient flies.Data from glycogen and protein contents were expressed as content per mg of fly. Triglycerides content by colorimetric assay was expressed in microgram per fly. Data from triglycerides content by thin layer chromatography were weighted according to the weight of the flies in each sample. Data from food intake were expressed as intake per fly. The intensity data from the triglycerides content by thin layer chromatography were transformed into the inverse of the mean grey value to obtain a higher value for darker chromatography spots . For all experiments, the effect of spermidine was tested using non-parametric t-tests. Sex and genotype were analysed separately.Figure S1Total fatty acid profile of males.A–D) Identification and quantification of total fatty acid content from wild-type and atg7−/− male flies. Fatty acids from male wild type flies left untreated (A) or fed 1 mM spermidine (C) and male atg7−/− mutant flies left untreated (B) or fed 1 mM spermidine (D) were derivatised with diazomethane to the corresponding fatty acid methyl esters (FAMEs), together with an internal standard (C17∶0). The samples were analyzed by GC-MS and the retention times and fragmentation patterns, compared with FAME standards. 3 independent replicates were monitored for each group.(TIF)Click here for additional data file.Figure S2Total fatty acid profile of females.A–D) Identification and quantification of total fatty acid content from wild-type and atg7−/− female flies. Fatty acids from female wild type flies left untreated (A) or fed 1 mM spermidine (C) and female atg7−/− mutant flies left untreated (B) or fed 1 mM spermidine (D) were derivatised with diazomethane to the corresponding fatty acid methyl esters (FAMEs), together with an internal standard (C17∶0). The samples were analysed by GC-MS and the retention times and fragmentation patterns, compared with FAME standards. 3 independent replicates were monitored for each group.(TIF)Click here for additional data file.Figure S3Phospholipid profile of males negative survey.A–D) Negative ion ES-MS survey scans (600–1000 m/z) of total lipid extracts from male wild type  and atg7−/− (B) flies as well as from spermidine-fed male wild type  and atg7−/− (D) flies.(TIF)Click here for additional data file.Figure S4Phospholipid profile of females negative survey.A–D) Negative ion ES-MS survey scans (600–1000 m/z) of total lipid extracts from female wild type  and atg7−/− (B) flies as well as from spermidine-fed female wild type  and atg7−/− (D) flies.(TIF)Click here for additional data file.Table S1Total fatty acids. Fatty acids levels in males and females normal or autophagy-deficient, fed or not 1 mM spermidine for one week. We observe for normal males a decline in C14∶1, C16∶0, C18∶0, C20∶4 and C20∶3 and an increase in C18∶2, C20∶2 and C20∶1 upon spermidine treatment. In normal spermidine-fed females, we see a decline in C14∶1, C14∶0, C20∶3 and C20∶2 and an increase in C18∶2. Lack of autophagy triggers a decrease in the levels of fatty acids C16∶0 and C18∶0 in males. We observe in atg7−/− males fed spermidine a decline in C18∶2 and C18∶0 and an increase in C14∶0, C18∶3, C20∶4, C20∶3 and C20∶2. In spermidine-fed at7−/− females, we see a decline in C14∶1, C20∶4, C20∶3 and C20∶2 and an increase in C18∶2.(DOCX)Click here for additional data file.Table S2Lipid species identified by mass spectrometry.(DOC)Click here for additional data file.