Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it’s near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset. In 1866, Gregor Mendel published his seminal research detailing the laws of inheritance . In the 3) group to the C5 position of the cytosine base in DNA resulting in generation of 5-methylcytosine (5mC)  catalyzed addition of a methyl  with a migration rate slightly greater than that of cytosine and suggested that the uncharacterized “epicytosine” might be 5mC . Ever siIn 2005, Song et al. reported a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)  based me4HCO3) as an additive to the mobile phase  dibromide)) [There have been many interesting reports on fluorescence-based epigenetic analysis owing to its simplicity for signal generation and detection. Wang et al. demonstrated a particle counting assay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts that were captured by immuno-magnetic particles . By amplromide)) . The occromide)) . Single romide)) , 61 but romide))  and thesAs one of the highly sensitive spectroscopic techniques, Surface plasmon resonance (SPR) known for its appeal in monitoring biomolecular interactions have also been applied in epigenetics evaluation Fig. . Nguyen Variations of electrochemical tools based on redox reactions have been introduced for detection of DNA methylation. Kurita et al. introduced methylated cytosine in DNA via ELISA with ECL detection in real genomic sample  Fig. 2e2e. Here,Microfluidic platform technology has several advantages over conventional analytical methodologies, enabling fast response, cost effectiveness and low consumption of reagents. Recently this method has been applied in the field of epigenetics to efficiently enhance performance of DNA methylation analysis. Cipriany et al. used fluorescently labeled Methylated DNA Binding Domain (MBD) proteins as probes to perform Single-Chromatin analysis at the nanosacle (SCAN) in DNA restricted to microfabricated nanofluidic channels Fig.  enablingNanopore technology offers a promising alternative to conventional DNA sequencing by measuring distinctive electric currents obtained from different bases and has been recently applied for epigenetic studies. Zeng et al. reported a α-hemolysin-based nanopore sensing method Fig.  for 5mC Nanoparticles that have been employed as tracers in many biosensor applications have also been employed in exploratory epigenetic research owing to its extraordinary physical properties such as photothermal effect, localized surface plasmon resonance which are based on electromagnetic field passing around the nanoparticle surface. The essence of this approach is the induction of particle aggregation to observe a shift in peak for detection by fluorescence or colorimetry. Ge et al. demonstrated a simple colorimetric method to detect DNA methylation . Here, mWang et al. developed a novel concept for enzymatic control of plasmonic coupling for DNA demethylation . Here, gThe challenge of quantifying global levels of DNA methylation derivatives can be gaged from the relative abundance of these epigenetic marks. In humans, 5mC makes up about 1% of the total DNA bases  and 5hmCIt is also conceivable that with the emergence of Next Generation Sequencing (NGS) technologies, quantification of global methylation derivatives along with the precise identification of localized sites undergoing these alterations will become prevalent. While this may in the foreseeable future help clinicians make informed choices pertaining to patient profiling and therapeutic management, standards will have to be developed to decorously interpret the disease risk imparted by global changes of the methylome. We realize that the infrastructural wealth available to scientists in biomedical settings may not be practical in a clinic and in this regards, and to address this challenge our lab and others have used lateral flow techniques that can potentially be used for onsite sensing , 86 in c


Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way. Arabidopsis thaliana. Primary miRNA transcripts (pri-miRNA) arise from the RNA polymerase II-dependent expression of independent transcriptional units. Their expression pattern is under the control of specific regulatory sequences as is the case for protein coding genes [AGO genes of which AGO1 [Control of gene expression is paramount for any organism in order to exist and transit through different developmental stages as well as to interrelate with their surroundings during their life cycle. All layers controlling gene expression are tightly regulated, from chromatin state to protein post-translational modifications, including mRNA stability. Small RNAs (sRNAs) have emerged in the last decades as central elements embedded in those regulatory layers. sRNAs come in several flavors depending on the source of RNA used for their biogenesis . MicroRNng genes . Pri-miRng genes . Proteinng genes ) are theng genes ) or DOUBng genes ), SERRATng genes ) and C-Tng genes ). The reng genes . Next, Hng genes ) is thouich AGO1  and AGO1ich AGO1 . Once loich AGO1 . Beyond ich AGO1 . Noteworich AGO1 .Plant miRNAs are involved in the regulation of a series of developmental and stress-related genetic programs , 16. NevIn order to circumvent such limitations and clearly discern how those parameters might influence miRNA performance, we used an artificial and highly sensitive miRNA reporter system and discrete and equivalent developmental points covering the entire plant life cycle . Our resThe mechanisms of miRNA-mediated attenuation of gene expression have been harnessed to silence specific genes with amiRs , 24. TheLUC) under the constitutive Cauliflower Mosaic Virus 35S promoter. Simultaneously, the expression of an artificial miRNA (amiR-LUC) driven by the very same promoter, specifically silences LUC expression. As control, we used a similar reporter system in which synonymous point mutations were introduced within the miRNA-complementary sequence in the LUC gene. Those point mutations rendered the LUC mRNA resistant (rLUC) to amiR-LUC regulation [RNA silencing has been described as an antiviral defense mechanism in both plants and invertebrates . Such degulation . Using tLUC or rLUC reporter systems were grown along at 16°C and 23°C. 16°C is closer to the temperatures Arabidopsis typically experiences in its normal habitats, while 23°C, despite being commonly used for Arabidopsis growth in controlled chambers, can be considered a stress temperature. Since the speed of Arabidopsis growth is temperature-dependent [Arabidopsis plants carrying either the ependent , we estaependent ). We alsependent ) and pooependent –32. As eependent , 19.To study accumulation of mature amiR-LUC, we assayed amiR levels by stem-loop qRT-PCR  and smali.e. leaves 4 and 7  and especially in inflorescence , Fig 2B.Discrepancies found between the amiR-LUC levels determined either by qRT-PCR or small RNA blot might be explained by the intrinsic properties of both techniques , Fig 2B.A simple reason for miRNA accumulation being temperature and stage-dependent could be differential expression of factors involved in miRNA production. We therefore assayed whether the expression of core factors involved in miRNA biogenesis was regulated by development and/or growth temperature. We focused on the core executor DCL1 and in its assistants HYL1, DRB2, SERRATE and CPL1 .DCL1 mRNA expression levels were similar across all samples, with the exception of a marked increase in inflorescences from plants grown at 23°C compared to their counterparts grown at lower temperature (perature .HYL1 levels at late vegetative development (leaf 7) compared to 16°C  led to decreased  to 16°C . DRB2 wa at 23°C . Likewis at 16°C .CPL1 expression in vegetative tissues that was also found for DRB2  is influenced by both development and environment. Our results support that the expression of several central players in miRNA performance also depends on development and temperature in which plants are grown.The view of the different pathways involved in sRNA production and action was initially rather simplistic and static . It was DCL1  under a mixture of cool and warm white fluorescent light at 16°C and 23°C and 65% humidity. reporter  and rLUCensitive  have beeensitive , 27.Total RNA was isolated as described in  using tiReverse transcription was performed with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) using 200ng of total RNA previously treated with DNase I (Thermo Scientific) following the protocol described in .-ΔCt in all assays except in -ΔΔCt method was applied to normalize LUC mRNA levels to the ones of rLUC. Expression levels were normalized to β-TUBULIN2 (At5g62690). Mature miRNA quantifications were performed by stem-loop RT-PCR as described [PCRs were carried out in presence of SYBR Green (Invitrogen) and monitored with the CFX384 Real-Time PCR Detection-System (Bio-Rad) in two technical and two biological replicates. Biological replicates were treated as independent samples. Relative expression changes were calculated using 2escribed .For small RNA blots, 3 μg of total RNA were used and two biological replicates performed. All primers used are listed in Proteins were isolated from the corresponding tissues from 15 randomized individuals per sample and biological replicate. After tissue homogenization, the resulting powder was resuspended in protein extraction buffer ). After centrifugation, 50 μl of protein were mixed with the same volume of Beetle-Juice (PJK) Firefly substrate. Luciferase activity from two biological replicates was measured in technical triplicates on a Centro LB 960 (Berthold Technologies) device. Protein concentration of two biological replicates was assessed using the Bradford protein assay kit (BioRad) in technical triplicates. From this, Luciferase activity per μg of protein was calculated and the average of both biological replicates was used for further analysis. Values were normalized to the ones from rLUC.Significance of the effect of temperature and development as observed on protein expression was assessed statistically for the data shown on Figs Analysis of variance was performed to investigate whether development had a global effect on the joint measurements across stages for each temperatures. For those cases in which the anova omnibus test revealed a significant effect of development, a post-hoc Tukey test was performed to identify the single stages responsible for that effect being significant while adjusting for multiple hypothesis testing.S1 Table(DOCX)Click here for additional data file.


En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability.Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers  in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration.  This process is essential for atrioventricular valve formation where EC expressing mesenchymal markers invade the cardiac jelly to form a cardiac cushion which subsequently forms the valves3. Recent studies have also implicated EndMT in the pathophysiology of vascular diseases including cerebral cavernous malformations4, pulmonary hypertension5, vascular graft remodelling6 and atherosclerosis10.Endothelial-to-mesenchymal transition (EndMT) is characterised by multiple morphological and physiological changes including loss of cell polarity, disruption of intercellular junctions, increased proliferation, delamination and migration of cells into surrounding tissue11. Low shear stress at branches and bends of arteries promotes atherosclerosis by inducing endothelial injury and proliferation15, which enhances permeability to cholesterol-rich lipoproteins16. By contrast, high shear stress protects arteries by inducing a quiescent, protected state in EC18. The endothelium at disease-prone sites constitutes a highly heterogeneous population of cells that display multiple pathophysiological features including inflammatory activation, proliferation, apoptosis and senescence15. Notably, recent studies revealed that low shear stress can also promote EndMT19. Moreover, EC with mesenchymal characteristics were identified overlying atherosclerotic plaques suggesting that EndMT may contribute to atherogenesis8. The signaling intermediaries that link mechanical forces to EndMT are only partially understood. Moonen et al. found that laminar shear stress protects EC from mesenchymal transition via activation of MEK5/ERK5 signalling which activates the transcription factors KLF4 and KLF28. On the other hand, we recently demonstrated that low shear stress promotes EndMT via activation of the transcription factors GATA4 and TWIST19. However, the mechanisms that promote EndMT downstream from TWIST1 in sheared EC remain uncertain and are an important focus of the current study. We assessed whether the transcription factor Snail fulfills this role because it is essential for epithelial-to-mesenchymal transition (EMT) during development22, but its role in mechanical EndMT has not previously been studied. Here we used cultured EC exposed to flow and animal models with varying levels of flow to demonstrate that Snail plays an essential role in the induction of EndMT in response to disturbed flow.Wall shear stress, imposed on the vessel lumen by the flow of blood, controls the localization of atherosclerotic lesions by regulating fundamental physiological activities in ECFor studies of human cells, experiments were approved by the University of Sheffield Research Ethics Committee (reference SMBRER310) and all subjects gave informed consent. Studies using human cells and tissues were used in accordance to the standards set by the Declaration of Helsinki. For animal studies, all procedures were approved by the University of Sheffield ethics committee and performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986 and in accordance with Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.Antibodies targeting human and murine Snail , human N-cadherin , human VE-cadherin , murine CD31  and Ki67  were obtained commercially. AlexaFluor-conjugated secondary antibodies and TO-PRO-3 were from Invitrogen.Pig aortas from 4–6 month old animals (approximately 80 kg) were obtained immediately after slaughter from a local abattoir. They were cut longitudinally along the outer curvature to expose the lumen. EC exposed to high (outer curvature) or low (inner curvature) shear stress were harvested using collagenase (1 mg/ml for 10 minutes at room temperature) prior to gentle scraping. RNA was extracted using an RNeasy MiniKit (Qiagen) and concentrated using an RNeasy MinElute Cleanup kit (Qiagen) and the purity and integrity of total RNA samples was assessed using a Bioanalyser (Agilent).15. EC at passage 3–5 were cultured until confluent in 6 well plates and exposed to flow using an orbital shaking platform  housed inside a cell culture incubator23. The radius of orbit of the orbital shaker was 10 mm and the rotation rate was set to 210 rpm. Previous computational fluid dynamic analysis revealed that these conditions generate low shear stress (approximately 5 dyn/cm2) with rapid variations in direction in the centre and high shear stress (approximately 15 dyn/cm2) with relatively uniform direction at the periphery23. In some experiments, cells were harvested by scraping from specific regions of the well using a standardized template to identify the high and low shear stress regions. Alternatively, EC were cultured on Ibidi gelatin-coated µ-Slides (Ibidi GmbH) until they reached confluency and exposed to low unidirectional (4 dyn/cm2), low oscillatory  or high unidirectional (13 dyn/cm2) shear stress using the Ibidi pump system9.Human umbilical vein EC (HUVEC) and porcine aortic EC (PAEC) were isolated using collagenase digestion and cultured. The purity of HUVEC was assessed by measuring the expression of VE-cadherin which was typically observed in >99% cells  or IgG1K-PeCy7 (eBioscience) for 30 min at 4 °C. After 2 washes, fluorescence was measured using a LSRII BD Bioscience flow cytometer.9. Non-targeting scrambled sequences were used as a control .Gene silencing was performed using siRNA against Snail1  using the Lipofectamine RNAiMAX transfection system as described15. Relative gene expression was calculated by comparing the number of thermal cycles that were necessary to generate threshold amounts of product. Fold changes were calculated using the ΔΔCt method.The levels of human or porcine transcripts were assessed by quantitative real time PCR (qRT-PCR) using gene-specific primers as describedThe expression levels of proteins were assessed by immunofluorescent staining using antibodies against Snail, N-cadherin or Ki67 and AlexaFluor488- or Alexafluor568-conjugated secondary antibodies. Nuclei were identified using DAPI (Sigma). Image analysis was performed using Image J software (1.49p) to calculate average fluorescence. Isotype controls or omission of the primary antibody was used to control for non-specific staining.24. Images were captured at 3 min intervals for 20 h after wounding. The distance migrated was determined by measuring the position of the monolayer edge in relation to the starting position using LAS_AF Software.Wounds were created on confluent EC monolayers using a pipette tip and migration of EC into the wounded area was visualised using time-lapse confocal microscopy (Leica AF6000 Timelapse microscope) as described9. HUVEC were cultured in Transwell inserts and then exposed to orbital shaking for 72 h or to static conditions as a control. The media in the upper compartment was then replaced with 10% serum-supplemented DMEM containing 1% BPA and rhodamine-labeled albumin (1 mg/ml). Media in the lower compartment was sampled at 1 h and fluorescence was measured using a fluorimeter  with excitation at 570 nm and emission at 600 nm.The permeability of EC monolayers exposed to flow was determined using rhodamine-labelled albumin as describedTwist1 (Tie2-Twist1KO) were generated by crossing Tie2-Cre expressing mice (Jackson Laboratory stock #004128) with Twist1 floxed mice (Twist1flox|flox)25. Constrictive cuffs were applied to the right carotid artery of isofluorane-anaesthetized mice following published methods26. Both halves of the cast are placed around the common carotid artery and secured with sutures. To minimise vascular injury the diameter of the internal groove of the cuff was carefully chosen to generate a stenosis of 75%. After 2 weeks mice were killed using Pentobarb (200 mg/kg i.p.). The expression levels of specific proteins were assessed in EC by en face staining as described15.Mice were housed under specific-pathogen free conditions. Mice with EC deletion of Coronary arteries from patients undergoing cardiac transplantation for ischaemic heart disease or dilated cardiomyopathy were obtained under the Sheffield Teaching Hospitals Institutional Review Board authorisation STH16346 (12/NW/0036). Sections made from formalin-fixed, paraffin-embedded tissues were incubated in xylene for 10 min, hydrated by sequential exposure to decreasing concentrations of ethanol (100% to 50%) and water. Heat-mediated antigen retrieval was carried out in tris-sodium citrate in a heated water bath. The sections were then blocked for endogenous peroxidase activity and incubated in 1% milk prior to overnight incubation with primary antibodies followed by HRP-conjugated secondary antibodies and substrate . Sections were then counterstained with hematoxylin and visualized by bright field microscopy.Differences between samples were analysed using an unpaired or paired Student’s t-test or ANOVA .All data generated or analysed during this study are included in this published article (and its Supplementary Information files).To determine whether shear stress regulates the expression of mesenchymal genes we exposed cultured EC to flow. qPCR revealed that the expression of several mesenchymal markers  was elevated in HUVEC exposed to low, oscillatory shear compared to high shear stress using a parallel plate apparatus Fig. . Similar27. We measured the expression of VE-cadherin in cultured EC exposed to flow using a combination of qRT-PCR, immunofluorescent staining and Western blotting. Interestingly, qRT-PCR revealed that VE-cadherin mRNA levels were enhanced in HUVEC exposed to low shear compared to high shear conditions  was elevated in EC freshly-isolated from a low shear (inner curvature) region compared to those freshly-isolated from a high shear region (outer curvature) of the porcine aorta  with Tie2-Cre transgenics (knockout mice are referred to as Tie2-Twist1KO). En face staining revealed that endothelial expression of Snail at the low shear stress site was reduced in Tie2-Twist1KO compared to Twist1flox/flox mice (Fig. Since Snail was induced by low shear stress rta Fig. . By contrta Fig. . En facerta Fig. . It is nice Fig. , indicatin vivo we modified flow in the murine carotid artery using a constrictive cuff. This causes tapering of the lumen to generate higher shear at the stenosis and low oscillatory shear downstream26. Cuff placement for 14 days led to enhanced expression of Snail at the downstream site indicating that low oscillatory shear stress is an activating signal for Snail in vivo (Fig. Atherosusceptible regions of arteries are associated with altered mass transport, inflammation and other physiological features as well as low shear stress. Therefore, to examine directly whether altered shear stress can induce Snail expression ivo Fig. .Figure 58. Given our observation that Snail promotes EndMT under low shear stress conditions, we wished to know whether it is involved in human atherosclerosis. Immunohistochemistry of coronary arteries from patients that underwent cardiac transplantation for ischemic heart disease revealed that Snail was expressed in luminal cells overlying coronary artery plaques and co-staining for von Willebrand Factor confirmed that they were of endothelial lineage (Fig. Previous studies demonstrated that EC overlying human atherosclerotic plaques express mesenchymal markersage Fig. . Moreoveage Fig. . Thus Sn3, recent studies have revealed that EndMT also contributes to several vascular pathophysiological processes including vascular malformations4, pulmonary hypertension5 and vascular graft remodelling6. Notably several laboratories, including our own, have shown recently that EndMT also contributes to the development of atherosclerosis10. Chen et al.7 revealed that EndMT in atherosclerosis is driven by an imbalance in TGF-β and FGFR1 signaling leading to the activation of central regulators of mesenchymal transition including Twist, Snail and Slug. EndMT is also sensitive to local hemodynamics; specifically in vitro and in vivo studies demonstrated that low shear stress is a positive regulator of EndMT whereas high shear stress is protective9. The mechanism involved ERK5, which was required for high shear stress-mediated protection8 and GATA4-TWIST1 signalling which drives EndMT in response to low shear9. In the current study, we further investigated the mechanism that promotes EndMT under low shear stress conditions and identified an essential role for the transcription factor Snail.In addition to its established roles in developmentin vitro. This revealed that Snail expression enhanced the permeability to macromolecules in EC exposed to low shear stress. Thus our observation implies that Snail may promote the build-up of cholesterol-rich lipoproteins and other biomolecules in the vessel wall at sites of low shear stress and has obvious implications for the initiation of atherosclerosis, which involves the accumulation of lipoproteins in the intima. Excessive mitosis of EC at atheroprone sites leads to the transient formation of intercellular gaps which enhances the permeability of endothelial monolayers to macromolecules16. Thus our observation that Snail positively regulates proliferation of EC provides an explanation for its ability to promote permeability in low shear conditions. Snail also promoted migration of EC exposed to low shear stress, a function that is characteristic of EndMT. Although migration of EC has been documented in the aorta28, its relationship to atherosclerosis is uncertain and requires further investigation. Since EndMT has been linked to increased vascular inflammation9 it will also be important in future studies to determine the effects of Snail on inflammatory pathways including NF-κB and mitogen-activated protein kinases.Our study demonstrated that Snail was expressed at sites of endogenous low shear stress in healthy murine and porcine arteries, and was induced by the imposition of low shear stress on carotid arteries. Thus we conclude that Snail is expressed at low shear stress regions that are predisposed to atherosclerosis and may therefore contribute to vascular injury and lesion formation. To determine the effects of Snail on EC function and its potential role in vascular dysfunction we carried out gene silencing experiments et al. implicated EndMT in plaque progression because the proportion of endothelial-derived mesenchymal cells in human plaques correlated positively with the severity of disease10. However, further work is required to determine the contribution of EndMT to plaque composition and its effects on the physiological and mechanical properties of rupture-prone plaques.Although it is established that EndMT accompanies atherosclerosis, the extent to which this process contributes to lesion initiation and progression remains uncertain. Evrard 31. EndMT can be reversed in some instances, for example mesenchymal-to-endothelial transition has been described following myocardial infarction29. Reversal may be more easily achieved in EC that undergo partial EndMT which involves the activation of mesenchymal genes and simultaneous retention of endothelial marker expression. Partial EndMT has been observed during sprouting angiogenesis in tip cells which lose apical-basal polarity, delaminate and migrate30. Although tip cells express markers of EndMT they retain expression of VE-cadherin and other EC markers and continue have contact with neighbouring EC. Because of these characteristics they have been described as undergoing partial EndMT30. Similarly, we observed in the current study that low shear stress induced mesenchymal genes whilst retaining expression of VE-cadherin which is consistent with a partial EndMT. The corollary is that atherosclerosis is associated with a partial form of EndMT that may be amenable to reversal towards an endothelial state. This hypothesis should be addressed in future studies to determine whether therapeutic targeting of Snail can treat atherosclerosis by reversing EndMT. Although it is possible that vascular diseases could be treated via EndMT reversal, this is unlikely to be achieved via inhibition of Snail because transcription factors are difficult to target therapeutically. Thus future studies should identify the target genes that are activated by Snail in sheared EC since some of them could provide a therapeutic target to prevent or reverse EndMT.Although it leads to a profound alteration in EC behaviour and appearance, EndMT represents part of a spectrum of EC phenotypes and an example of cellular plasticityper se can alter cell physiology because it involves electroporation and addition of nucleic acids. Indeed in this study we observed that cells transfected with a non-targeting control siRNA had reduced migration compared to untransfected cells (compare Fig. To assess the role of Snail in EC responses to low shear stress we silenced it by transfecting cells with specific siRNA sequences. It is plausible that the transfection process in vitro system that was used to apply flow to cultured EC. Using this system, EC monolayers cultured in 6-well plates were exposed to orbital shaking which generates relatively low shear at the centre of the well and high shear at the periphery. The regions studied were defined previously using computational fluid dynamics to calculate the shear stress distribution over the orbited well23. Moreover the altered flow at the centre and periphery was validated by particle imaging velocimetry13. Since shear stress is sensitive to changes in the orbital radius of the rotating platform, the frequency of rotation and the viscosity of the cell culture medium, it was ensured that these values were consistent between the current study and those used for computational fluid dynamic calculations13. We used a standardized template to identify the regions that were used for EC collection. Nevertheless, the approach is subject to potential sampling errors which could contribute to experimental variation. This was captured by carrying out multiple independent experiments with cells cultured from different individuals. It was observed that low shear stress had consistent effects on several physiological parameters; thus sampling errors were not high enough to mask these biological effects. Finally, since in vitro systems do not fully mimic the complex physiology of in vivo systems we validated our observations by studying Snail expression in murine, porcine and human arteries.Another important technical consideration relates to the orbital 26. One potential concern with this study is that cuff placement could injure the vessel. However we observed enhanced expression of Snail at a downstream region that was exposed to low oscillatory shear stress but was not exposed to the cuff itself. Another important consideration is the change in shear stress induced by the cuff. This has recently been calculated by Krams who used microCt and ultrasound data to inform computational fluid dynamic simulations32. Notably it was found that although shear stress is highly sensitive to geometry there was <10% variation in shear magnitude between cuffed arteries. Similarly, we observed that the expression of Snail at low oscillatory shear stress regions was also consistent between animals and was significantly higher compared to the high shear region or contralateral artery.Although our studies of the aortic arch in pigs and mice demonstrated that Snail expression was enriched in EC located at the inner curvature they did not reveal a causal relationship between shear stress and Snail expression. To address this we modified shear stress in the murine carotid artery using a constrictive cuff developed by KramsSUPPLEMENTAL MATERIAL


The current research developed and validated a Korean-translated version of the Personal Relative Deprivation Scale (PRDS). The PRDS measures individual differences in people’s tendencies to feel resentful about what they have compared to what other people like them have. Across 2 studies, Exploratory Factor Analyses revealed that the two reverse-worded items from the original PRDS did not load onto the primary factor for the Korean-translated PRDS. A reduced 3-item Korean PRDS, however, showed good convergent validity. Replicating previous findings using Western samples, greater tendencies to make social comparisons of abilities (but not opinions) were associated with higher PRDS (Studies 1 and 2), and participants scoring higher on the 3-item Korean PRDS were more materialistic (Studies 1 and 2), reported worse physical health (Study 1), had lower self-esteem (Study 2) and experienced higher stress (Study 2). Personal relative deprivation (PRD) refers to resentment caused by the awareness that one is deprived of desired and deserved outcomes compared to what others have –2. PeoplAlthough research has shown that the PRDS predicts a variety of important outcomes, with a few exceptions , most ofAmong East Asian countries that show collectivistic characteristics is South Korea –15. SeveThe purpose of Study 1 was to (a) develop a Korean-translated version of Callan et al.’s  PRDS andThe studies included in the current paper were approved by the ethics committee of the department of Psychology at Yonsei University.N = 224; 56.3% male; Mage = 24.10, SDage = 5.37) were recruited either for an exchange of course credits (n = 155) or via social media (n = 69). Participants filled out an online survey using a personal computer in a university laboratory. Fourteen additional participants were excluded because they failed an attention check item .Korean participants (disagree strongly to 5 = agree strongly). Individuals scoring higher on the INCOM tend to spend more time engaging in social comparisons ). excellent to 7 = very poor). This item was rescaled so that higher values indicate better global physical health.Participants reported their physical health using a back-translated version of a single-item measure  given astrongly disagree to 7 = strongly agree). Higher scores indicate higher materialism.To measure materialism, the 9-item Material Values Scale  was bacdid not finish high school to 4 = postgraduate degree). Korean-translated versions of all of the measures we employed can be found in the supporting information.Participants reported their annual household income before taxes given 11 categories . Income responses were converted into estimates of absolute income using Parker and Fenwick’s  median-br = -.21. Following Wong, Rindfleisch, and Burroughs’ ). Our results support that, in East Asian samples, PRD might play a crucial role in explaining the relation between social comparison tendency and a variety of beliefs and behavior that are relevant to health and materialistic values. For instance, people accustomed to Eastern culture tend to show overly relation-oriented self-representation and this has a negative effect on subjective well-being . NeverthS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.


A 40-year-old male presented to the ophthalmology clinic with a darkly pigmented infratemporal lesion in his right eye. The corrected visual acuity in both eyes was 6/6 and both pupils were equal and reactive. Slit lamp biomicroscopy showed a well-demarcated and heavily pigmented lesion in the peripheral iris between 6 and 8 o’clock. Ultrasound biomicroscopy (UBM) revealed a solid mass deriving from the iris stroma without ciliary body involvement, helping to classify the uveal melanoma and establishing the diagnosis of iris melanoma. Fine needle aspiration (FNA) confirmed melanoma with inactivation of the BAP1 gene. The patient was treated with brachytherapy using an I-125 plaque. Follow-up UBM, three years later, demonstrated significantly reduced dimensions of the tumour. UBM has become crucial to the differentiation of uveal melanomas from benign growths, and lesions <3 mm cannot be reliably visualised by other imaging modalities or localised to the correct uveal structure. Brachytherapy represents a safe and effective treatment option even in lesions that are BAP1 positive. Uveal melanomas are the most common primary intraocular tumour in adults  of which iris melanomas account for 3%–10% .A 40-year-old asymptomatic non-smoking Caucasian male with no past ocular history presented with a darkly pigmented infratemporal lesion in his right eye first noticed one year prior. The corrected visual acuity in both eyes was 6/6, measured intraocular pressure (IOP) was 15 and 16 mmHg in the right and left eyes respectively, and both the pupils were equal and reactive. Slit lamp biomicroscopy showed a well-demarcated and heavily pigmented lesion in the peripheral iris between 6 and 8 o’clock . Fundus Ultrasound biomicroscopy (UBM) revealed a solid mass deriving from the iris stroma without ciliary body involvement, suggesting a diagnosis of iris melanoma . FNA conThe patient was treated with brachytherapy using a 12-mm diameter I-125 plaque implanted over the ciliary body and iris at the 7’o clock meridian for one week for a total dose of 82 Gy.UBM, three years later, demonstrated the success of radiotherapy through the significantly reduced dimensions of the tumour . FollowiMalignancies without BAP1 inactivation generally have a favourable prognosis because of their lower rates of visual morbidity and metastasis and a five-year mortality rate of 2%–3% .UBM has become crucial to the differentiation of uveal melanomas from benign growths, as lesions <3 mm cannot be reliably visualised by other imaging modalities . As thesClinicians should have a low threshold to refer or work up cases of pigmented eye uveal lesions because of their similarity in appearance of nevus and melanoma, and UBM acts as an effective modality to elucidate a diagnosis in these ambiguous cases. The differential diagnosis for uveal lesions includes iris nevus, iris cyst, essential iris atrophy, and foreign body. In differentiating iris melanoma from nevus on UBM, common findings for melanoma include medium–high reflectivity, possible cavitation, and, most importantly, a loss of the posterior iris plane .


DBHCre/Crem−/−) or serotonergic (Creb1TPH2CreERT2/Crem−/−) neurons in a CREM-deficient background to avoid possible compensatory effects of CREM. Selective and functional ablation of CREB affected antidepressant-like behavior in a tail suspension test (TST) after antidepressant treatment. Contrary to single Creb1DBHCre mutants, Creb1DBHCre/Crem−/− mice did not respond to acute desipramine administration (20 mg/kg) on the TST. On the other hand, single Creb1TPH2CreERT2 mutants displayed reduced responses to fluoxetine (10 mg/kg) on the TST, while the effects in Creb1TPH2CreERT2/Crem−/− mice differed by gender. Our results provide further evidence for the important role of CREM as a compensatory factor. Additionally, the results indicate that new models based on the functional ablation of CREB in select neuronal populations may represent a valuable tool for investigating the role of CREB in the mechanism of antidepressant therapy.Evidence exists that chronic antidepressant therapy enhances CREB levels and activity. Nevertheless, the data are not conclusive, as previous analysis of transgenic mouse models has suggested that CREB inactivation in fact contributes to antidepressant-like behavior. The aim of this study was to evaluate the role of CREB in this context by exploiting novel transgenic mouse models, characterized by selective ablation of CREB restricted to noradrenergic (Creb1 Even though there are serious limitations to the monoamine theory as it does not provide a complete explanation regarding neither the mechanism of action of antidepressants, nor the basis of depression pathophysiology, it is supported by the fact that antidepressants that enhance the levels of two important neurotransmitters in the brain, noradrenaline and serotonin, alleviate depression symptoms3. Additional mechanisms contributing to the pathology of depression include dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis, inflammatory alterations, and epigenetic mechanisms2. Nevertheless, the treatment of choice for depression is primarily based on the modulation of noradrenergic or/and serotonergic signal transmission.Depression is a mental illness affecting complex cognitive and emotional functions with increasing prevalence in modern, highly industrialized societies. Decreased monoamine levels in the central nervous system represent a crucial element of the monoamine hypothesis of depression that currently dominates our understanding of the pathophysiological basis of this illness and its pharmacological treatment4, one of transcription factors binding to cAMP-responsive elements (CRE). CREB appears to be involved in both the mechanisms of antidepressant action and in the disease itself 5. Therefore, research has focused on the role of G-protein-coupled receptors and associated second messenger pathways, primarily involving the cyclic AMP (cAMP)-dependent pathway. Augmentation of cAMP leads to the activation of cAMP-dependent protein kinase A (PKA), and PKA activity is enhanced after chronic antidepressant treatment7. Alternative mechanisms implicated in the action of antidepressant drugs are related to Ca2±/calmodulin-dependent protein kinase II (CaMKII)9. CREB has been demonstrated to mediate the transcriptional activation of genes in response to both cAMP and Ca2+ influx signal transduction pathways11. For instance, according to the neurotrophic hypothesis of depression, levels of brain derived neurotrophic factor  are increased both by noradrenergic or/and serotonergic antidepressants, while exposure to stress is characterized by downregulation of BDNF expression in hippocampus12; this downregulation can be prevented by antidepressant treatment14. However, it has to be mentioned, that this effect is not specific and although enhancement of BDNF expression seems to exert antidepressant-like effects in the hippocampus, its actions might be opposite in other brain regions i.e. nucleus accumbens, where chronic social defeat stress increases BDNF protein levels15. BDNF also regulates serotonin signaling, as its main receptor, TrkB, can be found on serotonergic neurons as well16.There have been several attempts to identify a protein that could serve as a convergence point for antidepressant treatment, the most prominent example being the cyclic AMP response element binding protein (CREB) transcription factor17.The abovementioned monoamine systems exert mutual influence over each other. The serotonergic system may be inhibited by noradrenaline through action on α1- and β-adrenergic receptors on serotonergic neurons of the raphe nuclei, while serotonergic projections can inhibit the activity of the locus coeruleus5. In particular, chronic administration of desipramine and imipramine  increases expression of CREB mRNA and phospho-CREB (pCREB) in selected brain regions19. Similar effects have been observed after fluoxetine or citalopram  treatment20. On the other hand, adverse effects on CREB expression have also been observed after desipramine or fluoxetine treatment22. Furthermore, treatment with venlafaxine, a dual monoamine reuptake inhibitor, was shown to reduce pCREB in frontal cortex with no change in the total CREB expression23.Antidepressants can affect CREB in several different ways; however, the data are not conclusive. The generally accepted theory is that chronic antidepressant treatment enhances CREB levels and activity, thus implicating CREB as an important mechanism of antidepressant treatment25 or mice with a constitutive deletion of the gene28. Results from these models are counterintuitive, as the majority of studies have demonstrated that CREB inactivation contributes to antidepressant-like behavior. However, it should be emphasized that these loss-of-function studies possess many caveats, possibly making interpretation of the data difficult and misleading. Namely, (i) the experimental mutations generally targeted CREB in several brain structures; and (ii) compensatory effects of related CREB heterodimerization gene products  were not taken into consideration. The last caveat seems to be of particular importance, as it was shown that CREB is not the only mediator of cAMP-dependent transcriptional regulation and other nuclear effectors of the cAMP-dependent signaling pathway can compensate lack of CREB function29. In particular, CREB spatiotemporal knockouts are usually not as deteriorated regarding their phenotype as expected, as other c-AMP driven transcriptional activators (CREM and ATF-1) can compensate for each other, and various CREB deficient mice overexpress CREM29. This strong interdependence between CREB and CREM was also confirmed in the study of Mantamadiotis et al., showing that the embryonic mutation evoking loss of CREB in neural and glial progenitors (Creb1NesCreCrem−/− mice) was effective throughout the brains of these mutants only when both CREM alleles were also depleted30.Rodent models have made substantial contributions to advancing our understanding of depression and the mechanism of antidepressant treatment. Several models for studying the role of CREB are based on transgenic rats overexpressing CREB31, both lines were maintained in a CREM-deficient (Crem−/−) background.The aim of the current study was to evaluate the function of CREB in the mechanisms of antidepressant treatment by exploiting novel transgenic mouse models characterized by selective functional ablation of CREB restricted only to the noradrenergic or serotonergic neurons. Furthermore, considering the known compensatory effects of CREMDBHCre and Creb1TPH2Cre mice, respectively) was achieved by Cre/loxP recombination system. Transgenic mice (C57Bl/6N background) hosting Cre recombinase under the dopamine beta-hydroxylase (DBH) promoter (DBHCre mice)32 or tryptophan hydroxylase 2 (TPH2) promoter (TPH2Cre mice)33 were crossed with animals harboring the floxed Creb1 gene. The TPH2Cre line was created in inducible form (TPH2CreERT2)33. Induction of the inducible Cre recombinase was achieved by injection of 2 mg of tamoxifen  at least 4 weeks prior to any experimental procedure . Both lines were kept in a CREM-deficient (Crem−/−) background, as described previously30. We thus obtained two transgenic lines with functional deletion of CREB restricted to noradrenergic (Creb1DBHCre/Crem−/−) or serotonergic (Creb1TPH2CreERT2/Crem−/−) neurons. Genotyping was performed with a commercially available kit  according to the manufacturer’s protocol using the following primers: DBHCre: forward 5′-CTG CCA GGG ACA TGG CCA GG-3′, reverse 5′-GCA CAG TCG AGG CTG ATC AGC-3′; TPH2Cre: forward 5′ TGC AAC GAG TGA GG TTC-3′, reverse 5′-ATG TTT AGC TGG CCC AAA TG 3′; Creb1flox: forward 5′-TAT GTA AAG CAA GGG AAG ATA CTG-3′, reverse 5′-TAG ACA TAC TTG ACC CAT AGC ATT-3′; and CREM knockout: forward 5′-TGG ATT GTG CTG GGA GGT TGT TC-3′, reverse 5′-TCT TTG AGG GCC TTG AGT TCC TC-3′).Selective ablation of CREB in noradrenergic and serotonergic systems  littermates of the same sex in self-ventilated cages under standard laboratory conditions . All mice were 3–4 months old (approx. 12–16 weeks). This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Behavioral protocols were approved by the Animal Ethical Committee at the Institute of Pharmacology, Polish Academy of Sciences .Desipramine  and fluoxetine  were injected 30 min prior to the test. The control groups received 0.9% NaCl.Mice were sacrificed by cervical dislocation. Their brains were removed and fixed in 4% paraformaldehyde (PFA) overnight. After dehydration, the tissue was embedded in paraffin and coronally sectioned (7 µm) on a rotary microtome . Select sections from the corresponding region of the locus ceruleus (LC), dorsal raphe nuclei (DR), hippocampus, or frontal cortex in mutant and control mice were incubated overnight at 4 °C with primary anti-CREB , anti-NeuN , anti-Tph2  and anti-TH  antibodies. Antigen-bound primary antibodies were visualized with anti-rabbit Alexa-488, anti-sheep Alexa-594, and anti-mouse Alexa-594  coupled secondary antibodies. Stained sections were analyzed and acquired under a fluorescence microscope  equipped with a camera and specialized software .The OFT was performed to assess spontaneous locomotor activity. Mice were tracked by video camera using automated video tracking software  for 60 min in 40 × 40 cm white square boxes; the total distance moved was scored in 10-min intervals.The ROT was performed to assess motor coordination using an accelerated rotarod . The assessment was preceded by a training session 1 day before the experiment . During the experiment the time spent on the accelerating rod (4–40 rpm in a 5-min period) was measured.34.The TST was performed to evaluate depression-like and antidepressant behavior after drug treatment. We recorded the overall time that animals were immobile while suspended by the tail over a 6 min period. Scoring of immobility time was performed by means of automated video tracking software  as described previouslyData were analyzed using Statistica 12 software . All comparisons were performed using one-way analysis of variance followed by Fisher Least Significant Difference post-hoc test. Changes with p value lower than 0.05 were considered significant.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.DBHCreCrem−/− mice , the CREB protein was preserved in both transgenic lines  in the locus coeruleus (LC) of Creb1ice Fig.  as well ice Fig. . The DBHnes Fig. . These rDBHCreCrem−/− and Creb1TPH2CreERT2Crem−/− mice did not exhibit any visible impairments, nor was their body weight affected in comparison to their control littermates  and double mutant (Creb1DBHCreCrem−/− or Creb1TPH2CreERT2Crem−/−) mice. These results indicated that neither of the introduced mutations affect the general health or basal behavior of the studied animals, regardless of sex.Overall, behavioral screening of basal phenotypes did not reveal any differences between control, single mutant , Creb1DBHCreCrem−/− double mutants did not respond  evoked an antidepressant behavior on the TST; this response is shown by their shortened immobility time in comparison to saline treated animals  was sufficient to produce a drug-resistant phenotype after fluoxetine administration. The immobility time after fluoxetine injection did not change in these single mutants when compared to control littermates that did not receive the drug. Additionally, when the CREM protein was removed, animals reacted in a sex-dependent manner: Creb1TPH2CreERT2Crem−/− double mutant females did not react to fluoxetine treatment, while Creb1TPH2CreERT2Crem−/− double mutant males responded to fluoxetine similarly to control animals, exhibiting significantly decreased immobility times relative to saline-treated controls  background.DBHCre line has been previously validated in the central nervous system35. The current study confirmed this specificity in the double mutant line Creb1DBHCre/Crem−/− as well as in the newly created Creb1TPH2CreERT2/Crem−/− double mutant mice. In both cases, the selectivity of the mutation was confirmed by immunofluorescent staining. CREB expression was selectively and completely lost in regions expressing DBH and TPH2: the locus coeruleus  and double mutants  displayed no obvious alterations in daily cage behavior, weight gain  and selective serotonin reuptake inhibotor . Doses  were based on our previous experience and published literatureDBHCre/Crem−/− mice responded to acute desipramine treatment on the TST. This result differed from findings in single Creb1DBHCre mutants and was consistent with the initial hypothesis that CREM-dependent compensation for CREB function plays a role in antidepressant treatment. Apparently a single mutation was insufficient for disordering the mechanism of drug action, and the effects of desipramine on the TST were abolished only after concomitant CREM removal. This finding is also in line with other results highlighting the pivotal role of CREB in the action of antidepressant drugs targeting the noradrenergic system, in particular desipramine. Specifically, it has been proposed that the therapeutically relevant action of this drug may be related to attenuation of CREB-mediated gene transcription22. Recent studies have shown that desipramine improves depression-like behavior on the TST by upregulating p-CREB in the hippocampus40. Thus, the functional loss of CREB in Creb1DBHCre/Crem−/– mice potentially interferes with these proposed mechanisms.In the transgenic line targeting the noradrenergic system, neither male nor female Creb1TPH2Cre mice displayed resistance to fluoxetine treatment on the TST. Moreover, this effect was sustained only in case of Creb1TPH2CreERT2/Crem−/− female double mutants; in contrast, Creb1TPH2CreERT2/Crem−/− male double mutants responded to fluoxetine treatment in the same way as control mice. One of possible explanation that mires interpretation of obtained results might be the fact, that due to technical limitations and mice availability, in our experiments we compare constitutive (Creb1TPH2CreERT2/Crem−/−) vs inducible (Creb1TPH2CreERT2/Crem−/−) line. Therefore, before introducing drug factor, we thoroughly analyzed the basic behavior of both transgenic lines and did not find any discerning effects of introduced mutation in neither of them. Nevertheless, one cannot exclude that compensatory effects may emerge when triggered by external stimuli i.e. exposure to the drug. We speculate that in case of Creb1TPH2CreERT2 mice, CREM-dependent brain plasticity was insufficient to compensate for the single CREB ablation especially because the mutation took place in adult mice which eliminates the possible developmental compensatory mechanisms present in constitutive knock-out animals. This assumption is supported by prior findings that CREM overexpression in transgenic models is sometimes insufficient to prevent the CREB-deletion phenotype, particularly in the context of drug addiction31. Moreover, the precise mechanisms of interaction between CREB, CREM and ATF factors (including the extent and kinetics of mCREB dimerization) are not well understood41. On the other hand, the response to fluoxetine treatment observed in Creb1TPH2CreERT2/Crem−/− male mice could be interfered by different mechanism of action of drugs acting via the serotonergic system, particularly fluoxetine, whose effectiveness is additionally determined by various environmental factors42. This finding is compatible with observations of differential regulation of CREB expression by desipramine and fluoxetine and with the regional specificity of their effects43.Interestingly, we did not observe analogous effects in the second line targeting the serotonergic system after fluoxetine application. Regardless of sex, single-mutant Creb144. Differential responsiveness to antidepressants, including SSRIs, has been reported between men and women in clinics, although this finding remains controversial45. Furthermore, sex differences in the behavioral responsiveness of transgenic mouse models targeting the serotonergic system have been described in other studies as well47.In relation to the gender-dependent response to fluoxetine observed in serotonergic-specific mutants, interestingly, clinical studies have reported sex differences among prevalence of depression, particularly among perimenopausal women48, but overall this mutation does not appear to be phenotypically meaningful, being not associated with any profound impairment49. Namely, no significant difference were observed in daily cage behavior, spontaneous and amphetamine-induced locomotor activity, conditioned suppression of motility (reactiveness to stress)49. Crem−/− mice were characterized as slightly hyperactive only when analyzed in the dark phase, and by diminished anxiety behavior as revealed by elevated plus maze (EPM)49. However, this phenotype (even assuming exertion on obtained results) should in fact promote antidepressant behavior of double mutants analyzed in our experiments i.e. decreased immobility in TST at basal conditions, and such behavior was certainly not observed.Finally, it has to be clearly stated that all previous studies regarding the role of CREB in depression and antidepressant drug action have been carried out in multiple brain regions, while our mutation is focused on the origin sites of noradrenergic and serotonergic neurons in the central nervous system: in particular, the locus coeruleus (LC) and dorsal raphe (DR) nuclei. It remains uncertain whether and how CREB can influence other brain structures receiving inputs from noradrenergic and/or serotonergic projections. These structures are traditionally regarded as the most important areas in depression pathophysiology and the mechanism of antidepressants . Clarifying these influences will be our goal in future research. Due to technical limitations , we were unable to include a separate group of single-mutation CREM-deficient mice (Crem−/−). Crem−/− male mutants are known to be sterileThese initial observations, in particular those regarding the effects of fluoxetine, require confirmation in other behavioral paradigms. Nevertheless, these results provide overall confirmation of the crucial role of CREB in response to antidepressant treatment and clearly highlight CREM as an important compensatory factor, despite the different regulation observed in the serotonergic line. Additionally, these newly-created models based on functional ablation of CREB in select neuronal populations may represent a unique, valuable tool for investigating the role of CREB in the mechanism of antidepressants.


Conditional knockout of ASAP3 in mice caused elongation and stacking of microvilli in parietal cells, and substantially decreased gastric acid secretion. These were associated with active assembly of F-actin caused by a higher level of GTP-bound Arf6 GTPase. Consistently, a small molecular compound QS11 inhibited ASAP3 function and significantly reduced gastric acidity in vivo. Of note, the expression of ASAP3 was positively correlated with gastric acid secretion in 90 human cases, and high expression of ASAP3 was associated with reflux disease and peptic ulcer. These results reveal for the first time that ASAP3 regulates the microvilli structures in parietal cells. Our data also suggest ASAP3 as a feasible and drugable therapeutic target for gastric acidity-associated diseases.Gastric acidity-associated disorders such as peptic ulcer and reflux diseases are widespread, and the reported resistance and side effects of currently used medicines suggest an urgent requirement for alternative therapeutic approaches. Here we demonstrate a critical role of ASAP3 in regulating the microvilli structure of parietal cells  Insufficient gastric acidity may increase the risks of osteoporosis, rheumatoid arthritis, infectious diseases and malignancy, whereas excess acid secretion may also lead to widespread diseases such as peptic ulcer and gastro-esophageal reflux disease (GERD). Drugs that control gastric acid secretion, such as proton pump inhibitors (PPIs), are used in the treatment of many conditions such as dyspepsia, peptic ulcer, GERD, Barrett’s esophagus, eosinophilic esophagitis and so on. The PPIs are among the most widely sold drugs in the world and are in general well tolerated, but resistance and adverse effects such headache, nausea, diarrhea, abdominal pain, fatigue and dizziness have been reported. Thus, there is a need for discovering alternative approaches for controlling gastric acidity, which may provide therapeutic opportunity for patients who have unsatisfactory response to PPIs.Gastric acid secretion is required for the digestion of protein, absorption of vitamin B12, iron and calcium,in vivo studies have suggested the roles of Arf6, Hip1r in regulating gastric acid secretion, but these were thought to function through the vesicle transportation pathway. Similarly, an in vitro study based on isolated rabbit parietal cells indicated that ASAP3, an Arf GTPase activating protein, may affect gastric acid secretion by binding ezrin and inducing the fusion of tubulovesicles to apical membrane.The gastric parietal cells contain extensive apical membrane on the surface of microvilli, constituting a structural basis for transient proton pumping. Currently, it is poorly understood how the structure of microvilli is regulated. Several  The ArfGAP and ANK domains of ASAP3 have been found to bind Arf6. These structural domains indicate that ASAP3 may affect Arf6-dependent actin assembly near the curved apical membrane, supporting a potential role in regulating microvilli structure.ASAP3 contains the BAR  and PH domains in its amino portion and ArfGAP and ANK (ankryin repeat) domains in its carboxy portion . The PH in vivo, we generated a conditional knockout mouse model and applied a small molecular compound QS11 to suppress the function of ASAP3. The structural and functional alterations of ASAP3−/− parietal cells were studied in both resting and stimulated phases, and the long-term effect of ASAP3 disruption was examined by histopathology. The correlation between ASAP3 expression and gastric acidity was analyzed in 90 human cases. By these efforts, we aim to reveal the roles of ASAP3 in gastric acid secretion and clarify its therapeutic significance for gastric acidity-associated disorders.In order to investigate the roles of ASAP3 in parietal cells The protocol had the approval of the Ethics Committee of the Shanghai Jiao-Tong University School of Medicine, Renji Hospital, and the research was carried out according to the provisions of the Helsinki Declaration of 1975. Written informed consent was obtained from all participants involved in the study, and clinical information was collected with Institutional Review Board approval. The rate of GERD diagnosed (44/90) was related to the fact that all patients who underwent gastroscopy reported certain discomfort.Samples and gastric content were analyzed in patients who underwent gastroscopy in the Division of Gastroenterology and Hepatology, Shanghai Renji Hospital during 08:00–10:00 hours after fasted over 12 h. Patients with bile reflux, history of gastrointestinal tumor or surgery, or administration of PPIs or non-steroidal anti-inflammatory drugs were excluded for analysis. Gastric mucosal tissue specimens and gastric fluid were from 90 patients who underwent gastric endoscopy at the Shanghai Renji Hospital from 2013 to 2014. Gastric and duodenal ulcers were diagnosed by endoscopic examination. GERD was diagnosed by endoscopy combined with the Gastroesophageal Reflux Disease Questionnaire (GerdQ).g for 5 min, and the supernatant was recovered for measuring pH and titrating to pH 7.0 with 0.01 mol l−1 NaOH. Practically, it is difficult to acquire all the gastric fluid in the gastroscopic examination , thus the concentration of H+ in the gastric fluid sample was used to represent the gastric acidity.For detection of gastric acidity, the gastric contents collected by endoscopy was centrifuged at 500 Gastric body biopsy were routinely formalin-fixed, paraffin embedded and cut into 4-μm-thick sections then placed on polylysine-coated slides. After incubated in a 60 °C oven for 60 min, the slides were deparaffinized in xylene and dehydrated with graded ethanol, then washing with distilled water and phosphate-buffered saline. Endogenous peroxidase activity was blocked using 3.0% hydrogen peroxide for 15 min at room temperature. For antigen retrieval, the slides were placed in citrate antigen-repairing solution and heated in a high-pressure cooker in the gastric fluid sample was used to represent the gastric acidity. Immunohistochemistry of patient tissue samples until steam arose and then cooled off at room temperature for 40 min. After phosphate-buffered saline washing, the sections were blocked in goat serum for 30 min, then incubated with rat monoclonal antibody for human ASAP3  overnight at 4 °C. The slides were washed with phosphate-buffered saline and incubated with universal (anti-mouse/rabbit) detection reagent  as second antibody for 40 min at room temperature. After washing with phosphate-buffered saline, the immune reaction was demonstrated with 3,3'-Diaminobenzidine. The sections were then counterstained with hematoxylin, dehydrated and mounted. Tissue slides were visualized using an Olympus BX43F microscope and figures were captured using Olympus DP26 digital camera. A total of three fields selected from hot-spot areas (×400 objective lens) were acquired per slide. The integral optic density, representing the ASAP3 level of parietal cells in the field, and area of interest were counted and measured using Image-Pro Plus 6.0 software . Thus the optic density  represented the concentration of ASAP3 in each gastric body sample. A 4.7 kb fragment corresponding to the exons 2–4 of ASAP3 (EnsemblGene ID: ENSMUST00000047526) was cloned as the 5′ arm, followed by loxP-flanked exon 5 and FRT-neo-FRT site. The 4.1-kb-long 3′ arm overlapped with exons 6 and 7. Murine embryonic stem cells were electroporated with the linearized targeting vector and screened for homologous recombination by 300 μg ml−1 G418 and 2 μm ganciclovir for 8 days. Two correctly targeted embryonic stem clones were selected, expanded and injected into C57BL/6 mouse blastocysts to generate chimeric mice that were bred to obtain germ-line transmission. The experiments described in this study were performed using ASAP3 wild-type and knockout littermates on a C57BL/6 X 129SV mixed background in Shanghai Research Center for Model Organisms. The resultant ASAP3loxP/loxP mice were then crossed with UBC-cre/ERT2 mice, and adult offspring were induced by sequential peritoneal injection of tamoxifen for 7 days to knockout ASAP3. Animal procedures were approved by the Institutional Animal Care and Use Committees at Shanghai Research Center for Model Organisms and the Renji Hospital of Shanghai Jiao Tong University Medical School.The ASAP3 targeting vector bearing a gene-trap cassette doubly flanked by LoxP and FRT was constructed using the method described previously.+-K+-ATPase, ezrin, F-actin, Arf6, calcium pump pan PMCA ATPase and Ki67 were probed with the primary antibodies (dilution 1:50), followed by labeling with secondary antibodies (Alexa488-anti-rabbit or Alexa546-anti-mouse). After staining with DAPI (1:10 000), the coverslips were added with anti-fade reagent  and kept in the dark for 24 h. Images were acquired with a confocal fluorescence microscope . The colocalization analyses were performed using the microscopy software package Zen (Carl Zeiss) and ImageJ. Intensity profiles were analyzed and displayed using the ImageJ software package  cells, the ASAP3-deficient parietal cells displayed its overall fragmented and concentrated distribution, with visible sac-like pattern in magnified sections, which suggested abnormalities in acid-secreting membrane networks  has been found to regulate actin remodeling,ed still . On the ribution . Taken t+–K+-ATPase and secretory membrane as marked by a mouse monoclonal to calcium pump pan PMCA ATPase was found in stimulated ASAP3-deficient parietal cells, which was in contrast to high degree of their overlap in WT cells , suggesting its translocation to the apical membrane should be impaired. As a control, in WT parietal cells H+–K+-ATPase strongly overlapped F-actin/ezrin-occupied region, without apparent formation of cystic structure  and lipid bilayer, and found ASAP3 could induce the curvature of lipid bilayer with a radius of ~40 nm  were also with the radius of ~40 nm , 40 μm QS11 effectively evoked a decrease in the histamine- stimulated gastric acid secretion (To further validate the potential significance of ASAP3 in gastric acidity control, we employed a small-molecule ArfGAP inhibitor QS11 to mimic the ablation of ASAP3 ecretion . Althougecretion . In concR=0.615, P<0.0001, Helicobacter Pylori may be related to GERD, we compared ASAP3 expression in GERD and non-GERD cases who had no history of H. Pylori infection. Interestingly, the expression of ASAP3 was significantly higher in GERD patients than in non-GERD cases (To determine whether ASAP3 may affect gastric acid secretion in human, we measured ASAP3 expression in gastric endoscopic tissue specimens by immunohistochemistry and analyzed its association with gastric acid secretion. To this end, the gastric fluids of overnight-fasting patients were sampled through endoscopy and measured for pH and total acid content by titration. Of note, the expression level of ASAP3 strongly correlated with gastric acid content (Pearson RD cases . The subRD cases . These rin vivo investigation approaches, we provide the first evidence for ASAP3’s role in regulating the microvilli and apical membrane structures. These results also revealed ASAP3 as a feasible and drugable intervention target for controlling gastric acidity. Both targeted disruption of ASAP3 gene in mice and pharmaceutical inhibition of ASAP3 by QS11 small molecular compound demonstrated consistent effect on the suppression of gastric acidity. Moreover, the strong correlation between ASAP3 expression and human gastric acid secretion implicated ASAP3 as a potential therapeutic target in clinically refractory acid-related diseases.By combined in vivo study provided novel insight into the multifaceted ASAP3 functions in gastric parietal cells, which are involved in the regulation of microvilli and apical membrane structures. Transmission electronic microscopy of tissue sections from ASAP−/− mice revealed that microvilli were significantly elongated and stacked in resting parietal cells. The GTP-bound, activated form of Arf6 has been found to promote actin polymerization, thus ASAP3 disruption-induced Arf6 activation may contribute to the elongation of actin and microvilli in resting parietal cells (schematic representation in  potentially due to the in vitro isolation procedures that may disturb the ultrastructure of microvilli. In resting parietal cells, the mislocalization of H+–K+-ATPase seems to be caused by the occupational effect of the elongated and stacked microvilli structure. Although several members of the ArfGAP family have been found to modulate Arf6 activity, our data suggest that there is no redundancy of function in this instance: that neither ASAP1 nor any other ArfGAP can adequately compensate for loss of ASAP3 in parietal cells. Of note, the roles of ASAP3 in regulating apical membrane and microvilli structures have not been reported before, and our data suggest that ASAP3 plays multifaceted roles mediated by its interaction with apical membrane and the regulatory effect on Arf6 activity.Our −/− mice, therefore confirmed the previous observation on the requirement of ASAP3 for H+–K+-ATPase translocation. In addition, we also observed the expansion of canaliculi and the shrinkage/collapse of intracellular luminal space also contributed to the decreased gastric acid secretion. We propose that this may be due to the alteration of actin structure within the microvilli, which still deserves further investigation in future studies. Disordered apical membrane was also found in a previous ezrinkd/kd mouse model. Regarding the high mortality and severe growth retardation in ezrinkd/kd mice, our results suggested that ASAP3 should be a more feasible target for decreasing gastric acidity.The ASAP3-deficient mice also revealed significant ultrasturctural abnormality of apical membrane in stimulated parietal cells. We observed increased tubulovesicles in the cytoplasm of stimulated ASAP3 thereby making it important to identify alternative therapeutic targets for more robust and durable gastric acidity intervention. In our study, a small-molecule inhibitor of ASAP3 successfully decreased gastric acid secretion in vivo and mimicked the effect of ASAP3 knockout. Moreover, our data demonstrated the expression of ASAP3 significantly associated with the level of gastric acid secretion in human biopsies. These results collectively suggest that targeting ASAP3 by small molecular inhibitor such as QS11 may provide alternative strategy to control gastric acid-related diseases.As mentioned previously, deregulation of gastric acid secretion may result in some of the most prevalent diseases such as GERD and peptic ulcer. Among the compounds that inhibit acid secretion, acid susceptible PPIs, such as the substituted benzimidazoles, omeprazole, lanoprazole, rabeprazole and pantoprazole are the most prominent and effective ones. However, despite their claimed efficiency, phenomena of acid rebound and tolerance are major drawbacks to the use of these drugs that has recently come into light. It has been estimated that ~30% of GERD patients remain symptomatic on standard dose of PPI,In summary, our data suggest novel roles of ASAP3 in gastric parietal cells, and revealed ASAP3 as a feasible and drugable target for the control of gastric acidity and related disorders.


In this study, to enable the simple and non-invasive evaluation of autonomous circadian oscillation, we established a method for monitoring clock gene expression by performing ex vivo culture of whole hair root tissue. This method is extremely simple and imposes little burden on subjects. Results obtained using Cryptochrome-deficient mice support that circadian period length in hair tissue correlates with intrinsic period length observed in physiology and behavior. We then applied this method to old-old subjects with severe dementia, who showed abnormal circadian behavior, and found that their peripheral clocks autonomously oscillated in a manner similar to those of healthy or younger subjects, indicating that the effect of cellular senescence on the autonomous clock oscillator is limited at least in some cell types. Although further validation may be required, the hair tissue-based culture assay would be a tool to investigate intrinsic circadian characteristics in humans.Almost all organisms maintain a circadian clock from birth to death to synchronize their own physiology and behavior with the earth’s rotation. Because the  The circadian clockwork consists of cell-autonomous and clock gene-driven negative feedback loops of transcription4. The transcriptional feedback loops generate circadian expression of a wide range of numerous genes6, which in turn leads to circadian oscillation in diverse physiological processes8.Almost all living organisms exhibit circadian rhythms in physiology and behavior, which are driven by the internal circadian clockin vivo evaluation of individual intrinsic circadian characteristics in humans, either with constant routine or forced desynchrony protocols, is expensive and labor-intensive, evaluation of circadian characteristics by in vitro or ex vivo assays could present important advantages. Among efforts to realize this, Brown and colleagues reported an interesting method based on human epidermal skin biopsy9 which is useful for evaluating autonomous oscillation performance of clock gene expression in vitro. Importantly, although fibroblast circadian characteristics are not direct markers of physiological circadian characteristics, a few research groups have concluded that circadian characteristics in skin-derived fibroblasts reflects individual circadian preference, including chronotype10. However, epidermal skin biopsy is too invasive for elderly or non-healthy subjects, and cell dispersion or cell growth steps might potentially result in changing the original circadian characteristics.Because the ex vivo culture of whole hair root tissue. An age-related shortening of circadian period has been hypothesized to account for the circadian phase advance and early-morning awakening observed frequently in the elderly. We then applied this method to investigate the effect of cellular senescence on autonomous circadian gene expression in humans, who have a much longer lifespan than model organisms such as mice. To do this, we focused on old-old subjects with severe dementia who showed abnormal circadian behavior, and then used this method to investigate the correlation between disrupted circadian behavior and peripheral circadian characteristics.In this study, to enable the simple and non-invasive evaluation of autonomous circadian oscillation performance, including intrinsic period length, we established a method to monitor clock gene expression in real time by performing Healthy subjects and old-old subjects with severe dementia who showed abnormal circadian behavior were recruited. This study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review boards of Yamaguchi University and Dokkyo Medical University. Informed consent was obtained from each subject or subject’s family.luciferase gene driven by circadian promoter/enhancer elements controlling the hBmal1, mPer2, and hPer3 clock genes were constructed as follows. The transcription-regulatory region (−4799 - +171) of the human Period3 gene was excised from a BAC clone (RP3-467L1) using Xba I and Xho I, and subcloned into the pGL3 basic vector (Promega). The resulting vector was digested using Kpn I and self-ligated to produce hPer3 (−3124 - +171) -pGL3. In addition to hPer3, the previously constructed vectors, mPer2 (−2811 - +110) -pGL3 and hBmal1 (−3465 - +57) -pGL3, were used for adenovirus vector construction12. Together with the luciferase gene, the transcription-regulatory region of these three genes was excised from each pGL3 vector, and the inserts were subcloned into the pENTR-1A vector (Thermo Fisher Scientific). The restriction enzymes used for digestion of the pGL3 vectors were as follows: hPer3–luc, Asp718 (Kpn I) and Sal I; mPer2–luc, Asp718 (Kpn I) and Sal I; hBmal1–luc, Sal I  signal sequence and one inside hBmal1). The resulting vectors were hPer3 (−3124 - +171) -luc-pENTR-1A, mPer2 (−2811 - +110) -luc-pENTR-1A and hBmal1 (−1683 - +57) -luc-pENTR-1A. These entry vectors were deposited into the RIKEN BioResource Center . Before introduction into a destination vector, each pENTR-1A vector was transfected into NIH3T3 fibroblasts, synchronized by a dexamethasone shock (50 nM), and circadian bioluminescence rhythms were confirmed by real-time monitoring with a photomultiplier tube in the presence of luciferin. pENTR-1A inserts were introduced into the pAd/PL-DEST vector using LR recombination. After Pac I digestion, the resulting pAd/PL-DEST vectors were transfected into 293A cells to produce an adenoviral stock. After amplification, this adenoviral stock was used to express circadian-driven luciferase in hair follicle tissue. The recombinant adenoviruses were generated using the ViraPower Adenoviral Gateway Expression Kit , according to the manufacturer’s instructions. An adenovirus carrying the luciferase gene driven by the CAG promotor was purchased from the RIKEN bioresource center13. Adenovirus stocks  were added to culture medium at a 1:20 dilution.Adenoviruses carrying the Plucked facial or scalp hairs whose root surface was almost or fully covered with hair follicle cells were used for culture experiments. The experimental procedure for monitoring autonomous oscillation performance in clock gene expression is as follows: pluck a few strands of facial or scalp hair whose root surface is enriched with cells; immerse the whole tissue into DMEM  supplemented with 0.035% sodium bicarbonate, 10 mM HEPES, 4.5 g/l D-Glucose, 1% penicillin/streptomycin, 1 mM L-Glutamin, and 1 mM sodium pyruvate; maintain the sample in a keep-warm bag without CO2 during the transport from nursing homes to laboratories for approximately 10 h; transfer the tissue to the medium containing adenovirus and incubate for 22–24 h at 36.5 °C without dispersing cells; to avoid floating fix the hair shaft on the bottom of a 35 mm culture dish with silicone ; and cover the immobilized tissue with luciferin (0.1 mM)-containing DMEM  supplemented with antibiotics and incubate it at 36.5 °C with 5% CO2. Culture medium in all experiments contained phenol red except those for Fig. The data sets were detrended by subtracting the 24-h running average from the raw data. We calculated circadian robustness, circadian phase (angle), and circadian period length using the software Cosinor provided by Dr. Refinetti. Except for Fig. Per2::luc knock-in mice, Bmal1-Eluc transgenic mice and Cryptochrome knockout mice were a kind gift from Dr. Joseph Takahashi, Dr. Yoshihiro Nakajima and Dr. Takeshi Todo, respectively16. Mice were bred and maintained on a 12-hour light-dark (LD) cycle (lights on at 9:00 A.M.) and allowed ad libitum access to food and water. All protocols for animal experiments were approved by the Animal Research Committee of Yamaguchi University. Animal studies were performed in compliance with the Yamaguchi University Animal Care and Use guidelines.17. Briefly, after euthanasia, the both left and right mystacial pads were removed from mice, washed vigorously two times with 70% ethanol and three times with phosphate-buffered saline (PBS) and then placed in Dulbecco’s modified Eagle’s medium  supplemented with penicillin and streptomycin. Individual whisker follicles were carefully dissected under a dissecting microscope. Considerable care was taken to remove surrounding connective tissue but not to damage hair follicles. Whisker follicles were isolated and transferred on to a plastic Petri dish containing fresh DMEM. Hair cycle stages of whisker follicles were classified as anagen or catagen by morphology of the hair bulb and relative length of the hair shaft according to previous reports18. In mouse whisker follicles, termination of hair growth in the previous hair cycle and initiation of hair regeneration in the subsequent cycle partially overlap, therefore telogen is not obvious.Mouse whisker follicles were isolated by microdissection using methods previously describedTo avoid floating, the hair shaft was fixed on the bottom of a 35 mm culture dish with silicone , and the immobilized hair tissue was then covered with 0.1 mM luciferin-containing DMEM supplemented with antibiotics and incubated at 35 °C with 5% CO2. After 2-day pre-culture, circadian synchronization was performed with 100 nM dexamethasone (DEX) for a period of 2 h, and bioluminescence was measured in real time with a photomultiplier tube . Data sets were detrended by subtracting the 24-h running average from raw data.Subject A was an 87-year-old female diagnosed with Alzheimer’s disease, and had a Mini-Mental State Examination (MMSE) score of 0 points and a Functional Assessment Staging (FAST) of 7(e). Subject C was an 86-year-old male diagnosed with frontotemporal dementia, and had an MMSE score of 12 points and a FAST of 7(c). Subject D was a 94-year-old female diagnosed with frontotemporal dementia, and had an MMSE score of 0 points and a FAST of 7(c). Subject E was an 88-year-old female diagnosed with vascular dementia, and had an MMSE score of 2 points and a FAST of 7(c).ex vivo culture of whole hair root tissue. Adenoviruses carrying the luciferase gene driven by circadian promoter/enhancer elements controlling the Bmal1, Per2, and Per3 clock genes were constructed and used for gene transfer. The experimental procedure is extremely simple  among three adenovirus vectors in two subjects , and then bioluminescence was measured in real time in the presence of luciferin with a photomultiplier tube  or lengthens the period, respectively, of human physiological circadian rhythms at an individual level28, which is consistent with our present results in cultured hair tissue. Therefore, circadian characteristics in hair follicle tissue may correlate with those observed in physiology and behavior.To further confirm a correlation in circadian characteristics between clock gene expression in cultured hair follicle tissue and physiological or behavioral circadian rhythms at an individual level, we investigated whether the autonomous circadian oscillator in human hair tissue responded to caffeine and lithium in a similar manner to that at an individual level Fig. . Both thTo investigate the effect of aging and dementia on the human circadian clock machinery, old-old subjects with severe dementia, who showed abnormal circadian behavior, were recruited for analysis of circadian clock gene expression. The World Health Organization (WHO) defines the old-old as the people aged 75–84 and the oldest-old as 85+, although the definition varies among studies. We here classified subjects aged 75+ as the old-old. We did not consider sex differences because of the small sample size due to the difficulty of hair tissue collection from old-old dementia patients. The patients consisted of a man and four women, aged from 83 to 94 years. MMSE  score was two or less in four of five patients, and FAST  stage was seven in all patients. . Because their daily time schedule was strictly controlled indoors, all patients were exposed to similar environmental cues before and during the experimental period.Per3-luc, and bioluminescence rhythms were monitored in real time  were infected with an adenovirus carrying ime Fig. . To avoiime Fig. . Other tex vivo culture of a whole hair root tissue. As shown in Fig. Cry-deficient mice and caffeine and lithium-induced changes in circadian period length indicate that circadian characteristics in cultured hair follicle cells reflect those at an individual level. Although further validation may be required to elucidate how correctly ex vivo circadian characteristics in hair root tissue correlates with that in vivo, this method enabled ex vivo evaluation of autonomous oscillation performance of clock gene expression and could be an approach for the estimation and evaluation of human intrinsic circadian characteristics.Natural selection has favored endogenous circadian rhythmicity that autonomously persists with an intrinsic period close to that of Earth’s rotation in nearly all living organisms, including prokaryotes. In this study, to enable the investigation of intrinsic circadian characteristics in humans without using labor-intensive traditional experimental methods such as constant routine or forced desynchrony protocols, we developed a very simple and non-invasive method based on et al. based on human epidermal skin biopsy9. Importantly, those authors as well as others have concluded that circadian characteristics in peripheral cells such as skin-derived fibroblasts reflect individual circadian preference (chronotype). These interesting findings have been expected to make great contribution to the progress of human circadian biology. However, epidermal skin biopsy is too invasive for old or non-healthy subjects, and obtaining a sufficient number of fibroblasts for subsequent in vitro assays may be sometimes difficult, as cellular senescence attenuates fibroblast proliferation. In addition, cell dispersion or cell growth steps might potentially affect the original circadian characteristics. To compensate for these problems, we established a new method based on ex vivo culture of whole hair root tissue infected with adenovirus carrying the luciferase gene driven by circadian promoter/enhancer elements. This method exerts little burden on subjects, and the experimental procedure is extremely simple: pluck a few strands of facial or scalp hair, immerse them into a culture medium containing the adenovirus, and then place them into a device equipped with photomultiplier tubes.This approach has been feasible using a method previously reported by Brown in vivo, but this may be solved by adjusting the concentration of culture medium components, such as phenol red, as mentioned above . Nevertheless, this difference may not matter when comparing relative period length because the order relationship in period length among individuals is unchanged, as indicated in experiments using Cry-deficient mice in Fig. 9, suggesting that individual differences in circadian period length are enhanced under culture conditions. In addition to the limitation of short period length for the hair-tissue method, estimated circadian period length was unstable and variable among hair follicles when the Bmal1 and Per2 promoters were used. Consistent with this, in a previous report, we examined expression patterns of these three genes  in hair follicle tissue with realtime PCR, and found that only Per3 showed clear circadian rhythms in mRNA levels with good reproduction30. Although PER3 may not be an indispensable component of the core clock machinery, as has been reported in previous reports, it should be a reliable circadian molecular marker because the phase interval among circadian expression of clock genes including Per3 is nearly constant, not only in various tissues but also in cultured cells under different medium conditions19.However, the hair-tissue method has its own limitations: circadian period length in cultured hair tissue is slightly different from that see Fig. . Czeisleex vivo culture of a whole hair root tissue obtained from old-old dementia patients aged 83 to 94 with severe dementia who showed abnormal circadian behavior. Data obtained using this method showed that autonomous circadian oscillation performance in senescent cells obtained from dementia patients had no distinct abnormalities, suggesting no apparent effect of cellular senescence and dementia pathology on the autonomous clock oscillator machinery. As mentioned above, previous reports based on human epidermal skin biopsy have demonstrated that in vitro circadian period length in peripheral cells significantly correlates with individual human circadian preference10. Nevertheless, an unexpected finding was the lack of any significant difference in the peripheral circadian period between healthy subjects and dementia patients with abnormal circadian behavior. On the other hand, this result is consistent with a report that in vivo circadian period length is nearly the same between younger and older subjects29.An age-related shortening of circadian period has been hypothesized to account for the circadian phase advance and early-morning awakening observed frequently in the elderly. To examine the effect of cellular senescence on autonomous circadian gene expression in humans, organisms with a much longer lifespan than that of model organisms such as mice, we performed