Nematostella vectensis.Wound healing is the first stage of a series of cellular events that are necessary to initiate a regenerative response. Defective wound healing can block regeneration even in animals with a high regenerative capacity. Understanding how signals generated during wound healing promote regeneration of lost structures is highly important, considering that virtually all animals have the ability to heal but many lack the ability to regenerate missing structures. Cnidarians are the phylogenetic sister taxa to bilaterians and are highly regenerative animals. To gain a greater understanding of how early animals generate a regenerative response, we examined the cellular and molecular components involved during wound healing in the anthozoan cnidarian in situ hybridization to identify potential wound healing targets. We identified a number of genes directly related to the wound healing response in other animals  and suggest that glycoproteins (mucins and uromodulin) play a key role in early wound healing events. This study also identified a novel cnidarian-specific gene, for a thiamine biosynthesis enzyme (vitamin B synthesis), that may have been incorporated into the genome by lateral gene transfer from bacteria and now functions during wound healing. Lastly, we suggest that ERK signaling is a shared element of the early wound response for animals with a high regenerative capacity.Pharmacological inhibition of extracellular signal-regulated kinases (ERK) signaling blocks regeneration and wound healing in Nematostella. We characterized early and late wound healing events through genome-wide microarray analysis, quantitative PCR, and Nematostella wound healing, and provides a foundation for comparative analysis with other regenerative and non-regenerative species. We have shown that the same genes that heal puncture wounds are also activated after oral-aboral bisection, indicating a clear link with the initiation of regenerative healing. This study demonstrates the strength of using a forward approach (microarray) to characterize a developmental phenomenon  at a phylogenetically important crossroad of animal evolution (cnidarian-bilaterian ancestor). Accumulation of data on the early wound healing events across numerous systems may provide clues as to why some animals have limited regenerative abilities.This research describes the temporal events involved during  Wound healing is the process of cellular contraction, movement and re-adhesion immediately after injury and is the precursor to regeneration of lost structures. During these cellular dynamics, other components such as immunity, cell death or proliferation, and nervous system inputs all interact during the process of scar-free healing . Among aHydra, has long been a comparative model for regenerative study, bridging the gap between early animals, planarians, flies and vertebrates. The anthozoan cnidarian, Nematostella vectensis, is widely known as a comparative system for embryological studies, yet following bisection through their major longitudinal axis  both halves can regenerate into normal animals -S-phenylglycine t-butyl ester (DAPT), blocks head reformation  blocked regeneration with dramatic wound healing defects in both head regeneration Figure A,B and aNematostella wound closure.Using our puncture assay methodology, we found that inhibition of ERK signaling by U0126 caused aboral puncture wounds to remain open after six hours, the normal time for wounds to heal Figure E,F and eNematostella genome-wide microarray  to identify target genes involved in wound healing. We isolated mRNA from uninjured polyps, as well as from injured animals at one hour (early response genes) and four hours (late response genes). To determine how ERK signaling effects mRNA transcription during wound healing, we also collected U0126-treated animal mRNA one and four hours after injury for comparison. Genes organized in Figure Nematostella genome website [To determine the transcriptional input necessary for stimulating a wound healing response, we used a  website . Sox E1 (235335) and Runt (129231). A number of growth factor-related genes were also activated, including Wnt2 (242584), platelet-derived growth factor (239536) and sprouty (29671). Interestingly a number of genes related to mucus proteins or mucosa tissue were also highly up-regulated, including maltase-glucoamylase (224255) and mucin-1/2 precursors  or moderators of MAPK signaling including alpha-integrin (196726) and g-protein signaling . The primary transcription factors activated by injury were Fox O-related , in situ hybridization to confirm microarray results. Genes were selected for qPCR based on their genomic sequence size and if the desired PCR primer conditions could be met for qPCR. The seven genes shown in Figure A total number of 17 genes were analyzed by quantitative PCR (qPCR) and/or by uromodulin around the wound site, without reducing the endogenous expression around the aboral pole , also known as Tamm-Horsfall glycoprotein, was normally expressed at the most distal portion of the aboral pole of uninjured animals  belongs to the homeobox class of transcription factors and is normally expressed along the aboral ectodermal walls of the body column  was only expressed after injury and was localized to the ectodermal layer cells immediately around the site of injury Figure , row 3. The inhibitor of matrix metalloproteinase gene (238195) was broadly expressed throughout the endoderm and drastically reduced in U0126-uninjured animals  was lightly expressed compared to within injured animals, and localized to the aboral endoderm, tentacle tip endoderm and near the mouth Figure , row 5. We determined that removal of U0126 allowed injured animals to begin healing Figure F, therefin situ hybridization suggest that we identified several genes utilized during aboral wound closure. To determine if our identified targets also play a role in wound healing before regeneration, we conducted in situ hybridization of animals that underwent bisection along the oral-aboral axis in comparison to animals impaired by U0126  inhibitor and the maltase enzyme, all exhibited expression immediately after injury in both oral and aboral halves , but this activation appears hours after the initial injury. We have identified a number of early up-regulated MMPs that may act in releasing these growth factors to initiate synthesis. MMPs are a subset of peptidase enzymes that utilize calcium or zinc ions for activation and are known for their ability to release adhesion complexes between cells. Together with tissue inhibitors of metalloproteinases (TIMPs), which block MMP activity, cells are able to coordinate cell movement through regulation of adhesion molecules [Hydra[NvTIMP is expressed locally around the injury site  become factors in the regulation of the wound healing process. ERK signaling is known to play an important role in the regulation of apoptosis and bud formation in Hydra[Nematostella and is necessary for proper wound healing. Although ERK signaling acts as an early response element among these three taxa, several novel genetic components to the Nematostella wound healing program may provide a significant advantage compared to other systems that lack a high regenerative capacity, other than simply having highly proliferative I-cells (Hydra) or neoblast cells (planarians).The primary events taking place during early wound healing of  in Hydra-48, and  in Hydra. We haveNematostella  exhibited mesentery staining during wound healing , an enzyme utilized during the synthesis of vitamin B, a deficiency of which has been linked to wound healing defects . This getruction , and was Figures , 5 and 6. Integrins are mediators of focal adhesion complexes and numerous α- and β-subunits are activated or up-regulated during vertebrate wound healing [Nematostella and closely related coral species have fewer numbers of integrins than vertebrates [SoxE because these two genes are known to be in overlapping regions of the presumptive endoderm during gastrulation in Acropora millepora[Nematostella could be a valuable system to discern the functional relationship of integrins and wound healing.At four hours after injury, we began to see components of cell membrane signaling  as well as activation of growth factors healing . Nematostebrates . We idenmillepora,68. Due millepora, NematosNematostella. In Hydra and axolotl, the EF-hand calcium-binding are associated with the wound response [Nematostella gastrulation [The activation of integrin is closely associated with regulation of calcium and ionic balance  and a nuresponse ,41 and aresponse . EF-handresponse , and arerulation . AlthougPDGF (239536) has not been characterized in detail in Nematostella, but other fibroblast growth factor-related genes are thought to regulate axial patterning and cell specification [TGF-β gene was identified to be down-regulated in wild-type animals at one hour compared with U0126-treated individuals. Tight regulation of TGF-β and downstream targets is suggested as a possible means to resolve excess scar formation [Based on the timeline of gene activation Figure B, our refication -74. Inteormation . Data foormation . CompariNematostella the key transition from wound healing to a state of regeneration is the activation of cell proliferation. ERK signaling is necessary for the initiation of the early wound healing response in Nematostella and is closely linked to the activation of proliferative cells in other highly regenerative invertebrate model systems [Nematostella and is relatable to multiple model systems of regenerative biology.Wound healing and regeneration are separable developmental processes. Here we suggest that healing from a puncture wound takes roughly six hours after injury to allow for injured tissue to become functional again. Some time between 12 and 18 hours, signals from healed tissue activate a program initiating proliferation and regeneration . We have systems ,47,49,75Nematostella polyps raised in 1/3× seawater [Artemia between day seven and day fourteen after fertilization, and then were starved for seven days to minimize non-specific staining due to food particles in the body cavity. Juvenile polyps were used due to their small size and ease of visualization and because they can regenerate lost structures similar to adults. Animals were cut in 2 mm thick silicon-coated dishes . The silicon-coated dish was used to create a cutting/poking surface that did not damage the tools used. Glass needles for puncture assays were formed from capillaries (World Precision Instruments – TW100F4) using a needle puller . All transverse cuts were done with a cornea scalpel and were performed near the base of the pharynx. Cutting experiments were done in all regeneration experiments; puncture experiments were used to assay components of wound healing. A standardized protocol for injuring animals was developed, similar to puncture assays in Drosophila[Nematostella[Hydra.seawater  were colrosophila, to alloatostella, being tNematostella[Animals used in drug treatment experiments were soaked for one hour prior to injury. The drugs U0126  and DAPT  were both used at 10 μM concentration dissolved in 1% dimethyl-sulfoxide (DMSO). These two drugs have been shown to knockdown components of MapK (U0126) and notch (DAPT) signaling in atostella,51. For 2 to the 1/3× seawater; animals were fixed with 4% paraformaldehyde in 1/3× seawater for one hour at room temperature; animal tissue was washed five times with PBS with 0.2% Triton-X100 (PBT); fixed polyps were permeablized with proteinase K for 20 minutes as specified in the protocol; tissue was then washed twice with PBS and re-fixed with 4% paraformaldehyde in 1/3× seawater for one hour at room temperature; the tissue was washed five times in PBS; manufacturer’s protocol was followed for equilibration, biotinylation and SSC washes of the tissue; we added two extra 0.3% hydrogen peroxide washes  to help quench endogenous peroxidase activity; tissue was then washed twice in PBS; streptavidin horseradish peroxidase antibody was incubated overnight at 4°C; due to the larger amount of tissue used (rather than thin tissue sections), a larger volume of developing solution was needed, therefore signal was developed using DAB  rather than kit components. The developing reaction was stopped by washing with PBS. Samples were then cleared in an 80% glycerol solution containing Hoechst  to label nuclei. Samples were quantified by measuring a 50 × 50 μm area centered around the site of injury  and the number of nuclei in this region was compared to the number of DAB positive cells  to determine the distribution of apoptotic cells over time. Although the manufacturer’s protocol was designed for tissue sections, we performed the following for whole mount preparations: animals were relaxed by gently adding 7% MgClNematostella mucus with the histological stains Alcian blue (acidic mucins) and Periodic acid-Schiff reagent . These stains are used to visualize mucins present in mucosal tissue in vertebrates [2 to the 1/3× seawater. Samples were fixed by washing polyps three times with ice-cold 100% methanol for one hour at 4°C. Samples were rehydrated immediately after fixation in 60% methanol in distilled water (once), then 30% methanol (once), and finally PBT solution for five washes. Samples were transferred to glass three-spot dishes for subsequent staining. At this time samples were soaked in Alcian blue solution (pH 2.5) for 10 minutes, then washed three times in distilled water. Samples were soaked in 1% Periodic acid solution for 10 minutes, then washed three times with distilled water. Schiff’s reagent was added to each sample and was stained for approximately 20 minutes. Samples were then washed 10 times with distilled water, then dehydrated through a methanol series: 50% (twice), 75% (once), 85% (once), 95% (once) and 100% (twice). Samples were further cleared (three times) in Murray clear .We attempted to stain tebrates . SamplesWe found that after six hours puncture wounds were no longer visible by transmitted light and therefore we used confocal microscopy to determine the timeline of events leading up to the completion of the wound healing process Figure . SamplesWe utilized an antibody against phosphorylated-ERK,  to identify if p-ERK was activated during wound healing and regulated by U0126  attached to small transfer pipettes to gently create suction on the side of the polyp  camera and processed using Zen software. All figures were created using Adobe Illustrator (CS4).Time series images of regenerating head structures Figure B were taet al. [et al. [in situ hybridization and our other imaging studies.A total of 300 polyps (for microarray) or 100 polyps (for qPCR) were used for one biological replicate for each assay. RNA extraction techniques and cDNA synthesis were the same as described in Layden et al.  and Rött [et al. . To maxiNematostella genome with three replicate oligonucleotide probes per gene. Samples were normalized and fold-change calculations were produced using Nimblegen Inc. software according to previous work [in situ hybridization. In all drug treatment experiments, animals were pre-soaked for one hour prior to injury. A total number of 1,434 significant expression values exhibited a fold change of 2.5 or greater  to make sure their range was within the negligible value of 1.9 to 2.0. A total of three biological replicates consisting of 100 polyps per sample were analyzed. Relative fold-change values were calculated in Microsoft Excel and were standardized against our reference genes based on formulas from Livak and Schmittgen [qPCR samples were standardized with NvGADPH and NvRiboPro . This movie shows the interaction between mesentery structures and the puncture wound immediately within the first hour of injury.Click here for fileTime-lapse movie over the first three hours after injury. Photos were taken every 15 seconds. Movies are played at four times the normal speed. Animals were stained with acridine orange and fluoresced with 488 nm argon laser (with only 10% power). This movie shows the interaction of mesentery structures over the course of the first three hours after injury.Click here for fileTime-lapse movie over 15 minutes during the first hour after injury. Photos were taken every 10 seconds. Movies are played at two times the normal speed. Animals were stained with acridine orange and fluoresced with 488 nm argon laser (with only 10% power). This movie shows a sticky mucus-like residue left from the injury site as the animal migrates out of the focal view.Click here for fileMucin analysis during regeneration. Animals were stained with Periodic acid and Schiff’s reagent which contains the fluorescent compound fuchsin. (A) Scanning laser confocal images of the head  show large round bundles of cells that are heavily stained with fuchsin (white arrow). (B) Structures similar to those found in the pharynx are found throughout the ectoderm in the aboral part of the animal (white arrow). (C) The brightest concentration of staining occurs at the base of the mesenteries; (D) little to no staining was found along the tentacles. (E-E’) Uninjured animals appear to have less fluorescent labeling found throughout the animal when individuals are exposed to U0126. (F-H’) Time series of mucus staining during wound healing after head removal. (F-F’) One hour after injury fluorescent staining appears greatest near the wound epithelium. (G-G”) A brighter amount of staining appears present at four hours after injury, while U0126 animals show wound healing defects and less staining. (H-H’) By 12 hours, little mucin staining is visible in controls, where U0126 animals still exhibit wound healing defects, but appear to have elevated mucin levels.Click here for fileMaintained apoptotic regulation during wound healing. (A) Quantification of apoptosis and nuclei count of five different stages of juvenile polyps before and after injury. Samples exhibit relatively equal numbers of TUNEL-labeled cells, where uninjured animals express the lowest number. (B) Ratio of apoptosis compared to total nuclei count shows maintained apoptosis throughout early puncture events.Click here for fileInhibition of notch signaling disrupts head regeneration. Exposure of regenerating polyps to the notch inhibitor, DAPT, prevents head from developing new oral regions.Click here for fileThis file contains all microarray data. Each document is separated by a different tab. This is the excel file used to create Figure Click here for fileThe thiamine enzyme from Nematostella is likely a cnidarian-specific gene, derived from bacteria. This gene is found on scaffold 466 and spans positions 44637:52229. (A)Acropora digitifera genes closely related to the thiamine enzyme identified in Nematostella vectensis and their associated homology. (B) Alignment of Nematostella and Acropora sequences.Click here for filePrimer information for cloning.Click here for file


JAK2V617F mutated hematopoietic stem and progenitor cells are resistant to inflammation, and this gives the neoplastic clone a selective advantage allowing for its clonal expansion. Because inflammation plays a central role in MPN inflammation is a logical therapeutic target in MPN.Our understanding of inflammation's role in the pathogenesis of myeloproliferative neoplasm (MPN) is evolving. The impact of chronic inflammation, a characteristic feature of MPN, likely goes far beyond its role as a driver of constitutional symptoms. An inflammatory response to the neoplastic clone may be responsible for some pathologic aspects of MPN. Moreover,  Inflammation has a central role in myeloproliferative neoplasm (MPN). Excessive inflammation not only causes many of the debilitating symptoms associated with the disease but also drives the selective expansion of the neoplastic clone –3. Infla JAK2V617F clone is responsible for inflammation but exactly how the JAK2V617F clone induces inflammation is unclear. Excessive inflammatory cytokine production is not exclusive to the neoplastic cells in MPN. For example, the inflammatory cytokine tumor necrosis factor-alpha (TNF) is equally overproduced in both the JAK2V617F and the JAK2WT cells from MPN patients and in MPN mice (AF unpublished results). Moreover, mice injected with Ba/F3 cells expressing JAK2V617F have elevated production of inflammatory cytokines including TNF and IL-6, but the Ba/F3 JAK2V617F cells are not the source of these cytokines [ MPLW515L MPN model, but if STAT3 was deleted in the neoplastic cells but remained intact in the nonneoplastic cells, the disease phenotype remained robust [A deranged inflammatory cytokine profile is a shared characteristic of both humans with MPN as well as mouse MPN models. This suggests that theytokines . Ross Leytokines  and found robust . Taken t−/−) a JAK2V617F transduction-transplantation MPN model had no fibrosis, attenuated splenomegaly, and reduced leukocytosis yet erythrocytosis and excessive megakaryopoiesis were preserved . These data demonstrate that each cellular subset of the immune system affects distinct aspects of MPN pathophysiology.Inflammatory cells of both the innate and adaptive immune systems contribute to MPN pathology in mouse models, supporting the notion that some pathologic features of MPN are caused by the host's reaction to the neoplastic clone rather than a direct consequence of the neoplastic clone itself. For example, erythrocytosis was attenuated in MPN mouse models deficient in macrophages , 13. We  JAK2V617F clone could modulate the MPN phenotype  to test this hypothesis [It is possible that ruxolitinib's effect in MPN is via its anti-inflammatory properties and not necessarily through its inhibition of the mutant JAK2. Decreasing inflammation may reduce the selective advantage of thepothesis . Treatme JAK2V617F. How exactly MPN associated mutations may give hematopoietic stem cells a selective advantage in the setting of chronic inflammation is unknown, but the known consequences of chronic inflammation on hematopoietic stem cells provide us with some clues.Perhaps chronic inflammation produces an environment that is highly selective for hematopoietic stem cell clones with MPN associated mutations such as−/− mice by chronic bacterial exposure. This suggests that exposure to pathogens and the ensuing inflammatory response catalyzes HSC exhaustion. To further illustrate the role of inflammation in HSC exhaustion, a cross of the miR146a−/− mice onto a RAG1−/− background (which lacks T and B cells) rescued HSC from exhaustion. Together these data demonstrate that chronic inflammation leads to HSC exhaustion and promotes the development of myeloproliferation, marrow fibrosis, and extramedullary hematopoiesis.Chronic inflammation causes age-related hematopoietic stem cell (HSC) functional decline. The ability of hematopoietic stem cells to be quickly called into cycle is crucial for the rapid hematopoiesis required during times of stress, but HSC bombarded by chronic inflammation eventually exhausts. This HSC exhaustion is mediated by chronic cycling of HSC. In mouse model systems repeated exposures to inflammatory stimuli such as lipopolysaccharide (LPS)  negative−/− mice display accelerated cell-cycle kinetics and reduced transcriptional expression of negative cell-cycle regulators. This chronic cycling leads to HSC attrition, resulting in a 150-fold reduction of HSC in TPO−/− mice. Mutations which enhance MPL signaling result in superior hematopoietic stem cell reconstitution ability and self-renewal in mouse models. This includes mice deficient in negative regulators of MPL signaling such as Lnk [The thrombopoietin (TPO) signaling axis plays a central role in hematopoietic stem cell cycling . The hemh as Lnk  and MERIh as Lnk . MPL, JAK2, and LNK. It is currently unknown whether calreticulin mutations affect the MPL signaling pathway, but clues such as high expression of mutated calreticulin in MPN megakaryocytes [Activation of the MPL pathway is the common theme among mutations associated with MPN includingryocytes  implicatryocytes  demonstr JAK2V617F induced MPN in mouse models [ JAK2V617F transgenic mice onto a MPL−/− background which resulted in thrombocytopenia and lessened neutrophilia compared to JAK2V617F transgenic mice on a MPL wild-type background. Crossing of JAK2V617F transgenic mice onto a thrombopoietin deficient (TPO−/−) background however had a different effect. TPO−/− mice are thrombocytopenic, and expression of JAK2V617F restored platelets to a normal level. JAK2V617F mice on a TPO−/− background had larger spleens and increased numbers of megakaryocytes in the spleen compared to JAK2V617F transgenics on a wild-type background.Intact MPL signaling is required for development ofe models . HitchcoTPO−/−) delayed erythrocytosis, decreased spleen size, and reversed fibrosis. They found that loss of functional MPL ameliorated PV, with a decreased hematocrit, decreased leukocytosis, decreased platelet count, and reversal of fibrosis. Taken together, these data demonstrate that activation of the Tpo/MPL signaling pathway is crucial for the MPN disease phenotype. It is also attempting to speculate that activation of the MPL signaling pathway by JAK2V617F may also affect HSC cycling.Spivak's group also found that a functional thrombopoietin receptor is required for development of polycythemia vera in a mouse transgenic model of PV . In their hands, abrogation of TPO production  which is overexpressed in JAK2V617F cells [ JAK2V617F cells were resistant to Lcn2-induced growth suppression which conferred a relative growth advantage to JAK2V617F clones. Treatment of a mouse knock-in JAK2V617F model with NAC reduced erythrocytosis and splenomegaly [Increased ROS is a feature of MPN patients and mouse models.7F cells , 35. Nornomegaly  suggesti JAK2V617F neoplastic clone because JAK2V617F mutated hematopoietic progenitor cells are resistant to inflammatory cues which suppress normal hematopoietic progenitors , Crohn's disease (1.8), polymyalgia rheumatic (1.7), giant cell arteritis (5.9), Reiter's syndrome (15.9), and aplastic anemia (7.8) . Each of JAK2V617F.We are working to determine whether exaggerated production of inflammatory cytokines in response to immunogenic stimuli may be a predisposing factor to acquire MPN. We find that monocytes from MPN patients produce excessive amounts of TNF after stimulation through Toll-like receptors (TLR) and crucial pattern recognition receptors for microbial products . This excessive production of TNF is due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production. Both mutant and nonmutant monocytes from MPN patients overproduce TNF, demonstrating the excessive cytokine production is not directly mediated by JAK2V617F but many of the other pathologic features of the disease such as marrow fibrosis, constitutional symptoms, and cytopenias could be due to the host's inflammatory reaction to the neoplastic clone. It is clear that inflammation plays a critical role in the pathogenesis of MPN but the details are still evolving. A more in-depth understanding of why inflammation is high in MPN and how the host's immune system responds to the neoplastic clone will likely reveal fundamental insights into MPN disease pathogenesis and will also identify novel therapeutic targets in this disease.MPN is a unique hematologic malignancy with a multifaceted and complex pathobiology. The unrestrained expansion of mature myeloid cells can be directly attributed to the constitutive activation of JAK2 by


Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. Specifications TableValue of the data•The data provide a Nus protein fusion partner (tag) vector to increase the solubility of target proteins expressed in bacteria.•The data will allow for the ease of use of affinity purification-mediated separation of a target protein from the Nus tag.•The data will support the use of the Nus solubility tag in bacteria for target protein expression, solubility, and purification.1We engineered a Nus solubility tag-containing bacterial expression vector for expressing Nus solubility-tagged proteins and then removing the Nus tag from the target protein during protein purification . We have22.1Standard approaches for culturing bacterial cells were employed 2.22+ affinity chromatography The configuration of the pET43.1 Nus- and His-tag containing vector prevented thrombin or enterokinase-mediated separation of His-tagged target proteins from the Nus-tag, since the vector contained His-tags on both sides of these protease sites. Therefore, we modified the pET43.1b vector to interchange the position of the thrombin cleavage site and the N-terminal His tag. Following thrombin digestion, this new configuration allowed us to separate the target protein from the Nus tag using His/Ni2.3redoxCH). To generate Mical protein containing the redox and CH domains  in pET43.1bNG, a portion of the Mical protein containing the CH domain was PCR amplified using primers containing a 5′ Xho I restriction enzyme site (Forward: 5′–TGA CAG TCT GCT CGA GCC GTT TTG GCC CAC TGG ATC –3′) and 3′ Avr II restriction enzyme site (Reverse: 5′ – GTT TTA TCA GCC TAG GAA CGC CCA ACT TAA TTA ACA TTA GTG GTGGTG GTGGTG GTG CTC GAG TCT AGA GCG AAA CAA GTC GCA GAT CTG GTC C –3′) and after digestion of the appropriately sized PCR product with Xho I and Avr II, the fragment was inserted into the Xho I and Avr II sites of a previously generated pET43.1bNG vector containing the redox domain of Mical. This placed the CH domain of Mical, which we had previously found to be necessary in vivo for Mical׳s function redoxCH pET43.1bNG plasmid DNA using standard approaches as described above, digested to confirm the correct product, and sequenced on both strands.Our work with Mical has revealed that the oxidoreductase (redox) and calponin homology (CH) domains of Mical


However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes  An important advantage of differentiated adipose derived stem cells compared to mature adipocytes, is that, in contrast to mature adipocytes, they can be cryo-preserved and expanded. However, there are several important differences between differentiated adipose stem cells and freshly isolated human mature adipocytes - for example levels of maximal lipolysis in vitro drug screening, targeting adipocytes. In the search for improved differentiation protocols for human adipose stem cells many different approaches have been tested and there are now several established methods such as The development of new protocols and culture conditions for the differentiation of adipose derived stem cells to a more mature state would be beneficial for fundamental studies of adipocyte functions and mechanisms, as well as for in vivo-like surrounding in comparison to hard plasticcell culturing plates.Several, recent studies have demonstrated the importance of a cells structural habitat as a driver for phenotypic differentiation To further improve the differentiation of stem cells to adipocytes, efforts have been made to provide the cells with a more physiologically relevant environment by using surface-structure and 3D culture systems For this investigation, a culture system composed of electrospun fibers was selected. An electrospun fiber system is advantagous because it is similar in structure to collagen type I bundles which form the majority of the extra cellular matrix framework in adipose tissue An attractive material for 3D-culturing is the polymer polycaprolacetone (PCL), the properties of which already meet various medical and physical specifications for safety in clinical applications, and it is biodegradable PCL is suitable for electro-spun matrices and has already been demonstrated to be suitable for culturing of mesenchymal stem cells differentiated into bone Taken together, these reports encourage a first study of differentiation capacity and functionality of adipose derived stem cells when promoted towards a linage of adipocytes and cultured on electro-spun PCL matrices. This technique could lead to a viable method of achieving more adipocyte-like phenotypes in stem cell derived adipocyte models, than those currently in use.in vitro, high resolution microscopy techniques are favorable, that can optically investigate the cells whilst they reside in or on a matrix. The optical investigation of cells in a complex matrix is one of the main assets of Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which offers intrinsic chemical selectivity by targeting molecular vibrations To assess adipocyte phenotype The targeted vibrations are highly characteristic to certain molecules, as they vary with the chemical binding structures presented. This is useful for biological applications as C-H bonds, found in long alkyl chains of triglycerides and fatty acids, can be visualized.CARS microscopy works by an application of two pulsed lasers onto the sample, overlapping in time and space to drive chosen vibrations. The difference between the two laser frequencies defines the desired oscillation to image the chosen chemical structure. The molecular response can then be read out by a third laser pulse, generating a blue-shifted signal, which is easily separated and usable for laser scanning imaging.Since the signal is only targeted to a small area of the sample, where the two beams overlap, it is possible to perform 3D sectioning without the use of a pinhole, making this technique suitable for thick samples and cellular structures. The near-infrared light can penetrate further than conventional visible excitation beams used in fluorescence microscopy. Additionally, CARS occurs in the electronic ground state, minimizing potential photodamaging effects on the samples that can occur in more traditional imaging systems As well as these advantages, the label-free imaging capabilities of CARS microscopy circumvents the difficulty that lipophilic dyes used to stain lipids for visualization also readily binds to the PCL fibers The aim of this study is to investigate the effect of using aligned and randomly oriented PCL fibers matrices for large cell populations of stem cells differentiated to adipocytes by measuring proliferation, glucose uptake, gene expression and lipolysis. Furthermore, at single cell level, we investigate cell distribution in and on the scaffolds and intracellular lipid stores using CARS microscopy, making comparisons to conventional 2D cell culturing and freshly isolated mature adipocytes.Adipose derived stem cells were cultured and differentiated in tissue culture treated 96-well flat plastic plates , PCL fiber plates NanoAligned, thickness ∼10 µM  or NanoECM, thickness ∼20 µM . NanoAligned, is denoted aligned and NanoECM, is denoted random.14 Subject's; 12 females and 2 males with a mean age of 36.8 years (27–49) and a mean BMI of 25.8 (23.6–29.3), undergoing elective surgery, took part in this study. The subjects reported themselves as healthy with no prescribed medications. All subjects gave their written informed consent and all procedures were approved by the ethics board at Gothenburg University and were performed in accordance with the Declaration of Helsinki. N denotes biological repeats and n denotes replicate samples. Adipose-derived stem cells and mature adipocytes were not isolated from the same donors due to technical reasons.Danielsson et al.2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM Hepes (pH 7.4), 0, 1% (w/v) fatty acid-free bovine serum albumin, 100 nM phenylisopropyladenosine , 0.5 U/mL) adenosine deaminase  and 2 mM glucose.An isolation protocol for extraction of mature adipocytes was adapted from The stromal-vascular fraction was isolated from the filtered, collagenase digested tissue by centrifugation and purified by lysis of the red blood cells using a hypotonic red blood cell lysis buffer . The remaining cells were seeded (passage 0), expanded for two passages in BM-1 (ZenBio) supplemented with 10% heat inactivated calf serum , penicillin-streptomycin  and bFGF  and stored in −150°C prior to use.2 for 2D culture and 112 500 c/cm2 for the two categories of matrices in EGM-2NV Bullet kit medium  without gentamycin and cultured at 5% CO2 in 37°C. The seeding day was denominated as day 0. On day 1 the medium was changed to differentiation medium BM-1 supplemented with 3% fetal bovine serum , 1% Penicillin/Streptomycin, 100 nM insulin , 1 µM dexamethason , 1 µM pioglitazon  and 0.5 mM IBMX . On day 8 the medium was changed to maturation medium; BM-1 supplemented with fetal bovine serum, 1% penicillin/streptomycin, insulin and dexamethasone (same concentrations as before) and matured until day 14.Human adipose derived stem cells at passage 2 were seeded at 56 250 c/cmAdipose derived stem cells from three donors were seeded at indicated densities (on either 2D or on matrices), then differentiated using the described protocol, rinsed in phosphate buffer saline (PBS)  and fixed at indicated (in the graph) time points using 4% paraformaldehyde (PFA)  for 10 min, stained with Hoechst (350/461 nM) . Cells were imaged using a Cellavista system  and analyzed for nuclear number using the software provided by the manufacturer.Cells differentiated for a range of time durations, were stained with a live/dead kit containing both green-fluorescent calcein-AM to indicate intracellular esterase activity, and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity  and imaged using a fluorescence microscope.Differentiated adipose derived stem cells were fixed, using PFA as described above, at indicated time points and stained with oil-red-o  then imaged. Oil-o-red was eluted using isopropanol and the absorbance at 490 nm was measured using a Spectramax Pro .2 on inserts for 24-well plates with aligned or random PCL-fibers or at 56 250 cells/cm2 on glass cover slides (170 µm thickness) coated with rat tail collagen type 1  and differentiated as described above. The cells were fixed on indicated time points using PFA and stored in PBS until analysis.Cells. Human adipose derived stem cells in passage 2 were seeded in at 112 500cells/cm−1, were overlapped in time and space and focussed onto the sample plane of an inverted optical microscope  using an oil immersion objective  with a total laser power of 50 mW (at the sample position). The emission of the CARS signal is limited to the high-intensity region of the focal volume, resulting in a spatial resolution of ∼300 nm. Single photon counting detectors from Becker&Hickl GmbH were used to detect three different signals simultaneously. Beside the forward and back emitted CARS signals, intrinsic Multi-Photon Excitation Fluorescence (MPEF) signals were recorded between 495–530 nm. Dichroic mirrors and high optical-density filters were used to separate the signals before the detectors.Label-free imaging of cells and intracellular lipids was achieved by mapping densities of C-H groups using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. The custom-built setup is described in detail elsewhere CARS/MPEF images were collected with 512×512 pixel image sizes, 5.04 µs pixel dwell time, summed over 5 acquisitions. Multiple planes were sequentially acquired, forming 3D images of the distribution of cells and their lipid stores. The CARS-signals are colour-coded in red and the MPEF-signals shown in green.Lyn et. al. Image analysis was done using ImageJ  and the quantitative voxel analysis used by Lipid droplet diameters were binned in 1 µm bins and the resulting histograms were fitted with a Lorentizian distribution function using OrginPro9 .Cell positions on and in the PCL scaffolds were estimated via their MPFE intensity distribution along the z-axis, while the depth distribution of PCL fibers was determined via their CARS intensity. Both signals were normalized to the maximum. Evaluations were done for a representative z-stack for each scaffold morphology.3D reconstructions were done with the 3D Viewer tool provided by ImageJ.Overnight incubated mature adipocytes were washed in KRHG to remove media, suspended in KRHG at a concentration of 200 µl packed cells/mL and incubated 30 min with 10 nM (-)-N6-(2-phenylisopropyl)adenosine (PIA)  and 0.5 U/mL adenosine deaminase (ADA) . The cell suspension was subjected to centrifugation through diisonylphtalate and immediately frozen on dry ice until thawed by mixing with Ripa buffer , phosphatase inhibitors  and protease inhibitors  and stored at −20°C until analysis by western blot.Fourteenday differentiated adipocytes were washed in PBS and lysed in Ripa buffer and protease and phosphatase inhibitors then kept at −20°C until analyzed using Western blot.Cell lysate protein concentrations were assessed using a Pierce BCA kit  and an equal amount of total protein for each condition was subjected to SDS-PAGE using pre-cast 10% Bis-Tris gels , then transferred to a PVDF membrane  and immunoblotted with antibodies listed below. Each protein band was normalized to the corresponding anti-actin band which served as a loading control . For gene expression specifically, data was analyzed using Analysis of Variance (ANOVA) on the delta Ct values, defining treatment and donor as random and the interaction as random. Comparisons between groups were made using a Student's t-test following the ANOVA. Analyses were conducted using PROC MIXED in SAS 9.2 . Principal Components Analysis, PCA, was used to give an overview across genes using the R function prcomp . Data visualization and hierarchical clustering (UPGMA method with Euclidian method for distance measure) was done with TIBCO Spotfire version 5 . Normalization to undifferentiated samples was performed following guidelines from LifeTechnologies For all analyses, p<0.05 was considered to be significant and indicated with *, p<0.01 was indicated with ** and p<0.001with ***.Overnight incubated mature adipocytes were rinsed in KRHG and suspended at 3% cells (v/v) in KRHG. Equal cell volumes were then incubated with the stated concentrations of isoproterenol for 1 hour. Glycerol release in the medium was measured using a glycerol detecting kit  according to the manufacturer's protocol.Differentiated adipocytes were starved overnight in DMEM  supplemented with 1% BSA  and 1× penicillin-streptomycin , incubated with KRHG for 20 min. Indicated concentrations of isoproterenol  were added for 120 min and glycerol release in the medium was measured using a glycerol detection kit . The results were related to cell numbers counted with the Cellavista.14C] 100 µCi/ml  was added to the cells and they were left for a further 60 minute incubation at 37 °C. The incubation was terminated by centrifugation through diisononyl phtalate  and immediate freezing using dry ice. The cells were lysed in 100 µl PBS supplemented with 1% SDS and Complete Mini protease inhibitor mix. The lysate was subjected to both scintillation counting and protein concentration determination using a Pierce BCA kit .Mature overnight incubated adipocytes were washed in glucose free buffer, diluted to 200 µl packed cells/mL, incubated 30 min with PIA and ADA. Before the start of the experiment, the adipocytes were incubated with indicated concentrations (in the graph) of insulin or cytocalasin B  for 30 min in 37°C.A glucose mixture containing 18 mg/mL glucose and labeled deoxy-D-glucose 2-[1- 14C-marked D-deoxy-glucose was added and allowed to be taken up for 60 min. Cells were rinsed three times with PBS, then lysed in PBS with 1% SDS and protease inhibitors and subjected to scintillation counting and protein concentration determination using a Pierce BCA kit .Differentiated adipocytes were starved overnight in insulin free media and incubated 3 hours in glucose free DMEM  supplemented with 0.4% glucose-containing DMEM  and 25 mM Hepes . Cells were stimulated with indicated concentrations of insulin or cytocalasin B for 30 min. Adipose derived stem cells from three different donors were plated in a 24 w  in BM-1 supplemented with 3% fetal bovine serum and incubated in 37°C overnight. The cells were washed once in PBS and RNA was extracted using a RNA Easy Plus kit . These cells were considered to be undifferentiated adipose derived stem cells.Fourteen day differentiated adipose derived stem cells, were washed once in PBS and RNA was extracted using a RNA Easy Plus kit.The RNA concentration was measured using a Nanodrop  and 0.5 µg of RNA was used for retro transcription with High Capacity cDNA reverse transcription kit . 100 ng cDNA per sample was subjected to a custom made TaqMan Low Density Array . All samples were run in duplicates.Gene of interest – CtReference genes), modified from Livak et al Expression levels were acquired from the threshold cycle value (Ct) and all statistical calculations are based on deltaCt values  after differentiation, were then chosen for all subsequent experiments.We performed nuclear counts at several time points during differentiation and found significantly lower proliferation in the aligned and random fibers as compared to the 2D plated cells . A decreLive-dead staining of the differentiated human adipose derived stem cells indicated no difference in viability between the different culture conditions , this waPreadipocytes need to be withdrawn from the cell cycle before adipose differentiation can start Lipid accumulation shown with oil-red-o staining has been used as a golden standard for adipogenesis for years Oil-red-o pictures suggested more lipid accumulation in cells grown on aligned fibers than in 2D plates and random fibers . We, theOur results demonstrating increased lipid accumulation on cells grown on aligned fibers stands in contrast to findings in embryonic stem cells differentiated to adipocytes that have a similar lipid accumulation in 2D and PCL fiber cultures High resolution pictures acquired by CARS and MPEF microscopy allow the visualization and quantification of cell size (S3C), and of the size distribution of lipid droplets within individual cells . ExampleThe CARS signals can be separated from fluorescence signals by their wavelength, allowing individual analysis of both.The differentiated adipose derived stem cells – in contrast to the mature adipocytes – accumulate a large number of lipid droplets with a broad distribution of size range. There can be as many as 50 individual lipid droplets per cell after 14 days. The mature adipocytes have one large lipid droplet, filling almost the entire cytosol of the cell, with this as our target, our analysis focussed on the quantification of the bigger droplets in the cells. We found the largest droplets typically ranged from 6 to 20 µm in diameter. Thus, we analysed the abundance of droplets in this size range in 10 cells per condition. For cells in the 2D plates and on the aligned matrix we found on average 10 larger droplets per cell, while in cells grown on the random matrix there were less than 5 droplets per cell in the 6–20 µm size range, details can be found in This shift was observed in both the aligned and random matrices with the increase in droplet size being more pronounced in the latter. With a lower total droplet count in the random fiber matrices, leading to an reduced lipid accumulation, the overall lipid content  lower caTo further investigate lipid droplet differences in our culture systems, we measured perilipin the protein lining lipid droplets. Perilipin is important for lipid droplet formation in human adipocytes and its over-expression can promote formation of droplets Measurements of lipid accumulation are often used as a method to measure differentiation of adipose derived stem cells to adipocytes. However, lipid accumulation is not the only reporter for maturity of an adipocyte. To more accurately appraise differentiation we aimed to assess the functionality of adipose derived stem cells differentiated in 2D plates and on matrices, comparing them to freshly isolated human mature adipocytes.We first measured the insulin-stimulated glucose uptake of the differentiated adipocytes finding cells grown on random and aligned fibers are significantly more sensitive to insulin than control cells differentiated on 2D . The EC5The number of transported glucose molecules per cell, measured as cpm/cell was significantly higher in cells grown on random fibers than in the primary mature adipocytes, 2D control or aligned fibers, both basally and after 100 nM insulin stimulus .Insulin receptor (IR) levels are known to influence glucose uptake in human adipocytes, for example in type 2 diabetes We also investigated the phosphorlyation on serine 473 of protein kinase B (PKB) after insulin stimulation as a measure of activity in the insulin signaling pathway. The basal level of phosphorlyated PKB was significantly higher in random and aligned fibers, when compared to mature adipocytes . No signin vitro is increased however, when insulin is absent or very low, when compared to mature human adipocytes in vivoin vivo situation than cells differentiated on aligned fibers, when comparing glucose transport.In summary, the adipose derived stem cells differentiated on random fibers take up more glucose than mature adipocytes, both basally and after maximal insulin stimulation . The gluTo further investigate the impact of the different growth environments on differentiation of the cells, we performed gene expression profiling focusing on 20 genes central to adipocyte function . AdiposeInitial PCA analysis of the data set from the three groups with differentiated and undifferentiated cells clearly demonstrated that the donor-to-donor variation explained more of the differences in gene expression between samples, compared to the culture systems . The limThis data does show that there are no dramatic differences in expression levels of individual genes used as markers of adipocyte differentiation between the culture systems, especially compared to the normal variation between human subjects. The significant variation between human donors is a strong argument for using multiple donors when studying gene-expression. The fact that we find differences on the functional level and protein levels between cells on the 2D plates and the fiber matrices are unlikely to be due to changes in the expression profiles, but rather to differences in post transcriptional regulation. This clearly argues for the importance of not relying solely on gene-expression analysis for determination of maturation of cells, since small, undetected effects on global transcription might give rise to larger effects on the functionality when numerous proteins in a signaling network are involved. Further exploration of the relationship between gene expression and function in adipocytes is important but beyond the scope of this study.Despite the absence of dramatic differences on individual gene level, hierarchical clustering can be used to analyze which of the culture systems are most closely related, based on the expression profiles . UndiffeTaken together this data underlines the dramatic effect that differentiation itself has on gene expression, regardless of system, but also indicates that fibers induce some common, but small, transcriptional effects, regardless of orientation.To further investigate functionality of the differentiated adipocytes, lipolysis measured by glycerol release after isoproterenol stimulation was quantified. The dose response curve for isoproterenol stimulated lipolysis was similar for all differentiated adipocytes and mature adipocytes . This isThe levels of the rate-limiting enzyme; hormone sensitive lipase (HSL), were significantly higher on random fiber cultured and mature adipocytes in comparison to 2D cultured cells , whereasIn summary, we find decreased proliferation, increased lipid accumulation, increased HSL levels and more evolved glucose uptake in cells differentiated on PCL fiber matrices compared to conventional cell culture plastic. Potentially, the decreased proliferation can contribute to the increased maturation of the adipose derived stem cells on the fiber plates, as discussed above.The stiffness of the surface that human adipose derived stem cells adhere to during differentiation has also been coupled to the cells adipogenic potential; a surface mimicking the stiffness of adipose tissue increases adipogenesis in comparison to a harder surface There is a difference in how the matrices change the functionality in terms of glucose uptake and lipolysis, affecting one function and not the other. Potentially, lipid handling systems are affected more by environmental cues than the glucose uptake-regulating proteins.We investigated the effects on adipogenesis, of human adipose derived stem cells, being differentiated into adipocytes on PCL fiber matrices. We found decreased proliferation, increased lipid accumulation and a closer-to-mature adipocyte EC50 for insulin-stimulated glucose uptake in cells differentiated on aligned PCL fibers compared to 2D plates. Additionally, we found HSL levels and glucose uptake were higher in cells differentiated on random PCL fibers, compared to 2D. In summary these findings indicate enhanced maturation of adipose derived stem cells differentiated on PCL fibers, and we propose use of aligned PCL fiber plates for differentiation of adipose derived stem cells, when a model system is required for glucose uptake of human adipocytes.Figure S1Western blots for included antibodies. Example western blots for the included antibodies as indicated in the Figureure, dilutions can be found in the methods section. Loading control (actin) which was used for normalization is shown for each example blot.(EPS)Click here for additional data file.Figure S2Proliferation and viability of human adipose derived stem cells in PCL fiber matrices. A) Cells from donor 1 were seeded 43750 cells/cm2 (dotted lines), 56250 cells/cm2 (dashed lines) or 112500 cells/cm2 (solid lines) in 2D (black lines), aligned (green lines) or random (magenta lines) and differentiated the indicated times. Cells were fixed, stained and counted using the Cellavista. B) Cells seeded in 2D, aligned and random matrices as indicated, at indicated densities and differentiated the indicated times, were stained with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity and were imaged with a fluorescence microscope.(TIF)Click here for additional data file.Figure S3CARS images. 3D-image of 14-days differentiated human preadipocytes in aligned fibers. B) 3D-image of 14-days differentiated human preadipocytes in random fibers. In both images CARS signals are red-yellow and MPEF signals are blue. C) Cell sizes measured with CARS, n = 30 cells/condition.(EPS)Click here for additional data file.Table S1Gene-assay ID's.(DOCX)Click here for additional data file.File S1Contains cell location data in fiber matices.(XLSX)Click here for additional data file.File S2Contains all cell count, lipid content and cell size data.(XLSX)Click here for additional data file.File S3Contain example images for cell count.(EPS)Click here for additional data file.File S4Contains all glucose uptake and lipolysis data.(XLSX)Click here for additional data file.File S5Contains all western blot data.(XLSX)Click here for additional data file.File S6Contains gene expression data.(XLSX)Click here for additional data file.


Dystonia is a neurological disorder in which sustained muscle contractions induce twisting and repetitive movements or abnormal posturing. DYT1 early-onset primary dystonia is the most common form of hereditary dystonia and is caused by deletion of a glutamic acid residue (302/303) near the carboxyl-terminus of encoded torsinA. TorsinA is localized primarily within the contiguous lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), and is hypothesized to function as a molecular chaperone and an important regulator of the ER stress-signaling pathway, but how the mutation in torsinA causes disease remains unclear. Multiple lines of evidence suggest that the clinical symptoms of dystonia result from abnormalities in dopamine (DA) signaling, and possibly involving its down-stream effector adenylate cyclase that produces the second messenger cyclic adenosine-3′, 5′-monophosphate (cAMP). Here we find that mutation in torsinA induces ER stress, and inhibits the cyclic adenosine-3′, 5′-monophosphate (cAMP) response to the adenylate cyclase agonist forskolin. Both defective mechanins are corrected by the small molecule 4-phenylbutyrate (4-PBA) that alleviates ER stress. Our results link torsinA, the ER-stress-response, and cAMP-dependent signaling, and suggest 4-PBA could also be used in dystonia treatment. Other pharmacological agents known to modulate the cAMP cascade, and ER stress may also be therapeutic in dystonia patients and can be tested in the models described here, thus supplementing current efforts centered on the dopamine pathway. The DYT1 early-onset primary dystonia is the most severe form of hereditary dystonia and is caused by a mutation in the gene, TOR1A (DYT1), encoding torsinA protein To define therapeutic pathways to target dystonia, it is important to identify cellular processes affected in the brain. Our studies with dystonia animal models and fibroblast cells from DYT1 patients have implicated decreased intracellular levels of cAMP in these DYT1 dystonia models when compared to controls. Alterations of the second messenger cAMP affect normal brain function such as in synapses, neuronal memory, and striatal plasticity Dopamine is the pathway most extensively studied in dystonia. Genetic mutations of the GCH1 gene, which codes for guanosine triphosphate cyclohydrolase I (GTPCH I) Our previous studies suggest that the presence of torsinAΔE somehow contributes to ER stress load All human cell lines and protocols in the present study were carried out in accordance with the guidelines approved by institutional review boards (IRB) at Partners Human Research Committee (Protocol #: 2007P001632/MGH). Fibroblast cell lines received from Dr. Xandra Breakefield laboratory were previously collected through a Yale Medical School research protocol between 1974–1984. Individuals enrolled in this research protocol had skin biopsies taken, and signed consent forms. All animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Massachusetts General Hospital (MGH) Institutional Animal Care and Use Committee (IACUC) (Protocol Number: 2004N000271/MGH). All surgery was performed under anesthesia, and all efforts were made to minimize suffering.+/−) wt/ΔE) +/− and Tor1Awt/ΔE mice to generate timed pregnancies. The day of vaginal plug detection was considered embryonic day 0 (E0). Embryonic neuron cultures were prepared from each embryo on embryonic day 15 (E15). The genotype of each embryo was identified as reported previously Heterozygous Tor1A knockout mice  four days after being plated and the total media was changed after 24 hours. The density of immunoreactive cells positive for βIII tubulin , and GFAP (a marker of astrocytes) was performed in parallel at 5–8 days in vitro (DIV). Results showed that the majority of cells (99.5%) were positive only for βIII tubulin. Note that cultures from individual neurons (an average of 10 well in 96 plates per embryos) were maintained for 10 days, and were then used for cAMP assay.Mixed cortical and striatal neuron cultures were prepared from invidual E15 mouse embryos. Briefly, embryos were removed by Caesarean section from a pregnant mouse. Cortices and striata from embryonic mice were dissected and stored in ice-cold 1 x phosphate buffered saline (PBS). The cortices and striata of individual embryos were dissociated with 0.625% trypsin at 37°C in neurobasal media . Trypsin was removed and Dulbecco's Modified Eagle Medium plus nutrient mix F12  supplemented with 10% fetal bovine serum , N2 supplement and penicillin/streptomycin added to inactivate the trypsin. Cortices and striata were treated with 12.5 mg/ml DNase I  in DMEM/F12+ FBS for 5 min at 37°C and homogenized. Cell suspension was then diluted in NBM supplemented with with B27, 0.5 mM L-glutamine. The cells were counted and then plated on a 96-well plate coated with polyethyleneimine  in 0.15 M Borate Buffer at a density of 4×10MEF cultures were prepared as described Primary human skin fibroblasts of control fibroblasts  and of DYT1 patient's fibroblast (HF48) were generated in Dr. Breakefield ‘s laboratory as described previously SDS-PAGE electrophoresis and protein transfer were carried out as described Forskolin  was dissolved in DMSO at 10 mM concentration. Thapsigargin  was suspended in DMSO at 3 mM. The phenylbutyric acid or 4-phenylbutyrate  was dissolved in filtered sterile water at a 1 M stock concentration.4 cells/well (neurons) and 5×103 cells/well (fibroblasts) in 96-well plates. Fibrolast cells were incubated overnight before the assay, and neurons were culture for 10 days prior to the assay. After 30 minutes incubation with or without 10 µM forskolin, the cAMP levels were determined according to the manufacture's instructions. At least three independent experiments were performed for each assay. Data were normalized to the amount of protein in each sample, as determined using a dye-binding protein assay (Bio-Rad).We have optimized an ELISA cAMP assay  to determine cAMP intracellular levels in dystonia cell models. Briefly, neuron cells were seeded in a 4×10Neuronal and fibroblast viability was evaluated by CellTiter-Glo luminescent cell viability assay  to determine the number of viable cells in culture based on the quantitation of ATP levels, indicating the presence of metabolically active cells. Briefly, primary neurons and fibroblast cells were grown as described above and a volume of CellTiter-Glo Reagent was added to each well equal to the volume of cell culture medium. Then, the contents were mixed for 2 min on a shaker to induce cell lysis and the plates were incubated at room temperature for 10 min in the dark. Cellular luminescence intensity was measured using a Luminometer .12–18 primers, according to the manufacturer's instructions. RNA samples were treated with DNase I (Invitrogen) for the elimination of genomic DNA. Human spliced form of XBP1 mRNA expression  was determined by real-time qPCR using relative quantitation by the comparative threshold cycle number (Ct) method, iCycler, and SYBR Green Ready-mix  and normalized by glyraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Preliminary experiments were performed with each primer pair to determine the amplification temperature that provided an optimal correlation between template concentration and signal intensity. At least three independent experiments were performed for each assay.Total RNA of cells was extracted with RNeasy mini kit . The cDNA was prepared from total RNA using Superscript first-strand synthesis system (Invitrogen) with the oligo (dT)Statistical analysis was performed using GraphPad Prism software (version 3.0). Results are expressed as mean ± S.E.M. of at least three separate experiments unless otherwise indicated. Statistical significance of comparison between two groups was determined by two-tailed Student's t-test where indicated. Multiple comparison analyses of variance between the groups were performed by a one-way ANOVA test, followed by a Tukey's multiple comparison post hoc tests. Significant differences were considered at p values of less than 0.05.We first observed a potential relationship between mutant torsinA and adenylate cyclase during our studies using cholera toxin to examine the role torsinA might play in ERAD +/+), and homozygous (torsinA-/-) MEFs  MEFs , as wellnia mice . ResultsTo test whether these results translate to human cell models, we investigated whether cAMP levels were also altered in human fibroblast lines prepared from various dystonia DYT1 patients and healthy controls. At rest, there was no overall difference between control and DYT1 fibroblasts in basal levels of intracellular cAMP . After fWe previously reported that the absence of torsinA or the presence of malfunctioned torsinAΔE induces ER stress Furthermore, we investigated whether 4-PBA, a small molecule with chaperone-like activity, may decrease ER stress in our dystonia cell models. 4-PBA major function is to scavenge ammonia and glutamine In parallel experiments, gradual titrations of 4-PBA  were performed to assess the effects of PBA on ER stress in health fibroblast cells, and in DYT1 fibroblast cells. We observed, however, that treatment with concentrations 2.5 and 5 mM did not affect ER stress in health fibroblast cells, or in DYT1 fibroblast cells . HoweverHere, we addressed the involvement of torsinA in the response to ER stress and to the cAMP pathways. How these two pathways are linked mechanistically remains unclear, but it is possible though that the ER stress induced by mutant torsinA in patients with DYT1 dystonia contributes to neuronal dysfunction through a cAMP defect . In our Defective proteasome function and ER stress have a contributing role in various neurologic diseases Our findings linking ER stress and cAMP signaling in dystonia models, also highlights that there is much to consider about striatal signaling to cAMP and PKA. Benzodiazepines are frequently used in the treatment of dystonia. This drug has muscle-relaxant properties and alleviates pain caused by muscle spasms and various dystonias Numerous lines of evidence have linked dopaminergic dysfunction to dystonia Our results suggest that 4-PBA may have clinical utility in the treatment of dystonia. 4-PBA is a low molecular weight fatty acid already approved for clinical use as an ammonia scavenger in children with urea cycle disorders 4-PBA has high potential to treat neurologic diseases because it penetrates the blood–brain barrier, and exhibits significant neuroprotective effects in mouse models of neurodegenerative diseases, such as Alzheimer's disease (AD) The precise steps by which mutant torsinA leads to impaired cAMP accumulation are not known, but it may involve an ER dysfunction. Further studies are needed to test this hypothesis, and to determine the efficacy of treatment of DYT1 dystonia that targets both ER stress and cAMP cascade. More studies are also needed to elucidate whether 4-PBA is functioning as a HDAC inhibitor or as a molecular chaperone in our dystonia cell models. This will provide insightful information for the structural optimization of this compound. Nevertheless, therapies working through the ER stress and cAMP cascade in the striatum may have the potential to benefit dystonia patients by alleviating dystonic symptoms. Animal studies will determine whether 4-PBA can be used to treat, as well as prevent, DYT1 dystonia.Figure S1wt/wt, and heterozygotes torsinAwt/ΔE knock-in dystonia mice . Relative expression of sliced sXBP1 mRNA levels was measured by quantitative PCR. Results might indicate that the presence of torsinAΔE increases ER stress in neurons.Neurons expressing torsinAΔE has higher ER stress compared to control neurons. Total mRNA was prepared from a mixed neuronal culture of dissociated primary cortical and striatal at E15 from controls littermates torsinA(PDF)Click here for additional data file.Table S1Human fibroblast cells used in this study.(PDF)Click here for additional data file.


Corticosterone is known to accumulate in brain after various stressors including alcohol intoxication. Just as severe alcohol intoxication is typically required to impair memory formation only high concentrations of ethanol (60 mM) acutely inhibit long-term potentiation (LTP), a cellular memory mechanism, in naïve hippocampal slices. This LTP inhibition involves synthesis of neurosteroids, including allopregnanolone, and appears to involve a form of cellular stress. In the CA1 region of rat hippocampal slices, we examined whether a lower concentration of ethanol (20 mM) inhibits LTP in the presence of corticosterone, a stress-related modulator, and whether corticosterone stimulates local neurosteroid synthesis. Although low micromolar corticosterone alone did not inhibit LTP induction, we found that 20 mM ethanol inhibited LTP in the presence of corticosterone. At 20 mM, ethanol alone did not stimulate neurosteroid synthesis or inhibit LTP. LTP inhibition by corticosterone plus ethanol was blocked by finasteride, an inhibitor of 5α-reductase, suggesting a role for neurosteroid synthesis. We also found that corticosterone alone enhanced neurosteroid immunostaining in CA1 pyramidal neurons and that this immunostaining was further augmented by 20 mM ethanol. The enhanced neurosteroid staining was blocked by finasteride and the N-methyl-D-aspartate antagonist, 2-amino-5-phosphonovalerate (APV). These results indicate that corticosterone promotes neurosteroid synthesis in hippocampal pyramidal neurons and can participate in ethanol-mediated synaptic dysfunction even at moderate ethanol levels. These effects may contribute to the influence of stress on alcohol-induced cognitive impairment. Plasma levels of corticosterone, the major glucocorticoid in mice and rats, are increased by various types of stress, including alcohol intoxication. The rise in plasma corticosterone can result in increased corticosterone levels in brain  in the hippocampus is a cellular mechanism thought to underlie memory formation, and numerous studies have shown that stress can acutely and chronically impair both memory acquisition and LTP induction  modulate LTP and learning after various stressors  that enhance the function of γ-aminobutyric acid-A receptors  of the CA1 region for recording excitatory postsynaptic potentials (EPSPs) with electrodes filled with 2 M NaCl (5–10 MΩ resistance).For physiology studies, slices were moved to a submersion-recording chamber at 30°C with ACSF perfused at 2 ml/min. Extracellular recordings were obtained from the apical dendritic layer  pathway. Responses were monitored by applying single stimuli to the SC pathway every 60 s at half maximal intensity. After obtaining a control input-output curve and stable baseline recordings for 10 min, LTP was induced by a single 100 Hz × 1 s high frequency stimulation (HFS) at the same intensity stimulus. Input-output curves were repeated 60 min following HFS. In some experiments an IO curve was also repeated 20 min after HFS and appears as a gap in the graphs in Figures Slices for immunohistochemistry were screened for electrophysiological responses and were incubated with various reagents in separate 10 ml beakers as previously described . Finasteride was purchased from Steraloids . All other chemicals were purchased from Sigma Chemical Company . Finasteride and corticosterone were prepared as stock solutions in ethanol. Drugs were dissolved in ACSF at the time of experiment and administered by bath perfusion at the concentrations noted in the text. The concentrations selected for study and the durations of drug administration were based on prior studies examining their effects on synaptic transmission and synaptic plasticity in the absence of effects of baseline transmission in naïve slices.t-test was used for comparisons between groups. In cases of non-normally distributed data, the non-parametric Wilcoxon Rank Sum Test was used. Additionally, analysis of variance (ANOVA) was performed on between group means. Statistical comparisons were based on input-output curves at baseline and 60 min following HFS to determine the degree of change in EPSP slope at the 50% maximal point with p < 0.05 considered significant. Statistics were performed using commercial software .Data were collected and analyzed using PClamp software . Data are expressed as mean ± SEM 60 min following HFS, and are normalized with respect to initial baseline recordings (taken as 100%). A two-tailed Student’s N = 5, closed circles in Figure N = 3, data not shown). The degree of LTP in the presence of corticosterone did not differ significantly from control LTP in naïve slices .Because LTP inhibition by 60 mM ethanol involves local neurosteroid synthesis . A combination of 20 mM ethanol with corticosterone robustly enhanced neurosteroid immunostaining . A summary statistical analysis is shown in Figure p < 0.001 by one-way ANOVA). Cells in the pyramidal cell layer were positive with Dapi staining . The enhancement was clearly suppressed by both finasteride .To address possible mechanisms contributing to the effects of corticosterone and ethanol on neurosteroid synthesis, slices were pretreated with 1 μM finasteride or 100 μM D, L-amino-5-phosphonovalerate (APV) before administration of 1 μM corticosterone and 20 mM ethanol in a separate set of experiments. Again, the combination of corticosteroid and 20 mM ethanol enhanced immunostaining against 5α-reduced neurosteroids (Figure e Figure  and APV e Figure . A summaVarious chemicals and drugs including ammonia (Izumi et al., Numerous studies have shown that acute and chronic stressors can suppress LTP (Foy et al., stratum pyramidale were not positive for 5 α-reduced neurosteroids. We have previously observed that only some neurons in the pyramidal cell layer are immunopositive for neurosteroids when exposed to high ethanol or low micromolar NMDA (Tokuda et al., In our study, we found that exogenous corticosterone promotes LTP inhibition in the presence of 20 mM ethanol, but that neither agent was effective against LTP at the concentrations studied. This finding suggests that even more moderate alcohol consumption, in the absence of profound intoxication, may alter memory acquisition when corticosterone synthesis is increased by other stressors. Because the effects on LTP are blocked by finasteride, this LTP inhibition involves the synthesis of 5α-reduced neurosteroids including AlloP. Consistent with this, we found that corticosterone enhanced local neurosteroid immunostaining in the CA1 region. However, as we have shown previously, increases in neurosteroid levels alone are not sufficient to block LTP induction (Tokuda et al., The present study begins to provide information about mechanisms contributing to neurosteroid production in the presence of corticosterone and low ethanol. In prior work, we found that the ability of 60 mM ethanol to enhance neurosteroid production and to inhibit LTP was overcome by an NMDA receptor antagonist administered during the period of ethanol exposure (Tokuda et al., While we do not understand how ethanol and corticosterone interact in the hippocampus, the enhanced response in the presence of both agents could involve local production of corticosteroid or another messenger. Although it is unclear whether the brain can synthesize corticosterone locally, and prior work using ELISA measurements report the loss of corticosterone in rat brains after adrenalectomy (Gomez-Sanchez et al., YI and CZ designed the studies. YI and KO performed the experiments. YI and CZ analyzed data and wrote the manuscript. All authors approved the final manuscript.CZ serves on the Scientific Advisory Board of Sage Therapeutics. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ovarian cancer is the most lethal gynecologic malignancy and is the seventh leading cause of cancer deaths in women worldwide. Despite advances in surgery and chemotherapy, overall cure rate has remained very low. The poor clinical outcomes mainly come from the high percentage of cases being diagnosed at an advanced stage disease due to the lack of effective screening methods and frequent emergence of chemoresistance. Recent evidences have suggested that cancer stem cells may also contribute to the development of chemoresistance. However, there are still many questions on ovarian carcinogenesis and mechanisms of chemoresistance of ovarian cancer, which need to be resolved to improve the treatment outcomes of ovarian cancer.BRCA-associated ovarian cancer: from molecular genetics to risk management” by G. Girolimetti et al. demonstrated that ovarian cancer arising in BRCA 1 or 2 mutation carriers may have peculiar molecular, pathological, and clinical features. They also suggested that BRCA 1 or 2 mutational analyses would be helpful in tailoring ovarian cancer management based on BRCA status in the future.The research topics include molecular genetics of ovarian carcinogenesis, autophagic reaction in ovarian cancer, chemoprevention using phytochemicals, tumor heterogeneity issue, and dual carcinogenesis of the ovary (type I versus II). The paper entitled “Expression and clinical significance of the autophagy proteins BECLIN 1 and LC3 in ovarian cancer.”The work by G. Valente et al. showed that the positive expression of BECLIN 1 with a crucial role in the regulation of both autophagy and cell death was correlated with the presence of LC 3 positive autophagic vacuoles and was inversely correlated with the expression of BCL-2 inhibiting the autophagy function of BECLIN 1. They also suggested that a low level of autophagy might favor cancer progression and that ovarian cancer with upregulated autophagy might have a less aggressive behavior and be more responsive to chemotherapy in the paper entitled “Unique pattern of component gene disruption in the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in serous ovarian cancer.”The work by V. D. Martinez et al. assessed DNA copy-number loss (CNL), promoter hypermethylation, mRNA expression, and sequence mutation of KEAP1/CUL3/RBX1 complex as a regulator of the NFE2-related factor 2 (NRF2) pathway initiating response to oxidative stress in a cohort of 568 serous ovarian carcinomas form The Cancer Genome Atlas. They suggested that a remarkably high frequency of DNA and mRNA alterations may affect components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms in the paper entitled “Phytochemicals: a multitargeted approach to gynecologic cancer therapy” by L. Farrand et al. demonstrated molecular mechanisms of phytochemical action in cancer prevention and phytochemical-based approaches to overcome chemoresistance and phytochemical analogues and chemical modifications for greater efficacy. They suggested that high-throughput screening methods, rational modification, and developments in regulatory policies would accelerate the development of novel therapeutics based on phytochemical compounds, which would likely improve overall survival and quality of life for patients with gynecologic cancers.The paper entitled “ XRCC3 polymorphisms are associated with ovarian cancer risk in the paper entitled “Analyzing association of the XRCC3 gene polymorphism with ovarian cancer risk.” They found no association between XRCC3 rs861539 polymorphisms and ovarian cancer, whereas they observed a significant correlation with ovarian cancer risk using the homozygote comparison (T2T2 versus T1T1), heterozygote comparison (T1T2 versus T1T1), and the recessive genetic model (T2T2 versus T1T1 + T1T2). For XRCC3 rs1799796 polymorphisms, they also found a significant correlation with ovarian cancer risk using the heterozygote comparison (T1T2 versus T1T1).C. Yuan et al. performed a meta-analysis to examine whether theSingle amino acid arginine deprivation triggers prosurvival autophagic response in ovarian carcinoma SKOV3.” They also found that a significant drop in viability of arginine-starved SKOV3 cells was observed when autophagy was inhibited by either coadministration of chloroquine or transcriptional silencing of the essential autophagy protein BECLIN 1, suggesting that arginine deprivation-based combinational treatments that include autophagy inhibitors may produce a stronger anticancer effect as a second line therapy for a subset of chemoresistance ovarian cancers.G. Shuvayeva et al. demonstrated that single amino acid arginine deprivation triggered profound prosurvival autophagic response in cultured human ovarian cancer SKOV3 cells in the paper entitled “Epigenetic control of autophagy by microRNAs in ovarian cancer.” Furthermore, they emphasized that plasma and stroma-cell derived miRNAs in tumor-bearing patients could impact autophagy.The work by R. Titone et al. demonstrated that the mRNAs of several autophagy-related genes contain the target sequence for miRNAs belonging to different families with either oncosuppressive or oncogenic activities in the paper entitled “Recent concepts of ovarian carcinogenesis: type I and type II.” In this review, they demonstrated that low grade serous carcinomas may be thought to evolve in a stepwise fashion from benign serous cystadenoma to a serous borderline tumor while the serous tubal intraepithelial carcinomas of the junction of the fallopian tube epithelium with the mesothelium of the tubal serous undergo malignant transformation to high grade serous carcinomas due to their location and metastasize to the nearby ovary and surrounding pelvic peritoneum.The work by M. Koshiyama et al. mentioned a recent theory of dual carcinogenesis of the ovary in the paper entitled “Application of microRNA in diagnosis and treatment of ovarian cancer” by K. Banno et al. suggested that many miRNAs have altered expression in ovarian cancer compared to normal ovarian tissues and these changes may be useful for diagnosis and treatment. Thus, they expect that chemotherapy targeting epigenetic mechanisms associated with miRNAs may also be effective to reverse gene silencing.The paper entitled “Expression profiles of epithelial-mesenchymal transition-associated proteins in epithelial ovarian carcinoma” by M.-K. Kim et al. investigated the expression of Snail and Slug, the key regulators of epithelial-mesenchymal transition (EMT), in the primary ovarian cancer samples to assess the clinical significance of EMT-associated proteins. They found that Snail was differentially expressed according to the histologic subtype and was predominantly expressed in serous and endometrioid types. In the serous and endometrioid adenocarcinomas, the expression of Snail remained high across the stage and grade, suggesting its role in the early phase of carcinogenesis.The paper entitled “Oxidatively modified proteins in the serous subtype of ovarian carcinoma.” They showed that the levels of reactive protein carbonyl groups increased as stages progressed to malignancy, and the levels of protein carbonyls in serous ovarian carcinoma among African Americans are 40% higher reactive to Caucasian at similar advanced stages.S. Mehrabi et al. assessed the levels of oxidative modified proteins in 100 primary serous epithelial ovarian carcinomas and normal/surrounding tissues using spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two-dimensional gel electrophoresis, and Western blot analyses in the paper entitled “In summary, molecular genetics and autophagic reaction in ovarian carcinogenesis, multitargeted approaches using autophagic reaction and phytochemicals, and dual approaches considering types I and II ovarian carcinogenesis are of paramount importance. This special issue presents new perspectives on carcinogenesis and chemoresistance of ovarian cancer, which will be helpful in overcoming the limitations of diagnosis and treatment of ovarian cancer in the future.Yong Sang SongHee Seung KimDaisuke AokiDanny N. DhanasekaranBenjamin K. Tsang


It is well established that gross prefrontal cortex damage can affect an individual’s personality. It is also possible that subtle prefrontal cortex changes associated with conditions such as chronic pain, and not detectable until recent advances in human brain imaging, may also result in subtle changes in an individual’s personality. In an animal model of chronic neuropathic pain, subtle prefrontal cortex changes including altered basal dendritic length, resulted in altered decision making ability. Using multiple magnetic resonance imaging techniques, we found in humans, although gray matter volume and on-going activity were unaltered, chronic neuropathic pain was associated with reduced free and bound proton movement, indicators of subtle anatomical changes, in the medial prefrontal cortex, anterior cingulate cortex and mediodorsal thalamus. Furthermore, proton spectroscopy revealed an increase in neural integrity in the medial prefrontal cortex in neuropathic pain patients, the degree of which was significantly correlated to the personality temperament of novelty seeking. These data reveal that even subtle changes in prefrontal cortex anatomy may result in a significant change in an individual’s personality. It is well established that gross prefrontal cortex damage can alter an individual’s personality. Less recognized is the possibility that in individuals with conditions such as chronic pain, subtle changes in prefrontal cortex anatomy associated with the condition, may also result in subtle changes in an individual’s personality. The lack of recognition of a connection between personality and brain anatomy, likely stems from the fact that until recently, it was almost impossible to assess subtle changes in brain anatomy in living humans. The recent use of more sensitive brain imaging techniques has revealed that chronic pain is associated with subtle anatomical alterations in the thalamus, insular and cingulate cortices It has been proposed that cognitive changes associated with chronic pain may result from alterations in activity within cortical areas such as the prefrontal cortex The study aims to explore anatomical and biochemical changes within the mPFC and related brain regions in individuals with chronic pain and to determine if any changes are associated with an individual’s personality. Diffusion tensor imaging (DTI) and T2-relaxometry was used to detect subtle changes that indicate alterations in the physical organization and myelination and/or development of axons and dendrites Twenty two subjects with chronic pain  and 43 healthy controls without ongoing pain  were recruited at the University of Sydney, Australia. All chronic pain subjects were diagnosed with trigeminal neuropathy according to the Liverpool criteria Each chronic pain patient rated their on-going pain intensity with a vertical pencil stroke on a 10 cm horizontal line  three times a day for the week prior to the scanning session. These pain values were averaged to provide an indication of each subject’s chronic pain rating. Each chronic pain subject also completed a McGill Pain Questionnaire and drew a distribution map of their on-going pain.To assess individual subject’s temperament, 19 chronic pain  and 30 control subjects  completed the revised version of the temperament and character inventory (TCI-R) 2) covering the entire brain were collected using a 3T Philips Intera machine. Three T1-weighted images and 4 DTI series were collected separately for subsequent averaging. In 39 of the 43 control and 19 of the 22 chronic pain subjects, proton density and T2-weighted images covering the entire brain were also collected . Multiple echo times were used to create multiple images for the subsequent calculation of T2-relaxation maps. In 24 controls and 18 chronic pain subjects, a quantitative arterial spin labelling (QASL) series, encompassing the entire brain was collected  In all subjects, three 3D T1-weighted anatomical image sets  and four high-resolution diffusion tensor image sets  and 11 neuropathic pain subjects  returned for the second scanning session, the remaining subjects declined to return. Proton magnetic resonance spectroscopy (1H-MRS) was performed on the contralateral (to pain) mPFC and on the right mPFC in control subjects . The location of the region selected was based on the diffusion and T2 relaxometry results. Automatic shimming (pencilbeam auto first order option) was performed resulting in line widths of <10 Hz for all spectra. There were no significant differences in age  or gender composition  in any of the MRI analyses.All images were processed using SPM8 2 = (TE2–TE1)/ln(SI1/SI2) where TE1 and TE2 are the echo times for proton density and T2-weighted images, and SI1, SI2 represent proton density and T2-weighted image signal intensities, respectively. A ceiling threshold of 500 ms was applied to eliminate cerebrospinal fluid. In each subject, one of the T2-weighted images (TE = 60 ms) was spatially normalized and the parameters applied to the T2 map. The spatially normalized T2 map was smoothed (6 mm FWHM) and significant differences in T2 relaxation times between controls and chronic pain subjects determined . T2 relaxation times of significantly different clusters were extracted and significance verified . Significant correlations between T2 relaxation times and pain intensity, pain duration, and temperament values (novelty seeking etc) were determined (p<0.05). Significant differences in r-values in chronic pain compared with controls were determined using Fisher r-to-z transformation (p<0.05).The following equation was used to calculate T2 brain maps: TT1-weighted anatomical image sets were coregistered to one another, averaged, modulated, spatially normalized, segmented and smoothed (6 mm FWHM). Gray matter volumes from significant mean diffusivity and T2 relaxation clusters were extracted and compared between control and chronic pain subjects .QASL images were opened MRS data were analyzed in the time domain using the Java-based magnetic resonance user’s interface . The dominant water resonance was removed using the Hankel Lanczos Singular Valve Decomposition algorithm. All metabolite resonances were quantified using QUEST (containing a 29 ms TE metabolite basis set including N-acetylaspartate (NAA), Creatine Chronic pain subjects had on-going orofacial pain with mean pain intensity of 4.0±0.4 and mean pain duration of 5.7±0.9 years . ChronicChronic pain subjects had reduced T2 relaxation times in the contralateral (to pain) subgenual anterior cingulate cortex (ACC), contralateral ACC and medial prefrontal cortices, ipsilateral posterior insula, contralateral medial thalamus and primary somatosensory cortex bilaterally , Table 2Voxel based morphometry of T1-weighted anatomical images revealed that chronic pain was not associated with significant gray matter volume changes within the mPFC, ACC, mediodorsal thalamus, dorsal insula, or primary somatosensory cortex. Similarly, assessment of baseline regional cerebral blood flow using QASL revealed no significant difference in cerebral blood flow in chronic pain subjects compared with controls in the mPFC, ACC, mediodorsal thalamus, dorsal insula or primary somatosensory cortex , Table 2Finally, to assess biochemical changes within the mPFC, proton spectroscopy was performed. Within the contralateral mPFC, spectral analysis revealed that chronic pain subjects had significant elevated levels of N-acetyl aspartate/Creatine (NAA/Cr) compared to controls  . No signWithin the mPFC, ACC and thalamus, T2 relaxation times and mean diffusivity values in chronic pain subjects were not significantly correlated with either on-going pain intensity or pain duration. In contrast, within the mPFC and ACC, T2 relaxation times were positively correlated to novelty seeking  . That isIn chronic pain subjects NAA/Cr ratios within the mPFC were negatively correlated to novelty seeking (r = −0.55; Our results reveal that chronic pain is associated with significant anatomical changes within brain regions known to underpin an individual’s personality traits, i.e. the mPFC, ACC and mediodorsal thalamus. Strikingly, in chronic pain subjects only, the anatomical and biochemical changes within the mPFC and ACC were significantly correlated to the personality temperament of novelty seeking, with greater anatomical change associated with reduced novelty seeking. These data suggest that subtle change in brain anatomy may alter an individual’s personality characteristic.Metz and colleagues Although taken alone, the mean diffusivity and T2 relaxation changes in our chronic pain subjects may reflect alterations in glia or blood vessels, our spectroscopy data reveals that these changes are also associated with increased NAA and no change in myoinositol. NAA occurs almost exclusively in neurons and is widely accepted as a marker of neuronal viability and synaptic health It is becoming increasingly clear that chronic pain is characterized not only by an ongoing sensory and emotional experience, but also significant changes in an individual’s cognitive ability. For example, it has been reported that chronic pain subjects display impaired decision making in the Iowa Gambling Task, making disadvantageous choices that gained high immediate monetary returns at the risk of higher future losses, i.e., they were less sensitive to future consequences The mPFC and ACC receive dopaminergic inputs directly from neurons within the ventral tegmental area. In experimental animals, ventral tegmental area lesions also result in cognitive deficits such as decreased exploration of environmental stimuli and reduced investigative behaviour Our data reveal that subtle neural changes in the mPFC and ACC of individuals with chronic pain are associated with subtle differences in an individual’s personality profile. Of course it is well established that gross anatomical alterations in the prefrontal and cingulate cortices can drastically alter an individual’s personality; the case of Phineas Gage is a prime example. Although our data cannot implicate causality, it may provide an indication that even subtle brain alterations can change an individual’s personality. Interestingly, in healthy individuals the temperament Persistence has been shown to be associated with an overlapping circuit including the ACC, ventral striatum and the lateral orbital and mPFC