FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators.FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively.We assessed FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes. Of interest, FTO variant rs8050136, located ∼270-kb proximal to FTO (RBL2) gene expression. RBL2 is a key regulator of entry into cell division that may also play a role in adipogenesis FTO is widely expressed in human fetal and adult tissues, particularly the brain FTO gene in mice reduced white adipose tissue mass and adipocyte size, and protected from diet induced obesity In vitro studies and crystal structure analyses suggested a DNA/RNA demethylase function for FTO as a potential mechanism FTO in the development of obesity on the central and/or peripheral level. So far, there are only sparse data on how metabolic factors might modulate FTO expression in adipose tissue.Previously, various genes including the NAMPT expression by dexamethasone and isoproterenol in murine 3T3-L1 adipocytes has been reported NAMPT transcription by metabolic regulators in human preadipocytes compared to adipocytes still remains to be elucidated.NAMPT, also known as pre B-cell colony enhancing factor or “visfatin”, is another novel obesity related factor and has been shown to play a role in glucose homeostasis FTO expression during human adipocyte differentiation to assess whether the association of FTO with obesity may be directly related to adipogenesis. Furthermore, we investigated a potential transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators.Taken together, data from the literature suggest that FTO and NAMPT represent two novel candidates potentially involved in the pathophysiology of obesity and associated metabolic disorders. The regulation of these two factors in adipocytes remains less clear. We, therefore, aimed to evaluate Experiments were performed using the Simpson-Golabi-Behmel syndrome (SGBS) cell model which was a generous gift from M. Wabitsch . Cells were cultured in basal SGBS medium consisting of DMEM/Ham F12 medium  supplemented with 33 µM biotin and 17 µM pantothenic acid. Cells were differentiated into mature adipocytes as previously described Stimulation experiments were performed in SGBS preadipocytes and mature adipocytes at day 10 post-induction under serum-free conditions. Confluent preadipocytes were serum starved for 24 h and then cultivated in SGBS basal medium supplemented with either 100 nM insulin or 100 nM dexamethasone, 100 nM IGF-1 or 10 µM isoproterenol for additional 24 h for. For stimulation experiments in adipocytes we applied two different experimental approaches: i) the adipocyte cell medium was directly replaced by basal SGBS medium containing respective stimulants (see above) for 24 h (unstarved from adipogenic supplements) and ii) adipocytes were starved from adipogenic supplements in SGBS basal medium for 24 h prior to stimulation. Untreated cells incubated in basal SGBS medium for 24 h served as control (C). For stimulation with D-glucose the cell culture medium was replaced by DMEM adjusted to 0, 2.2, 4.4, 8.8, 17.5 or 35 mM D-glucose and supplemented with 33 µM biotin and 17 µM pantothenic acid for 48 h. Preadipocytes and adipocytes cultured for 48 h in DMEM adjusted to 17.5 mM glucose, which is consistent with the glucose concentration in basal SGBS medium, served as control.6] primers. FTO, RBL2, NAMPT and peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression was quantified by real-time PCR with TaqMan probe-based gene expression assay on the ABI 7500 Sequence Detection System . The housekeeping genes Hypoxanthine-guanine phosphoribosyltransferase (HPRT), ß-actin (ACTB), and TATA-box-binding protein (TBP) were quantified simultaneously. Sequence information of primers and probes is given in the FTO, RBL2, NAMPT and PPARγ in preadipocytes (d0), in untreated cells (C) or in cells incubated in 17.5 mM glucose was set 1.Total RNA was extracted using the RNeasy Mini Kit  according to the manufacturer's instructions. Reverse transcription was performed using 200 U M-MLV reverse transcriptase per 500 ng total RNA  with random hexamer [p(dN)t-test. The threshold for statistical significance was set at P<0.05. Statistical analyses were performed using Graph Pad Prism 4 .Data are presented as means ± SEM of at least 3 independent cell culture experiments, each performed in triplicates. Expression during adipogenesis was analyzed applying one-way ANOVA with repeated measurements and Dunnett's post test. The differences between means in stimulation experiments were analyzed by Student FTO mRNA expression is modulated during human adipocyte differentiation. Over the differentiation period of 12 days, FTO mRNA levels remained unchanged in the SGBS cell model , while incubation with insulin, dexamethasone and isoproterenol did not modulate FTO mRNA level (data not shown). Altering glucose concentrations did not affect FTO expression in preadipocytes or adipocytes .A levels . In adipA levels . In adipNAMPT expression, but complete glucose restriction stimulated NAMPT mRNA expression by 5.1±1.2 fold and 1.4±0.1 fold in preadipocytes and adipocytes, respectively (Increasing glucose concentrations had no impact on ectively .FTO and NAMPT as two novel candidates potentially involved in the pathophysiology of obesity and metabolic sequelae by metabolic regulators at the level of adipocytes.In this study, we evaluated the potential regulation of FTO is not significantly affected by adipocyte differentiation or metabolic regulators related to glucose and insulin metabolism in our cell models of human subcutaneous adipocytes. Variants in the FTO gene have been unequivocally and robustly associated with obesity risk FTO expression during human adipocyte differentiation. FTO mRNA expression was, however, not modulated during differentiation of preadipocytes into mature adipocytes suggesting that FTO expression in adipocytes may not be related to increasing adipose tissue mass. Also, mRNA levels of RBL2, that was suggested to mediate the biological consequences of a FTO variant on obesity risk PPARγ expression during adipogenesis confirmed efficient adipocyte differentiation, hence excluding a bias by suboptimal adipogenesis. Our results somewhat contrast the findings of previous studies demonstrating that FTO expression is decreased during differentiation of primary preadipocytes isolated from human subcutaneous adipose tissue γ after day 10–12 FTO expression at day 14 post induction in the study of Tews et al. FTO during adipogenesis in adipocytes obtained from other fat depots such as visceral adipose tissue still needs to be elucidated.We show that expression of the “obesity gene” FTO in the development of obesity was proposed by studies in mice showing that Fto deficiency resulted in reduced body weight and white adipose tissue mass A potential role for FTO expression in preadipocytes and adipocytes to mimic different metabolic states of energy excess and energy deficiency in preadipocytes and adipocytes. However, neither glucose or insulin nor dexamethasone or isoproterenol did affect FTO gene expression. Our results hence indicate that nutritional status does not influence FTO expression at the level of adipocytes in our models. FTO is widely expressed across multiple tissues including adipose tissue, liver, pancreas, and skeletal muscle. The highest abundance of FTO mRNA was, however, found in the brain, particularly in the hypothalamus FTO expression is nutritionally regulated in these hypothalamic regions FTO acts on the level of the central nervous system mainly in the arcuate nucleus of hypothalamus.To further evaluate the interaction with metabolic regulators, we investigated the impact of glucose, insulin as well as dexamethasone and isoproterenol, representing exogenous compounds that affect the stress axis, on FTO regulation by nutritional status. But the regulation of FTO by metabolic factors in adipocytes derived from different adipose tissue depots is still elusive.Overall, these results indicate that the brain rather than adipocytes are the primary site of NAMPT expression in human preadipocytes. In line with this, dexamethasone increased NAMPT mRNA levels in murine 3T3-L1 adipocytes NAMPT expression in the murine model NAMPT has been found to play an important role in the regulation of glucose metabolism, primarily through effects on beta cell function NAMPT expression was more pronounced in preadipocytes compared to adipocytes in our study. There are several possible explanations for that. First, NAMPT is mildly increased during adipogenesis Of interest, the effect of dexamethasone and isoproterenol on NAMPT mRNA levels. Complete glucose restriction, on the other side, induced NAMPT gene expression. Again, this effect was more pronounced in preadipocytes compared to adipocytes. Also other studies have reported that NAMPT expression is induced by glucose restriction and fasting Considering that NAMPT release has been reported to be enhanced by glucose in adipocytes NAMPT expression in subcutaneous adipocytes is not regulated by high insulin and glucose concentrations that represent common features of insulin resistance and type 2 diabetes. Interestingly, the stress axis affecting compounds dexamethasone and isoproterenol, that are known to induce insulin resistance in adipocytes NAMPT transcription. Our results indicate that NAMPT mRNA levels are enhanced in response to cellular stress but appear to be not regulated by nutrients in human adipocytes in vitro.In contrast to glucose, insulin had no effect on NAMPT synthesis in our cell model, which is consistent with data from studies in murine as well as primary human adipocytes FTO and NAMPT can not be generalized to the level of adipose tissue.A potential limitation of our study is that the SGBS cell model is a cell line that may not necessarily respond the same as normal adipocytes. Hence, our results on action and regulation of FTO gene expression is not related to differentiation and maturation of human adipocytes. Furthermore, we show that FTO expression is not regulated by factors known to impact glucose metabolism. NAMPT expression was induced by stress axis affecting compounds and complete glucose restriction supporting a potential role in compensation of cellular stress response in preadipocytes and adipocytes.In summary, we demonstrate that


Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown.Transmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids . The geographical range of bighorn sheep (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R154Q171 genotype). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R154Q171 genotype sheep.We acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.Our results indicate the  Ovis canadensis; BHS) were widely distributed and abundant in western North America [Mannheimia haemolytica, from domestic sheep (Ovis aires) to BHS and between BHS herds has caused numerous mortality events and has been modeled under experimental conditions [Odocoileus virginianus), mule deer (O. hemionus), wapiti  or moose  may also share disease with BHS [Prior to human settlement, bighorn sheep  . ScrapieC) is converted to a misfolded form (PrPTSE) that accumulates in the nervous system, is associated with disease and is a primary component of the infectious prion agent [C is completely degraded following PK treatment whereas the protease cleaves only the N-terminus from PrPTSE and leaves an infectious core intact [res) in a sample is a common diagnostic indicator of TSE infection [The infectious agent responsible for TSEs is thought to be a misfolded protein, termed a prion. During infection, the normal, host cellular prion protein  and the conversion efficiency ratio (CER) assay have all been used to assess TSE species barriers [C substrates are used in conversions [C in the substrate converted to PrPres by PrPTSE[C substrate is processed at pH 3.5 and can be converted to PrPres following incubation with any species of PrPTSE. Comparing PrPres levels in these two substrates provides a measure of the species barrier. Li et al. introduced this assay in an effort to predict the species barriers of numerous animals to wapiti CWD [Prnp genotype of 28 BHS in a tissue library and find no evidence of TSE in these tissues. We confirm that the CER assay correctly predicts known species barriers of laboratory mice to various TSEs and go on to use this assay to assess PrPC conversion in BHS substrates by domestic sheep classical scrapie, transmissible mink encephalopathy (TME) and CWD.For interspecies TSE transmission, the degree to which host PrPke place . While abarriers -23. In tversions . One of Prnp). A previous report and entry in GenBank (AAD48030.1) indicated that the prion protein from a group of BHS has the same amino acid sequence as scrapie-susceptible A136R154Q171 genotype domestic sheep [We isolated genomic DNA from 28 BHS in our tissue library and sequenced their prion protein gene .The sequence identity between domestic sheep and BHS prion proteins allowed us to use reagents tested in domestic sheep for analysis of prion protein in BHS tissues. We tested the BHS in our tissue library for evidence of preexisting TSEs by examining PK-treated brain homogenates for the presence of PrPg Figure A. Samplet Figure A, indicares in our library of BHS brain tissue, we screened retropharyngeal lymph nodes from the 28 BHS using the BioRad TeSeE ELISA test, an assay commonly used to test domestic sheep for scrapie. We found no positive samples (data not shown), consistent with the idea that our BHS tissues do not harbor preexisting TSEs.Retropharyngeal lymph nodes are among the first sites where abnormal prion protein can be detected in some preclinical TSEs and testing this tissue is useful in diagnosing domestic sheep scrapie . DespiteC substrates and TSE agents that have been used in previous mouse bioassays. Specifically, we used the RML strain of mouse-passaged scrapie, an agent adapted to passage in mice [The species barrier of laboratory mice to most TSEs is well-characterized . For tha in mice , domesti in mice  and CWD  in mice ,29.C substrates with these TSE agents and assessed conversion of PrPC to PrPres  agent was used as a control because there are multiple sequence differences between mink and BHS prion proteins . Both isolates of CWD caused PrPC to PrPres conversion in white-tailed deer .We calculated CER values by evaluating PrPC by PrPTSE from CWD and TME. We subjected dilutions of classical scrapie, white-tailed deer CWD and TME in A136R154Q171 genotype domestic sheep PMCA substrate to cyclic sonication . Additionally, the assay does not use radiation, reaction conditions are identical regardless of species or strain of TSE agent and, in our hands, the CER assay is robust and forgiving. Disadvantages of the assay include poor sensitivity compared to PMCA precluding the use of CER as a means to detect PrPTSE, the PrPres product of conversion reactions is not known to be infectious and, importantly, the CER assay is less well-established than other methods of assessing species barriers which makes interpreting reductions in conversion ratios in the absence of other corroborating data difficult. For example, the correlation between a 50% CER and TSE transmission parameters  following experimental challenge remains undefined and further work is needed to characterize this assay for use in species where bioassay data are not available. Nonetheless, in our current study we did find a similar pattern of PrPres formation when either CER or PMCA was used for conversion. Further studies comparing the two techniques is an interesting future direction.Using the CER assay, we found evidence that this method recapitulates known species barriers of laboratory mice to TSEs and data to suggest that BHS could be susceptible to classical scrapie and CWD, and less susceptible to TME. Our present investigation and previous studies by others ,24 sugge136R154Q171 sheep prion proteins, we must consider scrapie a potential risk to BHS. Efforts to keep domestic sheep and BHS separated, as are prudent to prevent transmission of other pathogens from domestic sheep to BHS [Ovis orientalis), another species of wild sheep [We are not aware of any studies examining natural transmission of scrapie from domestic sheep to BHS, but in light of the sequence identity of BHS and domestic Ap to BHS , are likld sheep .Prnp genotype V136R154Q171 have been considered to be most -susceptible to classical scrapie and selective breeding efforts have focused on reducing the numbers of these animals in domestic sheep flocks across the U.S. as a scrapie risk reduction measure. Recent research, however, by Gonzalez et al. strongly suggests that Prnp genotype of the recipient sheep is not the sole factor determining its scrapie susceptibility in vivo[Prnp genotype alone cannot account for the diversity of disease phenotypes observed and that the “scrapie phenotype in sheep results from a complex interaction between source, donor and recipient factors” [Prnp genetics using more geographically disparate samples.In Figure y in vivo. In carrfactors” . The susC to PrPres in either CER assays  have a lengthened CWD incubation period [132 appears to limit CWD, but not classical scrapie, susceptibility in a transgenic mouse model [C from BHS. In previous studies, we have also found that voles, which have mismatches in the “rigid loop” portion of the protein, are susceptible PrP misfolding and infection by cervid CWD [The finding that white-tailed deer CWD agent could convert sheep PrPs Figure  or PMCA s Figure  was notas Figure , includin period  and L132se model . Despitervid CWD ,38.et al. found that A136Q171 domestic sheep PrPC was not especially-well converted by CWD agent, but was the non-cervid substrate, among six tested species, that yielded the most PrPres[et al. intracerebrally challenged eight domestic sheep of various Prnp genotypes with mule deer CWD [136R154Q171) and one preclinical sheep (homozygous A136R154Q171) were identified at the conclusion of the study, indicating that sheep can be infected by CWD, although transmission is not especially facile.Previous work on the species barrier of sheep to CWD has been equivocal. Using cell-free conversion assays, Raymond st PrPres. In an adeer CWD . One cliin vivo work by Béringue et al. which indicate that V136R154Q171 ovinized transgenic mice challenged with wapiti CWD harbor high levels of splenic PrPres, indicating that sheep PrPC is susceptible to misfolding by CWD agent [et al., suggest that the lack of CWD transmission to BHS was not due to inability of BHS PrPC to be misfolded by CWD agent, but must derive from other factors.The results of our conversion assays appear to be supported by WD agent . At leasPrnp genes and our evidence that white-tailed deer and wapiti CWD can convert BHS PrPC to PrPres, we expect CWD from the various species to behave similarly. In a previous report, Li et al. found less PrPres generation in domestic sheep substrates when templated by wapiti CWD [res levels in sheep substrates.In our investigation, we used white-tailed deer and wapiti CWD, but have not yet investigated BHS prion protein conversion by mule deer CWD. Given the sequence similarity among cervid piti CWD  than we Prnp genotype. Our conversion reactions suggest that the species barrier protecting BHS from CWD may not be large and further studies, including in vivo experiments, are warranted. These animal challenge studies need not necessarily be performed in BHS, but could rather use Prnp genotype A136R154Q171 domestic sheep or existing transgenic mouse models [The results from our study suggest that the CER assay has the potential to be a useful tool to screen TSE species barriers. Further comparisons with PMCA and bioassays will clarify the best uses of the assay and help to define CER that are < 100%. We found that BHS are unlikely to have resistance to domestic sheep classical scrapie due to their e models . AdditioChemicals and other reagents were of the highest quality possible and were from either Sigma-Aldrich  or Fischer Scientific , unless otherwise specified.Animal work conducted at the National Wildlife Health Center was performed under institutional animal care and use committee protocol #EP080716. Brains from CD-1 Swiss mice intracerebrally-challenged with RML-strain TSE agent and Syrian hamsters intracerebrally-challenged with 263K TSE agent were used for control experiments. Healthy Swiss Webster mouse brains were purchased from Pel-Freez Biologicals  and healthy hamster brains were obtained from unchallenged animals.Prnp A136R154Q171 genotype (Genbank accession number: AAW88328.1) ewe maintained at the University of Idaho Caine Veterinary Teaching Center. Naïve control sheep brain was from Prnp A136R154Q171 genotype ewes from a certified scrapie-free facility acquired from Ovis Sheep . Isolates of CWD from clinically-ill white-tailed deer  or wapiti  were acquired from the Wisconsin Department of Natural Resources or the USGS Chronic Wasting Disease Positive Tissue Bank [96 genotype) was from a hunter-harvested animal acquired in 2010 in Vilas County, Wisconsin. Brain from a transmissible mink encephalopathy (TME)-infected mink (Neovision vision) was from an animal experimentally infected with the agent and control mink tissue was the generous gift of Dr. Maria Shank (Wisconsin Veterinary Diagnostic Laboratory). All infected brain was homogenized in phosphate buffered saline (PBS) at 10% w/v using separate Dounce homogenizers and stored at −80°C until use. All control brains tested negative for PrPres by immunoblot.Bighorn sheep brain tissues and retropharyngeal lymph nodes were collected from 28 animals of mixed gender and age from two herds culled in the state of Washington in 2010 by the United States Department of Agriculture . Sheep bsue Bank , respectPrnp gene [Prnp genes was performed using published primers and protocols [Genomic DNA from BHS was isolated using a Genomic DNA Tissue Micro-Prep kit  and was used to template PCR reactions using reagents from Marligen Biosciences  and the forward primer 5‘-TACGTGGGCATATGATGCTG-3‘ and the reverse primer 5‘-CTATCCTACTATGAGAAAAATGAG-3‘ specific to the extreme 5’ and 3’ regions of the coding sequence of the domestic sheep rnp gene . Sequencrotocols . Amplicorotocols .μg∙ml-1 for 1 hr at 37°C and subsequently prepared for NuPAGE and immunoblotting. To test the sensitivity of our immunoblot, a dilution series of 10% classical scrapie brain homogenate was prepared in 10% BHS brain homogenate, PK digested at 50 μg∙ml-1 for 1 hr at 37°C and prepared for NuPAGE and immunoblotting. Retropharyngeal lymph nodes from BHS were analyzed at the Wisconsin Veterinary Diagnostic Laboratory using the TeSeE ELISA kit and standard procedures . Brains from BHS used for conversion efficiency ratio assay substrates were also screened using PMCA as described in the PMCA section.Tissue from the obex region of the medulla oblongata of BHS was homogenized in PBS (10% w/v) and subjected to PK digestion at 50 in vitro conversion assay was prepared using the method described in Zou et al. [g for 30 min at 4°C. Pellets were resuspended in conversion buffer , briefly sonicated in a cuphorn sonicator, and the resultant conversion assay substrate was stored at −80°C until use.Uninfected brain homogenate used in the u et al. . Brieflyμl MAXYMum Recovery low binding PCR Tubes  by adding 5 μl of 10% infected brain homogenate to 95 μl of uninfected brain substrate that was previously denatured at either pH 3.5 or 7.4 as described earlier. Control samples lacking brain substrate contained only TSE agent and conversion buffer. Tubes were vortexed briefly then placed in a 96-well PCR tube thermo-shaker  for 24 hr at 37°C at 1000 rpm. After shaking, reactions were treated with sarkosyl to a final concentration of 2% w/v and digested with PK (100 μg∙ml-1) for 1 hr at 37°C. We found that adding sarkosyl to samples post-incubation aided PK digestion of prion protein in unseeded substrates (data not presented). Samples were then prepared for NuPAGE and immunoblotting. Assays with BHS substrate were performed using at least two individual animals and all assays were individually replicated at least three times.Conversion reactions were performed in 200 136R154Q171 genotype domestic sheep or BHS medullas homogenized to 10% w/v using Dounce homogenizers in ice-cold conversion buffer composed of PBS with 150 mM NaCl, 1% Triton X-100, 4 mM EDTA and mini-Complete protease inhibitor  at pH 7.4. Classical scrapie, white-tailed deer CWD or TME agents were diluted into the domestic sheep substrate at dilution factors of 10-2 or 10-3 of a 10% w/v brain homogenate. Substrate from BHS was left unseeded to test for pre-existing PrPSc. Samples were either frozen at −80°C or immediately subjected to PMCA with bead enhancement [μg∙ml-1) for 1 hr at 37°C prior to immunoblotting. Experiments were independently replicated twice and negative control samples lacking TSE agents were subjected to PMCA and were included in each experiment.Procedures for PMCA were based on those previously published for domestic sheep . Brieflyancement . SamplesPrior to NuPAGE, lithium dodecyl sulfate sample buffer and NuPAGE reducing agent  were added to samples to a final concentration of 1×. Samples were then heated at 95°C in a dry block heater for 5 min, cooled and loaded into 12% Bis-Tris NuPAGE gels. Proteins were transferred to polyvinyldifluoride membranes , blocked in non-fat dry milk and immunoblotted using SAF 83 or BAR 224 monoclonal antibodies  at a 1:5,000 dilution and polyclonal goat anti-mouse IgG  at a 1:10,000 dilution. Immunoreactivity was detected using SuperSignal West Pico chemiluminescent system  and an EC3 imaging system . For presentation purposes, some irrelevant lanes were excised from images of membranes and no further changes to brightness or contrast were made following excision. Data from separate gels presented in the same figure are presented in separate boxes.res were measured by densitometry using the VisionWorks LS software on the EC3 imaging system. The density of the pH 7.4 sample was divided by the density of the pH 3.5 sample and multiplied by 100 to produce a percentage. Data from replicates of experiment were compiled to generate a mean CER for a species with a given TSE agent ± standard deviation. An analysis of variance was performed using the Tukey-Kramer minimum significance differences method and Student’s t test performed with GraphPad software .Substrates at pH 7.4 and 3.5, which were prepared at the same time from the same brain homogenate starting material, were incubated and shaken with a TSE agent, PK treated, ran on the same gel and immunoblotted together. Levels of PrPOvis canadensis); Prnp: Gene encoding the prion protein; PrPC: Normal cellular prion protein; PrPTSE: Disease-associated prion protein; PK: Proteinase K; PrPres: Proteinase K-resistant prion protein; PMCA: Protein misfolding cyclic amplification; CER: Conversion efficiency ratio; TME: Transmissible mink encephalopathy; PBS: Phosphate buffered saline.TSE: Transmissible spongiform encephalopathy; CWD: Chronic wasting disease; BHS: Bighorn sheep  and native host conversion efficiency ratios substrates. Substrates prepared at either pH 7.4 or 3.5 from BHS, mink, domestic sheep or white-tailed deer (WTD) were shaken in the absence of TSE agents. No proteinase K-resistant prion protein was found by immunoblot with monoclonal antibody BAR 224.Click here for fileProtein misfolding cyclic amplification (PMCA) using domestic sheep (A136R154Q171) genotype substrate. The indicated TSE agents were diluted to 10-2 or 103 from 10% w/v stocks of brain homogenate into domestic sheep PMCA substrate and subjected to 96 cycles of sonication. Proteinase K (PK)-resistant prion protein levels were assessed by immunoblotting with monoclonal antibody BAR 224. As a control, substrate without TSE agent seed was subjected to PMCA cycling. Samples of sheep substrate containing the indicated TSE agents, but not subjected to PMCA, served to establish background levels of PK-resistant prion proteinClick here for file


Leber's hereditary optic neuropathy (LHON) is a common cause of inherited blindness, primarily due to one of three mitochondrial DNA (mtDNA) mutations. LHON, which has an unexplained variable penetrance and pathology, is characterised by disruption of the mitochondrial respiratory chain ultimately resulting in degeneration of the retinal ganglion cells.MAPT has been associated with a range of neurodegenerative disorders. Given the relationship between MAPT variation and altered mitochondrial respiratory chain function, we hypothesised that MAPT variation could contribute to the risk of blindness in LHON mtDNA mutation carriers.Phosphorylation of the tau protein is known to cause neurodegeneration and variation in MAPT variation in a large, well characterised LHON cohort, but were unable to find an association between MAPT genetic variation and visual failure in LHON mtDNA mutation carriers. Our findings suggest that genetic variation in MAPT is unlikely to make a major contribution to the risk of blindness among LHON mutation carriers.We studied  The diaAffected individuals develop focal degeneration of the optic nerve and present clinically with impaired color vision (dyschromatopsia), a dense visual field defect  and abnormal visual electrophysiology due to primary retinal ganglion cell loss (RGC) . AlthougMAPT gene on chromosome 17, has been shown to result in neurodegeneration through the accumulation of neurofilamentray tangles (NFT). Variation in MAPT has been associated with a range of neurodegenerative disorders, providing evidence that disrupting tau homeostasis is sufficient to cause neuronal degeneration  single nucleotide polymorphisms (SNPs):  in a European cohort consisting of 288 patients with LHON and 336 unaffected LHON family members (from 214 unrelated families) all harbouring a homoplasmic primary LHON mtDNA mutation. The clinical phenotype was determined by a local ophthalmologist, and all subjects were homoplasmic for one of the primary LHON mtDNA mutations . Mutation status was confirmed by direct sequencing of the MTND genes or PCR-RFLP analysis as previously described  substitutions predicted to affect  disease ; (2) pre disease . MAPT H1mination .The frequency of sequence variants was determined by primer extension of multiplex polymerase chain reaction products with the detection of the allele-specific extension products by matrix-associated laser desorption/ionization time of flight  mass spectrometry.Allele and genotype frequencies were compared using uncorrected Fishers exact test. Multiple logistic regression was performed to look for interactions between the variables. Age of onset was assessed as survivability using Kaplan–Meier, with comparisons made via Mantel–Cox log rank test. All analyses were carried out using SPSS software version 17.0 (SPSS Inc.). Statistical power was calculated using power for association with error (PAWE) .33.1MAPT H1/H2 haplotypes and genotypes in both affected LHON cases and unaffected LHON mtDNA mutation carriers was not significantly different to published population controls (MAPT H1/H2 haplotypes (P = 0.656) or H1/H2 alleles .The clinical penetrance of LHON is strongly associated with mtDNA haplogroup J . With thMAPT variants and haplogroup J by logistic regression, again controlling for gender, . There was no significant association identified with H-haplotypes , or common MAPT variants when limiting to non-J haplogroups.Similarly, we found no association between visual failure, the common 3.4MAPT common variants and age of onset .To assess the potential effect of H-haplotype and genotype on the age of onset of visual symptoms we compared H1 and H2 alleles to the age of onset of 250 symptomatic LHON patients . We were unable to identify a significant difference between H1 and H2 carriers (Mantel–Cox P = 0.623), further analysis showed no significant difference in age of onset when comparing 4MAPT variants previously shown to be associated with neurodegenerative disorders (MAPT gene is a major factor responsible for the incomplete penetrance in LHON. Previous studies have shown that the H1 haplotype is over represented in tauopathies (We conducted a large case control study of isorders ; based oopathies , demonstopathies , a factoopathies . HoweverMAPT SNPs, particularly rs1800547, rs2471738 and rs242557 (MAPT variants play a major role in determining the variable clinical phenotype in LHON families. It is conceivable that MAPT variation plays a minor role, perhaps in combination with other genetic and environmental factors, but this will be extremely difficult to demonstrate given that LHON is a relatively rare disorder affecting ~ 1 in 14,000 males. Collecting a large enough cohort to show more subtle genetic effects would be a major challenge.Previous studies have identified significant association to a number of rs242557 , to neur


VEGF and AFP mRNA determinations in the blood are promising prognostic factors for patients with HCC. This study explores their potential prognostic synergy in a cohort of HCC patients evaluated for potentially curative therapies.One hundred twenty-four patients with a diagnosis of HCC were prospectively enrolled in the study. Inclusion criteria were: (a) histological diagnosis of HCC and assessment of tumour grade and (b) determination of AFP mRNA status and VEGF levels in the blood before therapy.−1 (VEGF positive). Surgery was performed in 58 patients (47%), 54 (43%) had tumour ablation, and 12 had chemoembolisation (10%). Median follow-up and survival of the study group were 19 and 26 months , respectively. The association of AFP mRNA and VEGF proved to be prognostically more accurate than their single use in discriminating the risk of death  and survival probability . In particular, we identified 3 main molecular stages , and 35% had VEGFThe preoperative determination of AFP mRNA status and VEGF may potentially refine the prognostic evaluation of HCC patients and improve the selection process for potentially curative therapies. The biological history of HCC is closely related to the process of angiogenesis in neoplastic nodules During this complex process, a consensual de-differentiation of HCC and endothelial cells is of paramount importance in determining tumour aggressiveness as defined by the risk of vascular invasion and intra-extra hepatic metastasis. This physio-pathological model explains the crucial prognostic role of tumour grade and mVI as predictors of overall and disease-free survival in surgical and non-surgical series In current clinical practise, however, there are no accurate preoperative diagnostic tools available for tumour grade and mVI In this context, circulating molecular markers have the potential to be used as simple and effective tools to refine the prognostic prediction and treatment of HCC patients. Among the proposed circulating molecular markers VEGF is the best known angiogenic factor produced by tumours , but recent studies AFP mRNA is a specific marker of HCC cells in the circulation and several studies have shown a correlation with HCC-aggressive tumour features  The controversial prognostic accuracy of these biomarkers used alone, however, has not yet justified their introduction into clinical practise. We hypothesized that the combination of the most significant angiogenic factor (VEGF) and the most significant circulating HCC cell marker (AFP mRNA) could be used to improve the overall accuracy of HCC prognostic prediction and treatment decisions.On this basis, we started a prospective observational study for preoperative determination of VEGF serum levels and AFP mRNA in a cohort of HCC patients referred to our tertiary level surgically oriented unit in order to evaluate the indication for prognosis prediction in potentially curative therapies.In the study period, 124 HCC patients were enrolled. Baseline patient characteristics are described in The median size of the largest nodule was 3,5 cm (1 cm to 18 cm), and 72 patients (58%) had multi-nodular HCC.Radiologic diagnosis of gross vascular invasion was made in 31 patients (25%).All enrolled patients had histological evaluation of percutaneous/laparoscopic biopsies (60 patients) or surgical specimens (64 transplanted or resected patients). Poorly differentiated tumours (G3) were detected in 25 patients (20%). Staging procedures revealed 64 patients (52%) with Tumour, Nodes and Metastasis (TNM) III–IV tumours and 36 patients (29%) with BCLC C–D hepatic disease.−1 (positive) in 44 patients (35%).AFP mRNA determinations in blood preoperative samples yielded a positive result in 50 patients (40%). Serum VEGF values were higher than 23 pg mlLiver resection was performed on 46 patients: 17 patients had an anatomic resection (37%), the remaining had a wedge resection. 12 patients (10%) had LT. Six (5%) had laparotomic or laparoscopic radiofrequency thermal ablation. 48 (38%) had percutaneous ablation procedures . 12 had TACE (10%).After the initial therapy described in −1, TNM and BCLC staging systems, PSTγ-glutamiltransferase (GGT) values and to HCV positive ætiology. Only VEGF determinations were significantly related to PT values and to CRPH.Correlations are described in The median follow-up of the 124 enrolled HCC patients was 18,6months . During the follow-up, 65 patients (52%) died: 36 from tumour progression, 20 from cirrhosis-related complications, and 9 from non-hepatic-related deaths. Median survival of the study group was 26,4months  and 1, 3, and 5 year survival rates were 71, 46%, and 41%, respectively.−1, Child-Pugh B–C, bi-lobar tumour, surgery, PSTUpon univariate survival analysis (Cox model), the following variables  significThe Cox model and ROC curve analysis showed that the best interpretation of survival distribution was given by an association of AFP mRNA and VEGF in 3 stages –2.Upon multivariate analysis  of the oThe AFP mRNA/VEGF integrated variable was also tested in the following homogeneous subgroups of HCC patients: BCLC A patients, BCLC B patients and patients treated by resection or ablation procedures with a curative intent (BCLC A–B patients).The proposed molecular staging showed significant discriminatory ability  in BCLC A and B patients  but not esection  and ablaesection .This prospective observational study was specifically designed to evaluate the prognostic role of the preoperative determination of VEGF serum levels and AFP mRNA in a cohort of HCC patients in order to evaluate their indication for potentially curative therapies.The results of the present study should therefore be interpreted by considering the potential utility of these still experimental molecular markers in daily clinical practise.Prognostic factors may help the treatment strategy for HCC patients in different ways:in deciding the best therapy according to patient characteristics;in estimating individual survival perspectives;in designing clinical trials to test new therapies;in programming post-treatment follow-up and adjuvant therapy;Prognostic prediction and therapeutic decisions for HCC patients in most centres are currently based on macroscopic tumour characteristics detected by imaging studies (nodule size and number). In the past decade, the empirical rule of a single nodule smaller than 5 cm in diameter or multiple nodules (2 or 3) smaller than 3 cm has been used to define early HCC, with excellent outcomes achieved after LT These macro-morphological tumour characteristics have been incorporated in the BCLC classification BCLC staging is now considered the best available prognostic system for HCC patients Therefore, we agree that BCLC clinical variables  are crucial prognostic factors for patients with HCC. In addition, in this prospective study, these variables demonstrated significant discrimination of survival ability  on univaHowever, there is a substantial variation in prognosis among patients within the same BCLC stage The first result of this study, therefore, is that our proposed molecular staging may potentially refine the prognostic evaluation of patients with HCC. Circulating prognostic molecular markers (AFP mRNA and VEGF) showed significant ability in discriminating survival within BCLC stages, especially when used as an integrated variable .The prognostic heterogeneity makes BCLC staging alone relatively inaccurate for determining the best treatment strategy and estimating individual prognosis. As shown in recent studies Thus, in therapeutic decisions, negative prognostic factors do not necessarily coincide with absolute exclusion criteria for a specific therapy. For example, multi-nodularity, vascular invasion and portal hypertension The small sample size in this study does not allow us to reach definitive conclusions about the potential role of these molecular markers in treatment decision. On a purely speculative basis, the results of this study suggest the potential utility of these biomarkers in excluding potentially curative therapies for BCLC B patients with a poor prognosis  the Ethics Committee of our institution at the University Hospital of Padua did not specifically require an explicit approval for anonymized prospective observational studies. Patients were informed about the research, but no informed consent was requested since all data was anonymized.This prospective observational study started in January 2001. Inclusion criteria were: (a) histological diagnosis of HCC and assessment of tumour grade ; (b) baseline determination of AFP mRNA status and VEGF levels in the blood; and (c) a general consent for the use of a blood sample for research purposes. Exclusion criteria were: (a) fibrolamellar HCC; (b) concomitant extra-hepatic tumour; (c) HIV positivity; and (d) previous recent HCC treatment  An EDTA-K3-supplemented blood sample (7 ml) was obtained from all subjects and immediately stored at 4°C for no more than 3 h until processing. Total RNA was extracted and converted to cDNA. A nested polymerase chain reaction protocol was performed as previously reported −1, which was the minimum VEGF concentration with a coefficient of variation of less than 20%. The intra-assay precision for VEGF was less than 5% in the concentration range 87 pg ml−1 to 938 pg ml−1. The intra-assay precision for AFP protein was less than 5% in the range 20 ng ml−1 to 732 ng ml−1.A serum sample was also obtained from each subject and stored at −20°C until use in AFP protein and VEGF immunoassays . The sensitivity of the assay for VEGF was 23 pg mlAll patients were also tested for albumin, bilirubin, creatinin, AST, ALT, ALP, GGT and PT using standard methods.All enrolled patients were referred to our tertiary hepatobiliary surgery-liver transplantation unit to evaluate the feasibility of potentially curative therapies. Patients in BCLC A–B stages were considered for surgery or ablation procedures eventually associated with TACE: as previously reported Patients with advanced tumours  but good liver function (BCLC C) were eventually considered for surgery or ablation only from a palliative perspective.1. To investigate the correlation between AFP mRNA and VEGF determinations and clinical-morphological characteristics of enrolled HCC patients.2. To evaluate the prognostic role of AFP mRNA and VEGF determinations used alone or together as an integrated variable.−1 (sensitivity of the assay), this variable was considered a categorical variable identifying the 2 subgroups of patients with VEGF values−1 (positive) or −1 (negative). Comparisons between groups were analyzed by logistic regression, the The patients' baseline characteristics are expressed as medians (range) for continuous data and as frequencies (percentage) for categorical data. There were only 9 (7%) Child-Pugh C patients; thus, we considered only 2 stages, Child-Pugh A and B–C. Child-Pugh C patients were considered BCLC D only when they were without LT therapeutic perspectives. Because the TNM I stage included only 9 patients (7%) and the BCLC D stage only 4 patients (3%), the following variables were stratified in three main stages: BCLC A, B, C–D and TNM I–II, III, IV, respectively. Because only 44 patients (35%) had a serum VEGF value higher than 23 pg mlCox's univariate proportional hazards models were used to identify potential survival predictors in the study group. Cox regression and ROC curve analysis were used to evaluate the interaction between AFP mRNA and VEGF determinations and to determine the prognostic utility of a potential integrated variable, taking into account both molecular markers.A Cox's multivariate model was then created, including only variables with Probability curves for survival were calculated using the Kaplan-Meier method, and the log-rank test was applied to the Kaplan-Meier curves to identify potential predictors of survival.In order to reduce the confounding effect of treatment and staging on survival response variables, the prognostic impact of the molecular markers was also tested in subgroups of BCLC A, BCLC B, and BCLC A–B patients undergoing resection or ablation.Statistical significance was set at


The importance of the large number of thin-diameter and unmyelinated axons that connect different cortical areas is unknown. The pronounced propagation delays in these axons may prevent synchronization of cortical networks and therefore hinder efficient information integration and processing. Yet, such global information integration across cortical areas is vital for higher cognitive function. We hypothesized that delays in communication between cortical areas can disrupt synchronization and therefore enhance the set of activity trajectories and computations interconnected networks can perform. To evaluate this hypothesis, we studied the effect of long-range cortical projections with propagation delays in interconnected large-scale cortical networks that exhibited spontaneous rhythmic activity. Long-range connections with delays caused the emergence of metastable, spatio-temporally distinct activity states between which the networks spontaneously transitioned. Interestingly, the observed activity patterns correspond to macroscopic network dynamics such as globally synchronized activity, propagating wave fronts, and spiral waves that have been previously observed in neurophysiological recordings from humans and animal models. Transient perturbations with simulated transcranial alternating current stimulation (tACS) confirmed the multistability of the interconnected networks by switching the networks between these metastable states. Our model thus proposes that slower long-range connections enrich the landscape of activity states and represent a parsimonious mechanism for the emergence of multistability in cortical networks. These results further provide a mechanistic link between the known deficits in connectivity and cortical state dynamics in neuropsychiatric illnesses such as schizophrenia and autism, as well as suggest non-invasive brain stimulation as an effective treatment for these illnesses. The brain mediates behavior by orchestrating the activity of billions of neurons that communicate with each other through electric impulses. The transmission of these action potentials is surprisingly slow for a large fraction of these connections. Given the importance of precise timing of neuronal activity, the function of these slow connections has remained a puzzle. We here used computer simulations to investigate how slow connection speeds alter the overall activity patterns of two brain networks. We found that these connections enable the interconnected networks to generate distinct activity patterns such as different types of waves of electric activity. Our results therefore suggest that the slow transmission of electric impulses in the brain is not a “design flaw” but rather plays an important role in enabling the brain to generate a richer set of activity patterns. The ability of the brain to switch between different activity states is crucial to normal cognition, and abnormalities in switching behavior are associated with cognitive symptoms in psychiatric disorders such as schizophrenia and autism. It is therefore promising that we were able to control transitions between different activity states with non-invasive brain stimulation in our simulations, suggesting a novel approach to the treatment of these illnesses. Cognition emerges from the organized temporal structure of electric activity in large, interconnected cortical networks Systematic parameterization of network topology in computational models has demonstrated that connections between random pairs of distant, excitatory neurons within a network enhance temporal synchronization, whereas predominantly local connectivity between neighboring excitatory neurons facilitates macroscopic activity patterns such as oscillations and planar and spiral waves that propagate through the network Mathematical studies of the effects of delays on coupled oscillators have predicted diverse results as a consequence of delays. Foundational papers have found that delays between coupled systems produce stability under certain parameters in vivoWe hypothesized that slower long-range projections may enrich overall network activity by counteracting and disrupting the intrinsic, spontaneous dynamics of individual networks. According to our hypothesis, slower projections provide perturbations that are ill-timed to synchronize networks and therefore enable different activity trajectories that individual networks are unable to generate. To test this hypothesis, we used large-scale computer simulations to ask what role long-range projections with propagation delays may play in organizing the overall dynamics of two interconnected cortical networks with intrinsic spontaneous dynamics similar to isolated cortical networks To understand the effect of long-range projections (LRPs) on the dynamics of two interconnected cortical networks, we built a large-scale computational model of two networks connected by LRPs  where eaBoth with and without LRPs, UP states emerged as initially localized “regions of initiation” that then expanded through the local excitatory connectivity  state, with most PYs in the network firing almost simultaneously and the network as a whole demonstrating slow oscillatory behavior . HoweverNext, we asked how the occurrence of these three different macroscopic network states depended on LRP delays. We found that most interconnected networks followed an RF pattern, especially for short LRP delays . For lonIn order to further evaluate the robustness of this result, we also tested the effects of a distribution of delays. We ran two sets of simulations, the first with delays uniformly distributed ±20% of the mean and the second with delays uniformly distributed ±100% of the mean. Our results indicate that wider distributions resulted in fewer state transitions . AdditioWe then analyzed the transitions of individual simulations through these metastable spatio-temporal activity patterns over time , left. HTo further understand these different network states, we next applied perturbations to probe the stability of each state. Specifically, we simulated transcranial alternating current stimulation (tACS), which has recently emerged as a promising treatment for psychiatric and neurological illnesses because of its hypothesized ability to selectively manipulate temporal structure of cortical network activity We here used this stimulation modality to probe the dynamic properties of the different activity states that emerged from LRPs with propagation delays. We found that tACS at 3 Hz  not only enhanced the synchronization between the two networks but switched the two networks to the fully synchronized, RF state . NetworkInterestingly, a small fraction of the simulations did not show this enhancing effect of tACS. Rather, in these cases, tACS switched the networks from RF to either SW or SP states . In thisGiven these distinct effects of the same stimulation protocol in different simulations, we determined the relative occurrence of the different states and the state transition probabilities for all simulations  as a function of tACS. In the control condition before onset of stimulation , the majOverall, the state-dependent transition probabilities in the absence of tACS, at tACS onset, and at tACS removal  demonstrWe then compared how networks behaved together and found that in the absence of stimulation, both networks were in the RF state for the majority of simulations , where tHaving established that tACS affects the spatio-temporal activity of two interconnected networks, we next quantified the effect of tACS on the power of the network activity at the stimulation frequency (3 Hz). First, we looked at the effectiveness of tACS to entrain two networks during stimulation by comparing the power during stimulation to the power before stimulation. We found that tACS enhanced the power at 3 Hz of both PY networks during stimulation for most simulations, indicating its ability to entrain networks , left. NTo investigate how this outlasting effect of tACS related to the entrainment during stimulation, we compared the enhancement of power at 3 Hz during stimulation to the enhancement of power after stimulation , right. Although tACS typically entrained networks to a 3 Hz RF state, occasionally it had an opposite effect by disrupting RF during tACS and causing it to enter SW after tACS. We examined these network dynamics to determine which factors influenced such disruption. Networks that ended in SW after tACS were most often in SP or SW during tACS and only very rarely in RF . ConsequD; lower values indicate greater synaptic depression). The depression coefficient was indeed lower for networks that entered SW during tACS, and the normalized variance of D was greater for networks in SP or SW during tACS ) and lower LRP conductance (G(LRP)) both made networks more likely to enter a non-RF state, and these effects were increased with lower delays . After tThe relative prominence of SW after removal of tACS led us to measure the stability of the SW state. We first examined stability of SW in the absence of tACS and found that SW was a metastable state . Then wek-means clustering of their cross-correlograms. “Strong antiphase” behavior occurred when the two networks were individually entrained by their respective stimulation ) and higher LRP conductance made interspersed weak firing more likely  antiphase state . This stWe used simulations of two large, interconnected cortical networks to study how LRPs that connect the two networks affect the overall macroscopic dynamics. We found that introducing physiologically plausible delays to the LRPs greatly enhanced the repertoire of emergent dynamics, measured not only by synchronization between the two networks but also by the intrinsic spatio-temporal dynamics. Our results therefore suggest small-diameter and unmyelinated projection axons with propagation delays play an important role in enriching the landscape of cortical activity states. This finding contrasts with the traditionally assumed role of long-range connections to enable zero-lag synchrony between different cortical areas Our study exclusively utilized computer simulations and therefore has the same caveats as any modeling study. First, the level of abstraction for the model requires consideration. We used computationally efficient, yet biologically plausible model neurons since we were interested in studying the effect of connectivity without confounding the results with the effects of conductance-based, Hodgkin-Huxley-style neuron models, which could model more sophisticated intrinsic cellular dynamics. A reduced model investigating the bifurcations involved in state transitions would provide further insight into network dynamics, although for this study it would reduce the applicability of our findings to the development of novel brain stimulation paradigms. Second, any biologically plausible finding in a computer simulation needs to withstand tests for reasonable robustness to parameter variations. The entire data set presented in this study was based on multiple runs of every simulation with different instantiations of the randomized variables . Third, we believe that the value of most modeling studies can be readily assessed by the type of predictions they make that can then guide subsequent research, whether it be further computational work, wet lab bench studies, or even human preclinical trials. We therefore use the remainder of the discussion section to outline and discuss what we think are the implications and predictions of our results for the study of brain stimulation and network deficits in diseases with altered CNS connectivity such as schizophrenia, autism, and multiple sclerosis.in vitro work Brain stimulation, whether through implanted electrodes such as in deep brain stimulation Our study suggests that rather than reorganizing synaptic strength, tACS can induce a switch between different macroscopic activity states that are part of a repertoire of cortical states mediated by LRPs with propagation delays. Interestingly, we also found that the same stimulation paradigm had the opposite effect in a  subset of simulations where the stimulation reduced the synchronization; these results demonstrate that (1) the ongoing network dynamics (i.e. network state) and the underlying network topology determine the response to brain stimulation and (2) a global stimulus does not necessarily enhance synchronization. Antiphase tACS, a stimulus designed to disrupt synchronization, caused a set of new behaviors during stimulation, but in most cases failed to create antiphase structure between the networks as an outlasting effect. Consequently, the outlasting effects of stimulation are dependent on the phase of stimulation as well as the intrinsic network structure.As part of a computational model, conclusions drawn from our simulations of tACS are limited by the size of our networks and the fact that each PY receives the same magnitude of stimulation; however, simulated variance of tACS current amplitude has previously been found to have no effect on network response Pathological changes in connectivity in the central nervous system (CNS) are a hallmark of many neurological and psychiatric illnesses. For example, schizophrenia is often called a connectivity disorder due to the findings of aberrations in white matter and lack of functional connectivity in both functional MRI and electroencephalogram (EEG) studies We used computer simulations of large-scale, interconnected cortical networks in this study and found that long-range projections with physiological delays can play an unanticipated role in generating multistable network dynamics in cortex. Therefore, the so far neglected slow connecting fibers between cortical areas may not be a “flawed design” that prevents large-scale synchronization of cortical areas but rather enables the emergence of additional, qualitatively different network states that likely serve different neural computations. The ability of non-invasive brain stimulation to change these network states points to a promising treatment option for neuropsychiatric disorders involving abnormal connectivity and network dynamics.a, b, c, and d that determine the intrinsic dynamics. We used an Euler solver with a step width of Δt = 0.1 msec such that the update rule at every time-step of the stimulation to compute the new value of the membrane potential V′ is:V is the membrane voltage at the previous time-step, EAMPA = 0 mV is the excitatory reversal potential (AMPA), EGABA = −80 mV is the inhibitory reversal potential (GABAA), GEX and GIN represent the sums of all afferent excitatory (gPY) and inhibitory (gIN) conductances, ItACS and INoise are current injections to model transcranial alternating current stimulation (tACS) and to cause spontaneous background noise, and u is the slow recovery variable.We used the Izhikevich model a  and b (recovery sensitivity) were set to 0.02 and 0.2, respectively. We modeled regular spiking, intrinsically bursting, and chattering PY cells by setting the reset potential parameter, c, to values from −65 to −50 mV, and the recovery after an action potential, d, to values from 6 to 8. All values were drawn from generalized Pareto distributions . These distributions helped to bias the parameter values such that regular spiking cells were the most frequent PY cell type. For the INs, the parameters c and d were set to −65 mV and 2, respectively. To model both fast and low-threshold spiking neurons, parameters a and b were drawn from uniform distributions .For PYs, parameters GEX and GIN were the corresponding total conductances at the occurrence of the last presynaptic action potential, τEX = 2 msec and τIN = 3 msec were the synaptic decay time-constants, and Δtpsp was the time elapsed since the last presynaptic action potential. PY-PY connections exhibited short-term synaptic depression D  that exhibited an exponential recovery time-course (τD = 300 msec). PY-PY synaptic gPY-PY strength was calculated as:GPY-PY denoted the synaptic strength and D was updated for each presynaptic action potential for all PY-PY synapses:r = 0.6 represented the fraction of synaptic resources available immediately after vesicle release caused by an action potential.Synapses were model by conductances that were updated with a step in case of a presynaptic action potential and that were subject to exponential decay otherwise. All synapses of a given type were lumped together into a single synapse to increase computational efficiency of the simulations GPY-PY = 0.06, no autapses). Synaptic inhibition had global random connectivity both for PY-IN excitation  and feedback IN-PY inhibition . The global connectivity scheme for synaptic inhibition was chosen such that inhibition provided an overall activity-dependent reduction of PY firing rate without any extra spatial structure. The synaptic connectivity was chosen such that a 3 Hz endogenous oscillation occurred in the absence of long-range projections (LRPs). LRPs were configured by replacing a defined  fraction of local PY-PY connections with excitatory projections to random PYs in the other network . We evaluated the effect of a range of propagation delays for these LRPs .All simulations in this study consisted of two connected networks. Each network consisted of two layers, a PY network  and an IN network (200×200 model neurons arranged on a grid). The large number of neurons was motivated by the fact that tACS is likely to act as a global weak perturbation similar to the endogenous electric field INoise that was the sum of (1) a constant current injection ranging from 0 to 1.5  to create spontaneously firing PYs and (2) a variable current with a random value drawn at every time-step . Non-invasive brain stimulation with transcranial Alternating Current stimulation (tACS) was modeled with a small current injection  into PY cells that are susceptible to applied electric fields because of their elongated somato-dendritic axes All cells received a current injection The effect of the electric field resulting from tACS was modeled by injecting a small current into all PYs Network activity profiles were determined by the fraction of PY neurons that were firing over time. Both normalized cross-correlations and spectrograms were based on these activity profiles by network. Spectrograms were computed by Wavelet transformation with Morlet wavelets (0.5 to 10 Hz in 0.5 Hz step-width). Macroscopic spatio-temporal activity states were distinguished by the median PY activity peaks (percent PYs firing) in 1 sec bins. Peaks (UP states) were extracted with the Matlab findpeaks function . Rapid fire (RF) was assigned to peak values >60% of total number of PYs in the network, slow propagating (SP) was assigned to values 15–60%, and spiral wave (SW) was assigned to values <15%. Relative time spent in different states was determined over all simulations with the two networks considered together. State-dependent transition probabilities were determined for a 1 sec window before stimulation onset, 1 sec after stimulation onset, and last 1 sec window of simulation after stimulation.Data are reported as mean±s.e.m. Significance of correlations was determined by corrcoef function in Matlab with 0.05 as significance cut-off.Figure S1Long-range projections synchronized two cortical networks. (A) Traces of two PYs with LRP conductance of 0 and 0.06. With non-zero LRPs, UP states in PYs synchronize. (B) Power spectrum of PY network activity (red: G(LRP) = 0; blue: G(LRP) = 0.06). LRPs had little effect on overall structure of spectrum but modestly increased peak power.(PNG)Click here for additional data file.Figure S2Comparison of homologous and non-homologous LRPs (zero delay). (A) Activity snapshots. (B) Phase-plane representation. (C) Correlations between the two PY networks. (D–F) Same representation for non-homologous LRPs.(PNG)Click here for additional data file.Figure S3Wider variance of delays stabilized networks. Top: Narrow distribution (mean ±20%). Bottom: Wide distribution (mean ±100%). (A) Frequency of state transitions. (B) State distribution of networks. Wider delays result in fewer transitions and a reduced occurrence of non-RF behavior.(PNG)Click here for additional data file.Figure S4D . When Network 2 went through a transition towards decreased activity , UP states of Network 1 occurred during a period of strong synaptic depression in Network 2. When Network 2 transitioned towards increased activity , the effect of the input of Network 1 was increased due to the reduced synaptic depression allowing more neurons in Network 2 to fire.Mechanisms of state transitions. (A) IN activity plots; dashed lines represent example UP states in Network 1. (B) Top and middle: Time snapshots of PY activity in Network 1 and Network 2 for the UP states indicated in (A). Bottom: Synaptic depression variable (PNG)Click here for additional data file.Figure S5Behavior during tACS in unconnected networks. Distribution of behavior for two networks during tACS with no LRPs  = 1). Left: Before tACS. Middle: During tACS. Right: After tACS. Spiral waves can still be initiated by tACS even without LRPs, but they are not seen before tACS.(PNG)Click here for additional data file.Figure S6Percentage of time both networks were in the same state before, during, and after tACS. Left: tACS equalized the time spent in RF across delays. Middle: tACS also increased the likelihood that both networks were in a SP state. Right: tACS biased networks towards simultaneously being in SW (only seen during and after tACS). Values are normalized by the percentage of time spent in each state overall.(PNG)Click here for additional data file.Figure S7tACS abolished antiphase synchronization. (A) Example of slow antiphase coupling. Upper left: Cross-correlograms between networks before, during, and after tACS. Lower left: Network 2 displayed a state transition before entraining with Network 1 during stimulation. Right: Increased power at 3 Hz in both networks due to tACS. (B) Example of fast antiphase coupling suppressed by tACS. Same plots as in (A).(PNG)Click here for additional data file.Figure S8Synaptic depression influenced tACS behavior. Left: Depression coefficient at onset of tACS, grouped by behavior during tACS. Lower values indicate more synaptic depression; higher values indicate less synaptic depression. Networks entering SW during tACS had more strongly depressed networks than networks entering RF or SP. Right: Standard deviation of the depression coefficient normalized by the mean. Lower variance of depression correlates with stronger entrainment to tACS.(PNG)Click here for additional data file.Figure S9Persistence of spiral waves. (A) The three simulations that had constant SW behavior during original simulations, extended for another 7 seconds. One simulation (top) remained in SW except for a brief switch to SP, while the other two simulations (middle and bottom) stayed in SW for the entire time. (B) Persistence of SW in networks that ended with SW after tACS. Simulations ran for another 7 seconds. X-axis indicates the number of seconds SW persisted in the extended period. Many networks leave SW, but 22 networks (28.95%) remain in SW for the entire extended period. (C) Effects of parameters on SW persistence. Lower connectivity (left) correlates with longer persistence of SW. Conductance (middle) and delays (right) have no effect.(PNG)Click here for additional data file.Figure S10Antiphase induction of high-frequency behavior post-tACS. (A) PY activity during antiphase tACS. During stimulation, the network switches from in-phase ∼3 Hz firing to antiphase firing at 8.6 Hz, persisting upon removal of tACS. (B) Spectrogram shows change from 3 Hz firing to rapid high-frequency firing in both networks.(PNG)Click here for additional data file.Movie S1Example of rapid fire state. PY activity for two networks is shown in color, indicating the instantaneous firing rate. Most of each network became excited rapidly. Parameters: Delay = 0 msec, P = 0.99, G(LRP) = 0.06.(AVI)Click here for additional data file.Movie S2Example of slow propagating state. PY activity for two networks in SP for entire simulation. PY activity spread through the network by moving to proximal areas. Parameters: Delay = 50 msec, P = 0.95, G(LRP) = 0.03.(AVI)Click here for additional data file.Movie S3Example of spiral wave state. Network 1 was in SW for entire simulation while Network 2 was in RF for whole simulation. Spiral wave began with central rotor from which PY activity propagated, forming spiral pattern. Parameters: Delay = 10 msec, P = 0.99, G(LRP) = 0.015.(AVI)Click here for additional data file.Movie S4tACS entrained networks. Simulation presented in (AVI)Click here for additional data file.Movie S5tACS disrupted networks. Simulation presented in (AVI)Click here for additional data file.Table S1Multistability as a function of propagation delays .(XLSX)Click here for additional data file.Table S2Distributed delays reduced state transitions .(XLSX)Click here for additional data file.Table S32) of dynamics between the two interconnected networks (Correlation (Rnetworks .(XLSX)Click here for additional data file.Table S4Modulation of state dynamics by simulated tACS as a function of propagation delays .(XLSX)Click here for additional data file.Table S5Effects of simulation parameters on tACS behavior.(XLSX)Click here for additional data file.Text S1Synaptic depression contributes to state dynamics. An analysis of the effect of synaptic depression within a network on its response to input from the network connected to it.(DOCX)Click here for additional data file.


Cicindela sylvatica) as a model species, we relate presence/absence and abundance data of locally dispersing adults and sedentary larvae to abiotic and biotic variables measured in a multiscale approach within the geographic extent relevant to active conservation management. At the scale of hundreds of meters, fine-grained abiotic conditions  are fundamental determinants of the occurrence of both life stages, whereas the effect of biotic factors is mostly contained in the abiotic signature. The combination of dense heath vegetation and bare ground areas is thus the first requirement for the species’ preservation, provided that accessibility to the suitable habitat is ensured. At a smaller scale (centimetres), the influence of abiotic factors on larval occurrence becomes negligible, suggesting the existence of important additional variables acting within larval proximity. Sustained significant correlations between neighbouring larvae in the models provide an indication of the potential impact of neighbourhood crowding on the larval niche within a few centimetres. Since the species spends the majority of its life cycle in the larval stage, it is essential to consider the hierarchical abiotic and biotic processes affecting the larvae when designing practical conservation guidelines for the species. This underlines the necessity for a more critical evaluation of the consequences of disregarding niche variation between life stages when estimating niches and addressing effective conservation measures for species with complex life cycles.Detailed understanding of a species’ natural history and environmental needs across spatial scales is a primary requisite for effective conservation planning, particularly for species with complex life cycles in which different life stages occupy different niches and respond to the environment at different scales. However, niche models applied to conservation often neglect early life stages and are mostly performed at broad spatial scales. Using the endangered heath tiger beetle ( Ecological niche modelling (ENM) tools are increasingly applied to address a miscellany of challenges in species conservation biology and distributional ecology  et al.In this emerging field, the theoretical framework by Peterson Basically, the spatial extent and resolution of ENM should match the extent and resolution of the biological phenomenon under study and also take into account the species’ natural history and dispersal et al.Whether models for species with complex life histories should take more than a single life stage into account is a pending issue in ENM, as the persistence of viable populations is only feasible when the abiotic and biotic requirements of every life stage are fulfilled In this study we identify the factors that define life-stage ecological niches at fine resolution, by taking into account the life-history traits , and robust presence/absence and abundance data of co-occurring larvae and adults. We use tiger beetles as model organisms in a conservation approach to address the following questions: 1) Do co-occurring life stages exhibit identical fine-grained responses to the same environmental variables? 2) Are biotic factors  more relevant than abiotic ones in shaping life-stage distribution at fine spatial resolution? 3) Does the explicit inclusion of life-stage information affect the interpretation of species niches and distributional areas?Permits and approvals for the field campaign were obtained from the Regional Administration , and in consultation with all shepherds of the study sites.Cicindela sylvatica Linnaeus, 1758 (Coleoptera: Carabidae), is an endangered stenotopic species occurring in north, central and north-west Europe The heath tiger beetle, The species’ entire life cycle  takes place in the same location. Larvae go through three instars before pupation Cicindela campestris Linnaeus, 1758, a eurytopic species, widely distributed across the entire Palaearctic region In the study area, the target species co-occurs with the green tiger beetle, We surveyed two locations  in the Cantabrian mountain range (NW Spain), at the rear edge of the species’ distribution range . For cenCalluna vulgaris (L.) Hull heathland characterised by the combination of dense vegetation arranged in a cushion-like structure and large patches of bare ground, 2) BARE_SPOT: C. vulgaris heathland dominated by dense vegetation interrupted by small spots of bare ground, 3) DENSE: C. vulgaris heathland consisting of dense continuous vegetation, 4) GRASSLAND: upland siliceous grassland used as summer pasture, and 5) SHRUBLAND: shrub formation consisting of Cytisus purgans (L.) Boiss, Cytisus scoparius (L.) Link and Genista florida L. Habitat types in location B are: 1) BARE_PATH: C. vulgaris heathland consisting of dense vegetation interrupted by bare ground paths caused by cattle trampling, 2) BURNED: C. vulgaris heathland burned in September 2010 to create pastureland and improve forage quality, 3) DENSE, 4) GRASSLAND and 5) SHRUBLAND.We used a proportional random-stratified approach see  to gatheWe randomly placed a total of 120 sampling points in each location. The number of sampling points per habitat type was proportional to the expected relevance of each habitat for the species according to the literature and to the spatial extent of the habitat . In eachAdult beetles were captured by live pitfall trapping in July 2011. We placed one dry trap consisting of a large plastic cup  containing an inverted medium-sized plastic cup  per sampling point   locaEnvironmental information  was gathWe modelled the ecological niche of the species, separately for each life stage (adult and larva), and also, the partial niches of sexes  and larval instars  see . We analFor each data set, an identical group of environmental variables was evaluated as abiotic predictors of the occurrence of the target species . MultiplAssuming unlimited prey resources, we evaluated the main potential intra- and interspecific interactions (either positive or negative) between: 1) adults and larvae of the target species, 2) adults of the target and congeneric species , 3) larvae of the target and congeneric species , and 4) larvae and neighbouring larvae of the target species. The following variables were included as biotic predictors in the models : larva  to describe the ecological niche of the adult and the larva, and the partial niche of sexes and larval instars. Partial niche models yielded analogous outcomes and had lower qualities than the adult and larval models; they are thus not reported. We initially performed generalised linear mixed models (GLMMs) for the finest larval scale (25×25 cm), considering the identity of the 1×1 m square as random factor; however, the increase in model complexity did not improve predictive power and therefore only GLMs are reported.k, i.e., low degree of aggregation), using the log link function We modelled PA data following a binomial error distribution , using the logit link function; and AB data following a negative binomial error distribution  were considered in the models. Additionally, variable selection was based on the expected relevance of the predictors for the target species accounted for in the literature.For each data set, the same predictors were initially included in the ABIOT and BIOT models . The sta2R). For the ABIOT MAMs, we calculated the joint percentage of deviance explained by the predictors in each category . We checked for spatial autocorrelation in the model residuals by calculating Moran’s index (I). We tested Moran’s I significance by performing 1000 permutations and applying Holm’s correction to adjust for repeated testing. We found weak evidence for spatial autocorrelation in the species distribution, as only two out of 32 performed models had significantly clumped residuals (see Collinearity was further assessed in the models by examining the variance inflation factor (VIF) of the predictors uals see .vice versa). We evaluated the accuracy of the predictions of the BIOT models by IE. For PA response variable models, predictions were compared to observations by applying the area under the receiver operating characteristic (ROC) curve (AUC). The AUC is an informative measure of model accuracy when estimating the realised niche of a species and when true absence data are available et al.et al.ρρ, the following ranges were established, taking into account the critical value of the correlation coefficient with N>100 ρ>0.80; good 0.60<ρ<0.80; fair 0.40<ρ<0.60; poor 0.20<ρ<0.40; fail ρ<0.20.Two approaches were applied to evaluate the accuracy of the predictions of the ABIOT models ρ measures for PA and AB data, respectively. Transferability was assumed to fail when AUC<0.70 and ρ<0.40. Adult model transferability  was evaluated by assessingWe evaluated transferability (T) of the ABIOT MAMs between life stages (data sets 1–2 and 5–6) with the AUC and Spearman’s Location) by calculating the difference in model T  from location A to B and from B to A asStage) by calculating the difference in model T from adult to larva and from larva to adult in the same location asWe checked for asymmetrical transferability AT, see : 1) betw2R. The model with the lowest AIC value and the maximal deviance reduction indicates the best fit to the data. Following previous work 2R of the opposite set from the FULL model. The pure contribution of the abiotic and biotic predictors to the total explanatory power is defined by.We compared ABIOT, BIOT and FULL models using the AIC and Nagelkerke’s BAM diagram B), abiotic conditions (A), and movements  of the adult models ranged from 0.62 to 0.91. The fit of the larval models was highest at 1×1 m (0.81–0.94) but values decreased with increasing spatial resolution (0.67–0.83 at 50×50 cm and 0.34–0.55 at 25×25 cm).The fit of PA models was always lower than that of AB models . In 13 oThe number of abiotic predictors in the adult MAMs varied from four to seven , with COIn general, ABIOT MAM predictive accuracy measured by IE was higher than accuracy measured by EE. For adult PA models, IE accuracy values were above ‘good’ threshold values , while Eρ metric, only 75% of the adult and 50% of the larval models were satisfactorily transferable .For both PA and AB data, transferability (T) of the adult ABIOT MAMs was higher than T of the larval ones . When mevice versa . Life stages showed smaller AT than locations. Models transferred slightly better from adult to larva than vice versa, as the mean AUC and ρ metrics decreased 5% and 2% respectively when models were transferred from larva to adult.Models transferred better from location B to A than The fit of PA models was mostly lower than or equal to that of AB models . In 10 oNearly all adult and larval responses to biotic predictors in the BIOT MAMs were significantly positive . The majFor six out of eight adult and larval PA models, and for four out of eight AB models, IE accuracy values were above ‘fair’ threshold values .The inclusion of biotic predictors did not improve model fit substantially , and theNumerous processes acting at several spatial scales determine the niche and distributional area of a species Calluna vulgaris heathlands characterised by specific combinations of densely vegetated and bare ground areas. At larger modelling scales (1×1 m and 50×50 cm), the respective extents of these two elements in the suitable habitat constitute the main abiotic factor shaping life-stage occurrences. Bare, open ground devoid of vegetation provides essential sun-exposed surfaces where adults and larvae attain the high body temperature indispensable for prompt motion  and for prey detection Larval and adult stages of the model species inhabit Several studies on the environmental requirements of tiger beetles have demonstrated that larvae exhibit narrower tolerances than adults do BAM  diagram (BAM diagram represent areas in the geographic space (G) where biotic (B) and abiotic (A) conditions are suitable for occurrence, and that have been accessible to dispersal or colonisation over time (M) B∩A∩M represents the occupied area of distribution (GO), equivalent to the occupied niche BAM diagram , and because no substantial restrictions to adult dispersal exist , the principal constraint to adult occurrence is lack of favourable environments , but important restrictions to larval dispersal exist , such as the availability of proper oviposition sites and the limited larval motion, implying that not all suitable areas for the larvae are occupied , which may limit larval occurrence within the accessible region M. The key mechanisms of larval spatial aggregation  have yet to be understood, but for either positive  or negative  interactions a threshold number or density of neighbouring larvae may be needed for population survival We exemplify three configurations of the  diagram  to illus diagram . The reg diagram  illustra diagram  represen diagram  incorporTable S1Number of sampling points and number of captured adults and monitored larvae of the two tiger beetle species per habitat type in each location. Target species = Cicindela sylvatica, congeneric species = Cicindela campestris. Adult captures correspond to 63 and 49 marked individuals in locations A and B, respectively. Values in parentheses represent the number of sampling points in which adults and larvae were present. BARE_CUSHION = Calluna vulgaris (L.) Hull heathland characterised by the combination of dense vegetation arranged in a cushion-like structure and large patches of bare ground, BARE_SPOT = C. vulgaris heathland dominated by dense vegetation interrupted by small spots of bare ground, BARE_PATH = C. vulgaris heathland consisting of dense vegetation interrupted by bare ground paths caused by cattle trampling, BURNED = C. vulgaris heathland burned in September 2010 to create pastureland and improve forage quality, DENSE = C. vulgaris heathland consisting of dense continuous vegetation, GRASSLAND = upland siliceous grassland used as summer pasture, SHRUBLAND = shrub formation consisting of Cytisus purgans (L.) Boiss, Cytisus scoparius (L.) Link and Genista florida L.(DOCX)Click here for additional data file.Table S2Abiotic (ABIOT) minimal adequate model (MAM) results for each adult and larval presence/absence (PA) and abundance (AB) data set. Final GLM coefficient estimates  and standard errors (SEs) are indicated. Residual deviance is the amount of variation not explained by the predictors. P values smaller than 0.05 (χ2 distribution) are in bold face. Measures of model predictive accuracy (AUC and Spearman’s ρ) were derived by internal evaluation . For predictor abbreviations see (DOCX)Click here for additional data file.Table S3Biotic (BIOT) minimal adequate model (MAM) results for each adult and larval presence/absence (PA) and abundance (AB) data set. Final GLM coefficient estimates  and standard errors (SEs) are indicated. Residual deviance is the amount of variation not explained by the predictors. P values smaller than 0.05 (χ2 distribution) are in bold face. Measures of model predictive accuracy (AUC and Spearman’s ρ) were derived by internal evaluation . Target species = Cicindela sylvatica, congeneric species = Cicindela campestris. Larva_ 2 = second larval instar.(DOCX)Click here for additional data file.


Wound healing is a complex process involving a number of processes. Fetal regeneration has been shown to have a number of differences compared to scar-forming healing. This review discusses the number of differences identified in fetal regeneration. Understanding these differences may result in new therapeutic targets which may reduce or even prevent scarring in adult healing. Monodelphis domestica, have shown that fetal regeneration is not due to the moist, sterile environment of the uterus [Since the 1970s it has been well established that early human fetuses can heal cutaneous wounds without the formation of scar tissue [e uterus . Furthere uterus  whereas e uterus .Wound healing is an inherent response resulting in restoration of tissue integrity. It is a complex process involving cell migration, proliferation, differentiation, apoptosis, and the synthesis and remodelling of the extra cellular matrix (ECM). A number of factors are involved in the various stages of tissue repair including cell-cell interactions, cell-matrix interactions, a number of different cell types, and a large number of growth factors and cytokines. The regenerative phenotype of the fetus has shown a difference in a number of processes involved in wound healing, which may be manipulated to reduce or even prevent scarring.Fetal wound healing compared to adult wound healing has been shown to have a different and reduced inflammatory response , 6. The The proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) have been found to be decreased during scarless fetal repair even when fetal fibroblasts are stimulated with platelet-derived growth factor (PDGF) , 17. IL-Cyclooxygenase-2 (COX-2), part of the arachidonic acid cascade, is upregulated in response to an inflammatory response such as an injury. COX-2 functions by producing prostaglandins which control many aspects of inflammation. A murine model of scarless healing demonstrated low levels of COX-2 and prostaglandin-2 (PGE2), whereas the addition of exogenous PGE2 induced scar formation in a fetal model of wound healing . BlockinThe ECM is known to play an important role in wound healing as it can play a part in regulating growth factors and cytokines and alter cell behaviour . Fetal wα2, α3, α6, and β4, (laminin and collagen receptors) and neoexpression of α5, αV, and β6 (fibronectin and tenascin C receptors), and this may further explain the fetuses' ability to reepithelise wounds rapidly with a reduced presence of inflammatory cells [Glycoproteins, such as fibronectin, laminin, and tenascin C, bind integrins, collagen, and proteoglycans and are integral components of the ECM playing a role in cell adherence . Fibronery cells .β.The proteoglycans decorin and fibromodulin which are known to regulate collagen fibrillogenesis, growth factor activity, and cellular proliferation have shown variation in fetal wound healing. Decorin showed reduced expression in fetal fibroblasts and fetal skin compared to adult fibroblasts and skin . While dFetal and adult wounds show a number of differences in collagen synthesis; these differences include speed of deposition, variations in collagen ratios and quantity of collagen itself –53. Stud Fetal wounds show increased levels of the urokinase plasminogen activator and matrix metalloproteinases (MMPs) while their inhibitors (PAI-1 and TIMPs) are reduced during fetal wound healing –60. HighFetal studies have indicated that, unlike adult wound closure, fetal wounds close through an actin cable which acts like a purse string . This cain vitro study have shown that human fetal fibroblasts can differentiate into myofibroblasts when stimulated with exogenous TGF-β1 but again at earlier time points than postnatal fibroblasts [Granulation tissue is thought to play a considerable part in wound contraction in adult wound healing. Migrating adult fibroblasts are capable of generating some tensile strength to start contraction, and the myofibroblast  is the main cell responsible for wound contraction. Differentiation of fibroblasts to myofibroblasts requires a combination of growth factors, mechanical cues, and the presence of the EDA variant of fibronectin. The presence of myofibroblasts in fetal wounds remains controversial with a murine model showing no alpha smooth muscle actin expression (except associated with blood vessels ), which roblasts .Growth factors and their receptors play a vital role in wound healing with a number of aberrations associated with abnormal wound healing such as pathological scarring. A number of growth factors have shown different expression in fetal or scarless wound healing compared to adult or scarring wound healing .β family is multifunctional and is believed to be important in both tissue repair and scarring. The three isoforms of TGF-β are synthesized as latent precursors which require activation before they can exert their biological activity through binding to their heteromeric receptor complexes. Fetal wound healing has shown a rapid induction of TGF-β1 mRNA in fetal repair but at lower levels and with a more rapid clearance from the wound site compared to adult wounds [β2 levels, also considered to be profibrotic, was found to be lower in adult-like repair compared to fetal repair [β3, is expressed in adult animal wound healing [in vitro studies [β1 and TGF-β2 may reduce scar tissue formation [β3 has in some animal models shown improved scar formation [β3 if administered prior to the injury could reduce scarring [β3 had no effect in reducing scar tissue formation [β receptors identified , and variations in the TGF-β receptor expressions have been identified in fetal wound healing [The TGF-t wounds , 69, 70. healing , though  studies . Studiesormation , 73. Wheormation . Furtherscarring . Howeverormation . There h healing , 77.β it appears to have quicker clearance in fetal wound healing [Epidermal growth factor (EGF) is known to be involved in wound healing and is thought to be mitogenic for a number of cell types including fibroblasts and keratinocytes. EGF mRNA showed decreased levels with increasing gestational age (scarring) . Surpris healing . However healing . The fib healing . FGF iso healing . The FGF healing . While b healing .The role that angiogenesis and in particular VEGF has in scar formation remains unclear. Scarless fetal repair has not only shown reduced angiogenesis in fetal wounds , but groInsulin-like growth factors (IGF) are known profibrotic mitogens known to play a role in wound healing and fetal development. Treating wounds with exogenous IGF-I has been shown to accelerate wound healing through increased collagen synthesis and its mitogenic effect on keratinocytes and fibroblasts , 85. IGFβ1 is rapidly cleared from fetal scarless wounds. While others have shown that the phosphorylation of receptors and some intracellular signalling proteins differ between fetal and adult fibroblasts [β1 signalling pathway has been shown to be short lived in human fetal fibroblasts after stimulation with exogenous TGF-β1 [Fetal wound healing and fetal derived cells have indicated that there may be differences in intracellular signalling following the binding of the ligand (growth factor) to its receptor. Martin et al.  demonstrroblasts , 88, 89.s TGF-β1 , while os TGF-β1 , 91. VarWound healing requires the expression of a number of genes which are regulated by a number of transcription factors such as activator protein 1 (AP1) and the Hox genes. The AP-1 transcription factor is a heterodimeric protein composed of Fos, and Jun and activating transcription factor protein family members. AP-1 induction has been demonstrated in fetal mouse skin, while c-Fos protein was demonstrated to be upregulated in the epidermis after wounding , 92. TheHox protein activity is essential during embryogenesis, and the Hox genes have been implicated in limb regeneration , 95. A nβ1 in a number of experimental models [Gene expression in fetal fibroblasts shows difference gene expression compared to adult fibroblasts in response to TGF-l models , 102. Col models  showed tl models .A number of studies suggest that fetal fibroblasts proliferate more rapidly than adult fibroblasts . Though Apoptosis is an important process in wound healing occurring in inflammatory cells, myofibroblasts, and vascular cells, for example. Studies have shown that scarless healing shows no difference in total caspase 3 or any cleavage of caspase 3 compared to scarring healing in a murine model. However, scarless healing showed an increase in cleaved caspase 7 after wounding while scar-forming wounds showed no increase. Further scarless healing showed increased levels of cleaved PARP while scar-forming healing only showed a small amount of cleaved PARP . in vivo [in vitro studies have used human fetal-derived cells [A number of animal models have been used to study fetal wound healing in vivo , 67, 107ed cells , 88, 90.ed cells , 108. FuThe precise mechanism of fetal regeneration remains unclear with a number of differences identified between the fetal and adult wound healing . A numbeFurther work is required to understand how fetal cells promote regeneration and wound healing and if this can be promoted in adult wound repair. Work will also need to study the role that stem cells play in both adult and fetal wound repair. However, understanding fetal wound healing and regeneration will impact adult repair in the future and may lead to the reduction or even prevention in the formation of scar tissue in a number of organs.