In summary, the present data show that chronic GSB-106 treatment produces an antidepressant-like effect in the unpredictable chronic mild stress model, which is likely to be associated with the regulation of the BDNF-TrkB signaling.Induction of BDNF-TrkB signaling is associated with the action mechanisms of conventional and fast-acting antidepressants. GSB-106, developed as a small dimeric dipeptide mimetic of BDNF, was previously shown to produce antidepressant-like effects in the mouse Porsolt test, tail suspension test, Nomura water wheel test, in the chronic social defeat stress model and in the inflammation-induced model of depression. In the present study, we evaluated the effect of chronic per os administration of GSB-106 to Balb/c mice under unpredictable chronic mild stress (UCMS). It was observed for the first time that long term GSB-106 treatment  during ongoing UCMS procedure ameliorated the depressive-like behaviors in mice as indicated by the Porsolt test. In addition, chronic per os administration of GSB-106 resulted in an increase in BDNF levels, which were found to be decreased in the prefrontal cortex and hippocampus of mice after UCMS. Furthermore, prolonged GSB-106 treatment was accompanied by an increase in the content of pTrkB Major depressive disorder, commonly referred to as clinical depression, is a severe mental chronic disease that has a high prevalence in almost all developed countries, with more than 280 million people affected worldwide (approximately 4.4% of the population), and is expected to become the leading cause of disease burden by 2030 . Complic706/707 at the TrkB catalytic domain and the phospholipase Cγ-binding site (Tyr816), while leaving the phosphorylation of the Shc-interaction site Tyr515 unaffected [Although the neurobiology of depression is still not clearly understood, the monoamine-based, the glucocorticoid-dependent, the glutamate-driven, the neurotrophin signaling deficiency, the neuroinflammatory, the impaired brain circuit and networks hypotheses have been proposed ,7,8,9,10affected ,19,20. Aaffected . Casarotaffected . Overallaffected ,24 and raffected ,26. The given data indicate the importance of BDNF/TrkB pathway for depression, leading to several proposed approaches aiming to restore or enhance neurotrophin signaling, such as: a local delivery of neurotrophin by engineered cells or viral vectors; an increase of BDNF levels and the neurotrophin effects; a regulation of TrkB receptor synthesis and dimerization; an indirect Trk receptors activation via transactivation mechanisms; a modulation of individual steps in the desired signal transduction pathway; an interaction with TrkB receptor ; a functional mimicry ,27,28,29N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide; A crystallographic analysis, on par with mutagenesis studies and gain-of-function approaches based on chimeric recombination of BDNF protein, allowed to propose the amino acid residues, implicated in neurotrophin interaction with TrkB. In particular, residues 91–97 of the 4th loop of BDNF (Thr91-Met92-Asp93-Ser94-Lys95-Lys96-Arg97) have been recognized as one of the sites for neurotrophin/TrkB complex formation . OraGenerally, a panel of rodent tests and models is used to evaluate the potential antidepressant effect of the compounds. Based on its close resemblance to certain human depressive symptoms, unpredictable chronic mild stress (UCMS) is considered as one of the most translationally-relevant and valuable models for studying the pathophysiology of depression and the efficacy of antidepressants in animals . It has The purpose of this study is to examine the potential antidepressant-like effects of GSB-106 as demonstrated by an UCMS model of depression in Balb/c mice and to evaluate whether this effect is mediated through amelioration of BDNF/TrkB signaling.The GSB-106 compound administered to mice for 14 days in parallel with ongoing UCMS a dose-dep = 0.001) in immobilization time compared to non-stressed animals in the Porsolt test. A higher dose  of GSB-106 attenuated depressive-like responses in mice by 1.2  times compared to stressed mice treated with vehicle. Amitriptyline  reduced the dysphoric behavior of stressed animals by 1.5 times  compared to the control stressed mice. Thus, the preliminary stage of assessing the behavioral effects of substances in UCMS model indicated a dose-dependent anti-dysphoric effect of GSB-106. As shown in p = 0.0499) in the absence of UCMS. In the vehicle-treated mice group subjected to UCMS a 1.2 folds increase in immobilization duration was registered  compared to non-stressed animals treated with vehicle. In mice exposed to UCMS, chronic GSB-106 treatments decreased the immobilization periods by 2.9 folds , compared to the stressed mice treated with vehicle. Thus, the Porsolt test data indicate the anti-dysphoric effect of GSB-106 after 26 days of oral administration in Balb/c mice. At the same time, animals subjected to the stress procedure exhibited a deeper antidepressant effect of the substance compared to the corresponding control. After the effective dose (1 mg/kg) was determined, antidepressant-like activity and underlying mechanisms of GSB-106 were evaluated upon the prolonged use of the compound (26 administrations versus 14) b. As fol706/707, pTyr515, pTyr816) in the different brain regions following UCMS and GSB-106 treatment a Western blotting was performed. p = 0.001 and F = 27.06; p = 0.001 respectively) of mice exposed to UCMS, compared to the control non-stressed animals, which is consistent with the previous data [cortex = 18.5; p = 0.001 and Fhip = 27.06; p = 0.001) compared to that of the vehicle-treated UCMS mice. However, the administration of GSB-106 to animals that were not exposed to UCMS had no effect on BDNF content in these brain regions . To examine the protein level of BDNF and TrkB site-specific phosphorylation . Prolonged GSB-106 administration to UCMS subjected mice caused an increase in the pTrkB706/707 content in the prefrontal cortex  compared to UCMS animals, while the effect of GSB-106 on pTrkB706/707 level in both brain structures of non-stressed animals appeared to be statistically insignificant .Abundant evidence indicates that action mechanisms of pharmacologically diverse antidepressants implicate a phosphorylation and an activation/transactivation of the TrkB receptors in the brain regions is associated with the depression-like behavior . Our nex816 level was observed in the prefrontal cortex  and in the hippocampus  of UCMS mice compared to unstressed animals. Notably, data on TrkB816 phosphorylation in the prefrontal cortex of stressed animals shows bimodal distribution. However, none of the available data sets, other than those indicated, appear to be mathematically bimodal. At the same time, all our data pass the normality test . Conceivably, the bimodal distribution of pTrkB816 values might reflect the individual variability in response to GSB-106 treatment in the cohort of participating animals.As follows from the data presented in 816 site in the prefrontal cortex  and in the hippocampus  compared with control UCMS mice. Notably, in the absence of stress, GSB-106 caused a reduction of pTrkB816 in the prefrontal cortex , whereas in the hippocampus there was a non-significant trend for decreased TrkB phosphorylation at that tyrosine residue . We also found that UCMS produced a decrease in TrkB phosphorylation at Tyr515 site in the prefrontal cortex and the hippocampus  relative to control  of non-stressed mice after chronic GSB-106 treatment. However, no changes in pTrkB515 values were established in the prefrontal cortex and in the hippocampus of UCMS mice chronically treated with GSB-106 .Administration of GSB-106 for 26 days along with a continued stress procedure promoted a rise of TrkB phosphorylation at Tyr control d. FurtheY706/707 site) in the prefrontal cortex and induced a pronounced increase of TrkBY816 phosphorylation in the prefrontal cortex and the hippocampus of UCMS subjected mice. Overall, these results indicate that GSB-106 exhibits an obvious antidepressant-like effect in a mouse model of UCMS-induced depression that was mediated, at least partially, by an increase in BDNF level and site-selective phosphorylation of TrkBY706/707 and TrkBY816. The presented results for the first time show that prolonged per os GSB-106 administration  produced an antidepressant-like behavioral response during an ongoing stress exposure in UCMS-treated Balb/c mice, as evaluated by the Porsolt test. Furthermore, GSB-106 restored a decrease in BDNF level induced by the UCMS procedure in the prefrontal cortex and in the hippocampus. Additionally, chronic administration of GSB-106 resulted in a TrkB activation  and more GSB-106 treatments (26) were delivered. However, both experiments had one fundamental point in common, namely, they initially formed dysphoria-like chronic stress-induced behavior followed by treatment. Most studies utilizing this model of chronic stress have a similar design, where once the long-lasting stressful conditions are discontinued, recovery processes are likely to develop. In the present study, a 1 mg/kg dose of GSB-106 attenuated the depression-like responses of mice by 2.9 times following a 26-days treatment and by 1.2 times following a 14-days course, compared to the corresponding controls. 706/707) and a decrease of Tyr515 and Tyr816 phosphorylation in the prefrontal cortex and hippocampus of rodents subjected to UCMS, that is consistent with the depression-like behavior [515 and pTrkB816 content in the prefrontal cortex and the hippocampus of UCMS-treated mice was also revealed, while no changes in TrkB706/707 phosphorylation were observed in both brain structures of UCMS mice, generally indicating the alleviation of TrkB-dependent signaling in vulnerable brain regions of stressed animals. Overall, our behavioral and western-blot findings have demonstrated that UCMS produces depression-like symptoms (dysphoria), which are accompanied by the alterations of BDNF content and TrkB phosphorylation at specific tyrosine residues in the limbic brain structures, which are similar to those evidenced elsewhere. Importantly, the present study revealed that continuous UCMS procedure concomitantly with long-term per os GSB-106 treatment resulted in a BDNF content increase in the prefrontal cortex and the hippocampus and induction of site-specific phosphorylation of the TrkB receptor—at Tyr706/707 and Tyr816 sites, but not at Tyr515 site, in this way mimicking the pattern attributed for most of the conventional antidepressants. We consider that an increase in TrkB706/707 phosphorylation in the prefrontal cortex of stressed animals upon GSB-106 treatment might be a consequence of increased BDNF content, evoked by GSB-106, although it is quite difficult to determine the exact cause-and-effect relationships under the conditions of in vivo experiment with the multiple crosstalk and feedback loop mechanisms all of which suggest that this point requires additional investigations. Obtained data indicate the ability of GSB-106 to reverse depressive-like behavior. Simultaneously, GSB-106 normalizes the BDNF content, reduced due to a prolonged UCMS in animals, as well as the site-specific phosphorylation of TrkB receptor.It is well known that long-term exposure to stress, as well as other conditions that contribute to the depressive-like behavior in animals, is accompanied by a BDNF expression decrease in the prefrontal cortex and in the hippocampus, while an increase in the level of neurotrophin has been observed in the nucleus accumbens ,57. In pbehavior ,55,58. IIt has been experimentally established that the development of a depressive phenotype in UCMS is based on the elevated functionality of the hypothalamus-pituitary-adrenal (HPA) axis and its effects on the feedback systems of the limbic structures ,60. When a stressful event occurs, an activation of the HPA axis leads to the release of glucocorticoids and triggers a feedback mechanism, the main contributors of which are the hippocampus and the prefrontal cortex. As a result, the effects of stress are limited in time and the mechanism for restoring homeostasis and behavior is triggered. Prolonged activation of the HPA axis under chronic stress causes prolonged release of glucocorticoids which are toxic both for the hippocampus and the frontal cortex which leads to a decrease in the BDNF content ,62, deplIn contrast to the hippocampus and the prefrontal cortex, the amygdala provides positive feedback to the HPA axis. Prolonged UCMS with the loss of hippocampal inhibition over the HPA axis increases activity in the amygdala. UCMS has been found to cause an increased coherence of activity in the amygdala , along wIt is widely believed that chronic antidepressant treatment causes an increase in the synthesis or release of BDNF, activation of TrkB receptors and related signal transduction pathways, triggers up-regulation of genes associated with BDNF-induced plasticity ,24,70,71706/707 phosphorylation takes place within 30 min after a single dose, whereas no changes were detected at TrkB515 residue (Shc binding site) either under acute or chronic administration. Furthermore, an increase in Tyr706/707 phosphorylation was observed after sustained (21 days) antidepressant treatment as well [816 residue in the mice hippocampus was detected 1 h after acute administration of fluoxetine, riboxetine, citalopram, imipramine; a similar effect on the phosphorylation of Tyr816 was also established upon long-term administration (21 days) of fluoxetine [706/707), followed by an increase in Tyr816 (binding site for PLC-γ1). Notably, through preferential activation of TrkB-PLC-γ1-CREB-mediated signaling, antidepressants have been found to raise the level of synaptic plasticity in critical neural circuits to the degree required for relieving depressive symptoms, pointing out the significance of PLC-γ1/IP3/Ca2+ pathway for antidepressants response [706/707 and Tyr816 sites, but not at Tyr515) in the brain areas implicated in depression, caused by prolonged GSB-106 treatment in UCMS-exposed mice, are in line with the data observed on typical antidepressants, which provide TrkB-Tyr816/PLC-γ1 specific effects [Saarlainen et al. , Rantama as well . Howeveruoxetine . Altogetresponse ,79,80. O effects ,19. As w effects ,44,45,46706/707 residue, a recovery of the synaptic proteins PSD95 and synaptophysin levels in the prefrontal cortex in CMS-subjected animals [706/707 levels have also been demonstrated in a social defeat stress model and LPS-stimulated depressive-like behavior [Y816 but not TrkBY515 with subsequent activation of the PLC-γ1 pathway in primary striatal neurons and selectively reverted the decrease of pTyr816 but not pTyr515 in striatum of the chronically treated R6/1 transgenic mice [To our knowledge, this is the first report showing a site-selective phosphorylation of TrkB, elicited by small peptide or non-peptide BDNF analogs with antidepressant effects in the robust behavioral model of depression. Currently, several compounds with antidepressant properties were identified as putative ligands of the TrkB receptor, such as 7,8-dihydroxyflavone (TrkB agonist), ANA-12 (TrkB antagonist) and tetraterpenoid deoxygedunin. Zhang et al. demonstrated that chronic 7,8-dihydroxyflavone  treatment induce antidepressant-like effect in mouse chronic mild stress model, accompanied by a restoration of BDNF level, TrkB phosphorylation at Tyr animals . The antbehavior ,82. Anotnic mice . 816 phosphorylation in primary cortical neurons, whereas fluoxetine, imipramine, ketamine, R,R-HNK enhanced TrkB interaction with PLC-γ1 [Although the comprehensive evidence on the engagement of BDNF/TrkB signaling in antidepressant responses has been acquired, detailed mechanisms of this interaction remained unresolved until recently. Casarotto and colleagues reported the direct binding of both typical and rapid-acting antidepressants (in micromolar affinity) to the transmembrane region of TrkB dimers that promotes translocation and the synaptic localization of TrkB and facilitation of BDNF binding to TrkB and its activation. It is important to note that cholesterol has been shown to potentiate the effects of antidepressants and BDNF on TrkB signaling through the presumed impact on the conformation of TrkB dimers and the binding site of antidepressants. Furthermore, it was revealed that fluoxetine and ketamine increased Tyrh PLC-γ1 . To dateh PLC-γ1 . Whilst h PLC-γ1 ,84, neurh PLC-γ1 ,86 and ah PLC-γ1  effects h PLC-γ1 , and eluad libitum access to granulated chow  and water. All experimental procedures were in compliance with the 322 the EC Directive 86/609/EEC for animal experiments and were approved by the bioethics committee of the FSBI “Research Zakusov Institute of Pharmacology” (protocol No. 1 of 31 January 2021). The experiments were conducted on 128 male Balb/c mice weighing 22–25 g . The animals were group-housed under standard conditions (9–12 mice per home cage), with a 12-h dark-light cycle at a temperature of 22 ± 2 °C and GSB-106 ; (2) vehicle, stress (n = 12); (3) GSB-106 (0.1 mg/kg), stress (n = 12); (4) GSB-106 (1 mg/kg), stress (n = 12); (5) amitriptyline (10 mg/kg), stress (n = 10). The experiment started with 28 days of UCMS exposure after which, in conjunction with continuing stress, the test substances were administered over the following 14 days ; (2) GSB-106 (1 mg/kg), no stress (n = 10); (3) vehicle, stress (n = 9); (4) GSB-106 (1 mg/kg), stress (n = 9). Behavioral tests were carried out with the animals of the second experimental cohort under a similar schedule for the control and the experimental groups. The Porsolt test was performed 60 min after 26th administration of the vehicle or GSB-106. The second experimental series consisted of two parallel stages—a behavioral assay and BDNF and pTrkB protein contents assessment b. Both sThe antidepressant activity was studied in the Porsolt test in mice . Cylindr706/707) ; rabbit polyclonal anti-phospho-TrkB (Tyr816) ; anti-phospho-TrkB (Tyr515) ; mouse monoclonal anti-α-Tubulin . Secondary antibodies conjugated to HRP (used at 1:12000 dilution) were from Cell Signaling Technology  .Following primary antibodies were used in this study: rabbit polyclonal anti-BDNF ; rabbit monoclonal anti-TrkB ; anti-phospho-TrkB , weighed, and stored at −80 °C.−2 M Tris-Cl (pH 8.0), 10−3 M EDTA, 5 × 10−4 M EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.14 × 10−3 M NaCl, 1 × protease inhibitor cocktail (#P8340), 1 × phosphatase inhibitor cocktail 2 and 3 , 10−3 M PMSF  using Tissue LyserLT . Protein concentration was determined using BCA Protein Assay Kit . Lysates were loaded on SDS-PAGE, and separated proteins were transferred onto the nitrocellulose membranes . The membranes were blocked in 5% not-fat milk or 5% BSA in TBS-T for 60 min at room temperature, incubated with the appropriate primary antibody  and then with a secondary antibody conjugated with HRP (60 min at room temperature). Bands were visualized using SignalFire Plus ECL Reagent . Membranes were scanned using Amersham Imager 680  and quantified in the Image Quant TL v.8.1 . Measurement of each protein was then normalized on the related α-Tubulin loading control.Brain samples were homogenated and lysed with RIPA buffer (10www.graphpad.com). The normal distribution of the data was evaluated using the Shapiro–Wilk test. The results are presented as means ± SEM. The significance of intergroup differences was estimated by one-way analysis of the variance (ANOVA), followed by Tukey’s multiple comparisons test.Analysis was performed using the GraphPad Prizm 7.0 software  and at the PLC-γ1 interaction site TrkBY816  of UCMS mice might contribute to antidepressant-like mechanisms of GSB-106.Altogether, irrespective of the mode of GSB-106 interaction with the TrkB, prolonged per os treatment with dimeric dipeptide ameliorates depressive-like behavior in UCMS-stressed mice. Remarkably, our findings strongly suggest that an increase in BDNF in the prefrontal cortex and hippocampus, as well as the restoration of phosphorylation at the TrkB


An independent chronology was usually absent. Radiocarbon dating has recently begun to change this conventional model radically. The challenge, if an alternative, independent timeframe and history is to be created, is to articulate a high-resolution chronology appropriate and comparable with the lived histories of the Indigenous village settlements of the period. Improving substantially on previous initial work, we report here high-resolution defined chronologies for the three most extensively excavated and iconic ancestral Kanienʼkehá꞉ka (Mohawk) village sites in New York , and a fourth early historic Indigenous site, Brigg’s Run, and re-assess the wider chronology of the Mohawk River Valley in the mid-15th to earlier 17th centuries. This new chronology confirms initial suggestions from radiocarbon that a wholesale reappraisal of past assumptions is necessary, since our dates conflict completely with past dates and the previously presumed temporal order of these three iconic sites. In turn, a wider reassessment of northeastern North American early history and re-interpretation of Atlantic connectivities in the later 15th through early 17th centuries is required. Our new closely defined date ranges are achieved employing detailed archival analysis of excavation records to establish the contextual history for radiocarbon-dated samples from each site, tree-ring defined short time series from wood charcoal samples fitted against the radiocarbon calibration curve (‘wiggle-matching’), and Bayesian chronological modelling for each of the individual sites integrating all available prior knowledge and radiocarbon dating probabilities. We define (our preferred model) most likely (68.3% highest posterior density) village occupation ranges for Smith-Pagerie of ~1478–1498, Klock of ~1499–1521, Garoga of ~1550–1582, and Brigg’s Run of ~1619–1632.The timeframe of Indigenous settlements in Northeast North America in the 15 This early history is predominantly informed and told from the evidence of European documentary history [The 15 history –8). Acco history –14. This history . There ith to early 17th century era and beyond. The lack of, and necessity for, an Indigenous oral history perspective for American archaeology and history has been regularly noted  and hased (e.g. –29. Attees (e.g. ) are usues . Th. Thth toes . More evidence of European items, and especially the appearance of various types of glass beads and specific metal forms  placed sites into the late 16th and early 17th centuries. Among sites, comparison of changes in some artefact types were used to suggest apparent social and temporal relationships via seriation analysis (e.g. [14C) dating was long deemed not useful because a plateau/reversal in the 14C calibration curve created temporal ambiguity ~1480–1620, producing widely spread, or multiple, possible date range(s), and thus a lack of resolution. Dendrochronology was rarely practical on Indigenous sites in northeastern North America as recovered wood remains  typically had no more than a few decades of total tree-ring growth preserved.One critical step towards a history that is less affected by ethnocentric biases and assumptions is an accurate independent  timeframe. Here there is work to be done . The archaeology of northeastern North America during the last several centuries before European entradas has been the subject of intense research and is known in detail in terms of numerous sites of village occupations in various investigated areas e.g., , 35–44. is e.g. , 45–48) –48 th this ; improved 14C calibration curve resolution; and, in particular, the use of Bayesian chronological methods , and placing these into calendar time via correlation against the 14C calibration curve  s e.g., –61) enab enab14C  America –65. In patching’ , 67); ands e.g. , 68, 69), 6914C d14C dating effort in the region [The Mohawk River Valley in eastern New York forms one of the most intensively studied areas of Indigenous settlement in northeastern North America. The name refers to the association with the Kanienʼkehá꞉ka, or Mohawk Nation, one of the original Five Nations of the Haudenosaunee (Iroquois) Confederacy , 29, 70.e region . Notablee region , 76: Figth and 20th centuries. Results of the excavations at Garoga were first summarized by Ritchie and Funk [th century. They did not suggest a chronological sequence for the sites, although based on the amount of European-provenance metal found at Smith-Pagerie, they thought it might be more recent than Garoga. They proposed the sites could not have been occupied contemporaneously because one interpretation of ethnohistoric data suggested the Mohawk River basin was divided between clan territories, and they assumed that only three or four villages were contemporaneously occupied in the (wider) valley  of Smith-Pagerie, Klock, and Garoga with the Smith-Pagerie occupation initiating in the last two decades of the fifteenth century [Since Ritchie and Funk’s publication, the three sites have been considered a single community sequence during the 16ry (e.g. ). Based ry . Conver century .terminus ante quem (TAQ), terminus ad quem, and/or terminus post quem (TPQ) dates for each of the sites and refine the Bayesian models. We further obtained new dates on the Brigg’s Run site . Methodciations –65, 80.th to 17th century Indigenous archaeological record in eastern North America is only in its initial stages . The laes e.g. –65, 97)., 97.th tResearch on Native American settlement patterns in present-day New York was carried out in two extensive research programs in the 1960s through 1980s , 74. The14C dating of annual plant remains (maize) in northeastern North America. Combined with traditional relative-dating practices, including pottery type frequencies and the presence, amounts, and categories of European exchange items, this work enhanced understandings of the basin’s occupational histories, work that was later critiqued and clarified by Lenig [Snow  conducteby Lenig .14C measurements). Thus, in this present study, we have obtained a number of new radiocarbon measurements on samples from these four sites  constraints for the main site occupation phases of interest. Our aim was to achieve chronological resolution for each of these four sites in isolation—rather than from assumed site sequences or approximate dates—employing the 14C data from each site integrated with the prior information from site taphonomy and ethnohistoric and archaeological observations. This work permits us to resolve the date of each of the sites closely and clarifies their temporal relationships. We then further re-considered the chronological placements and relationships between these sites and a wider set of Mohawk River Valley sites  and USDA Plants (https://plants.usda.gov/) online databases. A LEO 1550 field emission scanning electron microscope (FESEM) was used for high magnification observation of anatomical micro-features and high-quality image capture.All wood charcoal fragments larger than 2 mm were fractured by hand or with a steel razor blade to create fresh transverse, radial, and tangential planes, in order to examine the wood anatomical structure and identify the taxon. After fracturing, wood samples were supported in a sand bath or modeling clay and examined under a Motic K-400P stereo microscope at x6–x50 magnification and an Olympus Bx51 polarizing microscope at x50–x500 magnification. Seeds were examined under the same set of microscopes. The macro- and micro-anatomical features of wood sections and seed samples were documented, photographed, and compared with those from modern reference collection materials in the Cornell Tree-Ring Laboratory, standard reference texts , 100, an14C dates employed are either new dates, all UCIAMS, published here  with Gas Bench input. Details on KCCAMS dating protocols are available on the facility’s website (https://www.ess.uci.edu/group/ams/facility/ams).The hed here , or prevterminus ad quem for the pits’ original functions. We focused especially on identifying wood charcoal samples from specific loci with a number of tree-rings preserved out to original waney edge/bark. The terminal tree-ring in these cases therefore provides a close date estimate for the human use of this sample in the episode represented in the context. The availability of a series of tree-rings leading to this event means that we can employ the 14C ‘wiggle-matching’ approach [The new samples for dating from the Garoga, Klock, and Smith-Pagerie sites were recovered from hearths or large pit features. The size and shape of the large pit features  and in mapproach , 67 to capproach , 98, 10514C Calibration curve resolution available. Inaccuracies may also occur at certain periods when the total length of the time series is sufficiently short that it may not be able to permit discrimination between multiple periods with similar atmospheric 14C levels and trajectories . Over the last 1000 years, such parallel instances of similar 14C levels and trajectories are typically distinguishable once time-series are greater than ~50 years. The latter issue of potential ambiguity is potentially relevant in the context of this article especially to the periods around ~1490–1535 and ~1600–1635. In such cases, additional constraints may be necessary to achieve satisfactory discrimination for very short time series. The former issue applies when, for example, single, annual, tree-rings only are dated  is not occurring at a time where there is a potential alternative calendar placement (contrast the case if the series was lying around either ~1490–1535 and/or ~1600–1635 when there could be an ambiguity). Instead, the mid to later 16th century calendar placement for this Klock time series is the only available placement for the series regardless of the evident variation, noise or offset.We note that cases of reported inaccuracies for short-sequence wiggle-matches at high-precision , 107 arees: e.g. –110), whes: e.g. , 112. Fonoted by , any insth century ([14C dates are consistent with placement in the first decades of the 17th century.Tree-ring sequences are described in terms of Relative Years (RY): the first measured (innermost) tree-ring  is RY 1001. In this case we sought to have more than one such sequence for each site with the view that achieving multiple compatible results reinforces confidence in the dates obtained. The samples from Brigg’s Run, in contrast, are from an avocational archaeologist’s collection donated to the New York State Museum with minimal associated documentation. Thus we make no intra-site history assumptions in this case. The key issue for this site, rich in European trade goods including types that, according to all understanding, can only date from the early 17century  for the earlier use and for the site as a whole.Garoga site Feature 36 was a cylindrical pit measuring 122 cm in diameter and 147 cm deep within Longhouse 9 ( at p.99)Klock Feature 84 was a large pit measuring 107 x 152 cm in plan and 122 cm deep  within LThe maize sample dated by ISGS-A0326 needs comment. This sample was reported in  as cominKlock Feature 135 was a pit measuring 123 cm in diameter located between Longhouses 3 and 5. Field notes indicate there was a lining of charred “vegetal material” at the base of the pit in which maize was present. This is the only mention of maize in the field notes and is the likely source of the AMS-dated maize kernel (UCIAMS-218475) reported in  representh century source indicates that pits were visible at sites like this at the time and would presumably therefore have been visible to ancestral Mohawk people after having been filled  as reported in . AMS dat filled ( at p.23)14C however is much older than any other dated element from the site . It is a ~98% probability outlier if assumed to be the first date from the site, and a 100% probability outlier if included in the subsequent main site Phase. This date is thus excluded from the dating models, whether as an unexplained outlier, or evidence of distinct and earlier human activity at the site locus.Smith-Pagerie Feature 60 Longhouse 5 was a small and relatively shallow pit (36" diameter and only 24" deep:  at p.61)14C calibration curve [https://c14.arch.ox.ac.uk/oxcalhelp/hlp_commands.html), such as Boundary, Date, Before, D_Sequence, Gap, Interval, LnN, Outlier Model, N, Phase, R_Combine, Sequence, are capitalized in our text. Individual outliers within site models were identified and down-weighted using the OxCal SSimple Outlier model within D_Sequence (tree-ring wiggle-match) sequences [Zea mays ssp. mays), B = animal bone, and R = residue (food/organic residue). Other samples are given descriptions. Each site is modelled within an overall site OxCal Phase between start and end Boundaries. Based on ethnohistoric information we regard the occupation of each village as likely being within a total range of 0–30 or 0–40 years with average site occupation lengths somewhere likely in the range of 10–20 or to 25 or so years [We employed the OxCal Bayesian chronological modelling software  using veon curve   a Log Normal distribution of LnN(ln(20),ln(2)); or (ii) a Normal distribution of N; or (iii) a Normal distribution of N. In each case the site Model 1 uses the LnN constraint; the site Model 2 uses the N20,10 constraint; and the Model 3 uses the N25,10 constraint see . Critica14C dated samples all from the site Phase–with no further information available as the material derives from a non-systematic avocational collection given to the New York State Museum–comprise four dates on short-lived samples (maize) belonging to the site Phase, and a wiggle-match series of dates on a 25-ring beech (Fagus grandifolia) sample ending in bark, reflecting a use date somewhere within the site Phase. There is relevant historical information ([terminus ante quem (TAQ) for the end of Brigg’s Run (on the north shore of the river) by 1635. Less definite but informative, is the rich assemblage of a range of materials recovered from Brigg’s Run which can only belong in the early 17th century, with some items indeed likely only post-1614 (and the founding of Fort Nassau) and even post-1624 –though materials likely dated from the years 1626–1635 are not specifically observed in Snow’s [th century.The rmation ( at pp.25rmation ( states tn Snow’s  review. n Snow’s  at pp. 2n Snow’s  at pp.9-n Snow’s  at pp.74Fagus grandifolia) sample and the last extant tree-ring from an elm (Ulmus sp.) sample are interpreted as providing a TAQ for the pit and so for Longhouse  9 at the site (with the date on the waney edge placed as the latest available evidence). There are seven other dates from House 9. Y-1381 is wood charcoal that may well include substantial in-built age, which we accordingly model applying the OxCal Charcoal Outlier model. AA-8370 on maize from F37 pit is a 100% probability outlier versus the site model–and is too old–for unexplained reasons. Either it is an erroneous measurement or represents previous, much older, activity at the site area. The other dates  come from the F2 pit. AA-7695 is a ca. 10% probability outlier in the model (whose date is slightly too old). We exclude AA-8370 and AA-7695. An intra-site Sequence for House 9 is modelled placing the data from the House before the wiggle match TAQ dates. A Difference query applied to the period between the start and end Boundaries for House 9 yields estimates for the duration of House 9 of 0–8 years at 68.3% hpd, and 0–21 years at 95.4% hpd. Other dates from Garoga come from pits in House 2, House 4, House 5 and House 12 and from F184 pit between House 1 and the stockade. We model the overall site Phase.As noted above, there are two wood charcoal samples evidently from a late high lobe on the south side of F36 pit. Wiggle-match dates for the waney edge of a beech (Ulmus sp.) branch preserving the entire stem from pith to bark with 25 rings (the last including latewood formation) came from what appears to be the last and final use of the Feature 84 pit–high-up in the pit just below plow-soil–and this last use is likely either approximately contemporaneous with, or after , the last use (post-closure) of the associated Longhouse  1 structure–since Feature 84 was under the area of a bed platform during the use of House 1 and thus this final activity in Feature 84 probably occurred after the removal of House 1. Thus the date on the waney edge of this sample offers a TAQ for House 1. The length of the ‘ante’ is not known. A tree-ring sequenced wiggle-match is available. Two 14C dates are on relative years (RY) 1001–1002 (innermost two rings of the sample: UCIAMS-226653 and 239720). Since these dates are on the identical tree-rings (and are very similar) we combine them as a weighted average. Another 14C date is on RY1010-1018 (UCIAMS-239721), ~12.5 years later, and a final 14C date is on RY1021-1025 (UCIAMS-226654), 9 years later again. The date of the last complete tree-ring (including the waney edge), RY1025, is 2 years after the mid-point of the RY1021-1025 sample.A small diameter elm  later, either 1592–1619 (UCIAMS-239721) or 1580–1622 (UCIAMS-226654). The net outcome versus IntCal20, adding in the constraints of the tree-ring derived spacing between the samples and trying to compromise what appear to be somewhat non-compatible data, is that the wiggle-match is pushed later with the RY1001-1002 weighted average placed most likely 1564–1589 (59.9% of 68.3% hpd) but at the price a poor OxCal individual Agreement value of ~44.6 < 60, since the modelled placement no longer corresponds with most of the likely non-modelled calibrated probability. At first sight, this result appears of concern. But it is important to consider that the IntCal curve is a narrow, modelled probability band trying to best-describe a large set of raw 14C calibration data available in this period. For example, one high-quality annual resolution time series of recent data (ETH dataset) shows considerably greater structure in the 16th century than represented in the smoothed IntCal20 record [14C age ranges of the Klock branch samples versus the raw IntCal20 dataset suggests greater potential flexibility. If we consider just the wiggle-match of this Ulmus sp. branch, the OxCal Amodel and Aoverall values for the data as reported ) yields Amodel and Aoverall values of ~70 and 79 with the Ulmus sp. RY1001-1002 sample with individual Agreement value ~68.The wiggle-match is not entirely straightforward as the urve see . The two0 record   dates its construction or start of use. The lens of charred maize cobs with kernels attached  date the primary use of the pit. We employ a weighted average 14C age for this apparently single episode/group. Another 14C date on a maize sample from Feature 116 pit is likely within this same earlier use horizon (UCIAMS-218476). There are then some 14C dates on samples and contexts from likely subsequent House 1 use. A date on maize (UCIAMS-190560) and a date deer bone (UCIAMS-190562) from hearths reflect use during the period of House 1. ISGS-A0326 is on maize from the Feature 50 pit, but, unlike the maize from the Feature 84 pit, this maize deposit was not at the bottom of the pit, and so probably represents use/filling after the pit was no longer serving its original/primary purpose and hence is from the main/later occupation versus initial/early occupation at the site. In addition to House 1 there are several other 14C dates on samples from other Klock contexts. One is an outlier that is clearly too old for unknown reasons  (AA-7204 on maize) which we exclude; the remaining dated samples belong to somewhere within the period of the Klock occupation . We consider the date on organic residue as potentially including in-built age and thus apply the OxCal Charcoal outlier model to this date. There is one sub-grouping. Two samples come from Feature 135 pit between Houses 3 and 5: a sample of maize (UCIAMS-218475) and a small ash (Fraxinus sp.) twig with 13 tree-rings ending in waney edge/bark. UCIAMS-239711 dates tree-rings RY1009-1013 of this twig, thus we can model the date of the waney edge and use of this twig as 2 years after this date (mid-point treated as RY1011).The last ring (including the waney edge) of the above Ulmus branch’s dated waney edge) and the Phase of other dates. Integrated into the overall site model and data, whether we use the quoted 14C values for the Ulmus sp. samples (a), or those with a 50% measurement error increase (b), makes very little difference other than to the Amodel values (~53 versus ~68/69) (considering Models 1a v. 1b and 2a v. 2b). The Date query for the overall Klock Phase is almost identical comparing the most likely 68.3% hpd ranges: (a) 1494/5-1521/3 and (b) 1496–1521.The overall Klock site Phase encompasses the House 1 Sequence . We exclude this date from the analysis. We also exclude UCIAMS-218491 from Feature 9 pit in Longhouse 1. We have no information on what material was in fact dated in the case . The δ13C value, -29.1‰, is also substantially different from all the others from the site, raising suspicions. The age is too recent and is a ~100% probability outlier if modelled in the site Phase. We thus do not include this date in the site model. This leaves the remaining Smith-Pagerie data from Feature 54 pit from Longhouse 1. As discussed above, maize samples from a mid-pit-depth deposit likely date use or residual material during the use period of the pit . The context of the charcoal samples  indicates that the wiggle-matched dates for the waney edge of these samples set a TAQ for the other material in the pit–with these later fills and re-use of the pit quite likely dating after (even well after) the main occupation of the site (and earlier use of the lower part of Feature 54 pit). The three wiggle-matched samples indicate dates for this subsequent use, re-excavation, and re-filling of the pit as represented in the Dark Lens charcoal likely from the mid-16th century to the early 17th century. Material higher in the pit fill may be even more recent (see comment above concerning the report in [Unburnt bone from Feature 127 (a hearth) in Longhouse  4 likely represents post-site-occupation deposition (since we would expect bone to be burnt from use of the hearth during the site occupation period). This sample is thus a TAQ for the site occupation. The latest dated occupation context for the site is the maize from the Feature 80 hearth from Longhouse 2. We regard this as likely a TAQ for the other data from the site in that we would expect periodic cleaning of hearths during a site’s occupation. Four dates are available on samples which can only be placed as from activity within the site Phase represented by Longhouse 1: UCIAMS-190565, UCIAMS-190563 and UCIAMS-190566 on bone from Features 11, 25 and 40 in Longhouse (House) 1, and ISGS-A0528 on organic residue from Feature 15 in Longhouse 1. One date on maize from Feature 60 pit in Longhouse 5, UCIAMS-218493, is an unexplained, much too old outlier (~100% probability if placed in the site Phase with the other data). This could be a sample or laboratory issue but may also reflect earlier activity at the locus. The Feature 60 pit is relatively small and shallow (36” diameter and 24” deep:  at p.61)eport in  at p.23)OxCal runfiles for each of the four sites discussed above—Brigg’s Run, Garoga, Klock and Smith-Pagerie—are set out with descriptive notes in 14C wiggle-matches to constrain and refine dating for each site [We considered each of the Smith-Pagerie, Klock, Garoga and Brigg’s Run sites in separate dating models. Previous assumptions and debates about site relationships among the Smith-Pagerie, Klock and Garoga sites (summarized above in Introduction) were specifically not included in our analysis, in order to obtain a neutral assessment based solely on the data available from each site, considered independently. Using the Bayesian chronological modelling approach as implemented in the OxCal software , 91, 92,ach site , 68; in th through early 17th centuries [As briefly discussed above, the one key further assumption made concerns the likely overall period of occupation at these sites. According to the specific observations made in early ethnohistoric accounts, as well as assessments made on the basis of the archaeological evidence and resource use estimates, we regard the occupation of each village as being less than ~40 years , and lienturies  at p.120enturies  at p.49)enturies  at pp.66enturies , 68, 117model and Aoverall values (>60) and the likely date range of each site is evident with minimal realistic ambiguity. There is no ambiguity in any of the most likely 68.3% hpd ranges. The one exception regarding the Amodel values is Klock Model 1a, Model 2a and Model 3a. As discussed in Methods (above), the wiggle-match of the dated tree-ring samples from the Ulmus sp. branch from Feature 84 is less than satisfactory given the available data and the IntCal20 smoothed calibration curve model andIn the case of Klock, one other possible variation in assumptions seems worth consideration. It might be argued as plausible that the samples found from (we may assume) the last use(s) of the hearth feature in House 1 (CIAMS-190562 on bone and UCIAMS-190560 on maize) reflect the last use of House 1 and so set a TAQ for the earlier material from the house and pits therein. If we revise Klock Model 1b to reflect this additional assumption, placing the dates on material from the hearth as the latest in the house Sequence, it only further solidifies the preferred date range for the site identified in each of Models 1–3: see 14C measurements and still yielded substantially older ages. In such circumstances, where no in-built age can be explained in terms of the sample , then it is possible that there was previous human activity at the locus—indeed, multiple uses of suitable, even strategic, loci through time have been observed or suggested in other work in the region . It. It14C mon (e.g. ). For exon  remove the less likely alternative probability regions. This reveals a likely approximate time-frame for this set of 10 sites. We compare the date ranges achieved against one set of previous date estimates for 11 of these 12 sites in We further considered the four sites dated in Figs us work  chronology for these Mohawk Valley sites. In this regard our findings here confirm, but importantly revise and refine, initial sed work . The calsed work , 121–123sed work ). It is th through early 17th centuries has conventionally been dated and described in terms of European history and the absence, presence and types of European trade goods. The first part of the title of a 1995 article perfectly captures the ingrained thought processes involved in most work until recently: “Untanglers of matters temporal and cultural: glass beads and the early contact period…” [th century). Thus, despite pioneering efforts to obtain large sets of radiocarbon dates, such as by [st century. It is only very recently that the combination of modern AMS 14C dating, allowing dates on small samples and with good measurement precision, allied with Bayesian chronological methods incorporating prior knowledge informing and constraining temporal relationships between variously samples, different contexts at a site, and overall occupation duration, have enabled temporal resolution at the scale of Indigenous village life-cycles and the associated human lived experience and history ch as by  for the ry e.g. , 68). In. Inth thry , 22th thnd space , 64, 80.The relatively refined and high-resolution definition of time for the sites focused on in this study highlight that the presence or absence of initial and limited quantities of European trade goods often cannot be used satisfactorily to define chronology –65, 69. th century, highlights how very little we know about European contacts and involvement with northeastern North America outside the few well-known episodes of the long-told standard narrative, such as Cartier landing in the St. Lawrence in 1534 and the establishment of Fort Nassau in New York from 1614. Earlier Norse contact and settlement is attested around 1000, and some other links between then and the 15th century are possible or hinted at [th through 16th centuries [th century (1604–1614). The recent re-discovery of William Weston and his voyage from Bristol to North America ~1499, is just one example of a rich, but largely hidden, history [th through 16th centuries from direct archaeological evidence there is thus an irony. In doing this we also throw additional important light, in reverse, via the independent dating of early European materials at Indigenous sites in North America—like Smith-Pagerie, Klock and Garoga [Detailed contextual study and additional radiocarbon dates confirm previous indications that the artefacts determined to be of European iron and copper, the latter by pXRF analysis, at Smith-Pagerie are presently the earliest well-dated examples of European metal circulation in Northern Iroquoia  Fig 5)..Fig 5). inted at , 125. Hiinted at , and a senturies –129, espenturies , 129, be history . In corrd Garoga —on an imS1 TableZea Mays ssp. mays are listed as “maize”. Similarly, we refer to white-tailed deer rather than Odocoileus virginianus. RY refers to Relative Year for tree-ring samples–this means the annual growth increments (tree-rings) within the sample. Note: the four Ulmus sp. samples listed as NYSM #45171.A1 are from the same small diameter branch sample. Two samples were mistakenly listed as Fagus sp. in the UCIAMS laboratory report but the samples sent (as part of two different sets of samples) were all Ulmus sp. and from the same branch. Wood charcoal is identified where it was part of the present study–samples and dates from previous work (e.g. the sample for date Y-1381) could not be identified if the relevant reports/publications did not provide this information. Missing δ13C values reflect our inability to find these in some older reports/publications. The δ13C values noted with an * were stated as estimated (versus measured) by the radiocarbon laboratory. The value replaced by ** was a case where the sample was too small for a separate IRMS assay; following normal UCIAMS laboratory protocols the δ13C was measured in the AMS for isotopic correction of the 14C age but this AMS-derived δ13C is not reported. Measurements in original reports converted to cm where previously in inches. Note: the reported date on a sample from Brigg’s Run, AA-8369 Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FileNote: in (DOCX)Click here for additional data file.S2 FileZea mays (maize) samples are identified as M, Odocoileus virginianus (white-tailed deer) samples are identified as B, and organic residue samples are identified as R. For details, see Fagus grandifolia (beech) are listed just as “Fagus”.Note: some of the labels/descriptions are edited for display in Figs (DOCX)Click here for additional data file.S3 FileZea mays (maize) samples are identified as M, Odocoileus virginianus (white-tailed deer) samples are identified as B, and organic residue samples are identified as R. For details, see Fagus grandifolia (beech) are listed just as “Fagus”.This model employs the site models (Model 1) for Smith-Pagerie, Klock (Model 1b), Garoga and Brigg’s Run from those listed in (DOCX)Click here for additional data file.


P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol Obesity, which has reached alarming levels worldwide, is associated with an increased risk of metabolic abnormalities including type 2 diabetes (T2D). T2D accounts for more than 90% of diabetes cases, while type 1 diabetes (T1D) accounts for about 5 to 10% of diabetes. Insulin deficiency in T1D as well as insulin resistance and insufficient insulin secretion from impaired pancreatic β cells in T2D result in increased glucose production in the liver, which is the major cause of hyperglycemia in diabetic patients , 4. Seve972 motif that helps the recognition and binding of the phosphotyrosine binding (PTB) domain in the insulin receptor substrate (IRS) proteins to the IR (P3 (PIP3) in cellular membranes and subsequent activation of downstream mediator AKT (Insulin binds to the extracellular α-subunit of the insulin receptor (IR), causing transphosphorylation of integral membrane β-subunit of IR by its intrinsic tyrosine kinase activity , 8. Phoso the IR . This seo the IR , adds ano the IR . IRS cano the IR , 14, 15.o the IR , 17, wheo the IR . The phoator AKT , 20, 21.ator AKT , suggestObesity and metabolic disorders are associated with increased blood bacterial lipopolysaccharide (LPS) level and its initiated low-grade of inflammation , and actA). In genetically obese db/db mice, there are drastic increases in total, cytosolic, and nuclear P300 protein levels in the liver compared with age-matched heterozygous lean mice (B–E). Consistently, immunostained liver sections by validated P300-specific antibody . Next, we examined P300 protein levels in two normal human liver samples and three obese patients and found that obese patients had significantly elevated liver P300 protein levels and large amounts of P300 in the cytoplasm of hepatocytes, contrasting with the mostly nuclear localization of this protein in normal humans (G and H). In agreement with our previous report (I and J) and in Hepa1-6 cells (A and B). In HFD-fed mice, treatment with A-485 for 2 weeks improved the hyperglycemia .Our previous report showed that high-fat-diet (HFD) feeding led to increased P300 protein levels in the liver . We valiFig. 1antibody  showed is report , inhibit-6 cells , A and BA and B). To test the effects of P300 acetyltransferase activity on insulin signaling, we treated Hepa1-6 cells with P300 acetyltransferase-specific inhibitor C646 and control inactive compound C37 (A and B), along with their mobility shift that may be caused by their tyrosine phosphorylation (C and D). Treatment with C646 significantly augmented AKT and GSK phosphorylation (C and D). However, the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ remained unchanged after treatment with C646 , indicating that C646-mediated activation of insulin signaling is not through augmenting the binding of IRS1 and 2 to the NPE(p)Y972 motif in IRβ . In both cytosolic and membrane fractions, C646 treatment increased the tyrosine phosphorylation levels in positions that correspond to the IRS1 and 2 proteins in the immunoblot (highlighted with the green square) (E). Of particular interest, C646 treatment significantly increased membrane-located IRS1 and 2  (F–H). C646 treatment caused the mobility shift of IRS1 and 2 in both cytosolic and membrane fractions, suggesting that the tyrosine residues of IRS1 and 2 were phosphorylated in both cellular compartments. To further prove that C646 can facilitate the membrane translocation of IRS1 and 2, primary hepatocytes from HFD-fed mice were prepared and subjected to serum starvation for 1 h before the addition of inhibitor C646. Indeed, C646 treatment increased the membrane located IRS1 and 2 in the absence of insulin treatment (I and J and H and I).To determine how C646 activates cellular insulin signaling in the absence of insulin, we prepared cytosolic and membrane fractions from Hepa1-6 cells treated with control inactive compound C37, or inhibitor C646, in the absence of both insulin and FBS treatment because the membrane translocation of IRS1 and 2 is required for the activation of insulin signaling  E. Of par%, IRS2) , F–H. C610 GC/IRS/mouse). The protein levels of expressed wild-type IRS1 and 2, and their mutant proteins were comparable with their corresponding endogenous protein levels in the liver of HFD-fed mice. At 19 days after the viral injections, liver tissues were collected. There were significantly increased liver AKT and GSK phosphorylation in mice with the expression of mutated KR proteins of IRS1 and 2 compared with mice with the expression of wild-type IRS1 and 2 proteins (A and B). Additionally, there were significantly higher levels of membrane located IRS1 and 2 proteins along with reduction of cytosolic IRS1 and 2 proteins in the liver of mice with the expression of mutated KR proteins of IRS1 and 2 (C and D). These results verified that acetylation of IRS1 and 2 has a negative impact on their membrane translocation.We found previously that seven lysine residues in IRS1 and 15 lysine residues in IRS2 can be acetylated and generated adenoviral expression vectors, in which these acetylated lysine residues in IRS1 and 2 were substituted with arginine, as mimics of nonacetylated lysine (KR mutants) . To examproteins , A and BS1 and 2 , C and DA–H and E and F). To test whether IR is required for C646 stimulated membrane translocation of IRS1 and 2, we generated liver-specific IR knockout (L-IRKO) mice by injection of AAV-TBG-Cre to HFD-fed floxed IR mice (A) and treated these mice with inhibitor C646 (20 nmol/g/day) via intraperitoneal injection for 2 weeks (E); in contrast, C646 treatment facilitated IRS1 and 2 membrane localization (F). In cytosolic and membrane fractions prepared from these L-IRKO mice (B), C646 treatment significantly increased the protein levels of both IRS1 and 2 in the membrane fractions, while significantly reducing protein levels of both IRS1 and 2 in the cytosolic fractions compared with their protein levels of vehicle-treated L-IRKO mice (G–J). These results indicate that IR is not required for C646 stimulated membrane translocation of IRS1 and 2. However, C646 was unable to improve insulin signaling in the liver of mice without IR (data not shown).C646 treatment activated insulin signaling and increased membrane located IRS1 and 2, while the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ remained unchanged A–H and , E and F IR mice A and tre 2 weeks . In the  2 weeks E; in conlization F. In cytRKO mice B, C646 tRKO mice , G–J. ThE–J), we reasoned that IRS acetylation may have a negative impact on IRS binding to phosphatidylinositolP2 (PIP2), which is also the substrate of immediate downstream mediator of PI3K. To test whether IRS acetylation can reduce their bindings to PIP2 in the membrane as a mechanism of the impairment of insulin signaling by P300, we conducted in vitro binding assays using acetylated IRS1 and 2. First, we used P300 to acetylate purified IRS1 and 2 proteins (K) in the presence or absence of acetyl-CoA (groups without the addition of acetyl-CoA served as controls) (L and N). Second, specific antibodies against IRS1 and 2 were used to immunoprecipitate acetylated or nonacetylated IRS1 and 2 to remove P300. Then, PIP2-fluorescein was added to the immunoprecipitates, and IRS-bound PIP2 was determined. We found that IRS acetylation by P300 reduced ∼70% bindings of PIP2 (M and O). Furthermore, the substitution of all identified acetylated lysine residues with arginine (pan KR mutation) in IRS1 or 2 abolished P300’s negative effect on the binding of PIP2 to IRS1 and 2 protein (Q–T). These results suggest that IRS acetylation may inhibit their membrane translocation through reduction of binding to PIP2. Since the acetylation of IRS1 and 2 could not affect their tyrosine phosphorylation by IRβ (C and D); therefore, P300 impairment of insulin signaling is mainly through the prevention of the membrane translocation of IRS1 and 2.Membrane translocation and binding to inositol compounds in the membrane can affect IRS′ function. PH domain in IRS can recognize and bind to membrane phospholipids , 14, 15.proteins K in the ontrols) , L and N of PIP2 , M and O protein , Q–T. Thn by IRβ , C and D972 motif that helps the recognition and binding of the PTB domain of IRS to the IR (A and B and A and B). However, in Hepa1-6 cells, C646 treatment did not increase the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ in the absence of insulin (A and B and E and F), but C646 treatment drastically increased the binding of IRS1 and 2 to IRβ (C). We reasoned that C646 may bind directly to IRβ, or IRS1 and 2, therefore, facilitating the interaction of IRβ with IRS1 and 2. To test this notion, we conducted Surface Plasmon Resonance (SPR) to determine whether C646 can bind to IRS1 and 2 or IRβ. We found that C646 can bind directly to unphosphorylated IRβ, but not to IRS1 and 2 , and C646 binds to IRβ with relatively high affinity (KD = 4.34 μM). Due to the fact that C646 can bind directly to unphosphorylated IRβ, we tested whether C646 has any effect on the binding of unphosphorylated IRβ with IRS1 or 2 using SPR. Strikingly, unphosphorylated IRβ can bind to either IRS1 or IRS2 with extremely high affinity  in the presence of C646 at nM concentrations (E and F). These data are consistent with the findings that C646 treatment drastically increased the association of IRS1 and 2 with unphosphorylated IRβ in Hepa1-6 cells . Thus, in the absence of tyrosine phosphorylation of IRβ by insulin, it is the C646 that directly promotes IRβ bindings to IRS.The tyrosine phosphorylation of IRβ at Y972 generates the NPE(p)Yo the IR . We vali2 to IRβ C. We reatrations , E and Fin vitro phosphorylation assays, in which IRβ and IRS1 or IRS2 were incubated with different concentrations of C646, followed by addition of ATP to initiate the phosphorylation of IRS. We found that C646 significantly increased the tyrosine phosphorylation of both IRS1 and 2 by IRβ (A–D). Having seen these data, we asked whether C646 can affect the enzymatic activity of IRβ. To test this hypothesis, we incubated the insulin receptor with different concentrations of C646, then determined its kinase activity on the phosphorylation of tyrosine residue in peptide substrate by measuring the production of ADP from ATP using InsR kinase assay. C646 did increase the kinase activity of insulin receptor by 50% at 100 nM concentration (E). The above data show that C646 not only promotes the binding of IRβ to IRS, but also activates the enzymatic activity of IRβ, leading to drastically increased bindings of IRβ to IRS1 and 2 (C) and tyrosine phosphorylation of IRS1 and 2 (A and B).To determine whether C646 promoting the binding of IRβ to IRS has any effect on the tyrosine phosphorylation of IRS by IRβ, we conducted 2 by IRβ , A–D. Hantration E. The abS1 and 2 C and tyrS1 and 2 , A and BF and G), suggesting that C646 cannot affect the transphosphorylation of IRβ. These results are consistent with the findings that C646 treatment could not change the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ (A and B and E and F). Another potent CBP and P300’s acetyltransferase inhibitor, A-485, was unable to affect the tyrosine phosphorylation of IRS1 and 2 by IRβ (H–K). These findings may explain why C646 has a stronger effect than A-485 on the activation of the insulin signaling pathway (compared C and D).However, interestingly, C646 had no effect on the tyrosine phosphorylation of IRβ itself , F and G2 by IRβ , H–K. ThL and M). It is possible that the cleft-sitting C646 can cause conformational changes carried out by insulin-mediated autophosphorylation of tyrosine residues in IRβ, allowing IRβ binding to IRS with high affinity and also increasing the enzymatic activity through reduction of activation energy.Since the crystal structure of unphosphorylated and low activity form of the insulin receptor kinase domain (IRK) have been determined , we usedob/ob mice and treated the primary hepatocytes with control inactive compound C37 or inhibitor C646 after serum starvation. We found that, in the absence of insulin, C646 treatment drastically increased the tyrosine phosphorylation levels in positions that correspond to IRS1 and 2 proteins in the immunoblot (A). These data are consistent with the results that C646 treatment increased IRS tyrosine phosphorylation in Hepa1-6 cells (A and E). C646 treatment also significantly increased PI3K activity and the phosphorylation of AKT and GSK in these primary hepatocytes in the absence of insulin (B–D) and significantly suppressed glucose production (E). Moreover, in obese ob/ob mice, C646 treatment through intraperitoneal injection (30 nmol/g/day) for 10 days significantly improved insulin sensitivity and hyperglycemia without significant changes in body weight (F and G). The above results show that the augmentation of ΙRβ activity, binding to IRS, and subsequent IRS tyrosine phosphorylation by C646 leads to the activation of PI3K-AKT signaling and the suppression of glucose production in the liver and alleviation of hyperglycemia.We further tested C646’s effect on insulin signaling in hepatocytes from mice with obesity and diabetes. We prepared primary hepatocytes from obese munoblot A. These -6 cells , A and E insulin , B–D andoduction E. Moreovy weight , F and GH–K). Also, considering that the acetylation of IRS1 and 2 by P300 had no impact on the total tyrosine phosphorylation of these proteins, these data suggest that the interaction of p85 in PI3K with IRS proteins is not the main C646 targeting site. However, it is possible that the acetylation of IRS1 and 2 may have a negative effect on the phosphorylation of tyrosine residue in the YMXM motifs, thus affecting the binding of p85 to IRS.The phosphorylation of tyrosine residues in IRS1 and 2 by IRβ will generate pYMXM motifs and lead to the docking of p85 in PI3K to the membrane located IRS proteins and activation of downstream mediators in the PI3K-AKT signaling. We therefore determined whether IRS acetylation could affect the docking of p85 protein in PI3K to IRS1 and 2 proteins. First, we used IRβ to phosphorylate the tyrosine residues in IRS1 and 2, then P300 protein was added in the absence or presence of acetyl-CoA, followed by Far-Western blot to examine the docking of p85 protein to IRS1 and 2. We found that the acetylation of either IRS1 or IRS2 had no effects on the docking of p85 to these proteins , H–K. Alvia reduction of the ubiquitination and degradation of P300, and elevated P300 impairs insulin signaling by acetylating IRS1 and 2 YThe mouse IRS1 and 2 genes were gifts from Ronald Kahn  , and theMouse primary hepatocytes were cultured in William’s medium E supplemented with ITS (BD Biosciences) and dexamethasone. After 24 to 48 h of seeding, cells were washed with PBS twice, and the medium was changed to FBS-free DMEM supplemented with 20 μM of either C37 or C646. After 3 h of serum starvation, cells were washed twice with PBS, and 1 ml glucose production medium was supplemented with 20 μM of either C37 or C646. After 3 h incubation with glucose production medium, both the medium and cells were collected. The medium was used to determine glucose concentrations with EnzyChrom Glucose Assay Kit , 42, 43.3VO4, and 10 mM MgCl2 at 30 °C for 15 min to 2 h in the absence or presence of 0.2 mM ATP. To acetylate IRS1 and 2, 2 μg of IRS1 or 2 was added to the reaction containing 50 mM Tris-HCl, pH 8.0, 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 1 mM PMSF, 10 mM sodium butyrate, 0.2 μg acetyl-CoA, and 1.5 μg P300 (Active Motif). Samples were incubated at 37 °C for 2 h.In phosphorylation assays, purified IRS1 and 2 proteins were incubated with 0.2 μg of IRβ  in reaction containing 25 mM Tris-HCl (pH7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Naob/ob mice were subjected to serum starvation for 2 h, then treated with 20 μM of C37 or C646 for 3 h. P110α antibody  was used to pull down PI3K, and its enzymatic activity was measured using the PI3K-Kinase Activity ELISA: Pico kit (Echelon Biosciences).Twenty-four hours after the seeding, primary hepatocytes from db/db mice, and ob/ob mice were purchased from the Jackson Laboratory. Floxed homozygous IR mice were fed on HFD  for 4 weeks, and AAV8-TGB-Cre (1 × 10ˆ11 GC/mouse) was injected via the jugular vein to generate liver-specific IR knockout mice. After viral injection, mice were treated with vehicle or C646 (20 nmol/g/day) for 2 weeks. To test C646’s effect on insulin sensitivity in obese ob/ob mice, 4-month-old ob/ob mice were randomly divided into two groups for which vehicle or C646 (30 nmol/g/day) was given through intraperitoneal injection for 10 days. Insulin tolerance test was performed after 6 h of fasting (0.8 unit/kg insulin). To test the effect of IRS1/2 mutants on their membrane translocation, 3-month-old homozygous double-floxed IRS1 and 2 mice were injected with adenovirus (1 × 1010 GC/mouse) through the jugular vein alone with AAV8-TGB-Cre (1 × 10ˆ11 GC/mouse). Two days after the viral injection, mice were placed on an HFD. After 19 days of HFD feeding, liver tissues were collected.All animal protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University. Male floxed homozygous IR mice, Liver tissues were obtained from the left lateral lobe of the liver of three obese patients  and immediately frozen on dry ice and stored at −80 °C. Normal human liver tissues were obtained through the Liver Tissue Cell Distribution System, which was funded by National Institutes of Health contract no. HHSN276201200017C .We firstly employed different amounts  of insulin receptor to determine the enzymatic activity needed in the InsR Kinase Assay (Promega) and found a linear curve of ADP production (consumption of ATP by insulin receptor). Subsequently, 1 μg of the insulin receptor was used in a 25 ul reaction containing different concentrations of C646, 0.2 μg of substrate peptide, 50 μM of ATP, and 1 × provided reaction buffer. The reactions were incubated at room temperature for 60 min. ADP-Glo Kinase Assay (Promega) was used to determine the ADP levels generated by the insulin receptor. Twenty-five microliters of ADP-Glo reagent was added to deplete residual ATP , then 50 ul of Kinase Detection Reagent was added to determine the ATP levels converted from ADP followed by the measurement of luminescence in BioTek Synergy H1 plate reader.Cellular lysates were passed 15 times through a syringe needle to break cells on ice. ΙRβ, IRS1 and 2 were immunoprecipitated using ΙRβ antibody  and IRS1 and 2 antibodies (Millipore). The reaction was incubated at 4 °C for 16 h, followed by the addition of protein G beads (Active Motif) to pull down the target protein and its associated proteins. Cytosolic and nuclear extracts were prepared using the CelLytic NuCLEAR Extraction kit (Sigma). Membrane, cytosolic, and nuclear fractions were prepared from Hepa1-6 cells using Subcellular Protein Fraction Kit for Cultured Cells (Thermo Fisher) following manufacturer’s instruction.et al. , and 0.05% Tween-20) for 1 h at room temperature. Subsequently, the membrane was incubated in protein-binding buffer , 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% skim milk powder, and 1 mM DTT, prepared freshly) plus 4 μg of p85 . After three washes with PBST buffer, the membrane was incubated with anti-p85  at 4 °C overnight.A far-Western blot was conducted using a modified method of Wu et al. . Purifiedb/db mice were fixed with 4% paraformaldehyde in PBS for overnight at room temperature, then incubated in 30% sucrose overnight and frozen in OCT. Tissues were sectioned. After antigen retrieval using a microwave, sections were permeabilized with 0.1 Triton X-100 (10 min) and blocked in 3% horse serum for 1 h at room temperature. Paraffin-embedded human liver samples were sectioned. Sections were incubated with P300-specific antibody  at 1: 20–200 dilution at 4 °C overnight.Frozen liver samples of heterozygous lean and homozygous via a Zeiss confocal microscope .Liver tissues were harvested, washed with cold PBS, and fixed immediately with 4% paraformaldehyde in PBS for 6 h at 4 °C, then incubated in 30% sucrose overnight, and frozen in OCT. The frozen sections were cut at 10 uM thickness. Sections were washed in PBS, blocked, and permeabilized in blocking buffer (5% goat serum with 0.3% TritonX-100 in PBS) for 1 h at room temperature. For primary hepatocytes, 24 h after the seeding of primary hepatocytes, cells were washed with PBS and cultured in FBS-free DMEM for 1 h, then C37 or C646 was added. 4 h later, medium was removed. Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 1% Triton X-100, and blocked with 3% horse serum in PBS. Antibodies against IRS1 (1:50), IRS2 (1:50), and Na/K ATPase (1:100) were used . Fluorescein-conjugated AffiniPure donkey anti-mouse IgG (1:150) and Cy3-conjugated AffiniPure donkey anti-rabbit IgG (1:250)  were diluted in PBS with 3% horse serum and incubated at room temperature for 2 h. Fluorescent images were acquired (These experiments were conducted in the Biosensor Core at the University of Maryland School of Medicine). For the determination of C646 bindings to IRβ, IRS1 and 2 proteins, these proteins were immobilized on the surface of a CM5 chip at flow cell 2, 3, and 4, respectively. Binding reactions were done in HBS-P buffer from Biacore (Cytiva Inc), containing 10 mM Hepes, 150 mM NaCl, and 0.05% (v/v) surfactant p20, pH 7.4. This buffer was filtered (0.2 μM) and degassed before use. In order to minimize mass transport effects, the binding analyses were performed at flow rate of 30 μl per minute at 25 °C. The analyte (C646) was dissolved in HBS-P buffer with 0.05% P20 and 2% DMSO and injected into flow cell 1, 2, 3, 4, and the association of analyte and ligands  was recorded respectively by Biacore T200 . Next, the surface was washed with buffer for 600 s to dissociate the analyte–ligand complexes. The signal from the Blank channel (flow cell-1) was served as a control to subtract from the channel containing IRβ, IRS1 and 2 proteins. The binding was removed by injecting 30 μl of glycine (pH 1.5) followed by injection of 30 μl of HBS-P (pH 7.4). For the determination of IRβ binding to IRS1 and 2 proteins, IRβ was immobilized on the surface of a CM5 chip, and the IRS1 or IRS2 protein was injected with different concentrations of C646. Sensorgrams of the interaction were generated by the instrument and analyzed using the software BIAeval 4.0 (Biacore Inc). The reference surface data were subtracted from the reaction surface data to eliminate refractive-index changes of the solution, injection noise, and nonspecific binding to the blank surface. A blank injection with buffer alone was subtracted from the resulting reaction surface data. Data were globally fitted to the Langmuir model for a 1:1 binding.t test and ANOVA test. Significance was accepted at the level of p < 0.05. Sample size (number of mice) was determined on the basis of our previous studies. At least three samples per group were chosen for statistical meaningful interpretation of results. Statistical significance (p < 0.05) was evaluated in GraphPad Prism using one-way ANOVA for multigroup comparisons or using Student’s t test for two-group comparison.Statistical significance was calculated with a Student’s heling@jhmi.edu) upon request.All data are contained in this manuscript, and supporting information is available from the author: Ling He (This article contains The authors declare that they have no conflict of interest with the contents of this article.


Cystatin M/E is a low molecular mass protein and a potent inhibitor of proteolytic enzymes . Cystatin M/E participates in epidermal homeostasis and its deregulation is associated with several skin diseases. Cystatin M/E was initially identified as a tumor suppressor in breast cancer. Its gene is epigenetically downregulated and its methylation status holds a prognostic significance. Moreover, it may also serve as a biomarker for clinical diagnosis. This tumor-suppressing role was documented in cutaneous carcinoma, melanoma, lung, cervical, brain, prostate, gastric and renal cancers. Conversely, increased levels of cystatin M/E in triple-negative breast cancer tissues correlate with a higher risk of metastasis and a lower disease-free survival rate. Beside its orthodox tumor-suppressing role, cystatin M/E may operate as a tumor-promoting effector as reported for thyroid, oral and pancreatic cancer and hepatocellular carcinoma. Given its seemingly contradictory results, in-depth analysis of the regulatory mechanisms of the expression and activity of cystatin M/E during tumorigenesis have to be expanded. Likewise, the possible involvement of cystatin M/E in signaling pathways, beside its protease inhibitor function, remains to be scrutinized.CST6 promoter region. The tumor-suppressing role of cystatin M/E was further reported in melanoma, cervical, brain, prostate, gastric and renal cancers, and cystatin M/E was proposed as a biomarker of prognostic significance. Contrariwise, cystatin M/E could have an antagonistic function, acting as a tumor promoter . Taking into account these apparently divergent functions, there is an urgent need to decipher the molecular and cellular regulatory mechanisms of the expression and activity of cystatin M/E associated with the safeguarding homeostasis of the proteolytic balance as well as its imbalance in cancer.Alongside its contribution in maintaining skin homeostasis and its probable involvement in fetal and placental development, cystatin M/E  was first described as a tumor suppressor of breast cancer. This review aims to provide an update on cystatin M/E with particular attention paid to its role during tumorigenesis. Cystatin M/E, which is related to type 2 cystatins, displays the unique property of being a dual tight-binding inhibitor of both legumain  and cysteine cathepsins L, V and B, while its expression level is epigenetically regulated via the methylation of the  Cystatins are reversible and competitive inhibitors of cysteine proteases operating both extracellularly and intracellularly . BaseAlmost simultaneously, cystatin M was characterized as the product of a downregulated gene in breast cancer , while ACST6) is located on the 11q13 region, while the cystatin C gene (CST3) is located on 20p11.2 [CST6 gene was first elucidated by Zeuween et al. [CST6 encompasses three exons within ~1.4 kb of genomic sequence from the translation start site to the translation termination site and a specific genomic organization within the cystatin 2 family, since the TATA box is lacking in the 5′-flanking sequence. Additionally, the two introns of CST6 (542 bp and 365 bp) are remarkably short compared to introns of cystatins C, D or S genes . The mouse CST6 ortholog was characterized by the same group and was assigned to the proximal end of chromosome 19. Mouse and human CST6 retain 69% amino acid identity and 82% amino acid conservation [The gene encoding cystatin M/E  and a main peptide chain (29–149). Mature cystatin M/E is composed of 121 amino acid residues . Its sequence has some unusual characteristics compared to cystatin C, the archetypal member of type 2 cystatins, specifically a five-residue insertion between amino acids 76 and 77, and a deletion of residue 91 (cystatin C numbering). Likewise, cystatin M/E displays two disulfide bridges at positions 73–83 and 97–117, respectively, and an N-glycosylation site at Asn108  and a ma,33.In a preliminary study, the three-dimensional structure of cystatin M/E was generated by homology modelling and refined by molecular dynamics simulation. The N-terminal region of cystatin M/E contributes equally to the inhibition of papain together with the conserved L1 and L2 loops . AlternaCystatin M/E was first reported as a potent inhibitor of papain Ki = 0.39 nM)  and was  nM  and Cystatin M/E demonstrates a compelling affinity (Ki = 0.0016 nM) for pig legumain . It appeThe binding of cystatin M/E to legumain is mediated by a reactive center loop (RCL) according to a canonical, substrate-like binding mode with Asn39 on RCL serving as P1 residue and stabilized via exosite interactions by the so-called legumain exosite loop (LEL), which makes contact in primed substrate binding sites . CystatiWhile it was shown that MDA-MB-435S, A375 and C8161 cells internalized substantial amounts of cystatin C, a low internalization of cystatin M/E was observed. Nevertheless, it may significantly affect intracellular legumain activity and cell migration . RecentlCST6 deficiency prevents the development of a multilayered epidermis in a 3D reconstructed skin model and may be incompatible with normal human foetal development [CST6 and profilaggrin, a major protein of keratohyalin granules in the granular layer [Under biological conditions, the expression pattern of human cystatin M/E is distinctive among type 2 cystatins, as elevated protein levels are chiefly restricted to epidermal keratinocytes, hair follicles, sebaceous glands and sweat glands ,45. The elopment . Cystatielopment ,46. A rear layer . Moreovear layer .CST6, was associated with hypotrichosis, eczema and impaired sweating. Functionally, the corresponding recombinant mutant cystatin M/E protein lacks its ability to inhibit CatV, CatL and legumain [Cystatin M/E deficiency in mice causes disturbed epidermal cornification, impaired barrier function and neonatal lethality because of excessive trans-epidermal water loss (for review: ). Harleqlegumain . Intereslegumain .Alternatively, Lyme disease results from infection with the spirochete Borrelia burgdorferi. The first and common clinical manifestation, called erythema migrans, corresponds to a circular, inflamed skin lesion. Proteomic analysis was conducted on an independent cohort of patients with erythema migrans, leading to the identification of some candidate molecules, including cystatin M/E, as putative acute-phase blood markers for the diagnosis of early Lyme disease .CST6 was identified as a pig pregnancy-related gene and dynamic changes in its mRNA level were observed in bovine endometrium [CST6 gene could be a candidate gene for prolificacy traits in sheep [In an early report, it was proposed that cystatin M/E in the amniotic fluid may participate in fetal development . During ometrium ,60. On tometrium ,62. Cystometrium . Additioin sheep . CST6 was identified as a disease-causing mutation of progressive myoclonus epilepsy of Unverricht-Lundborg type, a rare autosomal recessive disorder [Type 1 cystatins and cystatin C were proposed to play a role in Alzheimer’s disease according to their ability to form amyloid fibrils ,66,67. Idisorder . Yet, a disorder . Togethedisorder .CST6 was also strongly repressed, in addition to type 2 cystatin genes , which are all encoded by a gene cluster on 20p11 [In a seminal in vitro study, cystatin M was identified as a downregulated transcript in a metastatic breast cancer cell line when compared to a matched primary tumor cell line. Consequently, loss of cystatin M/E expression was proposed to be associated with the progression of human breast cancer . Transfeon 20p11 .CST6 was downregulated in bone metastatic (MDA-MB-231) derivative cell lines. Ectopic expression of CST6 suppressed proliferation, colony formation, migration and invasion in vitro. This overexpression rescued mice from osteolytic metastasis and death, while CST6 knock-down markedly enhanced cancer cell bone metastasis and shortened animal survival, sustaining that cystatin M/E is a bona fide suppressor of breast cancer osteolytic metastasis [Bone metastasis is a common cause of morbidity and mortality in breast cancer patients. Jin and colleagues demonstrated that tastasis .CST6 promoter hypermethylation in a panel of breast cancer cell lines  and in primary breast carcinomas, indicating that this aberrant characteristic is common in breast malignancies [CST6 [CST6 was also found in axillary lymph node metastasis in comparison to primary tumor tissue and adjacent normal tissue from the same breast cancer patients [CST6 transcription and expression, substantiating that cystatin M/E is an epigenetically-inactivated tumor suppressor gene [The loss of cystatin M/E expression has been strongly correlated with gnancies ,78,79. Hes [CST6 . An aberpatients ,82. Treasor gene ,78,79. CST6 and is associated with an aberrant overexpression of DNA (cytosine-5)-methyltransferase 3β (DNMT3b) [Basal-like breast tumors  contribute disproportionately to breast cancer deaths, as they have the propensity to display aggressive tumor characteristics . A cluster of tumors, identified as basal-like breast cancers, expresses a hypermethylator signature with a low expression level of six specific genes including (DNMT3b) . Seconda(DNMT3b) .CST6 as a cancer biomarker in breast tumors was statistically evaluated. Multivariate analysis showed that CST6 promoter methylation status was an independent prognostic factor for disease-free-interval (DFI) and overall survival (OS) [CTS6 hypermethylation may serve as a biomarker for clinical molecular diagnosis and is associated with unfavorable prognosis in operable breast cancer [CST6 promoter methylation status of circulating tumor cells (CTCs) isolated from peripheral blood of breast cancer patients was also analyzed. CST6 hypermethylation for both operable and metastatic breast cancer significantly differed from that of the control population [CST6 have a significantly longer progression-free survival compared to patients with corresponding enriched methylated CTCs [CST6, may enable breast cancer prediction and patient stratification, based on well-defined DNA methylation profiles, and might be a valuable biomarker for early detection of breast cancer [CST6 promoter is hypermethylated in cell-free DNA circulating in the plasma of breast cancer patients, but not in healthy individuals. Data support that CST6 promoter methylation in cell-free DNA could be a putative novel plasma tumor marker for breast cancer [CST6, was found for both patients with early and metastatic breast cancer [CST6 gene is influenced by the alteration of estrogen receptor (ER) and receptor tyrosine-protein kinase erbB-4 (HER4), and cystatin M/E was identified as a downstream target of ER and HER4 [The prognostic significance of the methylation status of val (OS) . Data sut cancer ,86. The pulation . Moreoveted CTCs . A panelt cancer . In a pit cancer . Alternat cancer . At the t cancer . Moreoveand HER4 .CST6 was identified as a repressed TBX2 (a T-box family oncogenic transcription factor) target gene through a mechanism involving Early Growth Response 1 (EGR1). An increase in TBX2 expression is known to drive breast cancer proliferation and metastasis. In contrast, the exogenous expression of cystatin M/E in TBX2-expressing breast cancer cells resulted in upregulated apoptosis. Remarkably, induction of apoptosis depends on the cystatin M/E legumain-inhibitory domain, but not on the cysteine cathepsin-inhibitory domain, emphasizing that legumain is a crucial oncogenic driver. Secretion or glycosylation of cystatin M/E is not a prerequisite to elicit cell killing effects. Thus, cystatin M/E represents an important barrier to breast tumorigenesis which could be by-passed by TBX2 transcriptional repression. Data also sustain that the “TBX2-cystatin M/E-legumain” pathway may represent a novel promising axis for the development of innovative therapies to target TBX2-driven breast cancers [CST6 was also repressed through a mechanism involving both TBX2 and EGR1 in rhabdomyosarcoma, a pediatric malignant cancer which is thought to arise from myogenic precursors in the skeletal muscle lineage [Cystatin M/E may modulate malignant properties of human breast carcinoma cells (MDA-MB-435S cells) partly through the downregulation of two major secreted signaling molecules, autotaxin/lyso-phospholipase D and interleukin-8, which combine mitogenic, motogenic and angiogenic properties . CST6 wa cancers . It coul lineage .−1 of protein), the estimated median level of cystatin M/E in NSCLC primary tumors is 14.6 pg.mg−1 of protein [CST6 is frequently transcriptionally downregulated in NSCLC tumors, while its ectopic expression suppresses NSCLC growth in culture [CST6 gene differed significantly based on smoking practice [Type I cystatins were proposed as independent prognostic markers for patients with non-small cell lung cancer (NSCLC), since high levels of both stefins A and B, but not cystatin M/E, are associated with a better survival probability. Compared to cystatin C  was fully hampered by promoter methylation, while the ectopic expression of cystatin M/E reduced TIC motility and invasion. Qiu et al. [CST6 epigenetic silencing may therefore represent a novel prognostic marker of gliomas. Interestingly, together with CST6, the apoptosis-inducer Bcl-2-interacting killer (BIK) is also a target of epigenetic silencing in malignant gliomas. BIK, which is a member of the BH3-only family of pro-apoptotic proteins, is known to suppress glioma cell growth in culture [Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children, and are characterized by a high morbidity and mortality. It was found that u et al.  proposed culture . The tre culture .CST6 and cystatin M/E protein expression, as well as its pathophysiological (deleterious vs. protective) relevance in cSCC metastasis. On the other hand, CST6 is involved in the suppression of proliferation, migration and metastasis of melanoma [Conversely to benign hyperplasia as observed during psoriasis vulgaris, a unique cancer-specific gene expression signature was identified for human cutaneous squamous cell carcinoma (cSCC) associated with a gene downregulation of granzyme B, and a differential gene upregulation (fold change > 3) of matrix metalloproteinase MMP-1, kallikrein 7, CatV and cystatin M/E . Contrarmelanoma . Interesmelanoma . Additiomelanoma . CST6 and increases cystatin M/E protein expression. Its overexpression significantly reduces prostate cell proliferation and inhibits tumor growth and the incidence of lung metastasis. The authors proposed that CST6 downregulation was associated with promoter histone modifications and may have participated in prostate cancer progression during the invasive and metastatic stages. Analysis of the methylation status of CST6 in gastric carcinomas and their paired adjacent non-tumor tissues revealed a loss of expression of cystatin M/E in 70% of gastric carcinomas due to promoter hypermethylation. Moreover, patients with hypermethylated CST6 had a significantly shorter survival time than those with hypomethylated promoter [CST6, may allow the classification of patients with gastric cancer into high-and low-risk groups with a significant OS difference [Expression of cystatin M/E is extensively decreased in metastatic prostate cell lines and prostate tumor tissues compared to normal human prostate epithelium . Treatmepromoter . In a clfference .CST6, exhibiting tumor-specific promoter region hypermethylation associated with transcriptional silencing [CST6 is associated with both a shortened progression-free and OS of patients with metastasized RCC undergoing anti-vascular endothelial growth factor (VEGF)-based therapy [Epigenetic study by high-density gene expression microarrays of renal cell carcinoma (RCC) cell lines identified eight genes, including ilencing . Hyperme therapy . Followi therapy  . Its constitutive expression in tic CatB .CST6 is induced subsequently to serine/threonine-protein kinase B-Raf (BRAF) activation and therefore may be downstream in the BRAF/MEK (mitogen-activated protein kinase kinase)/extracellular signal-regulated kinase signaling pathway in papillary thyroid carcinoma (PTC) [CST6, together with C-X-C motif chemokine ligand 14 (CXCL14) and secreted phosphoprotein 1 , is associated with PTC lymph node metastasis. ma (PTC) . MoreoveCST6 correlated with the OS and recurrence-free survival (RFS) of patients with hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) [CST6 may function as a prognostic biomarker for HCC, with a high CST6 expression being associated with poor survival of patients, in correlation with a previous report claiming that cystatin M/E could be a pancreatic tumor promotor [Survival analysis indicated that ted HCC) . CST6 mapromotor . Taken tpromotor .CST6 was initially identified as a tumor suppressor and an epigenetically downregulated gene in breast cancer. The methylation status of CST6 holds a prognostic significance and may also serve as a biomarker for clinical diagnosis. Afterward, this tumor-suppressing role was corroborated with other cancers  and the use of CST6 as a valuable biomarker was confirmed. Conversely, increased levels of cystatin M/E in TNBC tissues correlate with a higher risk of metastasis and a lower disease-free survival rate. Thus, beside its conventional tumor-suppressing role, cystatin M/E may act as a tumor-promoting effector. This outward antagonistic function was further testified for thyroid, oral and pancreatic cancer and hepatocellular carcinoma. Accordingly, molecular and cellular analysis of the regulatory mechanisms of the expression and activity of cystatin M/E associated with the maintenance of the proteolytic balance, as well its imbalance under pathophysiological conditions, has to be urgently deepened. Moreover, the putative involvement of cystatin M/E, during tumorigenesis, in signaling pathways independent of its protease inhibitor function, still remains to be deciphered.Cystatin M/E (type 2 cystatin) is a specific and dual tight-binding inhibitor of both cathepsins L, V and B (family C1) and legumain , via two distinct mechanisms of inhibition, which involve non-overlapping binding sites. Cystatin M/E plays a critical role in epidermal homeostasis, and its deregulation is associated with various skin diseases such as ichthyosis or psoriasis. Cystatin M/E is also probably involved in fetal and placental development and could participate in the pathogenesis of neurodegenerative progressive diseases, according to its ability to form amyloid fibrils.