Salvage of fractured abutment screw is one of the critical complications, which can be solved in different ways. In this study, an innovative approach is introduced to solve this problem. It is a relatively simple, non‐invasive technique, which would provide an excellent result when executed with ultimate care and precision. Implantologist knew the late failures arise from pathological processes involving biomechanics and host‐parasite interactions.Abutment screw fracture is a rare event, occurring <0.5%. Nonetheless, when a fracture occurs, it is very disturbing for the clinician.Every dentist knew that each fixture has a crest module, body, and apical region. The crest module of a fixture body is the portion designed to retain the prosthetic component. An antirotation feature is included in the crest module.Prosthetic screw: It is a fixture component that secures the fixture‐abutment or prosthesis to the fixture body. Loosening of the screw may result in the displacement of the prosthesis and eventual loss of prosthetic function.Torque is defined as the movement of a system of forces producing rotation.Occlusal loading is a multidirectional and variable magnitude force. There is a slight wobble of the abutment during functional loads.Fatigue fracture is caused by biomechanical overload, improper placement techniques, and a nonpassive fit of the suprastructure errors. Extended period of clinical use, repeated retightening of loose screw, poor design of the fixture‐abutment connection, inadequate tightening and screw joint movement, and metal fatigue after screw loosening also can induce these complications.Screw fracture is a challenge for the clinician due to the difficulty of removal of fractured screw fragments. A review of in vivo butt‐joint fixture studies about abutment screw loosening stated that screw loosens when the joint‐separating force is high. Forces acting to separate the screw joint are greater than the forces keeping the abutment and fixture together (clamping force).Additionally, the surface of a new metal screw has microscopic imperfections in the form of high spots, grooves, and irregularities, such that, when initial torque is applied, only the high spots in the system will be in contact. Flattening and wear of these high spots are described as screw settling and will result in the loss of the initial preload.Screw fragment retrieval, replaced by another screw.Fixture removal or discard and reimplantation in another site if applicable.Fabrication of a cemented cast post and core.There are several techniques for managing a fractured abutment screw.In lucky circumstances, the stock fractured end can be retrieved and replaced by a new abutment screw conservatively. Rotary instrumentation and/or ultrasonic instrumentation can be used to retrieve the fractured screw carefully. Damaged internal threads of the fixture are avoided in this study. Excessive heat generation at the fixture‐bone interface may cause irreversible bone injury and compromise osseointegration by local necrosis. Delay bone repair affects integration. As a result, clinicians might choose to either remove the fixture or replace it with a new one, or abandon the fixture and cover it with soft tissue.The expert challenge is required to retrieve this trapped piece. When the methods fail to retrieve the fractured segment, a fixture is considered useless for the patient. This means that another fixture might need to be placed if applicable. This usually involves increased expense, surgical removal of the bone‐integrated dental fixture, and additional procedures, which in turn also modifying the prosthesis design. Different alternatives have been described to deal with a nonrestorable fixture.In this study, we replaced the fabrication of metal post by the screw of the transfer cap of the fixture system itself after adjustment of the height of the piece. This relatively simple technique when executed with the ultimate care and precision would provide excellent result, and the non‐invasive nature of the procedure would also satisfy the patient. Damaged internal threads have been avoided in this procedure.2Cases with retrieved screw (two patients with an implant‐supported bridge):oIn the first case, male patient was presented with four fixtures in the upper jaw in the edentulous area from the right lateral to the left second molar bridge (9 units) with single fixture fractured screw that occurs after 7 years postdelivery of the prosthesis.oIn the second case, female patient was presented with 3 fixtures in the lower mandibular anterior region from left canine to right lateral bridge (5 units) with single fractured screw that occurs after 12 years postdelivery of the prosthesis.Eight patients were visiting the privet clinic seeking the solution for displaced fixed implant prosthesis. After clinical examination of the site, we discovered that one of the abutment screws in each case was fractured in different levels. Fixtures checked clinically and radiographically, which revealed that all fixtures were osseointegrated very well with a retained fractured screw piece in different grades. All patients have signed an ethical approval for the participation in the research. Table A case with a replaced fixture:In these two cases, there was previous mobility in the prosthesis occurred first. The screw was retrieved and changed with another one only. A screw was retrieved by an ultrasonic scaler. Care was taken not to touch the threaded walls to prevent any damage for the internal architecture of the fixture. After the fragment was removed, the screw hole was then flushed with dilute aqueous (10:1) sodium hypochlorite to remove any debris and fabrication of a new multiple supported bridges done which occlusally adjusted well.Cases with a new approach (five patients with a fixture‐supported bridge):oIn the first case, male patient was presented with 4 fixtures in the upper jaw in the edentulous area from the right canine to the 1st premolar left‐side bridge (7 units) with a single fixture fractured screw that occurs after 4 years postdelivery of the prosthesis.oIn the second case, female patient was presented with two fixtures in a lower mandibular right‐side region 2nd premolar, 1st, and 2nd molar bridge (3 units) with one fractured screw that occurs after 8 years postdelivery of the prosthesis.oIn the third case, female patient was presented with single fixture in 1st premolar where fracture screw occurs after six months of use.oIn the fourth case, male patient was presented with five fixtures in the fully edentulous mandible fixed bridge (10 units) with two fractured screws that occur after 9 years.oIn the fifth case, older female patient has two fractured screws in the fixed bridge supported by five fixtures in area that extends from left central to right upper first molar fixed bridge (7 units) that occur after 10 years.One female patient was presented with upper anterior fixed bridge supported by two fixtures in the two central and lateral left side (3 units) after 5 years postfunction. The fractured screw fixture was left, another fixture was placed in the lateral site, and new fixed prosthesis was then fabricated smoothly.A radiograph revealed a well‐integrated fixture with clinically insignificant cervical marginal bone loss by measuring the level of resorption radiographically, with a retained piece of the fractured screws in the opposition that cannot be retrieved. All these cases claimed that there was previous mobility in the prosthesis that occurs first, except one patient has no mobility anteceding the fracture.Discussions with the patients are maintained to reach the best solution and not to waste the effort to retrieve the fractured piece. The screw hole was flushed with dilute aqueous (10:1) sodium hypochlorite to remove any debris from the fixture.After discussion of treatment plans with each patient, we assured that it is applicable to use the screw of the transfer cap only to be inserted in the same injured fixture and fabricate the prosthesis. Ethical approval has been signed up by all patients underwent the new policy of treatment.After agreement with our patients, the following steps are carried out: The screw hole is prepared and flushed with dilute aqueous (10:1) sodium hypochlorite to remove any debris from the fixture; the internal serration of the fixture is checked; the correct diameter of the transfer cap screw check tightness is chosen to fit engagement and then cut the screw to correct height; and then light cure is build up on it. Then, the fabrication of a new prosthesis and postdelivery of the final fixed bridge are performed. Adjustment of the occlusion is done to eliminate any unwanted destructive forces. The patient's occlusal scheme and maximal bite force should be assessed for overload. The new abutment/screw is torqued into the fixture. The crown is occlusally adjusted and recemented for normal functioning. Assessment and correction of the occlusal scheme are important. All patients followed for 6 months to 2 years.3Abutment screw fractures Figure A cervical part fractured screw is easy to be removed. The removal is carried out either by special kits supported by the Fixtures Company's Suppliers Figure The most important need is the skill of the implantologist himself. Removal of fractured piece and change of screw are easily applicable methods in cervical part fractured screw type Figure When all the methods fail to retrieve the fractured segment, the fixture may be rendered useless. So we can solve that problem by placing another fixture according to the normally known protocol Figure For such a scenario, we used the transfer cap screw to seat in the space replacing the fractured part instead of the fabrication of metallic custom post and core. By this, we can salvage the near‐useless fixture Figure 4Abutment screw is the part connecting the abutment to the fixture. Fracture screw is one of the beard prosthetic complications in implant dentistry. It is most important as it affects the surgical part which is the fixture, too. We realize that it is rare to occur, but when this occurs actually, challenge is present. Both biomechanics effect and force of mastication should be understood well from the start even before placement of the fixture to avoid such complication.Apical and middle part fractures need a challenge as it is difficult to be retrieved. Treatments were available to solve this problem different according to many factors. These factors are patient's cooperation and agreement, site available, a cause of fracture, and number of fixture presence at the site. All these factors can change the decision.Salinas and Oh describe the mechanism in the occlusal loading events applied over the abutment and its retaining screw.There is a slight wobble of the abutment during functional loads. Mechanically fatigue fracture is the main cause of such complication. The crown will generally dislodge, and the apical end of the screw will remain in the fixture.Importantly, after an abutment screw has fractured, assessment of the occlusal scheme is mandatory. Flangan and Lipski advised that the occlusal overloading should be measured in fixture site. Cyclic loading can induce stresses causing an abutment screw fracture, preventing fracture complications can be minimized by occlusal scheme.The fixture screw hole should be flushed with a dilute solution of sodium hypochlorite (0.5%) and rinsed with water to ensure removal of any organic or particulate debris that may inhibit the removal of the fragment.Radiographical examination added to clinical examination is mandatory in preparation for treatment, to ensure osseous support quality of the supporting fixture and to detect screw‐level fracture. By apical radiographical examinationScrew fragment retrieval, replaced by another screw.Fixture removal or discard and reimplantation in another site if applicable.Fabrication of a cemented cast post and core.Seetoh and Yilmaz with their colleagues cite that different brands of fixtures have different abutment screw fractures.Harshakumar et alMany  fixture companies offer fracture screw removal kits (Help Kit). It is expensive and does not consistently remove the fracture segment.But when all the modalities fail to retrieve the segment, the clinician under such conditions might opt for removing the failed fixture and replacing it with a new one. This gives additional surgical trauma and financial burden to the patient. Thus, salvaging the fixture by other means appears to be a viable option in such situations. Custom cast post and core is presented to solve this complication.The injured fixture is not forbidden or discarded.There is no need for another surgical intervention to be replaced by a new fixtureThis technique requires less trauma and less cost.The fabrication of post and core needs more difficult technical work in both damaging the internal threads of the fixture and fabrication of metal post which is unaccounted future.This technique requires less time to fabricate the prosthetic part.In this study, we assured that it is applicable to use the screw of the transfer cap only to be inserted in the same injured fixture and fabricate the prosthesis. This can be beneficial for the following reasons:Five patients underwent this procedure from total eight, with good satisfaction of the patients with follow‐up ranged from six months to 2 years. Clinically stationary fixed prosthetic part is well present.5Challenge and luck are always present in such cases. A careful clinical and radiographical examination is mandatory before deciding which line of managing cases with fractured abutment screw is to be used to avoid any uncounted complications. Transfer cap screw can be used gently and cautiously if the fractured piece cannot be retrieved or seated deep in the fixture.No conflict of interest has been declared as all work has been done in private clinic for all authors.RYA‐R: was a senior specialist in maxillofacial surgery, was the owner of the idea with the official work and patients’ follow‐up, and wrote and published the manuscript. FAM: was a specialist in maxillofacial surgery, drafted and arranged articles, and copied the manuscript. BA‐GT: served as a maxillofacial consultant, critically revised the paper, and contributed to the scientific and professional review.


The cellular metabolic rate is markedly reduced for adapting to these conditions. AMP-activated protein kinase (AMPK) senses the cellular energy status and regulates metabolism. Therefore, we examined AMPK signaling in several brain regions and peripheral tissues in hibernating chipmunk. Eukaryotic elongation factor 2 (eEF2) is a downstream target of AMPK. Phosphorylation of eEF2, indicating its inactivation, is enhanced in the cerebral cortex of hibernating chipmunks. The study indicated that the sequential regulation of AMPK-mammalian target of rapamycin complex 1-eEF2 signaling was altered and protein synthesis ability was reduced in the cerebral cortex of hibernating chipmunks.During hibernation, mammalian cells are exposed to severe environmental stressors such as low temperature, lowered O In general, the cells of hibernators can tolerate low temperature and low O2 supply as well as energy deficiency, presumably reducing the metabolic rate of glucose, lipids, and proteins.Hibernation is an adaptive strategy of some mammals to survive the severe cold and food scarcity in winter. It is a drastic circannual phenotypic alteration in such mammals. In chipmunks, the body temperature falls to nearly 0 °C and heartbeat reduces dramatically during hibernation2, which suggests that hibernation is triggered in the brain. It is widely accepted that hibernation is associated with downregulation of metabolism and ensuing energy insufficiency, at least in the brain, although it is unclear whether these metabolic changes are the cause or the result of hibernation4. Therefore, this study aimed to analyze and compare the energy-sensing and anabolic signaling in the brains, and peripheral tissues of hibernating chipmunks.It has been proposed that the circannual hibernation of chipmunks is regulated by hibernating proteins (HPs) produced in the liver and worked in the brain during hibernation5. AMPK is activated when ATP levels fall in situation such as nutrient insufficiency and hypoxia. AMPK activity is coupled with phosphorylation of Thr172 residue of AMPKα subunit7. When activated by reduced cellular ATP levels, AMPK induces energy-generating catabolic pathways, such as autophagy, and inhibits energy-consuming anabolic pathways, such as fatty acid synthesis, gluconeogenesis, and protein synthesis, by suppressing mammalian target of rapamycin (mTOR) complex 1 (mTORC1)8. Our study also focuses on eukaryotic elongation factor 2 (eEF2), because it lies downstream of AMPK-mTORC19. eEF2 is a GTPase that translocates peptidyl-tRNA from the A-site to P-site of the ribosome, thereby controlling translation elongation. Upon phosphorylation by eEF2 kinase (eEF2K) at Thr56, eEF2 is unable to bind a ribosome and is thus inactivated10. eEF2K activity is regulated either negatively or positively by multiple kinases targeting different phosphorylation sites. For example, p70S6 kinase, a substrate of mTORC1, phosphorylates eEF2K Ser366 and inhibits its activity11. In contrast, AMPK phosphorylates eEF2K at Ser39812, and Ser491/49213 and enhances its activity, leading to eEF2 inactivation. Altered AMPK activity15, mTOR signaling16, eEF2 phosphorylation18, and protein synthesis18 have been reported in several tissues of several species during hibernation. However, changes of AMPK activity in the brain have been less focused. In squirrels, no change of AMPK activity in the brain was reported15. In addition, there has been no systematic analysis of these changes in the brain during hibernation in chipmunks. Considering that eEF2 activity is a major regulator of protein synthesis in neurons19, and protein synthesis is a major consumer of cellular energy, we analyzed sequential AMPK-mTORC1-eEF2 signaling and protein synthesis ability in the brain and peripheral tissues of active and hibernating chipmunks.AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine protein kinase, comprising a catalytic α subunit and regulatory β and γ subunits, senses the cellular energy status by detecting the AMP/ATP ratio and regulates metabolismThe surface body temperature of each animal was monitored over a year. Figure ad libitum) or genetic traits of the group of chipmunks sampled in this study. Chipmunks are not laboratory animals; our samples were obtained from the wild, therefore, their genetic background may vary. We observed a difference between euthermic and hibernating chipmunks in this study.Tissue samples were collected during active and hibernation periods based on the surface body temperature Fig. . InterboPhosphorylation of AMPKα Thr172), reflecting its activation, was examined in multiple brain regions of active and hibernating chipmunks by Western blotting. Among the brain regions tested, AMPKα phosphorylation was significantly enhanced during hibernation only in the cerebral cortex, but not in the hippocampus, cerebellum, or hypothalamus  and thus reduce protein synthesis11. eEF2K phosphorylation at Ser366 was decreased in the cerebral cortex of hibernating chipmunks , protein synthesis rate was lower in the cerebral cortex of hibernating chipmunks than in that of active chipmunks, whereas liver protein synthesis rate did not differ between the active and hibernation periods  an increase of AMPKα phosphorylation (indicating elevated AMPK activity), (2) suppression of mTORC1 activity accompanied with raptor phosphorylation, (3) phosphorylation (inactivation) of eEF2, resulting in (4) reduced ability of protein synthesis as evidenced by lower 15. Liver and skeletal muscles synthesize and store glycogen; therefore, AMPK activity may not quickly respond to falling blood glucose levels. Instead, AMPK activity may change depending on the period of torpor and fasting. Indeed, AMPKα phosphorylation in the liver and skeletal muscles of rats is not altered in a short-term fasting23. Further, long-term caloric restriction rather decreased phospho-AMPKα levels in rat liver24. In contrast to peripheral tissues, phosphorylation status of AMPKα and ACC was increased in the cortex of hibernating chipmunks, a result not observed in the whole brain of thirteen striped-ground squirrels15. The increase in AMPK activity observed in the cerebral cortex of ground squirrels may have been obscured by a lack of change in the rest of the brain. In the hypothalamus of golden mantled ground squirrels, phosphorylation of ACC, but not AMPKα, was increased14, which is in partial accordance with our finding of unaltered AMPKα phosphorylation in the hypothalamus of chipmunks. In the brain, glycogen stores and fatty acid beta-oxidation are limited and almost all energy is derived from blood glucose. Therefore, the reduction in blood glucose levels may rapidly affect AMPK activity in the cerebral cortex, but not in energy-storing peripheral organs. However, this does not explain why phosphorylation of AMPKα in other brain regions was unaltered. Further studies are required to reveal possible differences in the regulation of AMPKα phosphorylation status and its activity among brain regions.Among the brain regions tested, significant AMPK activation was observed only in the cerebral cortex of hibernating chipmunks. However, no changes in the liver and skeletal muscle were observed during either active or hibernation period. The lack of increase in AMPK in the liver and skeletal muscles is in accordance with previous results from studies of the ground squirrel13 and to activate indirectly through the suppression of the mTORC1 pathway11. In this study, both total and phosphorylated eEF2K at Ser366 were reduced in the cortex of hibernating chipmunks. Thus, direct phosphorylation of eEF2K by AMPK may contribute to the activation of eEF2K, thus inhibiting eEF2 activity.Along with AMPK activation, eEF2 phosphorylation (inactivation) was observed in the cerebral cortex, but not in the liver. AMPK is known to phosphorylate eEF2K directly18. Therefore, eEF2 inactivation in the brain may be a common mechanisms for reducing protein synthesis in hibernating rodents. It has also been reported the Decreased activity of protein phosphatase 2A, the dephosphorylation enzyme for eEF2 has also been reported in the brain of hibernating ground squirrels18. It may also occur in thecerebral cortex of hibernating chipmunks.Hallenbeck’s group has reported eEF2 phosphorylation, prolonged ribosomal transit time that indicates slowing elongation rate, and reduced protein synthesis in the brains of hibernating ground squirrels2+ dynamics may influence AMPKα and eEF2 phosphorylation. AMPKα is phosphorylated by calcium-calmodulin-dependent protein kinase kinase (CaMKK)27 and eEF2 is phosphorylated by eEF2K, 28. The activity of both kinases is Ca2+-dependent. Intracellular Ca2+ dynamics are known to be altered in cardiac muscle cells during hibernation29, suggesting that cell type-specific changes in Ca2+ mobilization lead to differential activity of AMPK and eEF2 among tissues. Indeed, cold-stress-induced eEF2K activation via Ca2+ upregulation was previously reported30.In addition to the cellular energy status, cell type-specific Ca2. These HPs have collagen-like domains in the N-terminal region and form a triple-helix31. Structurally, HPc is predicted to belong to the C1q protein family32. Adiponectin and CTRPs, other members of this family, are reported to induce AMPK activation34. Because HPc is suggested to act in the brain1, HPc in the cortex may directly activate AMPK.Another possible explanation for the cerebral cortex-specific activation of AMPK is the direct involvement of the hibernation protein complex (HPc) comprising HP20, HP25, and HP27Although the phosphorylation levels of the molecules tested were enhanced in the cortex during hibernation, this is not a general phenomenon . In fact, phosphorylation of Akt was decreased in the cerebral cortex  of hibernating squirrels in situ at 7 °C body temperature18.As previously reported35. Unfortunately, we have no data in this study; however it is an important point in hibernation studies.The remaining unsolved question regards the relationship between circannual (hibernation) and circadian rhythms. The changes in each signaling molecule and its phosphorylation status may have been modified by circadian rhythms because the animals in the current study were housed in constant darkness and thus their rhythms may have free-run. Indeed, circadian change in AMPK activity has been previously reported in ratsBased on the present study, we suggest that the increased activity of AMPK and decreased protein synthesis may contribute to the low-temperature resistance of brain cells during hibernation. Future studies are required to assess the involvement of hibernation proteins in these metabolic changes and low-temperature resistance.36. Validation of antibody specificity to chipmunk proteins is shown in Supplemental Fig. 35S]methionine and Protein G sepharose were purchased from Perkin Elmer and GE Healthcare, respectively.Anti-AMPKα, anti-phospho-AMPKα (Thr172), anti-ACC, anti-phospho-ACC (Ser79), anti-eEF2K, anti-phospho-eEF2K(Ser366), anti-Akt, anti-phospho-AktSer473), anti-p44/42MAPK, and anti-phosopho-p44/42MAPK (Thr202/Tyr204) were purchased from Cell Signaling Technology. Anti-mTOR was obtained from Immuno-Biological Laboratories. Anti-eEF2 and anti-phospho-eEF2 antibodies were prepared as described previously73, anti-38 fused with GST and rat eEF219 tagged with Flag were transfected to HEK293T cells and starved with serum to increase phosphorylated forms. GST-AMPKα and Flag eEF2 were pull-down with Glutathione-sepharose and anti-DYKDDDDK(Flag)-agarose, respectively. Western blots of the antigens were shown in Supplemental Fig. The target molecules analyzed in this study are well conserved among species, and tge amino acid sequences and phosphorylation sites of AMPKα1 in mouse, rat, rabbit, human and squirrel are shown in Supplemental Fig. Tamias sibiricus) less than one year of age were purchased from Arcland Sakamoto Co. Ltd. The chipmunks were housed individually at 23 °C under 12h-light/dark cycle conditions and provided a standard rat diet and water ad libitum. The animals were maintained for at least six months to acclimate to laboratory conditions. After checking general health conditions, they were transferred to the experimental condition of 4 °C and constant darkness. Periodical changes in the body temperature were monitored with an infrared irradiation thermometer once a day more than one year. Only individuals that exhibited periodical changes  were deeply anesthetized with carbon dioxide and sacrificed by decapitation and tissues were collected at 13:00–15:00 h from October to November. Tissue and blood samples were collected, immediately frozen in liquid nitrogen, and stored at −80 °C until further analysis.All the animal experiments were conducted in compliance with the protocol which was reviewed by the Institutional Animal Care and Use Committee and approved by the President of Niigata University (Permit Number: Niigata Univ. Res.258-1). Protocols were performed in accordance with the Guiding Principles for Care and Use of Laboratory Animals . Male chipmunks . After thawing the cryopreserved samples on ice, 0.3 μl blood was used for measuring the blood glucose levels.19. Briefly, frozen tissue samples were weighted and homogenized in ten volumes of lysis buffer (62.5 mM Tris-HCl (pH6.8), 2% SDS, Complete protease inhibitor cocktail(Roche Applied Science Ltd.), and PhosStop(Roche Applied Science Ltd.)). After centrifugation , supernatants were collected. The protein concentration of each sample was determined by Micro BCA (PIERCE Ltd.). Equal amounts of protein (50 μg/lane for indicated molecules and 10 μg/lane for β-actin) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to plolyvinylidene fluoride membranes. The membranes were cropped around the appropriate molecular size to save the amount of antibodies. The membranes were blocked in TNT , and 0.05% tween-20) containing 10% BSA and then incubated with the indicated primary antibody overnight. After washing with TNT, blotted membranes were incubated with horseradish-peroxidase (HRP)-conjugated anti-rabbit IgG  or HRP-conjugated anti-mouse IgG  for 1 h. After washing, peroxidase activity was detected by chemiluminescence reagents  and visualized on X-ray film . The quantity of protein expressed was quantified by “Image J” software for Macintosh OS X and standardized by β-actin. β-actin blot was performed to validate the protein concentration. It is impossible to reprobe with anti-β-actin in the same blots, for example AMPK because the amount of total applied protein is too much for β-actin blot. The actin signal is saturated and is not applicable for quantification. If the protein amount is adjusted to actin level, other molecules cannot be detected. All blots presented in this study are from the same samples so that actin blots are the same in all the Western blot experiments. In some cases, brightness and/or intensity of the gels were manipulated to clarify the signals. The manipulation covered the entire area of the blots to ensure that the changes were made equally to each band.Tissue extracts, electrophoresis, and Western blotting were performed as previously reported40. Tissue samples were homogenized in 20 volumes of lysis buffer , phosphatase inhibitor cocktail ) and centrifuged for 30 min at 4 °C. The protein concentration of the supernatant was determined and 800 μg of each sample was used for immunoprecipitation. Samples were pre-absorbed with Protein G Sepharose for 60 min at 4 °C, centrifuged for 3 min at 4 °C, then mixed with mouse anti-mTOR antibody (1 μg) and incubated overnight at 4 °C. To measure kinase activity more accurately, saturation amount of lysates and a small amount of antibody was used to precipitate same amount of mTOR from active and hibernating chipmunks. Protein G sepharose was added to the samples and incubated for 2 h at 4 °C. After washing, immunoprecipitates were mixed with kination buffer including recombinant GST 4E-BP and ATP, and incubated for 30 min at 30 °C. The reaction was stopped by adding SDS sample buffer and boiling for 5 min at 95 °C. In the same membrane, mTOR and P-4EBP was immunoblotted. After that, membrane was reprobed with anti-GST antibody. mTOR kinase activity was calculated by the signal intensity of (P-4EBP/GST)/mTOR.The kinase activity of mTOR in tissue samples was measured as in previous reports35S]methionine incorporation19. Samples of brain and liver were gently homogenized with 10 volumes of DMEM and protein concentration was determined. Each sample (50 μg) was incubated with 10 µCi of [35S]methionine for 30 min at 37 °C. Samples were then lysed in equal volumes of 1 M NaOH containing casein as carrier, and incubated for 30 min at 42 °C. An equal volume of 20% ice-cold trichloroacetic acid was added to each sample, and the mixture was incubated for 1 h at 4 °C. After centrifugation, free methionine and methionine incorporated into protein were estimated by determining the radioactivity of supernatants and the pellet, respectively. Protein synthesis was estimated based on the ratio of methionine in the pellet to total methionine.Protein synthesis was measured by [All values are presented as mean ± SE. Differences between groups were statistically determined by Student’s t-test when data were normally distributed or Wilcoxon rank-sum test when data were not normally distributed. Analysis were performed by the statistical software “R” (version 3.1.2). p < 0.05 were considered statistically significant.Supplemental Figures


Monoamine oxidase inhibitions are considered as important targets for the treatment of depression, anxiety, and neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. This has encouraged many medicinal chemistry research groups for the development of most promising selective monoamine oxidase (MAO) inhibitors. A large number of plant isolates also reported for significant MAO inhibition potential in recent years. Differently substituted flavonoids have been prepared and investigated as MAO-A and MAO-B inhibitors. Flavonoid scaffold showed notable antidepressant and neuroprotective properties as revealed by various and established preclinical trials. The current review made an attempt to summarizing and critically evaluating the new findings on the quercetin and related flavonoid derivatives functions as potent MAO isoform inhibitors. Depression and anxiety are estimated as incapacitating mental disorders which impose a huge health burden globally. According to the World Health Organization, major depression has now recognized as the fourth extensive cause of the worldwide in incapacity balanced life-years and could eventually turn into the second most critical cause by 2020 ,2,3. TreMoreover, neurodegenerative disorders constitute the third most essential health issue in different developed countries. Alzheimer’s disease is the most widely recognized neurodegenerative disorder followed by Parkinson’s disease. Along with the aging problem of human society, Alzheimer’s disease (AD) has become one of the biggest threats to the modernize population. Alzheimer’s disease is indicated by nerve cells die in the cerebral cortex and accounts for 60 to 80 percent of dementia cases which affected more than 25 million people worldwide in 2000 and may eventually to increase to 114 million by 2050 ,8,9. GenThe theory of MAO-B inhibitors for the prevention of neuronal damage is also accepted due to the reduction of hydrogen peroxide formation through inhibition of MAO. Clinical data suggests that patients with major depression have symptoms that are reflected changes in brain monoamine neurotransmitters, specifically serotonin (5-HT) and norepinephrine (NE) ,14,15. AMonoamine oxidase  is a flavin adenine dinucleotide (FAD) dependent enzyme which is mainly localized on the outer mitochondrial membrane, responsible for the oxidative deamination of monoamines, including neurotransmitters, such as norepinephrine, dopamine, and serotonin (5-hydroxytryptamine [5-HT]) ,24. The 2O2) and different reactive oxygen species has sufficient deleterious reactivity which accounts for associated health-related problems including neurological damage. The generation of H2O2 via MAOs is also reported to be a cytotoxic factor involved in oxidative stress, causes degeneration of nigral cells in Parkinson’s disease ..Uncaria Angelica keiskei Koidzumi (A. keiskei K.). Cynaroside showed notable MAO inhibition with IC50 values MAO-A400 µM and MAO-B 268 µM. Therefore, it is likely that that inhibition of MAO-B exerts antidepressant activity  was studied on rodent monoamine oxidase A and B . Me. MeMelasGinkgo Biloba was carried out on mouse brain or liver monoamine oxidase (MAO)-A and -B activity chromen-1-one with MAOA. Moreover, 31 hydrophobic inactions were established with the MAO-A binding cavity. The methanolic extract from leaves of hircinum . Quercetrodinger . The intforatum) . Design foratum) . The strforatum) . Visual H-chromen-4-ones, 3-benzylchroman-4-ones, and have been synthesized and benzylidenechroman-4-ones was investigated for in vitro as inhibition of h-MAOA and h-MAOB by Desideri et al. -6″-O-acetyl-β-d-gluco-pyranoside, xanthomicrol, isoscutellarein 7-O-[6′′′-O-acetyl-β-d-allopyranosyl-(1→2)]-β-d-gluco-pyranoside and salvigenin from Sideritis using recombinant hMAO isoenzymes. Docking experiments showed salvigenin as the most potent hMAO-A inhibitor by forming several van der Waals and electrostatic interactions within the active site of the hMAO-A where aromatic coumarin ring of the salvigenin established two π-π staking with TYR444 and TYR407 residues situated in the cavity. Moreover, the xanthomicrol has shown selective inhibitory interactions towards hMAO-A by forming five hydrogen bonds with the amino acids residues of the side chains of active site hMAO-A isoform . The aromatic coumarine ring of xanthomicrol was observed sandwiched between the TYR407and TYR444 amino acid residues, which established two π-π interactions with TYR407 and formed a hydrophobic cage within the binding pocket. More relevant molecular binding interactions among the natural leads and hMAO the docked complexes were analyzed by 2-dimensional methods. The docking profile of the selected compounds is given in the  and catechol-O-methyltransferase (COMT) from the seven selected protein targets as important targets for baicalein function by literature. For the study flavonoid, baicalein was isolated from the root extract of Scutellaria baicalensis Georgi. Docking calculations were carried out using Glide software for the comparison of binding energy of baicalein with the standard MK-801 binding. The authors validated and developed a consensus scoring formula for ranking of the targets of a titled compound.In a subsequent paper, Gao et al.  reportedstandard . Two catSivaraman and coworkers performed docking calculations to rationalize the MAO inhibitory potency of luteolin, quercetin, kaempferol, and apigenin by using Auto Dock tools . The binAchyranthes aspera seeds and computed molecular docking to get insight into the binding modes of 6-prenyl apigenin within the monoamine oxidase-A enzyme pocket. Molecular docking studies were carried out by using AutoDock [In a later work, Beula and coworkers  isolatedAutoDock , revealePsoralea corylifolia seeds. Psoralea corylifolia is a medicinal plant widely documented for its antiaging properties. These two unique prenylflavanones selectively inhibited MAO-B enzyme with the highest potential. Docking methodologies predicted the binding affinity for both flavonoids, genistein (GST) and bavachinin (BNN). Zarmouh and coworkers further explored their earlier studies in 2015 [Psoralea corylifolia L. for their human MAO inhibition. Docking studies were performed to validate the correct binding and mechanistic insight into docking poses depicted in , whereas their hydroxy-phenyl group was located to the hydrophobic active site entrance surfaces. Both isoflavones possessed crossed and similar orientation as compared with the standard. A best-matched docking pose of the standard was contributed by a slight pull of GST toward a hydrophilic zone at its C5-OH group. The docking studied observations are given in (The same group further studied the isoflavone genistein (GST) and its structural analog daidzein (DZ) as promising MAO-A and MAO-B inhibitors Zarmouh et al. . Moleculgiven in .Recently Gidaro and coworkers reported a computational method to generate the binding modes of quercetin and kaempferol to the active site of both hMAO isoforms . All the50 values against both MAO isoforms. Furthermore, the docking studies correlated in many experiments to explore the molecular mechanism of flavonoid at the MAO receptor level. This may give the idea for the structural activity requirement of different classes of natural flavonoids for the MAO inhibition. Compilation of overall SAR studied indicated some characteristics of flavonoid moiety (This deep exploration of the quercetin and related flavonoid derivatives highlights the enthusiasm of therapeutic science specialists towards finding new potent and selective monoamine oxidase inhibitors or useful targeting agents for neurological and mental disorders. The current review is aimed to demonstrate the tremendous pharmacological MAO inhibition profile of natural flavonoid derivatives. The experimental in vitro studies suggested that natural flavonoids showed micro- to nanomolar range ICd moiety . The gly


There are many downstream targets of mitogen-activated protein kinase (MAPK) signalling that are involved in neuronal development, cellular differentiation, cell migration, cancer, cardiovascular dysfunction and inflammation via their functions in promoting apoptosis and cell motility and regulating various cytokines. It has been reported that cyclic AMP response element-binding protein (CREB) is phosphorylated and activated by cyclic AMP signalling and calcium/calmodulin kinase. Recent evidence also points to CREB phosphorylation by the MAPK signalling pathway. However, the specific roles of CREB phosphorylation in MAPK signalling have not yet been reviewed in detail. Here, we describe the recent advances in the study of this MAPK-CREB signalling axis in human diseases. Overall, the crosstalk between extracellular signal-related kinase (ERK) 1/2 and p38 MAPK signalling has been shown to regulate various physiological functions, including central nervous system, cardiac fibrosis, alcoholic cardiac fibrosis, osteoclast differentiation, mucin production in the airway, vascular smooth muscle cell migration, steroidogenesis and asthmatic inflammation. In this review, we focus on ERK1/2 and/or p38 MAPK-dependent CREB activation associated with various diseases to provide insights for basic and clinical researchers. Three mitogen-activated protein kinase (MAPK) signalling pathways, including extracellular signal-related kinase (ERK) 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK), play significant roles in the inflammatory response involved in various human diseases. Among the many substrates of MAPK, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is a lesser-known critical player in inflammatory diseases associated with MAPK signalling. CREB was originally reported as a mediator of the cAMP signalling pathway ,5,6. OriCREB plays pivotal roles in learning, long-term memory and synaptic plasticity. PKA, CaMKIV, ERK and p38 MAPK are known to regulate CREB phosphorylation in neurons. Physiological roles of various kinase-dependent CREB phosphorylation has been investigated previously [High levels of cAMP are linked to the extensive axonal growth during development in the embryonic central nervous system . cAMP isAnother CREB kinase, CaMKIV, phosphorylates CREB in vitro . ActivitERK/p38 MAPK induced CREB phosphorylation distinct from the cAMP/PKA pathway in neuronal cells has been investigated. Xing et al. reported that nerve growth factor (NGF) activates ERK, which in turn activates RSK. NGF also activates p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in CREB phosphorylation in PC12 cells. Furthermore, the ERK/RSK and p38 MAPK/MAPKAP kinase 2 pathways co-ordinately contribute to CREB phosphorylation in NGF-treated PC12 cells because suppression of these pathways is necessary for completely inhibiting NGF-induced CREB phosphorylation. Retinoic acid is a potent regulator of neuronal cell differentiation and induces ERK1/2 phosphorylation, which results in CREB phosphorylation in PKA-deficient PC12 cells and primary neuronal cells via a PKA-independent pathway .2+ induces ERK-CREB phosphorylation through cAMP/PKA activation in PC12 cells and hippocampal neurons. RSK2 has been identified as Ca2+-activated CREB kinase in PC12 cells and hippocampal neurons [2+ release. Intracellular Ca2+ release activates the ERK/RSK pathway mediated by PKC, Rap1-B-Raf and PYK2 complex and then phosphorylates CREB in striatal neurons [Crosstalk between PKA and ERK associated with the formation of long-lasting neuronal plasticity has been reported in neuronal cells. Toropomyosin receptor kinases (Trk) is important for neurite outgrowth. Trk signalling leads to activation of the cAMP-ERK pathway, which promotes increased neurite outgrowth and regeneration in isolated cerebellar . Ca2+ in neurons . Activat neurons . Further neurons .In summary, it is known that PKA, CaMKIV, ERK and p38 MAPK can phosphorylate CREB upon stress and in the presence of neuronal growth factors and excitatory neurotransmitters. While PKA and CaMKIV phosphorylate CREB directly, ERK and p38 MAPK phosphorylate CREB indirectly in a process mediated by RSK and MAPKAP kinase 2, respectively. These pathways co-ordinately contribute to the development of the central nervous system mediated CREB phosphorylation.Alcoholic cardiomyopathy (ACM) is diagnosed by linking the dilation and impaired contraction of one or both myocardial ventricles with a history of heavy alcohol consumption . SeveralCardiac remodelling is defined as changes in the heart structure due to various pathologic events. Cardiac remodelling is a risk factor for chronic heart failure due to it causing reduced contractility and high mortality . The extDUSP8 gene inhibits cardiac ventricular remodelling, thereby suppressing ERK1/2 activity [It has been demonstrated that Ang II induces the production of periostin , a matriactivity . These sOsteoclasts are differentiated from the monocyte/macrophage lineage of hematopoietic cells. Bone homeostasis is regulated by bone formation and bone resorption activity. Osteoclasts that are responsible for bone resorption are involved in bone homeostasis together with osteoblasts, which constitute the bone matrix ,50,51. OVβ3 integrin is expressed in osteoclasts. αVβ3 integrin with c-Fms (CSF-1 receptor) collaboratively mediates osteoclast differentiation through the ERK1/2 and c-Fos signalling pathway with M-CSF stimulation. Interleukin (IL)-1α also promotes ERK activation for the survival of osteoclasts by preventing their apoptosis [c-Fos and NFATc1 during osteoclast differentiation through B-cell adaptor for phosphatidylinositol 3-kinase (Pl3K) (BCAP) or Ameloblastin (Ambn) [c-Fos and NFATc1 activation. Pharmacological inhibition of CaMK IV, as well as the genetic ablation of CaMK IV, reduce CREB phosphorylation and c-Fos expression [ERK activation is central for the survival of mature osteoclasts  and stabpoptosis . A recenn (Ambn) ,61. BCAPn (Ambn) . Ambn isn (Ambn) . A recenpression . Althougpression . Therefop38 MAPK is activated following MAPK kinase kinase 6 (MKK6) activation  upon RANTaken together, the ERK1/2 and p38 MAPK-CREB pathways play important roles in bone homeostasis. Although the underlying mechanism and the roles of CREB activation in osteoclast differentiation have not been fully elucidated, understanding of the ERK1/2 and p38 MAPK-CREB pathways will help develop therapeutic strategies for various bone diseases . Lung mucus is secreted by lung epithelial cells and plays a pivotal role in airway clearance and immunity in healthy lungs . Lung muMany stimuli, such as cytokines, epidermal growth factor receptor (EGFR) ligands, microorganisms and cigarette smoke induce MUC5 production by the activation of several signalling pathways and transcriptional factors. The pathways for MUC5 production are mainly through four cascades, EGFR signalling, cytokine signalling, toll-like receptor (TLR) signalling and reactive oxygen species (ROS) signalling. In the signalling pathways for MUC5 production, MAPK are the key molecules, except for the pathway through which IL-13 induces MUC5 production. MAPK signalling pathways result in the activation of several alternative transcription factors required for MUC5AC upregulation. Each stimulus activates a different MAPK signalling pathway.MUC5AC up-regulation [TGF-α and amphiregulin (a widely expressed transmembrane tyrosine kinase) bind EGFR and activate Ras or Raf and they in turn phosphorylate ERK1/2. ERK1/2 and activate transcriptional factors, such as activator protein (AP) 1, specificity protein (SP) 1 and CREB following the transcription of MUC5AC. Perrais et al. reported that the transcription factor SP1 is essential for EGF and TGF-α-mediated gulation ,73.MUC5AC gene expression. Furthermore, the activation of MSK 1 and CREB are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression [MUC5AC promoter might play an essential role in these processes by binding to CREB. LPA, LPS or TNF-α stimulation induces the production of cytokines, such as IL-8 and IL-1β, which is mediated by CREB phosphorylation. ERK1/2 or p38 MAPK facilitates MSK phosphorylation directly and thus phosphorylates CREB under these agonists [MUC5AC [MUC5AC through the activation of two signalling pathways. The first involves the activation of amphiregulin and EGFR. The amphiregulin binds EGFR and Ras or Raf is activated. MEK is phosphorylated following the phosphorylation of ERK1/2. The second involves ROS activation of Src and subsequently JNK. Both pathways activate Jun D and Fos-related antigen 2 (Fra-2) and thereby stimulate transcription of MUC5AC through AP1 [Several cytokines including IL-1β, tumour necrosis factor (TNF)-α and IL-13 induce MUC5AC production . Song etpression . This stagonists . Becauseagonists , the difagonists , increasagonists ,79. Micr [MUC5AC ,82,83,84 [MUC5AC . ROS stiough AP1 .EGFR, HER2 protein expression and poorer prognoses [® CRL-1848TM) and primary nasal epithelial cells stimulated with DEPs [Abnormal expression of MUC4 has been reported in non-small lung carcinoma and especially in adenocarcinomas. MUC4 is associated with male smokers, solid adenocarcinomas, negative TTF-1 expression, wild-type rognoses . In smalrognoses . Diesel ith DEPs .Collectively, MAPKs are the key molecules in the signalling pathways to produce MUC5. While p38 MAPK is essential for mucin production induced by IL-1β, TNF-α, the microbial components and DEPs via CREB phosphorylation, ERK1/2 is associated with TGF-α, IL-1β, TNF-α and cigarette-induced mucin production. JNK is activated by intracellular ROS induced by cigarette smoke. However, IL-13-induced mucin production is not related to MAPK signalling but with STAT6 phosphorylation .Rip increases MLCP activity by binding to myosin, actin and myosin phosphatase target subunit 1 [Accumulation of vascular smooth muscle cell (VSMC) in the intima of arteries plays a key role in atherogenesis . Upon enubunit 1 ,95.Other regulatory mechanisms of VSMC migration have been investigated. Ang II has various roles in regulating VSMC growth, apoptosis and migration. An earlier study has shown that Ang II activates ERK1/2 and JNK in VSMC . ExendinCREB activation has also been demonstrated to be important for VSMC migration. Ono et al. reported that TNF-α-induced VSMC migration is mediated by CREB activation via the p38 MAPK pathway. p38 MAPK inhibitor abolishes both TNF-α-induced both CREB phosphorylation and VSMC migration, while MEK, JNK, PI3-kinase or PKA inhibitor does not inhibit TNF-α-induced CREB phosphorylation. Transfection of adenovirus vector expressing dominant-negative of CREB results significantly inhibited TNF-α-induced VSMC migration and RAC1 protein expression, indicating that CREB activation mediates TNF-α-induced VSMC migration. The reaction is mediated by RAC1, a Rho family small GTPase . UTP, onChlamydophila pneumonia antigen also produces GM-CSF via p38 MAPK but without JNK, ERK1/2 or PI-3K activity [Granulocyte macrophage-CSF (GM-CSF) is important for the process of maturation of macrophages and granulocytes ,109. GM-activity . LPS indactivity . Though activity .Mechanisms of GM-CSF secretion have also been investigated in human lung fibroblasts ,125,126.IL33 promoter area activates the IL33 promoter by CREB phosphorylation via the p38 MAPK signalling pathway after TNF-α stimulation [ET-1 transcription, activates ERK1/2 and reactivates p38 MAPK in human airway smooth muscle cells. While activated ERK1/2 stimulates GM-CSF mRNA stabilization, reactivated p38 MAPK stimulates both ET-1 and GM-CSF secretion [The significance of CREB phosphorylation in TNF-α-induced GM-CSF secretion has been demonstrated by knocking down CREB expression using three specific siRNAs against CREB in human lung fibroblasts. The p38 MAPK inhibitor SB239063 inhibited TNF-α-induced GM CSF secretion by approximately 20% and CREB siRNAs inhibited both CREB mRNA expression and GM-CSF secretion equally. These results support that p38 MAPK and CREB are critical mediators of TNF-α-induced GM-CSF secretion . Recentlmulation . Quite rmulation . Interesecretion   and progesterone synthesis . Ang II cAMP induces CREB activation via ERK/p38 MAPK pathway. Forskolin-induced CREB phosphorylation is mediated by cAMP/PKA and by a time-delayed cAMP/PKA-dependent p38 MAPK/MSK1 pathway in NIH 3T3 cells. cAMP/PKA is the major pathway of CREB phosphorylation in forskolin-treated NIH 3T3 cells but PKA also phosphorylates CREB through p38 MAPK/MSK1. PKA-induced CREB activation via p38 MAPK-MSK1 is delayed compared to cAMP–PKA-induced CREB activation . Upon stThe roles of cAMP in CREB activation are varied depending on physiological conditions. The cAMP-PKA signalling pathway has an inhibitory effect on cardiac fibrosis. cAMP inhibits gene activation and TGFβ-Smad signalling by disrupting the interactions between Smad3 and co-activators  in a PKA-dependent manner . TreatmeRANKL increases the adenylate cyclase 3 expression and the intracellular cAMP levels in osteoclasts. The suppression of adenylate cyclase 3 enhances osteoclastogenesis in vitro and bone resorption in vivo . Wnt3a-iForskolin also increases MUC5AC production by elevating cAMP, indicating that cAMP-PKA signalling mediates IL-1β-induced MUC5AC mucin production in NHTBE . It has 2+ and VSMC migration [The roles of cAMP in VSMC migration are controversial . cAMP-PKigration .The inhibitory mechanism of GM-CSF secretion has been also investigated. Increased cAMP levels, by the treatment of phosphodiesterase 4 (PDE4) or the addition of cell-permeable cAMP, inhibits GM-CSF secretion without CREB inactivation . ForskolRoles of cAMP and ERK/p38 MAPK pathway and crosstalk between cAMP and ERK/p38 MAPK pathway on CREB-induced physiological functions are shown in Direct phosphorylation of CREB at Ser133 by PKA and CaMKIV has been reported ,15. RSK


HL addition line allowed the transcriptional impact both on 7HL genes of a non‐native genetic background and on the wheat gene complement as a result of the presence of 7HL to be assessed. Some 42% (389/923) of the 7HL genes assayed were differentially transcribed, which was the case for only 3% (960/35 301) of the wheat gene complement. The absence of any transcript in the addition line of a suite of chromosome 7A genes implied the presence of a 36 Mbp deletion at the distal end of the 7AL arm; this deletion was found to be in common across the full set of Chinese Spring/Betzes barley addition lines. The remaining differentially transcribed wheat genes were distributed across the whole genome. The up‐regulated barley genes were mostly located in the proximal part of the 7HL arm, while the down‐regulated ones were concentrated in the distal part; as a result, genes encoding basal cellular functions tended to be transcribed, while those encoding specific functions were suppressed. An insight has been gained into gene transcription in an alien introgression line, thereby providing a basis for understanding the interactions between wheat and exotic genes in introgression materials.Despite a long history, the production of useful alien introgression lines in wheat remains difficult mainly due to linkage drag and incomplete genetic compensation. In addition, little is known about the molecular mechanisms underlying the impact of foreign chromatin on plant phenotype. Here, a comparison of the transcriptomes of barley, wheat and a wheat–barley 7 Triticum aestivum) and barley (Hordeum vulgare) are the two leading temperate small‐grained cereals, and together supply, directly or indirectly, a major proportion of our calorific requirements. The major breeding priority over many decades has been to increase their productivity, and this goal was growing in importance in the face of a continuing expansion in the size of the human population, changes in the dietary habits and the threat of impending climate change  were differentially transcribed, comprising 233 down‐regulated (transcript abundance lower in CS + 7HL than in B) and 156 up‐regulated  and the total number of transcribed genes [R(not‐DT/Trans)], the ratios between the number of up‐ or down‐regulated and the total number of transcribed genes [R(Up/Trans) and R(Down/Trans)] and the mean log fold change (mean log FC). For the 7HL arm, the R(not‐DT/Trans) measure indicated that the central segment of the arm was enriched for not‐DT genes Figure . The RU. The disA comparison of the physical length of flow karyotyped mitotic copies of chromosome 7A suggested that the version harboured by CS + 7HL was approximately 3.0% shorter than the one harboured by CS Figure a. When Gvs CS + 7HL. As described above  and 7DL 610–636 Mbp) are homeologous to the 7AL deletion harbour, respectively, 143 and 122 genes. Of these, just six of the 7B genes and two of the 7D ones were transcribed at a higher level in CS + 7HL than in CS  were transcribed at a detectable level in CS + 7HL: 375 transcripts were represented by a count of 1–5 FPKM and 327 by >5 FPKM; of the latter, 92 were strongly transcribed (20–1360 FPKM). Around 60% of these genes had at least one transcriptionally active wheat homeolog, so are likely to contribute to the transcript pool. Among the set of highly transcribed 7HL genes, 47 were homeologs of a 7AL gene deleted in CS + 7HL; of these, 26 were at least half as abundantly transcribed in CS + 7HL as was their 7AL homeolog in CS  analysis of the set of wheat DT genes showed that among the 451 up‐regulated genes, there was an enrichment for 174 terms for biological processes and molecular functions, relating mostly to the response to biotic, abiotic and oxidative stresses. In particular, the GO terms ‘immune system process’, ‘response to biotic stimulus’, ‘immune response’ and ‘ubiquitin‐like protein ligase binding’ were prominent, as were terms relating to secondary metabolism synthesis such as ‘phenylpropanoid metabolic process’ and ‘secondary metabolite biosynthetic process’ Table . Meanwhivs CS + 7HL and B vs CS + 7HL.The RNA‐seq platform was used here to capture the transcriptomes of CS, CS + 7HL and B, and applied a more stringent set of criteria that is normally used for mapping reads to a genomic sequence. This was thought necessary to ensure discrimination between homeologs. Rather than aiming to maximize sequencing depth, it was considered a better use of resources to increase the level of biological replication. The approach allowed the transcript abundance of over 35 000 wheat and nearly 1000 barley (7HL) genes to be estimated in the CS + 7HL line, representing, respectively, 31.8% and 34.5% of the annotated gene content. The replication also provided a high measure of confidence to the identification of the 960 wheat and 389 barley genes classified as DT genes in two contrasts CS et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Wide hybridization is often associated with profound rearrangements of the host and exotic species chromosomal structure, along with alterations to both the DNA sequence and the epigenome; ultimately, these have a profound effect on the transcriptome of the wide hybrid . cDNA libraries were generated for each sample using a TruSeq RNA Sample Preparation Kit . The samples were randomly pooled by six and sequenced on 2 × 100 bp paired‐end flow cells using HiSeq2000 device (Illumina), generating ~30 million reads per sample. Ribosomal RNA sequences were removed with the help of sortMeRNA program , Betzes barley  and the CS/B addition line harbouring a pair of 7HL telosomes . Grain of CS + 7HL and of the six viable whole chromosome CS/B addition lines (+2H through +7H) were obtained from the National BioResource Project, Komugi (shigen.nig.ac.jp/wheat/komugi/strains/nbrpDetailAction.do?strainId=LPGKU2105). The mitotic chromosome number of each aneuploid line was checked to confirm the presence of the expected chromosome complement, following the Kopecký et al.  procedurRNA‐seq data have been deposited in the sequence read archive under the BioProject accession number PRJNA412872.et al., et al., et al., Each of the reads for each of the 18 samples  was mapped against the ‘High Confidence’ coding sequences of both wheat , R(Up/Trans)] were calculated. The mean logarithm of the fold change in transcript abundance (logFC) in the comparisons CS et al.  and 0.05 (7HL).Gene ontology terms were associated with each of the wheat and barley genes according to functional annotations provided by, respectively, IWGSC  and Mascet al. . Ontolog et al.,  was applThe authors declare no conflict of interest.Figure S1 All CS/B addition lines carry a major deletion in the 7AL chromosome arm. The same deletion as detected in the CS + 7HL ditelosomic addition line is present in the six CS/B whole chromosome addition lines, as shown by C‐banding. Chromosomes 7A are indicated on the pictures by red arrows.Click here for additional data file.Table S1 The RNA‐seq mapping fitness.Table S2 Molecular markers used to genotype 7AL deletion in CS and CS + 7HL.Table S3 The gene ontology terms enriched in the set of wheat up‐regulated genes in CS + 7HL.Table S4 The gene ontology terms enriched in the set of wheat down‐regulated genes in CS + 7HL.Table S5 The gene ontology terms enriched in the set of barley 7HL genes up‐regulated genes in CS + 7HL.Table S6 The gene ontology terms enriched in the set of barley down‐regulated genes in CS + 7HL.Click here for additional data file.


H1047R, or knocking down PTEN expression. We investigated eNOSS1177 phosphorylation, a major activating regulatory site, following mTORC1 inhibition. We also tested the sufficiency of mTORc1 activation to stimulate eNOSS1177 phosphorylation. Our data indicate mTORc1 activity is required for the phosphorylation of eNOSS1177, even in the presence of robust AKT activation. Moreover, we found that expression of RHEB, which functions in the absence of AKT activation to activate mTORc1, is sufficient to phosphorylate this site. Our data indicate that mTORc1, rather than AKT, may be the critical determinant of eNOSS1177 phosphorylation. As mTORc1 is a central regulator of cellular metabolism, the finding that this regulatory complex can directly participate in the regulation of eNOS provides new insights into metabolic uncoupling and vascular disease that often accompanies diabetes, high fat diets, and aging.Nitric oxide, produced by eNOS, plays critical roles in the regulation of vascular function and maintenance. Chronic PI3K signaling has recently been associated with vascular malformations. A well described substrate downstream of PI3K signaling is eNOS. Another critical downstream target of PI3K is the metabolic regulator, mTORc1. The relationship between mTORc1 and eNOS regulation, has not been determined. We generated cells with manipulated PI3K signaling by expressing the activating mutation, PIK3CA Nitric oxide (NO) can be generated by three isoforms of nitric oxide synthase (NOS), neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) , threonine (Thr), and tyrosine (Tyr) residues, the most heavily studied being the phosphorylation of Serine 1177. This site is a pivotal regulator of its enzymatic activity. Phosphorylation of this site is documented to be downstream of the phosphatidylinositide 3‐kinase (PI3K) pathway, specifically Akt  from pooled donors were purchased from Lonza and cultured as we have previously described , eNOS , and ERK2 (Santa Cruz Biotechnology SC‐154 1:1000). ERK2 is shown as a loading control. We have verified that expression of PIK3CA and RHEB, as well as knockdown of PTEN, does not alter the total protein expression of AKT, S6, or eNOS. All figures are representative of at least two independent HUVEC infections. Blots were imaged and quantified using a Fuji digital luminescence imager under nonsaturating conditions.t‐tests were used for comparing two groups. For all analysis, P ≤ 0.05 was considered statistically significant. Bar graphs are represented as mean ± SEM.Statistical analysis was performed using Sigma Plot. Two‐tailed Student's H1047R is a gain of function “hot spot” mutation within the catalytic subunit of PI3K shown to exist in cancer and vascular anomalies  as well as enhanced phosphorylation of S6 (S235/236), a downstream indicator of mTORc1 activity. Under these conditions, we found enhanced basal phosphorylation of eNOS (S1177), which was markedly augmented upon short‐term stimulation with human vascular endothelial growth factor (hVEGF)  Fig. . SimilarH1047R or PTEN‐KD. Under these maximal activation conditions, rapamycin had no effect on AKT phosphorylation, which remained high, but eNOSS1177 phosphorylation was nearly completely inhibited , in the absence of any significant change in AKT activation levels. Under growth factor‐free conditions, the expression of RHEB was sufficient to induce eNOS phosphorylation at levels comparable to VEGF. The addition of hVEGF to RHEB‐expressing cells results in a marked stimulation of phosphorylation of eNOS (S1177) (Fig. S1177 phosphorylation, demonstrating that the RHEB enhancement of phosphorylation is dependent upon mTORc1 activity (Fig. As mTORc1 was required for eNOS phosphorylation, we wished to determine if it was sufficient. We utilized lentiviral expression of an active RHEB of S6 S25/236, inS16H expression in HUVECs support these findings. These data underscore both the potential benefits, as well as the potential risks, of rapamycin or other mTORc1‐targeted therapies as a clinical intervention in cardiovascular disease.Our data demonstrate several important aspects of eNOS regulation including a novel mode of activation of eNOS phosphorylation at S1177. Both the pharmacological approach, with rapamycin, and genetic manipulation through mutant RHEBH1047R and RHEB expression conditions and is not significantly enhanced by VEGF. It will be critical to determine if the enhanced phosphorylation of eNOSS1177 following sustained mTORc1 activation is functionally coupled to changes in the endothelium or vasculature.Our data clearly demonstrate that eNOS phosphorylation downstream of the angiogenic agonist VEGF, as well as genetic augmentation of PI3K signaling, is not mediated directly by AKT. Rather, it seems likely that the principal role of AKT is the augmentation of mTORc1 activation, a process known to occur via modulation of TSC2 (Dibble and Cantley S1177 has been shown to stabilize the interaction between eNOS and the calcium‐binding protein, making it less dependent upon calcium (McCabe et al. Previously VEGF stimulation has been linked to increased intracellular calcium levels and given that eNOS is regulated by calcium (Hamdollah Zadeh et al. 2− generation. Its product NO is also sensitive to reactive oxygen species such as O2−‐forming OONO‐ yielding NO bioactively unavailable and ultimately further contributing to oxidative damage (Huang S1177 can augment superoxide generation, when uncoupled (Peng et al. Enhanced eNOS phosphorylation, does not inevitably result in enhanced NO production and improved endothelial function. eNOS uncoupling has been documented in patients with pathological endothelial dysfunction as a result of hypercholesterolemia, diabetes mellitus, and hypertension (Forstermann and Munzel ge Huang . ImportaVascular dysfunction is a universal feature of aging and aging is an associated risk factor for cardiovascular disease. Aging has long been associated with increased mTOR activity and treatment of mice with rapamycin enhances longevity and improves survival in obese mice on a high fat diet (Wilkinson et al. None.


In this study, we use an unprecedentedly high-resolution  ocean model that resolves shelf-sea and sub-ice-shelf environments in qualitative agreement with existing observations during austral summer conditions. We demonstrate that the waters reaching the Pine Island and Thwaites grounding lines follow specific, topographically-constrained routes, all passing through a relatively small area located around 104°W and 74.3°S. The temporal and spatial variabilities of ice shelf melt rates are dominantly controlled by the sub-ice shelf ocean current. Our findings highlight the importance of accurate and high-resolution ocean bathymetry and subglacial topography for determining mCDW pathways and ice shelf melt rates.In the Amundsen Sea, modified Circumpolar Deep Water (mCDW) intrudes into ice shelf cavities, causing high ice shelf melting near the ice sheet grounding lines, accelerating ice flow, and controlling the pace of future Antarctic contributions to global sea level. The pathways of mCDW towards grounding lines are crucial as they directly control the heat reaching the ice. A realistic representation of mCDW circulation, however, remains challenging due to the sparsity of  The bed topography of these glaciers slopes downward inland, to as deep as 2500 m below sea level, making the glaciers prone to marine ice sheet instability8 and thus further acceleration of grounded ice loss. These glaciers have been accelerating over the past two decades, hypothetically triggered by variable and relatively recent high basal melting of their ice shelves10.In the Amundsen Sea, the Pine Island and Thwaites Glaciers are the two fastest-flowing outlet glaciers11. The mCDW flows via submarine glacial troughs onto the continental shelf break, travels a few hundred kilometers southward, and flows into the deeper and inner parts of ice shelf cavities, where it meets ice in the vicinity of grounding lines  forming a buoyant mixture of mCDW and glacial meltwater. Ice shelf melt rates near grounding lines (1) are generally a few orders of magnitude higher than at other locations of the same ice shelves  and (2) significantly impact future evolutions of glaciers and thus their contributions to sea level rise17. Thus, it is necessary to understand mCDW pathways as well as processes determining ice shelf melt rates near grounding lines. However, existing observations are sparse in time and space and the importance of cavity circulation has only been inferred for Pine Island Glacier in previous studies12. Existing ocean simulations with realistic configurations typically either aim at representing (1) temporal variability and use horizontal grid spacings of ~1–2 km or coarser , or (2) the spatial distribution of melt and use idealized geometries or forcing or finer horizontal grid resolutions in small model domains .The main cause for high basal melting of Pine Island Ice Shelf (PIIS) and Thwaites Ice Shelf (TIS) is the relatively warm modified Circumpolar Deep Water 21, but with horizontal and vertical grid spacings of 200 m and 10 m, respectively, and with some adjustments to model parameters  pathways of warm mCDW from the continental shelf region (north of 74.24ºS) into the PIIS and TIS ice shelf cavities and towards their grounding lines and (2) processes that determine the magnitude of ice shelf melt near the PIIS and TIS grounding lines. For ice shelves, we assume a steady state for ice shelf thickness and cavity geometry and compute ice shelf melt rates following refs. 31. Although Dotson and Crosson ice shelves are in the model domain, they are located closer to the model boundary and we do not focus on these ice shelves for this study. We employ a passive tracer and Lagrangian particles (see Methods for details). We conduct a 60-day model simulation from 1 January 2010 to 1 March 2010, hereinafter CTRL after 30 days of spin-up. Sensitivity experiments with tides (TIDE) and without ice shelf melt flux (NOMELT) are also conducted  inflowing mCDW characterized by high temperature and high salinity is present in the outer part, while mCDW in the inner part is modified, being cooled and diluted  measurements under the PIIS cavity were carried out in 2009ted Fig. . We note34 provide time series of ocean currents and potential temperature .The mooring observations located at the PIIS front  observations were also obtained during 2010 in the Pine Island Bay region1 of the integrated melt rates for the PIIS, TIS, and the Dotson and Crosson ice shelves are 101 ± 8 Gt yr−1, 97.5 ± 7 Gt yr−1, and 84 ± 18 Gt yr−1, respectively, based on data collected between 2003 and 2009. Simulated time series of integrated ice shelf melt rates for the PIIS, TIS, and the Dotson and Crosson ice shelves are 64.8 Gt yr−1, 65.1 Gt yr−1, and 83.7 Gt yr−1, respectively, and are nearly stable throughout the simulation period  10–30 m yr−1 over most of the outer part of the cavity . Such features are well reproduced in our simulation. The observed ice shelf melt rates, however, peak near the grounding line at 100–200 m yr−1 while our simulated melt rates peak at 70–80 m yr−1 . These differences may be related to underestimated and poorly constrained heat and salt transfer coefficients21 and/or inaccurate ice shelf draft and ocean bottom bathymetry.Satellite-based estimates35. Both observed and simulated time series of ice shelf melt rates at the same location appear to have fluctuations with frequencies of 7–10 days . mCDW reaching the PIIS grounding line has a potential density of 27.75 kg m−3  enters the model domain from the northern boundary and flows southward along bathymetric contours . Sequential snapshots of tracer concentration representing mCDW (hereinafter mCDW tracer) released from the region north of 74.24ºS depict similar patterns to the potential temperature on the 27.75 kg m−3 isopycnal . These pathways basically follow the 60-day-mean current on the 27.75-kg mnal Fig.  and thusnal Fig.  and 16. 38.Vertical sections of 60-day-mean potential temperature and ocean speed along 101.1ºW below the PIIS show (1) warm mCDW stored at depth (below 600 m) and Winter Water (WW) or modified mCDW located above and (2) two main inflows, which are topographically constrained boundary currents, transporting warm mCDW towards the PIIS grounding line Fig. . Inflows40. Mean tidal currents based on tidal models42 are mostly small (<0.05 m s−1) over the Eastern Amundsen Sea continental shelf. Similar to a previous study40, the impact of tidal circulation on ice shelf melt rate is minor  high basal melt rates (>100 m yr−1) associated with both basal keels and apexes of channels in the inner cavity near the grounding line, (2) high basal melt rate (20–40 m yr−1) associated with keels over the middle to outer part of the ice shelf, and (3) low basal melt rate (0–10 m yr−1) associated with channels over the middle to outer part of the ice shelf. These features are well reproduced in our model  but note that they cannot be produced by the spatial pattern of potential temperature. The simulated potential temperature of ocean water at the uppermost model grid cell below the ice shelf is ~0.5 ºC below channels and ~1.5 ºC below keels  and thus ice shelf melt rates corresponding to this temperature difference fluctuates by only ~30%, which cannot explain the simulated spatial ice shelf melt pattern. Ocean velocity of the uppermost grid cell below the ice shelf, by way of contrast, shows small-scale features, consistent with previous studies43, that fluctuate from zero to as high as 0.3 m s−1 . It is likely that both glacial-melt-driven and wind-driven circulations change the ice base velocity at the ApRES location , with recent updates of more accurate bathymetry for the region near Pine Island, and the Crosson and Dotson ice shelves39. The trough extending towards the Pine Island Ice Shelf is well mapped39 and we do not find obvious differences among different bathymetry datasets. The simulated ice draft was obtained using high-resolution observations from commercial, sub-meter satellite stereo imagery for the PIIS15 and Antarctic Bedrock Mapping (BEDMAP-247) for the Thwaites, Dotson, and Cosson ice shelves. For ice shelves, we assume steady-state ice shelf thickness and cavity geometry and compute ice shelf melt rates following refs. 45.We use a regional configuration of the Massachusetts Institute of Technology general circulation model (MITgcm) with hydrostatic approximation, dynamic/thermodynamic sea-iceves Fig. . Nominal21, atmospheric forcing is provided by the ongoing ECCO LLC270 optimization48, which is based on ERA-Interim49 and has been adjusted using the ECCO adjoint-model-based methodology50. Lateral ocean boundary conditions are derived from an extended (2001–2016) ocean simulation based on ref. 13, with increased (70) vertical levels. Initial conditions are derived from a 30-day spin-up from an initial rest state with potential temperature and salinity from the January 2010 monthly-mean fields from the extended simulation based on ref. 13. There is no additional freshwater runoff, that is, all calving icebergs are assumed to be transported and melt outside the regional domain. We start our simulation without sea ice and sea ice does not form until the end of the model simulation.Similar to refs. 13, and no tracer restoring is applied. Particle release experiments are conducted offline using hourly outputs of ocean current using Octopus (https://github.com/jinbow/Octopus) and particles are released along 74.24°S at the depth of the 27.75 kg m−3 isopycnal.For the passive tracer representing mCDW, initial tracer concentrations are set to 1.0 for the region north of 74.24°S with potential temperature higher than 1.0°C, similar to ref. 42 are superimposed on the ocean lateral boundary condition. For the NOMELT case, heat and salt transfer coefficients at the interface between ice shelf and ocean are set to zero for all ice shelves in the model domain. The comparison of model and data  is shown in the main text and supplementary. Some of the figures are created with software Paraview and Ocean Data View53.Two other sensitivity experiments are also conducted. For the TIDE case, tidal currents from the CATS2008A inverse barotropic tide modelSupplementary info


Climate change is predicted to alter temperature, carbonate chemistry and oxygen availability in the oceans, which will affect individuals, populations and ecosystems. We use the fossil record of benthic foraminifers to assess developmental impacts in response to environmental changes during the Palaeocene–Eocene Thermal Maximum (PETM). Using an unprecedented number of µ-computed tomography scans, we determine the size of the proloculus (first chamber), the number of chambers and the final size of two benthic foraminiferal species which survived the extinction at sites 690 , 1210  and 1135 . The population at the shallowest site, 1135, does not show a clear response to the PETM, whereas those at the other sites record reductions in diameter or proloculus size. Temperature was similar at all sites, thus it is not likely to be the reason for differences between sites. At site 1210, small size coincided with higher chamber numbers during the peak event, and may have been caused by a combination of low carbonate ion concentrations and low food supply. Dwarfing at site 690 occurred at lower chamber numbers, and may have been caused by decreasing carbonate saturation at sufficient food levels to reproduce. Proloculus size varied strongly between sites and through time, suggesting a large influence of environment on both microspheric and megalospheric forms without clear bimodality. The effect of the environmental changes during the PETM was more pronounced at deeper sites, possibly implicating carbonate saturation.This article is part of a discussion meeting issue ‘Hyperthermals: rapid and extreme global warming in our geological past’. In high.6–4.8°C . At dept.6–4.8°C ,4). Warm.6–4.8°C , though .6–4.8°C . The res.6–4.8°C . However.6–4.8°C , but theEnvironmental factors influence an organism through developmental plasticity, thereby providing a target on which evolution can act to produce novel, potentially adaptive, phenotypes . Multi-gin situ pressures [The fossil record documents natural climate change and variability as well as preserving some species exposed to these environmental changes . The Palressures . Traditiressures  and a trressures , as wellressures ,24. Experessures  and, usiressures , unexpecressures .Many foraminifers grow by sequentially adding chambers and hence preserve their entire ontogeny in their morphology , which cHere, we use three-dimensional µ-computed tomography imaging to collect information on proloculus size, number of chambers and final size of two species of benthic foraminifera which survived the extinction at the PETM, at three locations, to quantify response by benthic foraminifers to the climatic and environmental perturbation. Based on our understanding of climate change impacts, competing stressors can impact growth in foraminifers. For example, dwarfing is a common physiological response to environmental stress . Altern2.(a)et al. [et al. [et al. [Nuttallides truempyi is absent in the lowermost peak CIE.Samples from three ocean drilling programme (ODP) sites were analysed to compare trends across the PETM in different environmental settings . Site 69et al. , for sitet al. , for sit [et al.  and for  [et al. . SamplesN. truempyi specimens were measured. All values are reported relative to the Vienna Pee Dee Belemnite (VPDB) standard. Analytical precision based on replicate analyses of in-house standard Carrara Marble and NBS-19 averages 0.04% (1 s) for δ13C and 0.07% (1 s) for δ18O. Carbon isotopes for site 1210 are from [Carbon isotopes for site 1135 were measured at the University of California Santa Cruz SIL facilities at Santa Cruz, CA, USA. From all samples, 10–15 are from  and for are from .(b)Bottom water temperatures at all sites were comparable before the CIE, with similar warming in response to the carbon injection –47. PalaBased on redox-sensitive elements in the sediments, Kerguelen Plateau may have seen suboxic conditions during the PETM ,52, wherThere are no direct measurements of carbonate ion changes in the deep ocean published to date. Surface ocean pH reconstructions based on boron isotopes in the South  and Nortet al. [δ13C minimum time at 30 kyr after the onset of the CIE. We first spun up the model under late Palaeocene boundary conditions, choosing an open-system run time of 200 kyr in order to bring the δ13C cycle into balance. The model temperatures and warming agree well with the proxy data. Carbonate ion concentrations at the onset and peak of the CIE are low and close to undersaturation at all sites, but especially at site 1210.To provide a larger granularity, we used cGENIE to estimate environmental change . The modet al. . We usedet al.  with the(c)Oridorsalis umbonatus and the extinct, probably epifaunal N. truempyi. The latter's descendent, Nuttallides umbonifera, lives epifaunally, and is adapted to deep-water environments with carbonate undersaturation [N. truempyi and O. umbonatus were picked from the greater than 63 residues of the sample of each time slice, with the majority containing greater than 10 individuals . For some specimens, overall size or even chamber number could be determined, but measuring proloculus size was impossible due to internal dissolution. Oridorsalis umbonatus was much less common in the studied samples (except for site 1135), and specimens were commonly not sufficiently well preserved to determine the chamber number (or proloculus size) reliably at site 1210.In total, we scanned and analysed 387 specimens. We focused on two species: the extant shallow infaunal ,56,57 Orturation  and highturation , thus iswww.tgs.com) to allow investigation of the internal features. As pixels are assigned a grey-scale value to represent the different X-ray attenuation properties of the materials making up the sample, the calcite test of the foraminiferal specimens could be isolated from the mount and any residual sediment infilling.The specimens were scanned using a Nikon XT H 225 ST CT scanner at 120 kV, a 58 µA current and an exposure time of 0.5 s. Each scan project consisted of 3141 projections, resulting in between 300 and 800 images (voxel size of 2.31 µm), which encompassed all the foraminifera of a single time slice. Slice data from the scans were imported into the three-dimensional visualization software Avizo N. truempyi are highly variable in all samples, though the averages in the populations are surprisingly stable (a). At the last site, the population contains some specimens with fewer chambers (minimum 13). There is no clear trend in the number of chambers associated with the core CIE: chamber number increases at site 1210, decreases at site 690  with a brief recovery followed by a second low, and shows no systematic change at the shallowest site 1135. Average chamber number in the population of O. umbonatus ranges between 17 and 22.Chamber numbers for y stable , rangingy stable a. At the(b)N. truempyi, the ranges of test diameters are similar at all sites, from 192 to 474 µm with mean values highest at site 1135 and lowest at site 1210 (b). Within the peak CIE, sizes at sites 690 are lower than below or above the event with a reduction from 334 µm prior to the event to 222 µm 39.4 kyr below the CIE; note that the size starts to decrease in the sample 9 kyr before the large change in carbon isotopes. By contrast, at the shallower site 1135, large sizes are present throughout.For ite 1210 b. WithinN. truempyi, O. umbonatus sizes are largest at the shallowest site 1135 (mean of 348 µm with little variation) and smallest at site 1210 . The average diameter in the population decreased by 100 µm at site 1210 and by 130 µm at site 690 N. truempyi range from 150 µm3 to approximately 30 000 µm3 in all sites, which is equivalent to diameters of 7–40 µm. In a few specimens, the proloculus is larger . At site 1210, N. truempyi proloculus sizes are small below and in the peak CIE, with an increase 205 kyr after the event (c). At site 690, small values dominate. Populations at site 1135 show the largest values below the onset of the CIE with a large drop within the CIE (c). Above the CIE, the mean values for each population are within error of each other. None of the N. truempyi proloculus size distributions in any of the samples shows a clear bimodality which would allow a clear separation of microspheric and megalospheric forms , with a clear bimodality before the CIE at site 1135 (ite 1135 . Unfortu(d)N. truempyi are not significantly correlated, nor is chamber number and final size. By contrast, the mean chamber number and proloculus size of N. truempyi are negatively correlated .One might expect that a larger number of chambers lead to larger size, but the number of chambers is not necessarily a good predictor of the final size in any of the assessed populations, even within a species . Across all sites and time intervals, mean proloculus size and final size chamber within the populations of 3. At site 1135, a large chamber number results in a large final diameter, though the statistical relationship is not significant due to the small number of specimens. The relation between number of chambers and final diameter appears more strongly controlled for specimens of both species below 250 µm  than N. truempyi to reach the same size in the core CIE and the recovery, but not in the post-CIE.In general, population proloculus size and diameter are statistically positively correlated, as small specimens have a small proloculus, whereas large specimens have a proloculus volume above 10 000 µmw 250 µm . Above 24.The main response in morphology during ontogeny of these benthic foraminiferal populations to the environmental changes at the PETM are (i) dwarfing during peak CIE at site 690 to values as low as those at site 1210, (ii) site-specific decreases (site 690), increases (site 1210) or no directional changes in chamber number associated with peak CIE, (iii) low proloculus values at sites 690 and 1210 at peak CIE, associated with a large variability, but no bimodality in the size distribution of the proloculus, and (iv) a lack of relationship between size of the first chamber, number of chambers and final size of the organism across sites and time.Size is the product of life history of a specimen, including factors such as growth rate (at specific food supply), reproduction and death . Among tper se is an unlikely cause for the dwarfism seen at the two deeper sites, but not at the shallowest site 1135. Increased temperatures lead to an increase in food demand, to support the higher metabolic rates. Food probably was most limited in the Pacific gyre (site 1210), where sizes were already small before the CIE, and where some authors suggested (though not quantified) dwarfing in other benthic foraminiferal species than the ones we investigated [Epistominella exigua . We . We per ua , making our interpretation speculative. The data from the populations at the three sites show different proloculus size changes. Sexual reproduction appears to be favoured at site 1210 up to 63 kyr after the CIE and site 690, whereas site 1135 shows a wide range of proloculus sizes. During the recovery phase of the CIE, both modes of reproduction may have been used at sites 1210 and 690.The increased complexity of sexual reproduction imposes inherent costs: mates have to be found at low standing stocks, special cell types formed and diploid genomes maintained . In asexIn some samples, two modes of size distribution are tentatively identifiable, but the large range in size, most clearly at site 1135, suggests that other factors than reproductive mode influence proloculus size. Proloculus size has been linked to environmental factors, e.g. a large proloculus at organic pollution (i.e. high food supply) , optimal5.per se, nor the increase in temperature, as these were the same at all three sites. We speculate that it was driven by a site-specific combination of food limitations and oxygen changes. Proloculus sizes vary strongly between sites and through time, suggesting a large influence of environment on both microspheric and megalospheric forms without clear bimodality.Our analysis of final size, number of chamber and size of the proloculus of deep-sea benthic foraminifera at three sites shows a highly variable response of morphology to the environmental changes across the PETM. The population at shallowest site 1135 does not show a directional response to the environmental impacts of the PETM, whereas the two other sites record reductions in proloculus size, and at site 690 in overall diameter. Some populations suggest resource limitations, at least seasonally, resulting in small sizes. The driver of this change was not temperature