Cachexia is a deadly muscle wasting syndrome that arises under conditions linked to chronic inflammation, such as cancer. Cytokines, including interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and their downstream effectors such as Signal Transducer and Activator of Transcription 3 (STAT3), have been shown to play a prominent role in muscle wasting. Previously, we demonstrated that Pateamine A (PatA), a compound that targets eukaryotic initiation factor 4A (eIF4A), could prevent muscle wasting by modulating the translation of the inducible Nitric Oxide Synthase (iNOS) mRNA. Here we show that hippuristanol, a compound that impedes eIF4A in a manner distinct from PatA, similarly inhibits the iNOS/NO pathway and cytokine-induced muscle wasting. Furthermore, we show that hippuristanol perturbs the activation of the STAT3 pathway and expression of STAT3-gene targets such as IL-6. The decreased activation of STAT3, which resulted from a decrease in STAT3 protein expression, was due to the inhibition of STAT3 translation as there were no changes in STAT3 mRNA levels. These effects are likely dependent on the inhibition of eIF4A activity since we observed similar results using PatA. Our results identify the inhibition of eIF4A-responsive transcripts, such as STAT3, as a viable approach to alleviate cachexia. The abnormal catabolic state found in cachectic patients arises from a combination of complex metabolic changes and dysregulation of certain humoral factors5. Cachexia is the primary cause of ~22% of cancer-related deaths6 and has been known for decades as being a major influence on mortality rate in cancer patients. In spite of the relevance of this syndrome as a contributor to cancer-related deaths, there are no widely employed therapeutics that effectively alleviate this disease7.Cachexia, a multi-factorial disease characterized by acute muscle wasting and weight loss, accompanies various inflammatory diseases such as cancer, sepsis and AIDS8. For example, the concurrent signaling of interferon γ (IFNγ) and tumor necrosis factor α (TNFα) (IT) can synergistically elicit muscle wasting by stimulating the activity of transcription factors including STAT3 and the heterodimeric NF-κB12. NF-κB signaling in skeletal muscle upregulates the muscle-specific E3 ligase MURF-1 and induces a loss of proteins integral for muscle fiber formation and maintenance, such as MyoD and Myogenin13. Moreover, we have shown that NF-κB can also mediate muscle wasting by collaborating with STAT3 to markedly increase the transcription of inducible nitric oxide synthase (iNOS), an enzyme that catalyzes the conversion of L-arginine to citrulline resulting in the release of nitric oxide (NO)12. Systemic interleukin-6 (IL-6) signaling is also crucial in inducing muscle wasting and has been shown to be involved in the pathophysiology of at least some models of cancer cachexia17. Chronic IL-6 exposure has been directly linked to the aberrant activation of autophagic and ubiquitin-proteasomal degradation systems in the muscle17. Furthermore, many studies have shown the importance of STAT3 in the muscle wasting process in a variety of IL-6-dependent models of cancer cachexia. These observations demonstrate that STAT3 is essential in cachexia driven by a multitude of cytokines including IFNγ, TNFα and IL-622. Attempts at interfering with cytokine signaling to impede cachexia progression have included the use of antibodies targeting TNFα or IL-6, however the success of these therapeutic approaches was very limited24. The disappointing outcomes in these trials could be due to the involvement of multiple distinct pathways, the cooperation of which is required for cachexia onset or due to redundancy in the downstream effectors of TNFα and IL-6, such as STAT312. In light of these results, therapies that can disrupt multiple pathways or target redundant factors downstream of these humoral factors may be a more fruitful approach to combatting cachexia.Despite the convoluted etiology of cachexia, some important mediators of its underlying pathophysiology have been identified. Accumulating evidence depicts certain pro-inflammatory cytokines and their downstream effectors as playing pivotal roles in the onset of cancer cachexia27. Compounds such as silvestrol, pateamine A (PatA) and hippuristanol (Hipp) mediate these effects by inhibiting the function of eIF4A, a RNA helicase component of eIF4F that unwinds complex secondary structures in mRNAs28. These compounds are believed to act in this manner by perturbing the translation of specific set of mRNAs containing complex secondary structures in their 5′ untranslated region (UTR) that hinder ribosomal recruitment31. Hipp is an allosteric inhibitor that prevents eIF4A binding to RNA32 whereas PatA and silvestrol deplete eIF4A from the eIF4F complex by causing eIF4A to clamp onto RNA34 thus disrupting the interplay between eIF4A and dependent transcripts35. In the past decade, these and other compounds that target the eIF4F complex have received considerable attention, with several in preclinical development25.Disrupting the initiation of eukaryotic mRNA translation, including the rate-limiting recruitment of the 40S ribosome via the eIF4F complex, has been shown to have anti-immunogenic, anti-oncogenic and anti-cachectic effectsin vitro and in vivo in a C26-adenocarcinoma tumour induced mouse model of muscle wasting27. Without affecting general translation, we found that this low dose of PatA selectively disrupts the translation of iNOS mRNA by increasing its affinity to eIF4A, suggesting that targeting iNOS via eIF4A may be an efficacious clinical strategy for alleviating cachexia. Although hindering iNOS translation likely contributes to the efficacy of PatA, the observation that impairing eIF4A is more efficacious than the iNOS inhibitor aminoguanidine (AMG) in preventing cachexia in vivo27, indicates that the expression of other eIF4A-dependent transcripts may also be altered by PatA. The fact that PatA action is irreversible and could be toxic34 put into question whether PatA could be a viable treatment option for cancer cachexia in humans. Herein, we tested whether other eIF4A-inhibiting compounds can, similarly to PatA, prevent cytokine induced-muscle wasting as well as whether this strategy impacts other pro-cachectic factors besides iNOS. We report that Hipp, which differs with PatA in its mode of inhibiting eIF4A function, prevented the onset of cytokine-induced muscle wasting due, in part, to curtailing the induction of the iNOS/NO pathway. Furthermore, we show for the first time, that compounds targeting eIF4A function modulate the activation of the STAT3 pathway. These drugs decreased STAT3 protein levels in IFNγ/TNFα -treated myotubes without a concurrent effect on the STAT3 mRNA suggesting that eIF4A regulates the translation of STAT3 mRNA under these conditions. Our study therefore indicates that targeting eIF4A function is a robust means of alleviating muscle wasting due to its ability to regulate the translation of specific pro-inflammatory mRNAs, such as iNOS and STAT3, which are involved in the activation of multiple downstream pro-cachectic pathways.The anti-inflammatory effects of compounds that alter eIF4A function prompted us to investigate their impact in cancer cachexia. Previously, we reported that low doses of PatA prevents cytokine-induced muscle wasting both 37, on our in vitro models of cachexia-induced muscle wasting. A popular in vitro model of cachexia involves the treatment of C2C12 myotubes with IFNγ and TNFα (IT) for 72 h, recapitulating many facets of the cachectic muscle38. In order to assess the impact of Hipp on cytokine-induced muscle wasting, we measured the fiber widths of myotubes treated with or without IT and/or Hipp for 72 hours27. We found that, like PatA, the presence of Hipp significantly prevented myotube wasting driven by IT  were grown in Dulbecco’s Modified Eagle Medium  with high glucose, L-glutamine, and sodium pyruvate, in addition to 20% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin antibiotics (Sigma-Aldrich). Cells were grown on tissue culture plates (Corning) with 0.1% gelatin (Sigma-Aldrich). Differentiation of the myoblasts into myotubes was triggered by switching to 2% horse serum (Gibco) and 1% penicillin/streptomycin in DMEM at 100% confluency49. Three days after the induction of differentiation, myotubes were incubated with or without IFNγ (100 U mL−1) and TNFα (20 ng mL−1) (IT) for 24 or 72 hours. Myotubes were incubated for 30 minutes at the beginning of IT treatment with or without Hippuristanol (100 and 200 nM), PatA (0.025 µM) and Silvestrol . Following this treatment, cells were washed with PBS and re-supplemented with media and IT.As described previously705-STAT3  and α-tubulin  were used.IFNγ and TNFα were obtained from R&D system. Myoglobin  iNOS (BD Pharmingen), total STAT3 , phosphotyrosine2, 0.1 mM sodium orthovanadate, and complete EDTA-free protease inhibitors (Roche Applied Science) as described previously50. Membranes were probed with antibodies against iNOS (1:5000), STAT3 (1:1000), phosphotyrosine705-STAT3 (1:1000) and Tubulin (1:1000). Western Blots were quantified using ImageJ.Western blots were performed using total protein extracts prepared in buffer containing 50 mm HEPES (pH 7.0), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 10 mm sodium pyrophosphate, 100 mm NaF, 1 mm EGTA, 1.5 mm MgClMyotubes were fixed with 3% paraformaldehyde and permeabilized in 0.5% Triton X-100/PBS. Cells were then incubated with antibodies against the promyogenic marker Myoglobin (Abcam) as well as DAPI to stain the nuclei. After washing, the cells were incubated with the appropriate secondary antibody and were visualized using an inverted Zeiss Observer.Z1 (40 × oil objective) and an Axiocam MRm digital camera. Myotube widths were measured using the Axiovision software. Myotube widths were obtained by taking measurements of two points along the fiber lengths. Three fields per condition in each experiment were measured.27. IL-6 in the supernatant of IT-treated myotubes was discerned using the Mouse IL-6 ELISA Ready-SET-Go!® Kit  as previously achieved51.Quantification of NO released was achieved using the GRIESS reagent48.Total RNA was reverse transcribed with the M-MuLV Reverse Transcriptase (New England Biolabs). Resulting cDNA was diluted 1/20 and quantified using Sso Fast EvaGreen Supermix (Biorad) as described previouslyPrimers used include: STAT3  IL-6 (Forward:5′-AACGATGATGCACTTGCAGA-3′ Reverse:5′CTCTGAAGGACTCTGGCTTTG-3′), RPL32Supplemental Information


The middle Eocene represents the Cenozoic onset of a dynamic cryosphere, with ice in both hemispheres during transient glacials and substantial regional climate heterogeneity. A more stable cryosphere developed at the Eocene-Oligocene transition, and is now threatened by anthropogenic emissions.Earth’s modern climate is defined by the presence of ice at both poles, but that ice is now disappearing. Therefore understanding the origin and causes of polar ice stability is more critical than ever. Here we provide novel geochemical data that constrain past dynamics of glacial ice on Greenland and Arctic sea ice. Based on accurate source determinations of individual ice-rafted Fe-oxide grains, we find evidence for episodic glaciation of distinct source regions on Greenland as far-ranging as ~68°N and ~80°N synchronous with ice-rafting from circum-Arctic sources, beginning in the middle Eocene. Glacial intervals broadly coincide with reduced CO 2 threshold of ~500 p.p.m.v.With rapidly disappearing ice, understanding the past behavior of the cryosphere is critical. Here, the authors indicate the initiation and disappearance of glaciation on Greenland and Arctic sea ice coincided in the past, synchronous with Antarctic ice and global ice volume, and a CO The timing and cause of this transition is intensely debated5, largely because of the paucity of sedimentary data from polar regions. Challenging conditions for sample recovery and glacial erosion mean there are few archives of past climate variability available. Studies of the Arctic have focused on a small number of sites and relied upon direct paleoclimate indicators to estimate the onset of ice, including the distribution and chemistry of ice-rafted debris (IRD)16, and the occurrence of microfossils that live in or near sea ice17. In addition, oxygen isotope (δ18O) records for different oceans provide an indirect proxy of ice volume and have been used to determine the timing of ice sheet appearance22. Other constraints come from proxy reconstructions of high-latitude temperatures26.One of the most prominent climate changes in our planet’s history involved the transition from early Cenozoic global warmth to the present glaciated state with ice in both hemispheres32. This paradigm argues for two major pulses of Antarctic ice growth, followed by two episodes of Northern Hemisphere ice growth ~20 million years later. The first major glaciation on Antarctica was believed to have occurred near the Eocene-Oligocene boundary, followed by deglaciation, and another major episode of widespread Antarctic ice sheet growth occurred nearly 10 million years later at the Oligocene-Miocene boundary2. Based on findings of glacially derived sediments on Iceland and in the North Atlantic and Pacific Oceans36, the onset of ice in the Northern Hemisphere was dated to the late Miocene, with local-scale glaciers developing between 14 and 6 Ma, and an expansion of ice growth to continental-scale glaciation at ~3 Ma.Based on these techniques, for decades geoscientists have suggested that activity of the cryosphere during the Cenozoic was initiated with the growth of an Antarctic ice sheet ~34 million years ago (Ma), while glacial ice in the Northern Hemisphere formed ~14–6 Ma13 in the Northern Hemisphere and Arctic sea ice37 as early as the middle Eocene to Oligocene (~47–34 Ma), possibly linked to declining CO213. Sediments recovered from the tropical Pacific Ocean provided detailed records that support simultaneous, but short-lived, glaciation at both poles during the middle Eocene to early Oligocene21. Specifically, the records show changes in the δ18O of marine carbonates, including benthic foraminifera and the bulk carbonate fraction of sediments largely comprised of coccoliths. Because carbonate δ18O is a marker for changes in temperature and water δ18O, these records reveal synchronous shifts in surface and bottom water hydrography in the tropical Pacific at the same time as changes in the carbonate compensation depth as early as ~44–435 or ~41.53 Ma. Benthic foraminiferal δ18O records were compared with coeval water-temperature estimates that were derived using Mg/Ca thermometry, a technique standard in Cenozoic studies41, in order to isolate the temperature and seawater δ18O components of the carbonate δ18O data5. Since the growth of ice sequesters 16O and leaves seawater more enriched in 18O, these results revealed evidence for transient episodes of ice growth beginning in the middle Eocene. Furthermore, using a global isotopic mass balance to calculate 18O partitioning between the oceans and ice sheets, these studies inferred the simultaneous presence of ice at both poles, with a minor but significant component of ice storage in parts of the Northern Hemisphere episodically from ~44 Ma5. However, one study argued the isotopic variations reported in Pacific sediments were not indicative of global ice volume, because such shifts were not observed in Atlantic sediments4. Yet it is more likely that δ18O records differ between the Pacific and Atlantic because of regional water mass effects and/or other localized artifacts .However, recently some investigations reported evidence for an earlier and more synchronous hemispheric onset of glaciation during the Cenozoic, with the episodic presence of glacial iceδ18O records has been supported by more direct evidence, such as the discovery in the Arctic of sea-ice associated diatoms17 and iceberg-rafted sediment16. Another piece of evidence has been the study of surface textures of quartz grains recovered from the central Arctic Ocean16, which include dissolution and conchoidal fractures, as both are consistent with transport by sea ice and/or glacial ice. The timing of Arctic IRD and sea ice development was also shown to be broadly consistent with a major decline in atmospheric CO213 inferred from one proxy reconstruction derived from boron isotope-based estimates of seawater pH42.The middle Eocene timing of the initiation of ephemeral Cenozoic bipolar glaciations inferred from Pacific 12. Interpretations of provenance have been limited because surface textures can only provide constraints on transport mechanism(s) and not source regions. Although whole rock element chemistry was used to argue for a possible East Greenland source of IRD between 38 and 30 Ma8, this technique is not considered conclusive at establishing provenance in the Arctic and elsewhere due to the impact of variable mineralogy on the elemental chemistry of bulk rock, and the complex and highly variable mineralogy and petrology of Greenland and circum-Arctic rocks and sediments. These previous interpretations are limited by the fact that the method used cannot prove that Greenland was the only possible source for these grains, which is why this method is generally not used in studies of Quaternary ice rafting. Therefore the provenance of IRD to the Greenland Sea and the extent of glacial ice on Greenland remains unknown.More recently, Eocene and Oligocene sediment from the Greenland Sea were shown to have IRD, including quartz grains with various surface textures indicative of ice transport43 to identify specific regions of origin for individual IRD grains. We constrain Greenland glacial ice and Arctic sea ice dynamics during the middle Eocene through early Oligocene. The occurrence of glacial ice on Greenland was synchronous with episodes of circum-Arctic ice-rafting and, in some cases, with proxies for ice volume and carbon cycle changes. Ephemeral glaciations occurred until the earliest Oligocene, marking the end of the transition from early Cenozoic hothouse conditions to a stable icehouse state.For this study, we apply a well-established chemical fingerprinting technique12. In total, we examined 100 sediment samples from Site 913 using the chemical fingerprinting method. Sediment sources were determined for ice-rafted grains that were present in 51 core samples. The remaining 49 samples did not contain Fe oxide grains.We examined 2029 detrital anhydrous Fe oxide grains from the sand fraction of middle Eocene to early Oligocene sediments recovered at Ocean Drilling Program (ODP) Site 913 , with the oldest sample (~47.1 Ma) containing grains that can be matched to sources in both of these regions  matched to two specific source areas on Greenland Figs. . In addi45. Many of the core samples contain an abundance of Fe oxide grains that can be traced to this small source region . Some, though not all, of these IRD peaks are also synchronous with changes in carbon cycle indices, including increases in the carbonate compensation depth, and low pCO2 (<500 p.p.m.v.) . Sea ice is the likely transport mechanism for grains from many of the circum-Arctic source regions.Our chemical fingerprinting approach quantitatively measured 14 elements12. Although sea ice may have been important locally along the Greenland coast for entraining sediments, we note the extent of sea ice in the Greenland Sea today is limited to the shelf along most of East Greenland46. During the middle Eocene through early Oligocene, when global climate was warmer than today, we assume the extent and thickness of sea ice in this region would have likely been substantially reduced. Modeling studies indicate a cyclonic circulation in the Greenland Sea region for the Eocene47, that sea ice was thinner or less extensive than today under these substantially warmer climatic conditions, and that sea-ice drift may have also been significantly faster48. The circuitous route required to reach Site 913 from source areas #42, #43, and #46 makes it likely that grains from these Greenland source areas were dominantly transported by icebergs the >400 km distance from the coast to Site 913 , were glaciated. The presence of IRD at Site 913 that was simultaneously delivered from source areas #42, #43, and #46 indicates that glacial ice came from widespread areas across East and Southeast Greenland. Fe oxide grains measured in this study matched to every sample cataloged in the database from these three source areas . The abundance of Fe oxide grains in many samples from Site 913 indicates that at times, large numbers of icebergs were produced, consistent with the intermittent occurrence of glaciations, with ephemeral glacial maxima that at times spread far inland and supported calving at the coast  climate. In addition to temporal variability, there would also have been significant spatial heterogeneity in conditions due to dynamical processes  that today exert a dominant control over regional differences in climate. During transient glacial maxima, if the calving glaciers that supplied icebergs extended inland to the divide, then glacial ice covering these three areas would encompass about 4.3 × 10). These findings are indicative of synchronous ice-rafting in from multiple shelf regions around the Arctic. Several of these sources are represented by significant numbers of Fe oxide grains in Site 913 samples. Sources include Banks and Ellef Ringnes Islands in North America, and Franz Josef Land in Russia , our slightly favored explanation, given that the ocean-climate-cryosphere system would have exhibited variability over a range of timescales including millennial and orbital-scale during the Eocene and Oligocene, just as it has throughout Earth’s history. Another possibility is that directly correlating our data from Site 913 to the published records from ACEX is hampered by limitations on age control. These issues can only be addressed through the recovery and study of more complete sedimentary successions from different regions during future research expeditions.We note that alkenone-based sea surface temperatures derived for the ACEX core from the central Arctic Ocean have been interpreted to reflect summer temperatures of between 6–20 °C during the middle Eocene (~47–38 Ma)The provenance data for Site 913 pinpoints the origins of IRD in the Greenland Sea to source regions in East Greenland, Southeast Greenland, and the entire circum-Arctic Ocean, which episodically contributed IRD in the middle Eocene and became more stable by the early Oligocene  samples we examined contain IRD, indicating that at times ice may have disappeared completely . Determining the timing and extent of glaciations in the Northern Hemisphere can help to constrain the tectonic and environmental drivers of climate change2. Some have argued that tectonically forced changes in ocean currents circumscribing Antarctica triggered glaciation during the Eocene and Oligocene63, while a later onset of Northern Hemisphere glaciation occurred largely because of current-driven heat transport changes resulting from the emergence of the isthmus of Panama64. An earlier timing for the onset of ice in the Northern Hemisphere indicates ocean heat transport was not likely the sole or direct trigger for the Cenozoic initiation of the first glaciations on Greenland, although the oceanic meridional overturning circulation may have served as a feedback mechanism for ice growth and/or a driver of carbon cycle changes.Ultimately, our results are consistent with episodic bipolar glaciation and circum-Arctic ice-rafting beginning in the middle Eocene and throughout the early Oligocene. Significantly, results suggest a broadly concurrent Cenozoic onset and history of Arctic sea ice, glacial ice on Greenland, and glacial ice on Antarctica Fig. . Determi65, in part based on the timing of ice growth relative to reconstructions of CO2 fluctuations. During the Cenozoic, falling pCO2 may have resulted in temperatures at both poles dropping below critical thresholds for the formation of sea ice and glacial ice13. Some studies have hypothesized the polar regions in both hemispheres had relatively similar CO2 thresholds for the onset of glaciation13. Alternatively, substantially different CO2 and temperature thresholds may exist for glaciation on Greenland and Antarctica due to differences in land mass, latitude, and solar insolation66. Our data allow us not only to test whether there is evidence for CO2 serving as a critical component of the climate system governing cryospheric evolution during the Cenozoic, but also to evaluate these differing views of thresholds for glaciations.Others have argued that greenhouse gas levels may have had a role in driving these transitionspCO2 derived from multiple marine and terrestrial proxies . Thus, our results may support an occurrence of glaciations with broadly similar CO2-equivalent and temperature thresholds in both hemispheres13.The IRD source fingerprinting results coincide at times with fluctuations reported in reconstructions of 2 include insolation and lapse rates. For example, during intervals with relatively low CO2 values and orbital parameters favoring cold polar summers, given thermodynamically constrained values for lapse rates, these times would have been associated with precipitation falling as snow at elevation in the surrounding region, including Greenland, leading to the presence of glacial ice. Figure 2 values that provide a dynamically plausible mechanism for Northern Hemisphere ice during the hothouse-icehouse climate transition of the early Cenozoic.Other factors that link glaciation to COSignificantly, the source fingerprinting method provides evidence that the initiation of glacial ice on Greenland, with a possible large ice cap or even small ice sheet, occurred earlier than previously suggested, and that the occurrence of glacial ice in the middle Eocene to early Oligocene was synchronous with pan-Arctic sea ice. These new observations allow us to critically reevaluate our prior understanding, suggesting that previous theories were only working hypotheses and allowing us to work towards a more complete interpretation. In fact, the Cenozoic initiation of glacial ice on Greenland, and sea ice in the Arctic, was close in timing to the development of Antarctic ice. We find evidence for ephemeral glacial ice and sea ice in the Northern Hemisphere, and times of ice-free conditions, until the Oi-1 glaciation, and for more stable glacial conditions in the early Oligocene. Future studies will provide insights into the history of Northern Hemisphere ice and bipolar glaciations during the Eocene and Oligocene.2 regimes.To more fully resolve glacial and sea ice dynamics, given coring gaps in current sedimentary archives and limitations with existing age models, it is critical to recover new and complete Cenozoic sedimentary successions from the Arctic and Southern Ocean. Continuous successions will also enable the development of high-resolution records that could provide constraints on the role of orbital forcing in governing cryospheric stability. The ability to generate paired records of IRD and carbonate stable isotope ratios in a single core, which is not possible at Site 913 due to the lack of preserved carbonate, may shed light on past changes in the extent of Greenland ice and on the relative timing of glaciation in the Northern Hemisphere compared to fluctuations in global ice budgets during the Cenozoic. An extension of the Arctic source database to include presently undocumented regions, including in Northeastern Greenland, would place more complete constraints on the spatial extent of ice on Greenland in the past, although some sources may have been eroded away. Such research would shed light on the behavior of an ancient Greenland ice cap or ice sheet under warm and polythermal glacial regimes that presumably existed during the middle Eocene through Oligocene. In addition, the application of the IRD source fingerprinting method and other techniques including weathering proxies, to new and existing sediment core sites, will allow us to gain a more complete picture of Greenland glacial ice and Arctic sea ice dynamics under higher CO43. By characterizing multi-element signatures of detrital iron oxide minerals that occur in core samples, we are able to isolate sources of IRD over time, determine the sources and shed light on the relative extent of glacial ice on Greenland, and decipher contributions of sea ice from specific circum-Arctic sources during a given interval. We constrain Greenland glacial ice and Arctic sea ice dynamics using data from Site 913 during the middle Eocene through early Oligocene interval (47–26 Ma). We also compare our results with published data, including other proxies for sea ice, glacial ice, and ice volume. Our results are compared to extensive carbon cycle proxy data sets that enable us to study the relationship between Northern Hemisphere cryosphere dynamics and atmospheric pCO2, and to investigate causal mechanisms, such as whether fluctuations in CO2 may have driven the transition to a Cenozoic climate characterized by glacial maxima in both hemispheres, i.e., episodic bipolar glaciations.To critically test the hypothesis that glacial ice occurred ephemerally on Greenland beginning in the middle Eocene and became more stable after the Eocene–Oligocene transition, we applied an accurate, precise, and well-established geochemical fingerprinting technique that allows us to identify the specific regions of origin for IRD37. These records are also compared to a global benthic δ18O and δ13C synthesis, a seawater δ18O synthesis, estimates of the carbonate compensation depth, and reconstructed atmospheric pCO2 from multiple proxies58. A full list of publications that are the sources of data used for comparison to our results, are listed in the Supplementary Methods.Results were compared with published data on IRD, including mass accumulation rates, dropstone occurrence, and Arctic sea ice diatom occurrence43.Iron oxides are dominant accessory minerals in most sediments and rocks and are highly durable. Their diverse geochemistry  gives rise to unique regional chemo-geographic patterns. Therefore the chemistry of individual mineral grains can be used to pinpoint each grain’s region of provenance43. The anhydrous detrital Fe oxide grains (Fe grains) were separated magnetically using a hand magnet and the Frantz magnetic separator on the sieved 45–250 μm size fractions. These grains were then mounted in epoxy plugs, polished, and photographed so that each Fe grain could be identified as to location on the photo and mineral type for later analysis on the Cameca SX100 electron probe microanalyzer (EPMA). The mineral identifications were checked after EPMA analysis to insure that the microscopic identification done with a reflected light microscope (1000×) were correct. Each grain was analyzed for 14 elements . Each grain was then matched to grains of the same mineral type from the entire Arctic database of over 39,000 Fe grain analyses for all 14 elements for nearshore and onshore samples taken from 284 sample sites43. These Fe oxides formed in igneous and metamorphic rocks that almost always predated the Eocene (most are much older) and have been eroded and deposited in Pleistocene and younger deposits where they were sampled for the database. Each element has to be within 2 standard deviations of multiple analyses of hundreds of selected grains with high values of each element.Samples were measured and data analyzed blindly at Old Dominion University. The method for precise source determination uses the chemical signature of 14 elements in nine types of iron oxide minerals43. By characterizing all of the grains in a sediment sample, and by knowing the exact source for each grain, we can determine the multiple regions that contributed IRD present in a sediment sample. When more than one source Fe grain matched to an Fe grain from a sample, the sources were pro-rationed according to how close the analyses were to all 14 elements. This provenance tool has also been compared to the use of lithic grains for source determination in several studies with compatible but far more precise results that are cited in the Supplementary Methods.This technique enables us to trace each grain to a specific source region Fig. . The unc43. Sample coverage for Northeastern Greenland between source areas #35 and #46 is limited, although this region is not known to have recently produced many icebergs. Source area #46 consists of an inner shelf core (PS2641) with 147 samples from every few centimeters down to 266 cm. All Fe oxide grains matched to sources north of Fram Strait were removed from this database in order to ensure that only Fe oxide grains from East Greenland are represented.Thus, we can ascertain the geographic distribution of glacial ice and sea ice that rafted grains to Site 913. We note that while it is possible that matched Fe oxide grains are from unsampled source areas, the probability of Fe oxide grains having the same chemical fingerprint as grains represented in the source database is low (<0.01)The complete match of all Fe grains in Site 913 samples to Greenland and circum-Arctic sources can be found in Supplementary Data sets 67.The sediments recovered from Site 913 have been dated using a reliable age model based on paleomagnetics and biostratigraphy69 that yield different ages and place the first appearance of IRD in ACEX a few million years apart, and produce different ice-rafting histories for that site. Therefore we show the results of using each of these age models in Fig. There are data sets for two different sites used to place constraints on Arctic sea ice as shown in Figs. 37.Estimates for number of ACEX IRD grains that can be matched to a source region comes from a publicationEstimates for Arctic sea ice onset come from multiple publications. Full list of citations in Supplementary Methods.12.Estimates of IRD mass accumulation rates at Site 913 come from a publicationEstimates for Antarctic ice storage come from multiple publications. Full list of citations in Supplementary Methods.δ18O and δ13C are a compilation from multiple publications. Full list of citations in Supplementary Methods.Benthic foraminiferal δ18O from multiple publications. Full list of citations in Supplementary Methods.Reconstructed seawater Reconstructed tropical Pacific and equatorial Pacific CCD from multiple publications. Full list of citations in Supplementary Methods.2 synthesis contains data from multiple publications. Full list of citations in Supplementary Methods.Proxy atmospheric COThe data sets generated or analyzed during this study are included in Supplementary Data Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


The purpose of this study was to evaluate the radiologic outcome of percutaneous medial hemi-epiphysiodesis using a transphyseal screw for the management of caput valgum associated with developmental dysplasia of the hip (DDH).Eighteen hips (18 patients) having caput valgum treated with screw hemi-epiphysiodesis were followed for more than 2 years, and were included in this study. The mean age at the time of the index operation was 8.3 years  and age at the latest follow-up was 12.2 years . The screw in 5 hips was changed into a longer one at postoperative 21.8 months  because the proximal femur outgrew the screw. The screws in 11 hips were removed at the mean age of 10.9 years . We retrospectively analyzed the change in various radiologic parameters over time.p = 0.001). The mean difference of the HEA between the operated and contralateral sides was 16.9 ± 15.1° preoperatively, which decreased to 2.4 ± 12.4° at the latest follow-up (p = 0.008). The mean articulo-trochanteric distance of the operated side, which was 3.2 ± 5.5 mm longer than that of the contralateral side preoperatively, became 5.6 ± 9.1 mm shorter at the latest follow-up (p = 0.001). The ratio of femoral neck length of the operated side to that of the contralateral side decreased over the follow-up period. Acetabular shape as measured by the Sharp angle and acetabular roof angle and femoral head coverage as measured by lateral center-edge angle did not change significantly by the index operation. The ratio of medial joint space width of the operated side to that of the contralateral side did not change significantly.The mean Hilgenreiner-epiphyseal angle (HEA) of the operated side was 5.1 ± 11.3° preoperatively, and increased to 20.6 ± 11.3° at the latest follow-up (Screw medial hemi-epiphysiodesis can effectively correct caput valgum associated with DDH. However, this technique remains coxa brevis and does not seem to significantly affect acetabular morphology or reduce subluxation. Osteonecrosis of the capital femoral epiphysis, resulting in growth disturbance of the proximal femur, is a common and major complication secondary to treatment of developmental dysplasia of the hip (DDH) –5. Of foAlthough Kim et al. reported that lateral growth disturbance was not necessarily associated with poor acetabular remodeling , caput vProximal femoral varus osteotomy (PFVO) could be a treatment option to correct caput valgum deformity caused by lateral growth disturbance. However, because PFVO is usually performed at the level of the lesser trochanter, away from the center of rotation of angulation, it occasionally requires a great amount of varization to correct deformity and leads to greater trochanter overriding. In addition, it is sometimes difficult to decide on the amount of varus when performing PFVO because it is hard to predict whether further deformity develops until skeletal maturity .Guided growth of the proximal femur using a transphyseal screw across the inferomedial aspect of the proximal femoral epiphyseal plate is an alternative treatment option for caput valgum. Although guided growth using a transphyseal screw has been widely used after introduced by Métaizeau in 1998, a few animal studies reported the effect of medial hemi-epiphysiodesis using a transphyseal screw in skeletally immature hips –16. To tThe purpose of this study was to evaluate the radiologic outcome of percutaneous medial hemi-epiphysiodesis using a transphyseal screw for caput valgum associated with DDH.We collected cases of proximal femoral lateral growth disturbance associated with DDH presenting with progressive caput valgum that were treated with percutaneous medial hemi-epiphysiodesis using a transphyseal screw. Between August 2009 and December 2014, they were treated in a tertiary-care children’s hospital and followed for more than 2 years. Hips associated with neuromuscular disease, skeletal dysplasia or congenital anomaly of other organs/systems were excluded. There was no teratologic hip dislocation or hip dislocation combined with arthrogryposis. Based on these criteria, 18 hips (18 patients) became the subjects of this study and medical records and serial radiographs were reviewed and analyzed.There were 14 female (78%) and 4 male (22%) patients. Fourteen hips (78%) were unilateral DDH, and three hips (17%) were bilateral DDH with only one hip developing caput valgum. The remaining patient had bilateral DDH, whose both hips developed caput valgum and were treated with screw medial hemi-epiphysiodesis. One side was randomly selected and included in the study. Of 14 unilateral cases, 9 (64%) were right hips and 5 (36%) were left hips. The mean age (± standard deviation) was 8.3 ± 1.9 years  at the time of screw placement, and 12.2 ± 2.0 years (9.4 to 16.4) at the time of the latest follow-up. The duration of follow-up averaged 3.9 ± 1.4 years (2.0 to 6.9).Surgical procedures which were performed before, concurrent with, or after screw medial hemi-epiphysiodesis, are listed in Table Anteroposterior radiographs of the pelvis were taken with the hip positioned in neutral rotation and neutral abduction/adduction at each visit. We measured indicators of pelvic alignment in all radiographs. The quotient of pelvic rotation indicating pelvis position in the horizontal plane was 1.01 ± 0.18 (0.68 to 1.46) and the symphysis os-ischium angle, indicating the pelvis position in the sagittal plane, was 99.9° ± 8.7° (90.0° to 115.1°), which was considered acceptable , 20.For evaluating proximal femoral morphology, the Hilgenreiner epiphyseal angle (HEA) , head-shLeg-length-discrepancy (LLD) was evaluated by iliac crest height difference measured on standing AP radiograph of the pelvis. Four patients with bilateral DDH and two patients who underwent epiphysiodesis of the distal femur concurrent with screw medial hemi-epiphysiodesis of the proximal femur were excluded from the analysis of LLD. The patient who underwent epiphysiodesis of the distal femur at the latest follow-up was included in the analysis.To determine intra-observer reliability, measurements were made by one of the authors (W.K.H.) on two different days, 2 weeks apart. To determine the inter-observer reliability, the same measurements were made by another author (C.H.S.) after a consensus building session to define the radiographic measurements. Intra-observer and inter-observer reliability were evaluated by intraclass correlation coefficients (ICCs), which were calculated assuming absolute agreement and a single measurement with a 2-way-random-effects-model. In the intra-observer reliability test, ICCs were 0.988  for HEA, 0.957  for HSA, 0.917  for NSA, 0.909  for Sharp angle, 0.973  for acetabular roof angle, 0.780  for LCEA, and 0.961  for MJS width ratio. In the inter-observer reliability test, the ICCs for the same parameters were 0.871 , 0.830 , 0.903 , 0.919 , 0.826 , 0.715 , 0.762 , and 0.886 , respectively.P values <0.05 were considered significant.The Mann-Whitney U test was used to evaluate the significance of the difference between mean values between operated and contralateral sides. The Wilcoxon signed-rank test was used to evaluate the significance of change of parameters over time. p = 0.001). The change of HEA ranged from −6.1° to 34.6°. However, that of the contralateral hips did not change significantly. The difference of HEA between the operated and contralateral sides decreased significantly (p = 0.008) , while the NSA was not significantly different between the sides (p = 0.085). The HSA of the operated side significantly decreased after the index operation but that of the contralateral side did not . The NSA significantly decreased on both sides after the index operation, but the amount of change was not significantly different between the two sides (p = 0.285). At the latest follow-up, the means of both HSA and NSA became close to zero degrees.Preoperatively, the operated side had significantly larger HSA than the contralateral side (p = 0.001). Meanwhile, the ATD of the contralateral side changed little. As a result, the mean ATD of the operated side was 5.6 ± 9.1 mm shorter than that of the contralateral side at the latest follow-up. Femoral neck length ratio also decreased in 11 hips (79%) (p = 0.056).The mean ATD of the operated side was 3.2 ± 5.5 mm longer than that of the contralateral side preoperatively which significantly decreased during follow-up (p = 0.401)  Tables .The screws were changed into longer ones, in five of the hips during the follow-up period postoperatively at 21.8 ± 4.7 months , because the proximal femora outgrew the screws. Patient’s age at the index operation was 6.1 ± 1.6 years (4.3 to 7.7). The HEA increased until the revision surgery by 13.1° ± 5.7° (4.9° to 19.1°) and increased until the latest follow-up by 10.1° ± 5.8° (2.8° to 16.6°) over the mean period of 34 ± 25 months (2 to 60).Screws were removed in 11 of 18 hips at the mean age of 10.9 ± 2.0 years (8.0 to 14.5). Six of the 11 hips underwent removal of the screw before skeletal maturity at the mean age of 9.6 ± 1.4 years (8.0 to 11.3) and were followed for more than 6 months after screw removal. In these six hips, HEA and HSA did not significantly change from the time of screw removal to the latest follow-up over the mean period of 24.5 ± 12.4 months (6 to 42).p = 0.006).The mean length of the operated legs was 12.6 ± 6.2 mm (0 to 22) longer than that of the contralateral legs preoperatively and 3.2 ± 8.8 mm (−11.6 to 19.7) longer at the latest follow-up. The mean growth of the operated legs significantly diminished compared to that of the contralateral legs over the follow-up period . Next, although radiographs were taken with the pelvis in acceptable positions and the hip in patella facing forward, the shape of the proximal femoral physis varied by femoral rotation, which may affect measurements of some of the radiologic parameters. And a femoral head which is a three-dimensional organ was evaluated two-dimensionally only using X-ray. Lastly, although it was the largest study to date investigating screw medial hemi-epiphysiodesis, the sample size was relatively small for powerful statistical analysis , 17, 18.. Next, aPercutaneous medial hemi-epiphysiodesis using a transphyseal screw can correct caput valgum associated with DDH effectively and safely. However, this procedure does not correct coxa brevis deformity and does not appear to make a significant impact on acetabular morphology and subluxation.


Conversely, late Aptian (∼115 Myr ago) and Maastrichtian (∼70 Myr ago) continental configurations set the stage for cooler climatic conditions, favouring possible inception of Antarctic ice sheets under CO2 concentrations, respectively, about 400 and 300 p.p.m. higher than for the Cenomanian–Turonian. Our simulations notably emphasize that palaeogeography can crucially impact global climate by modulating the CO2 threshold for ice sheet inception and make the possibility of glacial events during the Cenomanian–Turonian unlikely.The historical view of a uniformly warm Cretaceous is being increasingly challenged by the accumulation of new data hinting at the possibility of glacial events, even during the Cenomanian–Turonian (∼95 Myr ago), the warmest interval of the Cretaceous. Here we show that the palaeogeography typifying the Cenomanian–Turonian renders the Earth System resilient to glaciation with no perennial ice accumulation occurring under prescribed CO Indirect evidence indicates the surprising occurrence of glacial events during the peak warmth of the Cretaceous world. Here, based on coupled climate-ice sheet model simulations, the authors show that such events were likely thwarted by palaeogeographic reorganisations and complex ocean-atmosphere feedbacks. Many studies have indeed demonstrated that palaeogeographic changes could generate substantial global and/or regional climatic variations323326342 levels are prescribed  to cover the estimated range for the end of the Mesozoic23242538392 (between 280 and 750 p.p.m.) are then performed to better constrain the CO2 threshold for glacial inception over Antarctica for each palaeogeography.We perform numerical experiments with three different palaeogeographies2 is decreased to 840 p.p.m., small isolated ice caps develop over Antarctica's highest peaks in the Aptian configuration but their sizes are too modest to remain perennial (Methods). In contrast, a further drop to 560 p.p.m. allows a substantial amount of ice to accumulate over the Antarctic continent in the Aptian and Maastrichtian configurations while the Turonian Antarctic persists in an ice-free state  demonstrate that the Antarctic ice sheet CO2 threshold is crossed, respectively, between 840 and 750 p.p.m. and between 750 and 650 p.p.m. for the Aptian and Maastrichtian configurations  at 500 hPa are consistently about 3 °C (0.5 g kg−1) higher than Aptian MAT (Q) and about 2 °C (0.3 g kg−1) higher than Maastrichtian MAT (Q). In particular, the global warming during the Turonian displays regionally non-uniform patterns, with a significant polar amplification and strong seasonal variations  Antarctic summer temperatures in the Cenomanian–Turonian configuration. Even though future work is needed to confirm these modelling results—as absolute CO2 thresholds for glacial inception remain model-dependent—and to refine proxy-based estimates of CO2 concentrations and their relative evolution through the Cretaceous, we suggest that Aptian and Maastrichtian ice sheets are indeed likely to have developed during times of favourable orbital forcing and atmospheric CO2 levels. On the contrary, our study supports the vision of a super-hot, ice-free Cenomanian–Turonian, epitomizing the term of ‘Climatic Optimum'.In conclusion, our findings suggest that the atmospheric COet al.Our palaeogeographic reconstructions are taken from ref. 545455−2 for the Aptian, 1,354 W  m−2 for the Cenomanian–Turonian and 1,357.4 W m−2 for the Maastrichtian.We impose the same boundary conditions except the time-dependent solar constant, which is calculated by reducing the modern by 1% per 100 million years2 levels2 threshold for the glaciation and of temporal evolution of the ice sheet. This method, however, needs relatively well-documented variations of atmospheric CO2 levels and orbital parameters and unfortunately suffers from exceedingly expensive computational costs.Several recent studies have shown that complex coupling methods between general circulation models (GCMs) and ice sheet models (ISMs) were required to provide relevant estimates of the ice volume, which can be accommodated over the high latitudes262 levels are affected by large uncertainties and remain poorly resolved temporallySeveral issues presently preclude the use of such complex methods on the Cretaceous. First, due to the chaotic nature of the solar system2 levels required to trigger hundreds of thousands of years to million year-long stable ice sheets. We have applied a relatively simple one-way coupling method, which allows us to test the broad range of boundary conditions we dispose of, and which is explained with more details below. Moreover, it should be noted that another recent study has attested of the relevance of simple one-way coupling between climate and ice sheets regarding the estimation of atmospheric CO2 thresholds for stable glacial onsetIn this study, we follow a different approach. Our purpose is to compare the sensitivity of three Cretaceous palaeogeographies to glacial onset and to provide estimates of the CO2 levels with the high-resolution fully coupled IPSL-CM5 model is prohibitive, we use the mixed resolution  fully coupled Fast Ocean Atmosphere Model (FOAM)2, we integrate the FOAM GCM for 2,000 years without deep ocean acceleration or flux corrections. During the last 100 years of each simulation, there is no apparent drift in the upper ocean and <0.05 °C per century change in globally averaged ocean temperature. Then, we average the SST field over the last 100 years and use it to force the higher-resolution AGCM LMDz for 20 years with identical boundary conditions. In both GCMs, orbital parameters are maintained constant to a favourable configuration for the inception of a glaciation in the Southern Hemisphere . Temperature and precipitation fields are subsequently averaged over the last 5 years of simulation and used to drive the ISM GRISLI2. The integration time of the ISM is chosen by approximating that, over a full precession cycle, favourable conditions for ice accumulation over the high latitudes occur during about half a cycle (∼10 kyr) and unfavourable conditions during the other half.The different climatic states used to force the ISM are obtained following ref. 2 levels: 1,120, 980, 840 and 560 p.p.m. These levels bracket the CO2 threshold for a perennial Antarctic glaciation derived with our more complex method. This value of ∼925 p.p.m. is close to the CO2 threshold obtained with other models and is also in agreement with palaeo-CO2 data reconstructions  is larger than what would be the minimal size required for the ice sheet to survive the changing orbital conditions and become perennial .For each of these CO forcing . The sim2 threshold for perennial glaciation for each Cretaceous palaeogeography. For simplicity, and because the exact minimal ice sheet size that will remain perennial cannot simply be extrapolated from the EO 980 and 840 p.p.m. sizes, we assume here that to trigger the onset of a perennial ice sheet, the size obtained with the Cretaceous palaeogeographies must be larger or equal to the 840 p.p.m. size obtained with the EO palaeogeography .Supplementary Figures 1-13 and Supplementary Table 1


Peumus boldus, is known to inhibit telomerase at non-toxic concentrations. Cytotoxicity of N-benzylsecoboldine hydrochloride (BSB), a synthetic derivative of boldine, was determined using the MTT method in MCF7 and MDA-MB231 cells. Aliquots of cell lysates were incubated with various concentrations of BSB in qTRAP (quantitative telomere repeat amplification protocol)-ligand experiments before substrate elongation by telomerase or amplification by hot-start Taq polymerase. The crystal structure of TERT, the catalytic subunit of telomerase from Tribolium castaneum, was used for docking and molecular dynamics analysis. The qTRAP-ligand data gave an IC50 value of about 0.17 ± 0.1 µM for BSB, roughly 400 times stronger than boldine, while the LD50 in the cytotoxicity assays were 12.5 and 21.88 µM, respectively, in cells treated for 48 h. Although both compounds interacted well with the active site, MD analysis suggests a second binding site with which BSB interacts via two hydrogen bonds, much more strongly than boldine. Theoretical analyses also evaluated the IC50 for BSB as submicromolar. BSB, with greater hydrophobicity and flexibility than boldine, represents a promising structure to inhibit telomerase at non-toxic concentrations.Telomerase, the enzyme responsible for cell immortality, is an important target in anti-cancer drug discovery. Boldine, an abundant aporphine alkaloid of  Peumus boldus) [Lindera aggregata [3 receptors [Boldine  is an aporphine alkaloid found in several plant species and is the main alkaloid in the bark and a relatively abundant component of the leaves of the boldo tree  activation . BoldineOur previous studies have shown antiproliferative effects of boldine on several cancer cell lines including HepG2, MCF7 and MDA-MB-231 at non-toxic concentrations, and identified its promising potential in telomerase inhibition ,12. TeloN-benzylsecoboldine hydrochloride  hydrochloride has LD50 = 16.25 and 21.88 µM against the MCF7 and MDA-MB-231 lines, respectively. Also, microscopic observation showed that the survival percentage of cells in the presence of BSB was significant and was dose-dependently reduced. The MTT test showed that T) to telomerase activity measurements when the compound was added before the amplification step (TAP) obtained from q-TRAP-ligand assay. Telomerase activity was reduced to ≤50% of untreated samples, at BSB concentrations ≤0.5 µM, although not completely suppressed at higher doses. The inhibitory effect of boldine on telomerase activity was dose-dependent so that in the presence of 150 µM boldine telomerase activity fell to around 10% of the control reaction. However, the technique calculated 0.17 ± 0.1 and 68 ± 2.5 µM as IC50 values for BSB and boldine, respectively. The differences between untreated control samples and the treated samples are significant, as analyzed using one-way ANOVA with the post-hoc Tukey HSD test (p < 0.01). The q-TRAP-ligand assay showed that BSB strongly inhibits telomerase in a direct interaction  might exist. Therefore, we looked for binding site(s) on two levels: focused and blind docking.nDD) [T. castaneum telomerase has three conserved aspartic acid residues  which coordinate to Mg2+, and Lys372 provides the base for the deoxynucleotide condensation reaction [Among animal species, telomerase enzymes have highly conserved motifs A and C in the RT palm region and have signature conserved amino acids in their sequences, especially in the catalytic active site (KXD(X)nDD) . In humanDD) . The catreaction ,15. FocuBlind docking was performed to detect the possible binding mode and sites of boldine and BSB on TERT. Blind docking results showed that both ligands may bind to TERT in a binding box different from the active site with inhibition constants of 9.20 μM and 130.61 nM, respectively . In the In the molecular docking position of TERT, boldine bound to the active site with a binding energy of −6.87 kcal/mol and an inhibition constant of 9.15 μM, and compound BSB did so with a binding energy of −9.08 kcal/mol and an inhibition constant of 221.08 nm. In the molecular dynamics simulation resulting from the blind docking position in TERT, boldine exhibited a binding energy of −6.64 kcal/mol and an inhibition constant of 9.15 μM. However, BSB binds much more strongly: binding energy = −9.39 kcal/mol and inhibition constant = 0.130 μM. Molecular dynamics simulations were performed to investigate the dynamics of boldine and the BSB interactions with TERT at the atomic level.To investigate the stability of the whole system during the molecular dynamics simulations, the system energy for the different systems was calculated . Both coThe dynamic stability of boldine and BSB was studied by calculating the root mean square deviations (RMSDs) during the molecular dynamics simulations. Results of RMSDs of the model structures are shown in The lower average of RMSDs of boldine with the initial position obtained from the blind docking screen in comparison with the binding box of boldine at the initial position obtained from the focused docking screen, during the 10 ns molecular dynamics simulations, confirmed that boldine in the new binding box located on Lys179, Pro180, Arg181, Gly182, Arg205, Asn286 and Asn290 has a more stable interaction with TERT . Anti-proliferation experiments on boldine against MCF7 and MDA-MB-231 cells exhibited LD50 values of 160 and 150 µM after 48 h treatment, respectively. Similar experiments with BSB gave roughly 10 times lower LD50 values than boldine, as the most potent boldine derivative tested against breast cancer cell lines MCF7 and MDA-MB231. The greater hydrophobicity and more flexible structure of BSB in comparison with boldine and other derivatives preserving the aporphine skeleton may play critical roles in enhancing the interaction with its molecular targets inside the cell.Boldo leaves have been used in various preparations and formulas in traditional medicine. Several beneficial effects including anti-oxidative, anti-pyretic, anti-proliferative and hepatoprotective have been recorded for boldine, erroneously assumed to be the main alkaloid of the extract. We previously found an additional mechanism for the anti-proliferative effects of boldine, based on telomerase inhibition. In the present study we investigated the strength of this effect in a synthetic derivative of boldine, 50 value as 0.17 ± 0.1 µM against Tribolium castaneum telomerase. Considering the IC50 value of 68 ± 2.5 µM for its parent natural compound, boldine, BSB showed roughly 400 times stronger direct inhibition of telomerase activity. This may be explained by stronger interactions between BSB and telomerase. It is known that boldine does not interact with its substrate, the oligonucleotide of the telomeric repeats [qTRAP-ligand assays in the presence of BSB determined its IC repeats . HoweverT. castaneum (3KYL). In our focused and blind docking studies, boldine interacted well with TERT through two different binding sites: one inside the enzyme active site where it accepts the substrates and another in the vicinity of the active site. In general, interactions of the ligands with the second binding site were evaluated as moderately stronger than with the active site. However, molecular dynamics studies showed that neither boldine nor BSB reside stably in the active site but are released and attach to the second binding site, as will be explained in more detail here.No crystal structure of human TERT is available. Considering the highly conserved function and structure of this enzyme among species, we investigated whether boldine and the derivatives bind with the crystal structure of telomerase from T. castaneum TERT, we obtained its available 3D structure for our modeling studies [T. castaneum telomerase, but human telomerase has two additional α-helices (residues 415–456) [nDD) in its active site and DNA primer grip. This sequence in human TERT includes the catalytic triad of Asp712, Asp868, Asp869 in addition to Lys 710, while in T. castaneum it is composed of Asp343, Asp344, Asp251 and lys372 [Telomerase enzymes contain two essential subunits; a catalytic protein (TERT) and an integral RNA component (TER) ,16. TERT studies . The TRB415–456) . The cend lys372 ,15,16. Td lys372 ,23.T. castaneum TERT and relocate to a new box. Since there are conserved active site residues in T. castaneum and human TERTs, it is suggested that boldine and BSB may similarly inhibit human telomerase as non-competitive inhibitors.Based on our focused docking and molecular dynamics simulations, boldine and BSB release the active site in T. castaneum TERT. Boldine inhibits telomerase by connecting to a Gln190 amide hydrogen via two hydrogen bonds in a binding pocket surrounded by Lys406, Gln190, Thr140, Asn142, Lys147 and Pro408 and Arg181 and Asn290 in the finger domain of T. castaneum TERT. This location conforms to the small loop beside conserved motif 2 of the RT fingers domain. Steczkiewicz et al. show two residues, K626 and R631, in motif 2 in the RT domain while K902 of motif D in the RT palm may interact with both sugar rings and phosphate groups of the base of the telomeric DNA substrate in human TERT [T. castaneum TERT. Boldine locates at a position beside motif 2, in the finger domain of T. castaneum TERT, and accordingly it may also locate at the correlated site in human TERT.Our results showed that boldine and its derivative BSB have a strong inhibitory effect on man TERT . ResidueN-benzylsecoboldine) strongly interacts at sub-micromolar concentrations with the catalytic subunit of T. castaneum TERT. It docks close to the active site in a binding pocket that is surrounded by Asp251, Thr 371, Lys 372, Arg 381, His 382, Gln 384, and Arg 374 in the palm domain of T. castaneum TERT. This lies in front of the site that interacts with telomeric DNA in motif 2 (K626:K189 and R631:R194) in the RT domain, with K902:K372 from motif D in the RT palm of human and T. castaneum TERT, respectively. As these regions are highly conserved in human and T. castaneum TERT, our results suggest that BSB probably interferes with the substrate-enzyme interaction as a non-competitive inhibitor.In conclusion, BSB  bark as its 1:1 complex with chloroform [N-Benzylsecoboldine hydrochloride (BSB) was prepared from boldine free base by successive N-benzylation and Hofmann elimination of the obtained N-benzylboldinium chloride, as described in the literature [1H NMR. The chemical structure of both compounds is shown in All chemicals except those mentioned separately were obtained at extra pure quality from Sigma-Aldrch or Merck (Germany). Boldine hydrochloride was prepared from boldine isolated from loroform , recrystloroform . N-Benzyterature . Both co2 at 37 °C  and sub-cultured routinely after reaching almost 80% confluence. Cell viability was evaluated using the trypan blue exclusion method [The breast cancer cell lines MCF7 and MDA-MB-231 were grown in Dulbecco’s modified Eagle’s medium supplemented (DMEM High Glucose with stable glutamine) with 10% fetal bovine serum (FBS gold), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere containing 5% COn method . Boldine4 cells/well and treated with each compound at serially diluted concentrations. After various incubation times, MTT at 0.5 mg/mL final concentration was added and incubated for 4 h to be reduced to formazan by viable cells. The concentration of this blue dye, after dissolving in DMSO containing 10% SDS and 1% acetic acid, was measured by absorbance measurement at 570 nm using a plate reader , and then cell viability was calculated using Gen5 software version 1.06. The assay was carried out at least in three independent logical repeats, each of which included samples in triplicates. The concentration of boldine and the derivatives that caused cell growth to decrease to 50% of untreated controls, IC50, was determined from the dose–response curves. The results were analyzed using one way ANOVA with post-hoc Tukey HSD test and reported as means ± SEM. Cell viability was evaluated using the MTT -2,5-diphenyl- tetrazolium bromide)  assay . Briefly2, 1 mM EGTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 5 mM beta-mercaptoethanol, 0.5% CHAPS and 10% glycerol and centrifuged at 14,000× g. The protein concentration of the collected supernatant was measured using the microBradford assay. Q-TRAP-ligand assay is based on a SYBR-Green quantitative telomere repeat amplification protocol (q-TRAP) [Ct) determined for each sample by using Rotor-Gene 6.01 software (https://www.qiagen.com/us/resources/) was compared with those of the standards generated from serially diluted cell lysates of the untreated MCF7 control. This experiment was performed at least three times with each repeat, including triplicate samples for each concentration of the desired compound. The probable traces of RNase contamination which potentially might give rise to false positive results were checked by incubating total RNA with aliquots of boldine or BSB for 30 min at room temperature followed by electrophoresis in agarose gel.Direct treatment of telomerase in cell lysates with boldine and the derivatives was performed using the q-TRAP-ligand assay as described earlier . Briefly(q-TRAP)  with som(q-TRAP) ,30. In gTribolium castaneum telomerase (containing TRBD and RT domains) alone [T. castaneum telomerase (PDB code: 3KYL) was taken from the Protein Data Bank (http://www.rcsb.org/pdb) and utilized for docking runs in complex with boldine and its derivative. To prepare the proteins for docking, all non-protein related molecules including water were deleted and hydrogen atoms were added using Autodock tools 4.2 (ADT) [The ribonucleoprotein telomerase is composed of a template containing RNA and a catalytic subunit TERT. The catalytic subunit of telomerase structures from different species collectively contain conserved components including two very important domains: an RNA binding domain (TRBD) and a reverse transcriptase domain (RT). The amino acid sequence identities between human telomerase domains TRBD and RT and the corresponding structures in the Protein Data Bank (PDB) are 22 and 24%, respectively . No 3D ss) alone  and the s) alone . Here, t.2 (ADT) .To optimize the Tc-telomerase structure, 10 ns MD simulations were carried out with the GROMACS 4.6.5 package. The GROMOS96 force field ,34 was aDocking calculation was performed using Autodock Tools 4.2 software with autodock4 and autogrid4 programs ,43. The ftp://ftp.gromacs.org/pub/gromacs/gromacs-5.0.4.tar.gz) as mentioned above in The ligands, i.e., boldine and/or its derivative docked with telomerase, were used to perform explicit solvent MD simulations. MD simulations were carried out with the GROMACS 4.6.5 package (


New materials are needed for achieving major improvements. Crystalline materials, displaying unidirectional and single-sized pores, preferentially with low pore tortuosity and high pore density, are promising candidates for membrane synthesis. Herein, we study hexagonal ice crystals as an example of this class of materials. By slowly growing ice crystals inside capillary tubes we were able to measure the permeation of several gas species through ice crystals and investigate its relation with both the size of the guest molecules and temperature of the crystal.Gas separation using porous solids have attracted great attention due to their energetic applications. There is an enormous economic and environmental interest in the development of improved technologies for relevant processes, such as H Membrane separation is growing as a low-cost and energy-efficient alternative to traditional methods, such as adsorption or cryo-separation. Most membranes in use in industry are polymeric [Separation or purification of light gases is crucial in many industrial activities. In fact, there is an enormous economic and environmental interest in the development of better technologies for relevant processes such as Holymeric , and desolymeric ,4 and peolymeric ,6,7,8 arolymeric  or mixedc-axis and that they can be used as single crystal membranes able to distinguish between different gas molecules. We observed that the gas molecules cannot escape through the ice-glass capillary wall interface, which enables an easy scale-up by simply using perforated solid plates to promote multiple crystal growth. Moreover, we found that the flow rate of the gas species is severely regulated by ice temperature, expanding the number of potential species that may be separated.It is well known that ordinary water and several organic compounds  solidifyc-axis relative to the capillary were determined by X-ray diffraction  and applying different temperature decreasing rates. The ice crystal structures and the alignment of the tion see . We obtatructure  with an −3 µL) of ultrapure water (milli-Q water) was introduced inside a glass capillary. The ice crystal was obtained by cooling at temperature rate of 10 K/hr until 260 K followed by 360 K/hr towards the predetermined temperature. A standard cryostream cooler for X-ray diffraction was used. Pressure of the feed gas chamber was monitored and permeabilities were determined through a mass balance on the chamber volume.Single-crystal permeation experiments were performed against the atmosphere using a pressurized feed gas chamber  Å @150 K to 4.492(4) Å @240 K, uncertainties in parenthesis) and an increase in the c crystal lattice (7.290(3) Å @150 K to 7.335(7) Å @240 K). Overall, there is a negative thermal expansion of the cell volume (128.79(8) Å3 @150 K to 128.16(19) Å3 @240 K) as it was formerly obtained by Dantl (1962) by single crystal experiments [In addition, we also investigated the temperature effect on ice structure by single-crystal X-ray diffraction. It has already been observed that above 100 K there is a residual thermal expansion, virtually isotropic, of powdered ice Ih.  Hexagonae  crystals . The estIce phase transitions and ice–gas interactions (gas hydrate stability and gas diffusion mechanisms) are deeply investigated but poorly understood. Neverthless, our results are intriguing in light of the global perspective in the field.c–ice Ih transition temperature is located between 160 K and 205 K [The ice Ind 205 K  and the et al. [2, diffuse without distorting the ice lattice while large molecules, such as O2, N2, CO2 and CH4, diffuse by a bond-breaking mechanism. However, Demurov and colleagues [2 through defect-free hexagonal ice at 200 K and observed no evidence of diffusion. Alavi and Ripmeester [2 can diffuse out of the hydrates through the hexagonal rings, or even through the smaller pentagonal rings. Mitlin et al. [et al. [Gas molecules with molecular dimension lower than 0.9 nm can be incorporated in water crystalline inclusions—known as clathrate hydrates. Most hydrates belong to three structural families, two cubic forms and one hexagonal form ,19,20,21et al. ,28,29 arlleagues  reportedpmeester  on the on et al.  argued t [et al.  observed [et al.  showed t2, O2, N2, CH4 and Ar can diffuse through the hexagonal ice structure. Diffusion of these compounds was already predicted theoretically by other authors [−3µL) is too small to measure gas sorption uptake. Neverthless, adsorption isotherms of light gases, such as H2, N2 and CO2 in hexagonal ice, were already reported in the literature [Here we show that H authors . Howeverterature ,36.Ice was already used as a chromatographic stationary phase to separate enantiomers . Here, w


The effects of amplitude and type of the El Niño-Southern Oscillation (ENSO) on sea surface temperature (SST) predictability on a global scale were investigated, by examining historical climate forecasts for the period 1982–2006 from air-sea coupled seasonal prediction systems. Unlike in previous studies, SST predictability was evaluated in different phases of ENSO and for episodes with various strengths. Our results reveal that the seasonal mean Niño 3.4 index is well predicted in a multi-model ensemble (MME), even for four-month lead predictions. However, coupled models have particularly low skill in predicting the global SST pattern during weak ENSO events. During weak El Niño events, which are also El Niño Modoki in this period, a number of models fail to reproduce the associated tri-pole SST pattern over the tropical Pacific. During weak La Niña periods, SST signals in the MME tend to be less persistent than observations. Therefore, a good ENSO forecast does not guarantee a good SST prediction from a global perspective. The strength and type of ENSO need to be considered when inferring global SST and other climate impacts from model-predicted ENSO information. The El Niño-Southern Oscillation (ENSO) is the most dominant forcing factor of inter-annual climate variability124110Because of their importance, the ENSO phenomenon and global SST changes are constantly monitored by most operational centres. Some centres, including the Japan Meteorological Administration (JMA), National Centers for Environmental Prediction (NCEP), Korean Meteorological Administration (KMA), Australian Government Bureau of Meteorology (BoM), APEC Climate Center (APCC) and others, even provide forecasts of their evolution and the associated climate anomalies. Although numerous studies of the predictability of ENSO and its climate impacts have been conducted, the possible roles of the intensity and type of ENSO on SST predictions are still not well established. It has been found that a model’s skill at ENSO prediction is higher during El Niño/La Niña than in normal periods161819202123The aim of this study is to assess the skill of CGCM-based seasonal forecast systems at predicting ENSO occurrences and their associated SST changes for all seasons using the APCC multi-model ensemble (MME). ENSO events were identified based on the Niño 3.4 index, which is defined as the area-averaged anomalous SST within 5°S–5°N, 170°–120°W. The index is considered a key indicator of ENSO by most operational centres ). An El Niño is defined when the 5–month running mean of this index exceeds 0.4 °C for at least six monthsFirst, we considered ENSO predictability based on MME by comparing the observed Niño 3.4 index with the ones derived from one-month and four-month lead MME mean forecasts. In view of the relationship between the ENSO amplitude and SST predictability, ENSO events were classified according to the strength of the Niño 3.4 index (see Methods). For the warm ENSO phase, three out of the four strong events correspond to canonical El Niño, whereas all four weak events could be classified as El Niño Modoki within the 1982–2006 period (see Tables S1 and S2). In contrast to El Niño cases, it is noteworthy that only two out of five weak La Niña events corresponded to La Niña Modoki. During strong La Niña years, lower than normal ocean temperature was observed across the eastern-central Pacific. However, in weak La Niña periods, anomalous SST along the equatorial Pacific did not exhibit a tri-pole-like (i.e. warm–cold–warm) pattern. Also, notice the significantly warm SST in the north western Pacific during strong La Niña; for weak events SST signals were much weaker in the same region. The correlation between the strong (weak) La Niña SST pattern and the SST regression map based on the Niño 3 (El Niño Modoki) index was only −0.6 (−0.38). It is clear that a weak La Niña does not always correspond to a La Niña Modoki.Model simulated SST patterns for strong and weak ENSO events are now examined. In general, SST anomalies during strong ENSO events from all of the climate models considered compare well with their observational ones . On the other hand, some models seem to have difficulty in reproducing SST patterns associated with weak ENSO events. To further investigate SST predictability and its relationship with the ENSO strength, the skills of the Niño 3.4 index and SST pattern forecasts for each categorised ENSO events and for both boreal summer and winter are compared see . In geneThe strength of ENSO events is even more crucial in determining a model’s skill at capturing the accompanying global SST patterns. During strong El Niño, the global SST was well forecasted (with pattern correlation between model and observations up to 0.66). It should be mentioned that this was not true for weak El Niño (with pattern correlation of ~0.34 to 0.5). This is consistent with our previous results regarding strong and weak El Niño SST forecasts see . Global Effects of the strength and type of ENSO on global SST predictability in the APCC MME seasonal forecast system were assessed based on hindcasts targeting all seasons for the period 1982–2006. In general, seasonal SST predictability depends strongly on the phase as well as the intensity of ENSO. The Niño 3.4 index is well predicted by coupled models. However, they tend to perform better in capturing variations of the Niño 3.4 index during strong ENSO events, than in periods when ENSO is weak in their long-lead (4-month lead) predictions. It is probably due to the fact that models’ ability in capturing ENSO evolution is mainly determined by ocean dynamics; strong events can lead to stronger anomalous states in the ocean component, which might be conducive to better ENSO simulations as the lead time increases. The global SST prediction skill is even more sensitive to the strength of ENSO. The strongest El Niño events identified in the hindcast period belong to canonical El Niño, whereas all weak events were found to correspond to El Niño Modoki. During those strong events, SST changes around the globe were well predicted by models. However, a number of models failed to reproduce the SST anomalies typically associated with an El Niño Modoki , resulting in relatively poor skill by the MME in capturing global SST variations in weak El Niño years. When La Niña is weak, models tend to give central-to-eastern Pacific SST signals, which are less persistent than observations, and global SST forecasts also perform poorly compared to those during strong La Niña cases. Because of this sensitivity of SST prediction skills to the type of ENSO, more studies are needed to determine the various mechanisms that trigger and sustain the two types of ENSO events, and to assess how well these mechanisms are reproduced in state-of-the-art coupled models. It has been suggested that a strong or canonical ENSO is related to tropical atmosphere–ocean coupling and tied to thermocline variations. On the other hand, weak El Niño or El Niño Modoki is related to the Pacific subtropical high and the Asian–Australian monsoon system, and could be more influenced by atmospheric forcing and local air-sea coupling rather than basin-wide ocean dynamicsOur results also highlight the problem of adopting a single index for ENSO prediction. Although it is the most widely used ENSO index, especially within the seasonal climate prediction community, the use of the Niño 3.4 index only might risk mixing the two types of ENSO. Using only SST over the central equatorial Pacific, and without taking into account conditions farther east, the Niño 3.4 index cannot distinguish between canonical and atypical El Niño events. The different types of El Niño are also known to produce different teleconnection patterns and hence very different climate impacts across the globeIn this study, the optimum interpolation (OI) of SST data313233To determine the ENSO phase and amplitude, the 3-month running mean Niño 3.4 index was used. Warm (cold) events were identified whenever the standard deviation was greater (less) than 0.65 (−0.65) for at least 4 months see . Among tHow to cite this article: Sohn, S.-J. et al. How do the strength and type of ENSO affect SST predictability in coupled models. Sci. Rep.6, 33790; doi: 10.1038/srep33790 (2016).