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The catalyst rearranges propane, which contains three carbon atoms, into other molecules, such as butane (containing four carbons), pentane (with five carbons) and ethane (with two carbons). "Our aim is to convert lower molecular weight alkanes to valuable diesel-range alkanes," said Manoja Samantaray from the KAUST Catalysis Center. At the heart of the catalyst are compounds of two metals, titanium and tungsten, which are anchored to a silica surface via oxygen atoms. The strategy used was catalysis by design. Previous studies showed that monometallic catalysts were engaged in two functions: alkane to olefin and then olefin metathesis. Titanium was chosen because of its ability to activate the C-H bond of paraffins to transform them to olefins, and tungsten was chosen for its high activity for olefin metathesis. To create the catalyst, the team heated silica to remove as much water as possible and then added hexamethyl tungsten and tetraneopentyl titanium, forming a light-yellow powder. The researchers studied the catalyst using nuclear magnetic resonance (NMR) spectroscopy to show that the tungsten and titanium atoms lie extremely close together on the silica surfaces, perhaps as close as ≈0.5 nanometres. The researchers, led by the Director of the center Jean-Marie Basset, then tested the catalyst by heating it to 150°C with propane for three days. After optimizing the reaction conditions— for example, by allowing the propane to flow continuously over the catalyst—they found that the main products of the reaction were ethane and butane and that each pair of tungsten and titanium atoms could catalyze an average of 10,000 cycles before losing their activity. This "turnover number" is the highest ever reported for a propane metathesis reaction. This success of catalysis by design, the researchers propose, is due to an expected cooperative effect between the two metals. First, a titanium atom removes hydrogen atoms from propane to form propene and then a neighboring tungsten atom breaks open propene at its carbon-carbon double bond, creating fragments that can recombine into other hydrocarbons. The researchers also found that catalyst powders containing only tungsten or titanium performed very poorly; even when these two powders were physically mixed together, their performance did not match the cooperative catalyst. The team hopes to design an even better catalyst with a higher turnover number, and a longer lifetime. "We believe that in the near future, industry can adopt our approach for producing diesel-range alkanes and more generally of catalysis by design," said Samantaray. Explore further: A new catalyst to transform propane into propene More information: Manoja K. Samantaray et al. Unearthing a Well-Defined Highly Active Bimetallic W/Ti Precatalyst Anchored on a Single Silica Surface for Metathesis of Propane, Journal of the American Chemical Society (2017). DOI: 10.1021/jacs.6b12970


Obesity, already a global epidemic, is on the rise. Over one third of the U.S. population is currently afflicted, according to the Centers for Disease Control and the monetary costs alone are approaching $150 billion dollars annually. Causes of the epidemic include changing diets and greater sedentism, though environmental factors may also contribute. A new study compares the two most common surgical therapies for obesity, known as Roux-en-Y gastric bypass (RYGB), and laparoscopic adjustable gastric banding (LAGB). The results demonstrate that RYGB--the more aggressive of the two surgeries-- produces profound changes in the composition of microbial communities in the gut, with the resulting gut flora distinct from both obese and normal weight patients. The results are likely due to the dramatic reorganization of the gut caused by RYGB surgery, which increases microbial diversity. The new research paves the way for new diagnostics and therapies for obesity. The gamut of adverse health effects associated with obesity is broad, including such devastating illnesses as type 2 diabetes, coronary artery disease, stroke and certain forms of cancer. Patients often suffer loss of mobility, social isolation and inability to work. Currently bariatric surgery is the most effective treatment for morbid obesity, in terms of significant and sustained weight loss. In the new study, appearing in the current issue of the Nature Publishing Group journal International Society for Microbial Ecology (ISME), Zehra Esra Ilhan, Rosa Krajmalnik Brown and their colleagues at the Biodesign Institute at ASU, along with researchers from Mayo Clinic, and Pacific Northwest National Laboratory, explore microbial communities in the human gut following RYBG and LAGB surgeries. The results confirmed their earlier research with a smaller sample size, showing that in the case of the more aggressive and irreversible RYGB surgery, microbial communities underwent a profound and permanent shift following weight loss. The resulting post-surgical composition of gut microbes observed for RYGB patients was distinct from both normal weight and obese patients, and displayed the high microbial diversity associated with a healthy gut. The current study also applied the technique of nuclear magnetic resonance (NMR) to examine the metabolome--a composite of the metabolites produced by the various microbes in the gut, again noting significant alterations as a result of the RYGB procedure. In the case of the alternate treatment, LAGB, changes in the gut microbiota were mild and accompanying weight loss was less pronounced. "This is one of the first studies to show that anatomically different surgeries with different success rates have different microbiome and microbiome-related outcomes," notes Ilhan, lead author of the new paper. Further, the results indicate that correction of obesity tends to improve related metabolic conditions, including diabetes and high cholesterol. "One of the key findings of the paper confirms what we had already observed in earlier research. RYGP gastric bypass had a huge effect on the microbial community structure," Krajmalnik-Brown says. This fact may have profound implications for both the understanding and management of obesity. The millions of bacterial microbes in the human gut perform a vast range of critical functions in the body and have even been implicated in mood and behavior. Among their critical responsibilities are the micro-management of nutrients in the food we digest, hence their central place in the regulation of body weight. A tell-tale indicator of pathology in obese patients has been found in the gut, where a markedly lower diversity of microbial communities is observed. As Krajmalnik-Brown explains, diversity of gut microbes is essential to good health. "Diversity is good because of what we call functional redundancy," she says. "If you have 10 workers that can do the same job, when one of them gets sick, the job still gets done." Low microbial diversity in the gut, by contrast, is associated not only with obesity but a range of ailments including inflammatory bowel disease, ulcerative colitis and autism. (Earlier research by Krajmalnik-Brown and her colleagues demonstrated diminished diversity in the gut microbiome of autistic children and in a more recent study, improvement in the symptoms of autism was demonstrated following transplantation of beneficial microbes.) Competition in diverse microbial networks in the gut helps provide a system of checks and balances. Should diversity fall, a delicate democracy can be shattered and tyranny may prevail, as populations of microbes like Salmonella or Clostridium difficile--usually subsisting at low levels in the gut --expand and take over. The study sought to explore long-term changes in the gut in patients who had undergone either of the two surgeries at least 9 months prior, comparing them with normal weight and pre-bariatric obese patients. While the reasons for the sharp disparity of results between RYGB and gastric banding are not entirely clear, the results indicate that simply reducing the size of the stomach through gastric banding is not sufficient to induce the large changes in microbial communities observed for the RYGB group. One hypothesis the authors put forward is that RYGB alters the physiology of the gut to such a degree that microbes formerly unable to survive conditions in the obese gut are able to flourish in their surgically-modified surroundings. "One of the things we observe from the literature is that the oral microbiome community composition is very similar to the colon microbiome composition after bariatric surgery," Ilhan says. "You're giving new microbes a chance to make it. Most of the species are acid sensitive, which supports the idea that changes in stomach pH levels may permit these microbes to survive and make it to the colon." According to John DiBaise, a gastroenterologist at Mayo Clinic, Scottsdale and co-author of the new study, "These new data on microbial community structure and function significantly expand our knowledge on how the microbiome is associated with weight loss following bariatric surgery." While it seems clear that RYGB surgery produced permanent changes in bacterial communities in the gut, the resulting microbial community may also act to help maintain weight loss over the long term. Experiments have shown that transplantation of beneficial microbes from mice that have undergone RYGB surgery into obese mice induces dramatic weight loss. While these results have yet to be replicated in humans, the findings open the door to the eventual use of healthy microbial communities to treat obesity. Although the RYGB surgery has been quite successful for many patients suffering from morbid obesity, it is a serious, invasive procedure that is not without risks. Further, some patients are not successful and regain the weight they have lost post-surgery, perhaps because they lack the favorable microbes necessary for permanent weight loss. As Ilhan says, "a probiotic that would replace surgery would be great. Another positive outcome would be if we can find a microbial biomarker that will identify the best candidates for surgery and sustained weight loss." Research reported in this 24 publication was supported by the National Institute of Diabetes and Digestive and Kidney 25 Diseases of the National Institutes of Health under Award Number R01DK090379. Distinctive microbiomes and metabolites linked with weight loss after gastric bypass, but not gastric banding Zehra Esra Ilhan1,2, John K DiBaise3, Nancy G. Isern4, David W. Hoyt4, Andrew K 5 Marcus1, Dae-Wook Kang1, Michael D. Crowell3, Bruce E Rittmann1, and Rosa Krajmalnik-Brown1 1Swette Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, 85287, U.S.A., 2 School of Life Sciences, Arizona State University, 85287, U.S.A, 3 Mayo Clinic, Division of Gastroenterology, Scottsdale, U.S.A., 4 Environmental and Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, 12 WA, U.S.A.


News Article | May 24, 2017
Site: www.nature.com

To understand the contribution of α-amino acid metabolism to the cancer progression of CML, we analysed blood amino acid levels in mouse models that recapitulate the chronic and blast crisis phases of human CML3, 4. Using amine-specific fluorescent labelling coupled with high-performance liquid chromatography (HPLC), 16 amino acids were quantified in the blood plasma from leukaemic mice (Extended Data Fig. 1a–d). Mice bearing blast crisis (BC)-CML showed moderate but significant elevations of plasma glutamate, alanine and the BCAAs (namely valine, leucine and isoleucine) compared with chronic phase (CP)-CML mice, indicating hyperaminoacidaemia (Extended Data Fig. 1e). Intracellular levels of BCAAs and proline were higher in BC-CML, whereas intracellular glutamate and alanine were comparable in the two disease phases (Fig. 1a). These results suggest that increased BCAA uptake or metabolism may contribute to CML progression. We analysed the gene expression and found no significant upregulation of known BCAA transporters in BC-CML compared with CP-CML (data not shown). Leucine import into BC-CML cells was not greater than into CP-CML cells (Extended Data Fig. 1f), indicating that increased BCAA uptake does not explain the higher BCAA levels in BC-CML. To examine the possibility of altered intracellular BCAA metabolism, we next analysed the expression of genes encoding amino acid metabolic enzymes and found that the branched-chain amino acid aminotransferase 1 (Bcat1) was more highly expressed in BC-CML than in CP-CML at both the messenger RNA (mRNA) and protein levels (Fig. 1b–c and Extended Data Fig. 1g–h). In contrast, normal haematopoietic stem/progenitor cells (HSPCs) from healthy mice had very low levels of Bcat1 expression (Lin− Sca-1+ c-Kit+ (LSK) population; Fig. 1b), and normal tissues did not show detectable Bcat1 expression except for the brain and testis (Extended Data Fig. 1i). Bcat1 encodes an evolutionarily conserved cytoplasmic aminotransferase for glutamate and BCAAs, constituting a regulatory component of cytoplasmic amino acid and keto acid metabolism5 (Fig. 1d and Extended Data Fig. 1j). Bcat2, a paralogue encoding the mitochondrial BCAA aminotransferase, and alanine and aspartate aminotransferases did not show differential expression between CP- and BC-CML (Extended Data Fig. 1g, k–l). Although BCAT1 catalyses transamination in both directions, the breakdown of BCAAs is the predominant reaction in most cell types6. For BCAT1 to generate BCAAs via the reverse reaction, the corresponding branched-chain keto acids (BCKAs), as well as glutamate, must be present as substrates. We found that all three BCKAs, keto-isovalerate (KIV), keto-isocaproate (KIC) and keto-methylvalerate were present in both the blood plasma and leukaemia cells (Extended Data Fig. 2a–d). In BC-CML cells, BCKAs were present at concentrations equivalent to 22–55% of the corresponding BCAAs, suggesting that intracellular BCKAs can serve as substrates for BCAA production (Extended Data Fig. 2e). Next, we examined whether BCAAs are produced through BCAT1 transamination reactions in leukaemia cells by stable-isotope tracer experiments with [13C]valine or [13C]KIV. Intracellular 13C-labelled metabolites in K562 human BC-CML cells were analysed using one- and two-dimensional 1H–13C heteronuclear single-bond correlation (HSQC) analysis by high-field NMR spectroscopy (Fig. 1e–h and Extended Data Fig. 3). HSQC analysis detects only metabolites that have incorporated 13C isotope. To determine whether KIV is converted to valine, cells were cultured in media supplemented with uniformly labelled [(U)-13C]KIV and non-labelled valine at physiological concentrations (30 and 170 μM, respectively), and analysed for intracellular 13C-labelled metabolites. After 15 min of labelling, the generation of [13C]valine was clearly observed, indicating the efficient intracellular production of valine from KIV (Fig. 1f, h). In contrast, [13C]KIV formation was barely detectable in the cells cultured with non-labelled KIV and [(U)-13C]valine (Fig. 1e, g). Our observation of intracellular [13C]valine signals indicates its transport into BC-CML cells. We also detected robust signals for [13C]KIV when present (Extended Data Fig. 3d, f). The formation of valine from KIV, but not the breakdown of valine to KIV, was also observed when we used equal concentrations of KIV and valine in the labelling media (170 μM each; Fig. 1g, h). We did not detect KIC formation from [13C]leucine either (Extended Data Fig. 3g–i). These results indicate that little, if any, BCAAs are catabolized to BCKAs in leukaemia cells. To provide further evidence for the intracellular BCAA production through transamination, we performed alternative labelling experiments to track the fate of the amine group of glutamate. We cultured K562 cells with [15N]amine-labelled glutamine, which is metabolized to [15N]amine-glutamate by glutaminase upon cellular intake, and analysed the subsequent labelling of BCAAs via 1H NMR and 1H–15N heteronuclear multiple-bond correlation. This analysis detects only metabolites that have incorporated 15N, whereas 1H NMR detects any compounds containing protons (Extended Data Fig. 4a–f). At 29–72 h after labelling, we detected 15N-amine-labelled BCAAs, indicating transamination from glutamine to BCAAs (Fig. 1i). By 72 h, the 15N-amine-labelled BCAAs had accumulated to fractional abundances ranging from 24% to 39% (Extended Data Fig. 4g), indicating a significant contribution of transamination to the intracellular BCAA pool. Lentiviral BCAT1 knockdown resulted in greater than a 50% decrease in the amount of intracellular BCAAs produced (Fig. 1j). These data demonstrate that BCKA transamination by BCAT1 contributes to the BCAA pool in leukaemia cells. Given that Bcat1 is highly expressed and augments intracellular BCAAs in BC-CML, Bcat1 may functionally contribute to the acute properties of BC-CML. To test this possibility, we inhibited Bcat1 expression using a short hairpin RNA (shRNA)-mediated gene knockdown approach. We sorted the immature lineage-negative (Lin−) cells from primary BC-CML samples, a population that contains the leukaemia-initiating cells of this cancer, and introduced two independent retroviral shRNA constructs (Extended Data Fig. 1j; shBcat1-a and shBcat1-b). Both constructs inhibited Bcat1 expression in BC-CML compared with a non-targeting negative control shRNA (shCtrl) (Extended Data Fig. 5a–c). Bcat1 knockdown resulted in significantly smaller colonies and a 40–60% reduction in the colony-forming ability relative to a control (Fig. 2a). The co-introduction of a shRNA-resistant Bcat1 cDNA rescued the reduced clonogenic potential (Extended Data Fig. 5d). As an alternative approach to gene knockdown, we treated BC-CML cells with gabapentin (Gbp), a chemical inhibitor of BCAT1. Gbp is a structural analogue of leucine and specifically and competitively inhibits the transaminase activity of BCAT1 but not that of BCAT2 (ref. 7). BC-CML cells plated with Gbp formed smaller colonies and showed a dose-dependent impairment in clonogenic growth (Fig. 2b). In contrast, normal HSPCs were only minimally affected by gene knockdown or Gbp treatment (Extended Data Fig. 5e, f). These data suggest that BCAT1 inhibition may selectively impair the propagation of leukaemia without affecting normal haematopoiesis. To examine whether Bcat1 loss affects the propagation of BC-CML in vivo, Lin− cells expressing shBcat1 were transplanted into conditioned recipient mice. Whereas 75% of the recipients transplanted with control cells succumbed to the disease within 30 days, only 47% (shBcat1-a) and 31% (shBcat1-b) of the mice transplanted with Bcat1-knockdown cells developed the disease, and more than half of these mice survived even when followed out to 60 days (Fig. 2c). Among the mice that developed disease with Bcat1 knockdown, most had leukaemia that was characterized by differentiated granulocytes and lower levels of immature myeloblasts (Fig. 2d and Extended Data Fig. 5g). They also displayed a lower frequency of immature Lin− cells than control leukaemia (Extended Data Fig. 5h), indicating that the loss of Bcat1 induced differentiation and impaired the leukaemia-initiating cell activity. Consistent with these phenotypes, serial transplantation of the leukaemia cells revealed that while all the control leukaemia cells propagated the disease, none of the mice transplanted with Bcat1-knockdown leukaemia cells succumbed to the disease (line ‘1k’ in Fig. 2e). In addition, we established a doxycycline (Dox)-inducible Bcat1 knockdown system (i-shBcat1) and examined the impact of Bcat1 loss on the disease maintenance. Ten days after transplantation with BC-CML cells infected with i-shBcat1, leukaemic engraftment was assessed in each recipient, and Dox treatment was initiated (Extended Data Fig. 5i, j). While almost all the mice that were transplanted with control cells and the non-Dox-treated mice developed leukaemia, more than half of the Dox-treated i-shBcat1 mice remained disease-free (Extended Data Fig. 5k), indicating that Bcat1 is required for the continuous propagation of BC-CML. At the cellular level, we did not observe enhanced apoptosis or a decrease in actively cycling cells by Bcat1 knockdown (Extended Data Fig. 5l, m). These results demonstrate that Bcat1 is critical for the sustained growth and maintenance of leukaemia-initiating cells in BC-CML. We next examined whether the enforced expression of Bcat1 could drive blastic transformation in haematopoietic cells. Although we observed a significant increase in Bcat1 expression compared with the vector control, Bcat1 expression alone did not enhance the colony-forming ability of either LSK or Lin− c-Kit+ haematopoietic cells isolated from normal bone marrow (Extended Data Fig. 6a, b). To determine whether BCR–ABL1 cooperates with Bcat1 overexpression to confer an aggressive growth phenotype, we transduced normal HSPCs with Bcat1 and BCR–ABL1. Compared with the vector control, the combinatorial expression promoted clonogenic growth in vitro (Extended Data Fig. 6c), and the transplantation of the cells led to significantly elevated leukaemia burdens (Extended Data Fig. 6d, e), splenomegaly and increased mortality in the recipient mice (Fig. 2f), with a concomitant increase in plasma BCAA levels (Extended Data Fig. 6f). Accordingly, leukaemia that developed in response to Bcat1 overexpression exhibited a highly immature myeloblastic morphology compared with the control (Fig. 2g and Extended Data Fig. 6g). These data indicate that activated Bcat1 mediates the blastic transformation of CP-CML cells. Our results demonstrate that Bcat1 is essential for the development of BC-CML in mice, while normal bone marrow HSPCs show a very limited dependence on this metabolic enzyme. To investigate the contribution of BCAT1 to human leukaemia, we looked at a panel of 13 peripheral blood samples from healthy and leukaemic individuals and found human BCAT1 expression was higher in BC-CML than in either normal or CP-CML cells (Fig. 3a). To determine whether this expression pattern reflects a general trend in human CML, we analysed BCAT1 levels in a Gene Expression Omnibus (GEO) dataset of 113 cases of CML8. This focused analysis revealed a significant elevation in BCAT1 expression as the disease progresses from the chronic to the accelerated phase and then to the blast crisis phase (Fig. 3b). On average, BCAT1 expression was 15-fold higher in BC-CML than in CP-CML. We did not find significant changes in BCAT2 expression, which is consistent with the results from the mouse models (Fig. 3c and Extended Data Fig. 1g). These data indicate that activation of BCAT1 is a shared characteristic in the progression of human CML. Lentiviral BCAT1 knockdown or Gbp treatment markedly inhibited the colony-forming ability of K562 human BC-CML (Extended Data Fig. 7a–d) and patient-derived primary leukaemia cells (Fig. 3d, e and Extended Data Fig. 7e, f). Interestingly, we observed BCAT1 activation in primary human acute myeloid leukaemia as well (AML; Fig. 3f), and Gbp effectively inhibited the clonal growth of human AML cell lines and primary de novo AML cells (Fig. 3g and Extended Data Fig. 7g–i). Moreover, BCAT1 expression levels predict disease outcome in patient cohorts. Cases from The Cancer Genome Atlas (TCGA) AML dataset were divided into quartiles on the basis of BCAT1 expression levels (Extended Data Fig. 7j), and we found that the median survival time was 46% shorter in the BCAT1-high group (427 versus 792 days; Fig. 3h). These results demonstrate an essential role for BCAT1 in the pathogenesis of a wide array of human myeloid malignancies. To understand how the BCAT1-driven change in metabolism promotes leukaemia growth, we analysed intracellular amino acid concentrations upon BCAT1 inhibition and found that all three BCAAs were significantly reduced by shBCAT1 or Gbp treatment compared with the controls (Extended Data Fig. 8a, b). Interestingly, the addition of BCAAs, but not alanyl–glutamine (GlutaMax), functionally suppressed the reduction of colony-forming ability caused by BCAT1 knockdown (Fig. 3i), suggesting that BCAT1 enhances clonogenic growth through BCAA production via BCKA reamination. BCAAs, particularly leucine, activate the mTORC1 pathway via cytosolic leucine sensor proteins, which integrate multiple signals from nutrient sensing and growth factor stimuli to promote cell growth9, 10, 11, 12. Thus, we examined whether reduced BCAA production by BCAT1 inhibition results in the attenuation of the mTORC1 signal. Indeed, BCAT1 blockade by either shRNA or Gbp treatment significantly reduced the phosphorylation of S6 kinase (pS6K), a downstream target of mTORC1 kinase (Fig. 3j), suggesting BCAT1 activation of the mTORC1 pathway. We observed no apparent changes in the levels of phosphorylated AKT upon BCAT1 inhibition, suggesting a predominant contribution of BCAA nutrient signals to the activation of mTORC1 (Extended Data Fig. 8c). Consistently, the mTORC1 inhibitor rapamycin reversed the BCAA-induced suppression of colony formation (Fig. 3i) and the BCAA-induced increase in pS6K (Fig. 3k). To further investigate the BCAT1-mediated regulation of CML progression, we performed gene correlation analyses using tumour gene expression datasets available in the GEO and TCGA databases. We found that BCAT1 and MSI2 are often co-expressed in several types of cancer, including leukaemias, colorectal and breast cancers (Extended Data Fig. 9a, b). MSI2 is a member of the evolutionarily conserved Musashi RNA binding protein family, which regulates cell fates during development and in multiple adult stem-cell systems in metazoans13, 14, 15. At the molecular level, Musashi proteins bind to r(G/A)U AGU sequences (MSI binding elements, MBEs) and post-transcriptionally regulate gene expression via mRNA binding16, 17. Importantly, MSI genes are aberrantly activated in human malignancies, such as gliomas and breast and colorectal cancers18, 19. In human BC-CML, the MSI2 gene is upregulated and functionally required for the progression of this leukaemia20, 21. To determine whether BCAT1 is a direct target of the MSI2 RNA binding protein, we analysed the BCAT1 mRNA sequence and found 40 putative MBEs in the 3′ untranslated region (3′ UTR; Extended Data Fig. 9c). To test whether MSI2 binds to the BCAT1 transcripts, we expressed a Flag-tagged MSI2 protein in K562 cells and performed RNA immunoprecipitation (RIP). Flag–MSI2 co-precipitated the BCAT1 transcripts with a greater than 1,500-fold enrichment relative to the vector control (Fig. 4a). In contrast, when RIP was performed with a mutant MSI2 protein in which three phenylalanine residues essential for RNA binding were replaced with leucine16, the amount of the BCAT1 mRNA recovered was markedly diminished (Fig. 4a, RNA binding defective mutant (RBD)), indicating that the co-precipitation of BCAT1 transcript requires the RNA binding activity of MSI2. The transcripts for β-2-microglobulin (B2M) or c-Myc oncogene (MYC) contain only one copy of a putative MBE in their 3′ UTRs (data not shown), and MSI2 RIP did not enrich B2M or MYC mRNAs as efficiently as BCAT1 (Fig. 4a). Furthermore, RIP with an anti-MSI2 antibody showed that endogenous MSI2 proteins bound to BCAT1 transcripts, while B2M or MYC mRNAs exhibited minimal enrichment relative to that of an immunoglobulin-G (IgG) control (Fig. 4b), indicating that MSI2 is specifically associated with the BCAT1 transcripts. Because MSI2 knockdown reduced the levels of BCAT1 protein and p-S6K (Extended Data Fig. 9d), the binding of MSI2 to BCAT1 mRNA positively regulates BCAT1 translation and mTORC1 activation. Importantly, BCAT1 overexpression (Fig. 4c) and BCAA supplementation (Fig. 4d) effectively suppressed the attenuation of the colony-forming ability caused by MSI2 knockdown, with a concomitant increase in pS6K levels in a rapamycin-sensitive manner (Fig. 4e). The levels of AKT phosphorylation were unaffected by shMSI2 (Extended Data Fig. 8c). Collectively, our work presented here demonstrates an essential role for the MSI2–BCAT1 axis in myeloid leukaemia and provides a proof-of-principle for inhibiting the BCAA metabolic pathway to regulate CML progression (Fig. 4f). The upregulation and functional requirements of BCAT1 have been reported in glioblastoma and in colorectal and breast tumours22, 23. Interestingly, Musashi proteins also regulate the same spectrum of cancers including myeloid leukaemia18, 19, 20, 21, 24, 25, suggesting a highly conserved role for the MSI–BCAT1 pathway in multiple cancer types. Despite the conservation of this pathway, the metabolic role of BCAT1 seems distinct and dependent on the tissue of origin; in the brain, BCAT1 catalyses BCAA breakdown and glutamate production to enhance tumour growth in glioblastoma23, whereas it promotes BCAA production in leukaemia. Two different types of tumour, specifically pancreatic ductal adenocarcinoma and non-small-cell lung carcinoma, were recently shown to exhibit different usages of BCAAs26. Despite the same initiating events of KRAS activation and TP53 deletion, non-small-cell lung carcinoma cells actively utilize BCAAs by enhancing their uptake and oxidative breakdown to BCKAs, whereas pancreatic ductal adenocarcinoma cells display decreased uptake and thus little dependency on BCAAs. Consistently, BCAT1 and BCAT2 are required for tumour formation in non-small-cell lung carcinoma but not in pancreatic ductal adenocarcinoma. Although BCAT1 is functionally required for tumour growth in a broad range of malignancies, these reports and our studies highlight the context-dependent role of the BCAT1 metabolic pathway in cancer.


News Article | May 25, 2017
Site: physicsworld.com

A new way of boosting the resolution of quantum magnetic sensors has been developed independently by three teams of physicists. The technique has already been used to achieve a huge improvement in nuclear magnetic resonance (NMR) spectroscopy. Quantum sensing is used to measure frequencies in multiple areas of physics, but for a quantum sensor to measure anything, it must interact with its environment. This degrades its quantum properties very quickly – and this limits the measurement accuracy. Now, however, three research groups have independently synchronized multiple quantum measurements using a classical clock, allowing frequency measurements up to 100 million times more accurate than previously possible with a quantum sensor. One group then went on to demonstrate unprecedented accuracy in micron-scale NMR spectroscopy. All three groups – at ETH Zürich in Switzerland, Ulm University in Germany and Harvard University in the US – made use of negatively charged nitrogen-vacancy (NV) centres in diamonds. These occur when two adjacent carbon atoms in a carbon lattice are replaced by a nitrogen atom and a vacant site. The spin states of NV centres can be controlled and measured using light, and are also exquisitely sensitive to magnetic fields. Whereas the traditional coil detectors used in NMR spectroscopy and magnetic resonance imaging (MRI) require bulk samples, atomic-scale NV centres can be placed right next to molecules in "nano-NMR" experiments, which are becoming widespread. In 2016, the Harvard and Ulm researchers detected individual protein molecules on the surface of an NV-implanted diamond and even inferred some structural features by studying changes in the frequencies of the fields detected by the NV centres. To determine the structure of large molecules using nano-NMR requires even better spectral resolution to allow more precise measurement of the precession frequencies of nuclei, and thus their chemical environments. "The length of time over which you can sample a signal limits the resolution with which you can determine its spectrum," explains Kristian Cujia, a member of the ETH Zürich team. Unfortunately, the coherent quantum state of an NV centre collapses after a few microseconds because of environmental interactions. Such a short measurement carries significant uncertainty. Worse still, to improve the spatial resolution of diamonds, researchers often implant NV centres more densely or place them closer to the surface. This brings the NV centres closer to the sample, making them more sensitive to its magnetic field, but it also makes them less isolated, causing decoherence to occur more quickly, further reducing the spectral resolution. Researchers can improve the magnetic sensitivity of NV centres by simply making multiple measurements. As the errors on successive measurements are uncorrelated, the precision improves as more measurements are made. However, the spectral resolution does not improve with such repeated, uncorrelated measurements. The three teams have surmounted this problem by synchronizing repeated NV magnetic measurements to an external clock. This allows them to keep track of time even after decoherence occurs. "Normally, you would have to take your next measurement as an independent measurement," explains Ulm's Liam McGuinness. "When we did our next measurement, we already had a clock that was keeping track of time. That let us stitch together a sequence of measurements." Indeed, the researchers could make a measurement on an NV centre that could be monitored indefinitely, effectively eliminating the limitation of NV decoherence. All the groups were able to measure megahertz-scale frequencies with sub-millihertz precision – nearly a million times better than the spectral resolution of other NV measurement protocols. McGuinness and colleagues used their measurement protocol to perform NMR spectroscopy on a nanometre-sized sample of polybutene. However, the researchers encountered a problem: "Our molecules diffuse past our NV centre," explains McGuinness. This restricted the length of time the researchers could observe a single molecule, preventing them from obtaining a resolution better than about 1 kHz. The Harvard group, however, came up with a solution to this problem by getting the measurement protocol to work for ensembles of NV centres in the same diamond. This means that their sample volume is slightly larger (micron sized) and their measurements suffer much less from the effects of molecular diffusion. "With current technology, you can't use the synchronized readout technique usefully for high-spectral-resolution NMR at the nanoscale, because of the random fluctuation of the sample's spin polarization [which impedes coherent detection of the small NMR signal]," says Harvard's Ronald Walsworth. "At the micron scale you can." The Harvard researchers obtained resolutions as good as 3 Hz – nearly 100 times smaller than ever seen before in NMR using NV centres. They also observed many of the crucial features used to interpret NMR signals for the first time – including J-couplings. "That opens up a whole new world of micron-scale NMR – potentially for intracellular NMR, for example," says Walsworth. The next step, says Walsworth, will be to try to perform genuinely new science using NV-centre NMR. McGuinness says the new sensing protocol is a "general technique" and could find application well beyond NV centres and NMR. "We draw parallels to heterodyne, or beat-note, detection. If you have a weak laser and you want to measure its frequency, you take another very strong laser, join them together and measure the beat note. Here, instead of taking a classical laser, we take a quantum sensor." Theoretical physicist Andrew Jordan, who was not involved in the research, says that the ETH Zürich and Ulm University papers represent “a nice advance in this field...Maybe the most important parameter we have is frequency, because that sets the precision of our timekeeping devices. I think this is going to be an important technique going forward, if nothing else to calibrate people's systems before they go on to do other applications”. He declined to comment on the Harvard research because it has not yet been through the peer-review process. The ETH Zürich and Ulm University papers are published in Science. The Harvard research is described in a preprint on arXiv.


News Article | May 25, 2017
Site: www.eurekalert.org

A molecule produced by a Thai liver parasite could be the solution to those non-healing wounds Every day 12 Australian diabetics have a limb amputated because of a non-healing wound. Globally, it's one every 30 seconds. A molecule produced by a Thai liver parasite could be the solution to those non-healing wounds - and scientists from the Australian Institute of Tropical Health and Medicine (AITHM) are now able to produce a version of the molecule on a large enough scale to make it available for laboratory tests and eventually clinical trials. The molecule is granulin, one of a family of protein growth factors involved with cell proliferation. "It's produced by a parasitic liver fluke, Opisthorchis viverrini, which originally came to our attention because it causes a liver cancer that kills 26,000 people each year in Thailand," parasitologist Dr Michael Smout said. As part of their work on a potential vaccine to protect people from the parasite, Dr Smout and colleagues established that the granulin it produces has a hidden talent - it supercharges healing. "We realised the molecule, discovered in worm spit, could offer a solution for non-healing wounds, which are a problem for diabetics, smokers and the elderly," he said. With fellow researchers from the AITHM at James Cook University in Cairns, Dr Smout has been investigating ways to produce granulin in sufficient quantities for larger-scale testing. The team first tried recombinant DNA techniques, effectively inserting granulin into bacteria, with the aim of producing plentiful supplies of a reliable copy of the molecule. "Unfortunately, granulin didn't perform well when we introduced it to E. coli bacteria, so we couldn't use recombinant techniques to produce a testable supply," said Professor Norelle Daly, whose research involves exploring the potential of peptides as drug candidates for therapeutic applications. "We had to go back to the drawing board and find a way to synthesize part of the molecule - to build our own version of designer worm spit," she said. The researchers worked to establish which parts of the molecule were critical to wound healing, and to find a way to reproduce the active parts of granulin molecules (peptides). Nuclear Magnetic Resonance (NMR) spectroscopy revealed the molecule's complex shape: a string of amino acids bent into a twisted 3D shape that includes hairpin bends. "In biology the shape and fold of a molecule can be critical to its function," Dr Smout said. "Getting the fold right is important - it can be like the difference between throwing a well folded paper plane, or tossing a crumpled ball of paper." After testing different segments and structures, the team concluded that those hairpin bends were the key. "They're held in the twisted 3-D shape by disulfide bonds, and surprisingly we've found that by introducing an extra, non-native, bond we can produce peptides that hold the right shape to promote healing," Professor Daly said. "You could say we've found an extra fold that helps our peptide paper plane fly straight and target wounds." The lab-produced granulin peptides have shown great promise in tests, driving cell proliferation in human cells grown in lab plates, and demonstrating potent wound healing in mice. Now that they can mass-produce perfectly folded, wound-healing peptides, the researchers are looking for potential partners as they progress towards further testing and eventually clinical trials. "We have plenty of work to do before clinical trials, but we're confident we have a very strong contender for what could one day be a cream that a diabetic could apply at home, avoiding a lengthy hospital stay and possible amputation," said Professor Alex Loukas, whose work includes the investigation of hookworm proteins to treat autoimmune and allergic diseases. "A take-home cream would be a great step forward for those with chronic wounds, and it would also save our health system a great deal of money. "One in every seven diabetics in Australia will have a non-healing wound at some point, and many suffer amputations as a result. It's estimated the long hospital stays involved in treating chronic wounds cost our healthcare system AU$3.7 billion per year." The research is published in the latest edition of the Journal of Medicinal Chemistry.


News Article | May 26, 2017
Site: www.biosciencetechnology.com

Every day 12 Australian diabetics have a limb amputated because of a non-healing wound. Globally, it's one every 30 seconds. A molecule produced by a Thai liver parasite could be the solution to those non-healing wounds - and scientists from the Australian Institute of Tropical Health and Medicine (AITHM) are now able to produce a version of the molecule on a large enough scale to make it available for laboratory tests and eventually clinical trials. The molecule is granulin, one of a family of protein growth factors involved with cell proliferation. "It's produced by a parasitic liver fluke, Opisthorchis viverrini, which originally came to our attention because it causes a liver cancer that kills 26,000 people each year in Thailand," parasitologist Dr Michael Smout said. As part of their work on a potential vaccine to protect people from the parasite, Dr Smout and colleagues established that the granulin it produces has a hidden talent - it supercharges healing. "We realized the molecule, discovered in worm spit, could offer a solution for non-healing wounds, which are a problem for diabetics, smokers and the elderly," he said. With fellow researchers from the AITHM at James Cook University in Cairns, Dr Smout has been investigating ways to produce granulin in sufficient quantities for larger-scale testing. The team first tried recombinant DNA techniques, effectively inserting granulin into bacteria, with the aim of producing plentiful supplies of a reliable copy of the molecule. "Unfortunately, granulin didn't perform well when we introduced it to E. coli bacteria, so we couldn't use recombinant techniques to produce a testable supply," said Professor Norelle Daly, whose research involves exploring the potential of peptides as drug candidates for therapeutic applications. "We had to go back to the drawing board and find a way to synthesize part of the molecule - to build our own version of designer worm spit," she said. The researchers worked to establish which parts of the molecule were critical to wound healing, and to find a way to reproduce the active parts of granulin molecules (peptides). Nuclear Magnetic Resonance (NMR) spectroscopy revealed the molecule's complex shape: a string of amino acids bent into a twisted 3D shape that includes hairpin bends. "In biology the shape and fold of a molecule can be critical to its function," Dr Smout said. "Getting the fold right is important - it can be like the difference between throwing a well folded paper plane, or tossing a crumpled ball of paper." After testing different segments and structures, the team concluded that those hairpin bends were the key. "They're held in the twisted 3-D shape by disulfide bonds, and surprisingly we've found that by introducing an extra, non-native, bond we can produce peptides that hold the right shape to promote healing," Professor Daly said. "You could say we've found an extra fold that helps our peptide paper plane fly straight and target wounds." The lab-produced granulin peptides have shown great promise in tests, driving cell proliferation in human cells grown in lab plates, and demonstrating potent wound healing in mice. Now that they can mass-produce perfectly folded, wound-healing peptides, the researchers are looking for potential partners as they progress towards further testing and eventually clinical trials. "We have plenty of work to do before clinical trials, but we're confident we have a very strong contender for what could one day be a cream that a diabetic could apply at home, avoiding a lengthy hospital stay and possible amputation," said Professor Alex Loukas, whose work includes the investigation of hookworm proteins to treat autoimmune and allergic diseases. "A take-home cream would be a great step forward for those with chronic wounds, and it would also save our health system a great deal of money. "One in every seven diabetics in Australia will have a non-healing wound at some point, and many suffer amputations as a result. It's estimated the long hospital stays involved in treating chronic wounds cost our healthcare system AU$3.7 billion per year." The research is published in the latest edition of the Journal of Medicinal Chemistry.


A molecule produced by a Thai liver parasite could be the solution to those non-healing wounds - and scientists from the Australian Institute of Tropical Health and Medicine (AITHM) are now able to produce a version of the molecule on a large enough scale to make it available for laboratory tests and eventually clinical trials. The molecule is granulin, one of a family of protein growth factors involved with cell proliferation. "It's produced by a parasitic liver fluke, Opisthorchis viverrini, which originally came to our attention because it causes a liver cancer that kills 26,000 people each year in Thailand," parasitologist Dr Michael Smout said. As part of their work on a potential vaccine to protect people from the parasite, Dr Smout and colleagues established that the granulin it produces has a hidden talent - it supercharges healing. "We realised the molecule, discovered in worm spit, could offer a solution for non-healing wounds, which are a problem for diabetics, smokers and the elderly," he said. With fellow researchers from the AITHM at James Cook University in Cairns, Dr Smout has been investigating ways to produce granulin in sufficient quantities for larger-scale testing. The team first tried recombinant DNA techniques, effectively inserting granulin into bacteria, with the aim of producing plentiful supplies of a reliable copy of the molecule. "Unfortunately, granulin didn't perform well when we introduced it to E. coli bacteria, so we couldn't use recombinant techniques to produce a testable supply," said Professor Norelle Daly, whose research involves exploring the potential of peptides as drug candidates for therapeutic applications. "We had to go back to the drawing board and find a way to synthesize part of the molecule - to build our own version of designer worm spit," she said. The researchers worked to establish which parts of the molecule were critical to wound healing, and to find a way to reproduce the active parts of granulin molecules (peptides). Nuclear Magnetic Resonance (NMR) spectroscopy revealed the molecule's complex shape: a string of amino acids bent into a twisted 3D shape that includes hairpin bends. "In biology the shape and fold of a molecule can be critical to its function," Dr Smout said. "Getting the fold right is important - it can be like the difference between throwing a well folded paper plane, or tossing a crumpled ball of paper." After testing different segments and structures, the team concluded that those hairpin bends were the key. "They're held in the twisted 3-D shape by disulfide bonds, and surprisingly we've found that by introducing an extra, non-native, bond we can produce peptides that hold the right shape to promote healing," Professor Daly said. "You could say we've found an extra fold that helps our peptide paper plane fly straight and target wounds." The lab-produced granulin peptides have shown great promise in tests, driving cell proliferation in human cells grown in lab plates, and demonstrating potent wound healing in mice. Now that they can mass-produce perfectly folded, wound-healing peptides, the researchers are looking for potential partners as they progress towards further testing and eventually clinical trials. "We have plenty of work to do before clinical trials, but we're confident we have a very strong contender for what could one day be a cream that a diabetic could apply at home, avoiding a lengthy hospital stay and possible amputation," said Professor Alex Loukas, whose work includes the investigation of hookworm proteins to treat autoimmune and allergic diseases. "A take-home cream would be a great step forward for those with chronic wounds, and it would also save our health system a great deal of money. "One in every seven diabetics in Australia will have a non-healing wound at some point, and many suffer amputations as a result. It's estimated the long hospital stays involved in treating chronic wounds cost our healthcare system AU$3.7 billion per year." The research is published in the latest edition of the Journal of Medicinal Chemistry. More information: Paramjit S. Bansal et al. Development of a Potent Wound Healing Agent Based on the Liver Fluke Granulin Structural Fold, Journal of Medicinal Chemistry (2017). DOI: 10.1021/acs.jmedchem.7b00047


News Article | May 25, 2017
Site: www.sciencedaily.com

Every day 12 Australian diabetics have a limb amputated because of a non-healing wound. Globally, it's one every 30 seconds. A molecule produced by a Thai liver parasite could be the solution to those non-healing wounds -- and scientists from the Australian Institute of Tropical Health and Medicine (AITHM) are now able to produce a version of the molecule on a large enough scale to make it available for laboratory tests and eventually clinical trials. The molecule is granulin, one of a family of protein growth factors involved with cell proliferation. "It's produced by a parasitic liver fluke, Opisthorchis viverrini, which originally came to our attention because it causes a liver cancer that kills 26,000 people each year in Thailand," parasitologist Dr Michael Smout said. As part of their work on a potential vaccine to protect people from the parasite, Dr Smout and colleagues established that the granulin it produces has a hidden talent -- it supercharges healing. "We realised the molecule, discovered in worm spit, could offer a solution for non-healing wounds, which are a problem for diabetics, smokers and the elderly," he said. With fellow researchers from the AITHM at James Cook University in Cairns, Dr Smout has been investigating ways to produce granulin in sufficient quantities for larger-scale testing. The team first tried recombinant DNA techniques, effectively inserting granulin into bacteria, with the aim of producing plentiful supplies of a reliable copy of the molecule. "Unfortunately, granulin didn't perform well when we introduced it to E. coli bacteria, so we couldn't use recombinant techniques to produce a testable supply," said Professor Norelle Daly, whose research involves exploring the potential of peptides as drug candidates for therapeutic applications. "We had to go back to the drawing board and find a way to synthesize part of the molecule -- to build our own version of designer worm spit," she said. The researchers worked to establish which parts of the molecule were critical to wound healing, and to find a way to reproduce the active parts of granulin molecules (peptides). Nuclear Magnetic Resonance (NMR) spectroscopy revealed the molecule's complex shape: a string of amino acids bent into a twisted 3D shape that includes hairpin bends. "In biology the shape and fold of a molecule can be critical to its function," Dr Smout said. "Getting the fold right is important -- it can be like the difference between throwing a well folded paper plane, or tossing a crumpled ball of paper." After testing different segments and structures, the team concluded that those hairpin bends were the key. "They're held in the twisted 3-D shape by disulfide bonds, and surprisingly we've found that by introducing an extra, non-native, bond we can produce peptides that hold the right shape to promote healing," Professor Daly said. "You could say we've found an extra fold that helps our peptide paper plane fly straight and target wounds." The lab-produced granulin peptides have shown great promise in tests, driving cell proliferation in human cells grown in lab plates, and demonstrating potent wound healing in mice. Now that they can mass-produce perfectly folded, wound-healing peptides, the researchers are looking for potential partners as they progress towards further testing and eventually clinical trials. "We have plenty of work to do before clinical trials, but we're confident we have a very strong contender for what could one day be a cream that a diabetic could apply at home, avoiding a lengthy hospital stay and possible amputation," said Professor Alex Loukas, whose work includes the investigation of hookworm proteins to treat autoimmune and allergic diseases. "A take-home cream would be a great step forward for those with chronic wounds, and it would also save our health system a great deal of money. "One in every seven diabetics in Australia will have a non-healing wound at some point, and many suffer amputations as a result. It's estimated the long hospital stays involved in treating chronic wounds cost our healthcare system AU$3.7 billion per year." The research is published in the latest edition of the Journal of Medicinal Chemistry.


News Article | May 29, 2017
Site: cen.acs.org

Oxygen-17 NMR spectroscopy is one tool that chemists can use to study the structure and reactivity of various organic and inorganic compounds. But 17O’s low natural abundance—merely 0.04%—requires enriching samples with the isotope, a process that is often costly and time-consuming. One solution to the challenge of 17O labeling may be mechanical: Combine a reagent with a stoichiometric amount of 17O-enriched water and grind the mixture in a ball mill, suggests a team led by Danielle Laurencin of the Institut Charles Gerhardt Montpellier (Angew. Chem. Int. Ed. Engl. 2017, DOI: 10.1002/anie.201702251). Grinding reagents in a ball mill to induce reactions is a form of mechanochemistry that has gained popularity in recent years as a relatively quick and convenient way to make some organic and inorganic compounds. As the balls collide in the mill, effects such as shear stress and increased temperature may help stimulate chemistry at the interfaces between particles. Laurencin and colleagues produced 17O-enriched metal oxides by combining a metal hydroxide with less than two equivalents of 17O-enriched water, grinding the reagents for 30 minutes, then heating the material to convert it to the metal oxide. Enriching 60 mg of Mg(OH) or Ca(OH) to 17O levels suitable for solid-state NMR analyses cost the team about $10. For 17O NMR of organic compounds, the researchers focused on carboxylic acids, which frequently turn up in biomolecules and metal ligands, such as those in metal-organic frameworks. The researchers first ground the organic compounds with 1,1’-carbonyl-diimidazole to activate the carboxylic groups and then milled the material with 17O-enriched water. The whole procedure took less than two hours.


To understand the fundamental mechanism behind acid dissolution, Zhang et al. from the Institute for Chemical Research at Kyoto University encapsulated HF, as well as HF•H O and H O within a C fullerene. They found that in order to force the molecules into the open fullerene cavity, the molecules required "pushing from the outside" using high pressure conditions, and "pulling from the inside" via molecular interactions between HF and H O. They were able to identify how hydrogen bonding occurred between these two molecules. Their work appears in Science Advances. Prior work by Zhang et al. showed that the C fullerene could be opened in a three-step process that involved the addition of a pyridazine derivative either to the alpha or beta bonds on the C . This created a 13-member ring opening that formed slightly different compounds, denoted by α-13mem and β-13mem. Dehydration of both compounds resulted in a 16-member ring opening. The ring could be closed again via hydrolysis and a two-step process. β-16mem was large enough to capture H O, but α-16mem was not. Given these results from previous studies, for the current study, Zhang et al. used α-16mem to try to encapsulate HF. Instead, they found three different possibilities within the fullerenes: HF@C , (HF•H O)@C , and H O@C . Their reaction conditions required high pressure (9000 atm) to "push" the guest molecule into the α-16mem cavity. Time-dependent studies showed that HF filled the cavity first, followed by H O•HF, and then H O. Notably, the open cage did not entrap H O when HF was not present, indicating that the interaction between H O and HF prompted H O encapsulation. Further studies showed that HF is "pulling" H O into the cavity while the high pressure environment "pushes" it into the cavity. This process allowed the authors to study the interaction between H O and HF within a confined environment using 1H NMR. NMR analysis showed that that the (H O•HF)@C was down-shifted from H O@C and HF@C , which indicated hydrogen bonding. Furthermore, shift and coupling values indicated that oxygen was acting as the hydrogen-bond acceptor. Using single-crystal x-ray diffraction, Zhang et al. demonstrated the structure of the (HF•H O)@C , and report the first x-ray structure for doubly encapsulated C . These analyses and experimental studies confirmed that the H+ ion in HF forms a linear hydrogen bond with the O in H O. Additionally, compared to theoretical calculations of free H O and HF, the studies of the encapsulated molecules revealed close contact with hydrogen and oxygen that may be characteristic of H3O+•F-. The C fullerene derivative provides an excellent nanoenvironment for studying isolated chemical species, something that has not been available to chemists in the past. This isolated environment allowed the authors to investigate the interactions of two compounds without interference from the surrounding environment and provided important insights into a ubiquitous chemical process. More information: Rui Zhang et al. Isolation of the simplest hydrated acid, Science Advances (2017). DOI: 10.1126/sciadv.1602833 Abstract Dissociation of an acid molecule in aqueous media is one of the most fundamental solvation processes but its details remain poorly understood at the distinct molecular level. Conducting high-pressure treatments of an open-cage fullerene C70 derivative with hydrogen fluoride (HF) in the presence of H2O, we achieved an unprecedented encapsulation of H2O·HF and H2O. Restoration of the opening yielded the endohedral C70s, that is, (H2O·HF)@C70, H2O@C70, and HF@C70 in macroscopic scales. Putting an H2O·HF complex into the fullerene cage was a crucial step, and it would proceed by the synergistic effects of "pushing from outside" and "pulling from inside." The structure of the H2O·HF was unambiguously determined by single crystal x-ray diffraction analysis. The nuclear magnetic resonance measurements revealed the formation of a hydrogen bond between the H2O and HF molecules without proton transfer even at 140°C.

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