St Vincents Institute of Medical Research

The protein sequenator is an instrument for the automatic determination of amino acid sequences in proteins and peptides. It operates on the principle of the phenylisothiocyanate degradation scheme. The automated process embraces the formation of the phenylthiocarbamyl derivative of the protein and the splitting off of the N‐terminal amino acid as thiazolinone. The degradation proceeds at a rate of 15.4 cycles in 24 hours and with a yield in the individual cycle in excess of 98%. The material requirements are approximately 0.25 μmoles of protein. The thiazolinones are converted to the corresponding phenylthiohydantoins in a separate operation, and the latter identified by thin layer chromatography. The process has been applied to the whole molecule of apomyoglobin from the humpback whale, and it has been possible to establish the sequence of the first 60 amino acids from the N‐terminal end.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.

Motivation: Single-cell RNA sequencing (scRNA-seq) is increasingly used to study gene expression at the level of individual cells. However, preparing raw sequence data for further analysis is not a straightforward process. Biases, artifacts and other sources of unwanted variation are present in the data, requiring substantial time and effort to be spent on pre-processing, quality control (QC) and normalization. Results: We have developed the R/Bioconductor package scater to facilitate rigorous pre-processing, quality control, normalization and visualization of scRNA-seq data. The package provides a convenient, flexible workflow to process raw sequencing reads into a high-quality expression dataset ready for downstream analysis. scater provides a rich suite of plotting tools for single-cell data and a flexible data structure that is compatible with existing tools and can be used as infrastructure for future software development. Availability and Implementation: The open-source code, along with installation instructions, vignettes and case studies, is available through Bioconductor at http://bioconductor.org/packages/scater . Contact: davis@ebi.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

The conversion of an epithelial cell to a mesenchymal cell is critical to metazoan embryogenesis and a defining structural feature of organ development. Current interest in this process, which is described as an epithelial-mesenchymal transition (EMT), stems from its developmental importance and its involvement in several adult pathologies. Interest and research in EMT are currently at a high level, as seen by the attendance at the recent EMT meeting in Vancouver, Canada (October 1-3, 2005). The meeting, which was hosted by The EMT International Association, was the second international EMT meeting, the first being held in Port Douglas, Queensland, Australia in October 2003. The EMT International Association was formed in 2002 to provide an international body for those interested in EMT and the reverse process, mesenchymal-epithelial transition, and, most importantly, to bring together those working on EMT in development, cancer, fibrosis, and pathology. These themes continued during the recent meeting in Vancouver. Discussion at the Vancouver meeting spanned several areas of research, including signaling pathway activation of EMT and the transcription factors and gene targets involved. Also covered in detail was the basic cell biology of EMT and its role in cancer and fibrosis, as well as the identification of new markers to facilitate the observation of EMT in vivo. This is particularly important because the potential contribution of EMT during neoplasia is the subject of vigorous scientific debate (Tarin, D., E.W. Thompson, and D.F. Newgreen. 2005. Cancer Res. 65:5996-6000; Thompson, E.W., D.F. Newgreen, and D. Tarin. 2005. Cancer Res. 65:5991-5995).

Single-cell RNA sequencing (scRNA-seq) is widely used to profile the transcriptome of individual cells. This provides biological resolution that cannot be matched by bulk RNA sequencing, at the cost of increased technical noise and data complexity. The differences between scRNA-seq and bulk RNA-seq data mean that the analysis of the former cannot be performed by recycling bioinformatics pipelines for the latter. Rather, dedicated single-cell methods are required at various steps to exploit the cellular resolution while accounting for technical noise. This article describes a computational workflow for low-level analyses of scRNA-seq data, based primarily on software packages from the open-source Bioconductor project. It covers basic steps including quality control, data exploration and normalization, as well as more complex procedures such as cell cycle phase assignment, identification of highly variable and correlated genes, clustering into subpopulations and marker gene detection. Analyses were demonstrated on gene-level count data from several publicly available datasets involving haematopoietic stem cells, brain-derived cells, T-helper cells and mouse embryonic stem cells. This will provide a range of usage scenarios from which readers can construct their own analysis pipelines.

The strength and integrity of our bones depends on maintaining a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. As we age or as a result of disease, this delicate balancing act becomes tipped in favor of osteoclasts so that bone resorption exceeds bone formation, rendering bones brittle and prone to fracture. A better understanding of the biology of osteoclasts and osteoblasts is providing opportunities for developing therapeutics to treat diseases of bone. Drugs that inhibit the formation or activity of osteoclasts are valuable for treating osteoporosis, Paget's disease, and inflammation of bone associated with rheumatoid arthritis or periodontal disease. Far less attention has been paid to promoting bone formation with, for example, growth factors or hormones, an approach that would be a valuable adjunct therapy for patients receiving inhibitors of bone resorption.

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy demand is matched to energy supply. The AMP-activated protein kinase (AMPK) alphabetagamma heterotrimer has emerged as an important integrator of signals that control energy balance through the regulation of multiple biochemical pathways in all eukaryotes. In this review, we begin with the discovery of the AMPK family and discuss the recent structural studies that have revealed the molecular basis for AMP binding to the enzyme's gamma subunit. AMPK's regulation involves autoinhibitory features and phosphorylation of both the catalytic alpha subunit and the beta-targeting subunit. We review the role of AMPK at the cellular level through examination of its many substrates and discuss how it controls cellular energy balance. We look at how AMPK integrates stress responses such as exercise as well as nutrient and hormonal signals to control food intake, energy expenditure, and substrate utilization at the whole body level. Lastly, we review the possible role of AMPK in multiple common diseases and the role of the new age of drugs targeting AMPK signaling.

The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.

MOTIVATION: Mutations play fundamental roles in evolution by introducing diversity into genomes. Missense mutations in structural genes may become either selectively advantageous or disadvantageous to the organism by affecting protein stability and/or interfering with interactions between partners. Thus, the ability to predict the impact of mutations on protein stability and interactions is of significant value, particularly in understanding the effects of Mendelian and somatic mutations on the progression of disease. Here, we propose a novel approach to the study of missense mutations, called mCSM, which relies on graph-based signatures. These encode distance patterns between atoms and are used to represent the protein residue environment and to train predictive models. To understand the roles of mutations in disease, we have evaluated their impacts not only on protein stability but also on protein-protein and protein-nucleic acid interactions. RESULTS: We show that mCSM performs as well as or better than other methods that are used widely. The mCSM signatures were successfully used in different tasks demonstrating that the impact of a mutation can be correlated with the atomic-distance patterns surrounding an amino acid residue. We showed that mCSM can predict stability changes of a wide range of mutations occurring in the tumour suppressor protein p53, demonstrating the applicability of the proposed method in a challenging disease scenario. AVAILABILITY AND IMPLEMENTATION: A web server is available at http://structure.bioc.cam.ac.uk/mcsm.

BONE is a complex tissue in which resorption and formation continue throughout life. This process is called bone remodeling. Osteotropic hormones such as 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], PTH, and calcitonin preferentially modulate the process of bone resorption to maintain bone remodeling. The bone tissue contains various types of cells, of which the bone-forming osteoblasts and bone-resorbing osteoclasts are mainly responsible for bone remodeling. Osteoblasts are believed to be derived from undifferentiated mesenchymal cells, which further differentiate into osteocytes and are embedded in calcified tissues. Osteoclasts are multinucleated cells present only in bone. It is believed that osteoclast progenitors are of hemopoietic origin, and they are recruited from hemopoietic tissues such as bone marrow and circulating blood to bone. Osteoclast progenitors then proliferate and differentiate into mononuclear preosteoclasts and fuse with each other to form multinucleated osteoclasts. Osteoclasts have a unique morphology and function to resorb calcified bone by making resorption pits (Howship's lacunae). Because of the inaccessibility and fragility of osteoclasts studies on their function have been hampered. Furthermore, it is extremely difficult to obtain a large number of mammalian osteoclasts.

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKαThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB-/- mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), treatment activates AMPK by αThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase,arelargelyinhibitedbytheCa2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKα and CaMKKβ. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, αThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKα and CaMKKβ. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1-/- murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo. The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKαThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB-/- mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), treatment activates AMPK by αThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase,arelargelyinhibitedbytheCa2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKα and CaMKKβ. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, αThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKα and CaMKKβ. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1-/- murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo. The AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKK, AMPK kinase; MEF, mouse embryo fibroblast; ACC, acetyl-CoA carboxylase; CaM, calmodulin; CaMKK, CaM-dependent protein kinase kinase; siRNA, small interfering RNA; HRP, horseradish peroxidase; ANOVA, analysis of variance; 2-DG, 2-deoxyglucose; MAP, mitogen-activated protein; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside. regulates many aspects of cellular metabolism, especially in response to metabolic stress (1Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (729) Google Scholar). AMPK is a serine/threonine protein kinase and a member of the Snf1/AMPK protein kinase family (1Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (729) Google Scholar). It is an αβγ heterotrimeric protein, consisting of an α catalytic subunit, a β subunit important both for enzyme activity and for targeting, and a γ regulatory subunit, which binds the allosteric activator, AMP. The activity of AMPK absolutely requires phosphorylation of the α subunit on Thr-172 in its activation loop by one or more upstream kinases (AMPKK) (1Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (729) Google Scholar). The major breakthrough in identifying AMPK upstream kinases came from the study of the regulation of the AMPK ortholog, Snf1, in Saccharomyces cerevisiae, in which Pak1 was shown to act as a Snf1p kinase kinase (2Nath N. McCartney R.R. Schmidt M.C. Mol. Cell. Biol. 2003; 23: 3909-3917Crossref PubMed Scopus (126) Google Scholar). Subsequently, it was shown that three closely related kinases, Pak1p, Tos3p, and Elm1p, needed to be deleted to generate the Snf1- phenotype (3Hong S.P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 4Sutherland C.M. Hawley S.A. McCartney R.R. Leech A. Stark M.J. Schmidt M.C. Hardie D.G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Sequence comparison revealed that the human LKB1 tumor suppressor kinase was the most closely related mammalian kinase. LKB1 was subsequently identified by several groups as being an important upstream kinase active on AMPK (5Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 6Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). Several lines of evidence point to the presence of non-LKB1 AMPKKs. Multiple AMPKK activities are separable during chromatography of extracts from rodent heart (8Altarejos J.Y. Taniguchi M. Clanachan A.S. Lopaschuk G.D. J. Biol. Chem. 2005; 280: 183-190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Baron S.J. Li J. Russell III, R.R. Neumann D. Miller E.J. Tuerk R. Wallimann T. Hurley R.L. Witters L.A. Young L.H. Circ. Res. 2005; 96: 337-345Crossref PubMed Scopus (90) Google Scholar). Murine fibroblasts obtained from LKB1-/- embryos by two different groups still demonstrate residual AMPKT172α phosphorylation and AMPK activity, albeit not as responsive to the usual activators of AMPK, such as the nucleoside AICAR (5Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). Partially purified Ca2+/CaM-dependent protein kinase kinase (CaMKK) from pig brain has been shown to be active in vitro on AMPK, but it was concluded that the kinetics of phosphorylation by CaMKK were weaker than those for a partially purified AMPKK and that CaMKKs were unlikely to function as AMPKKs in intact cells and tissues (10Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Although this view has been widely accepted (28Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar), Nath et al. (2Nath N. McCartney R.R. Schmidt M.C. Mol. Cell. Biol. 2003; 23: 3909-3917Crossref PubMed Scopus (126) Google Scholar) suggested that CaMKKβ may be an AMPKK based on homology with yeast PAK1. Recently, CaMKKα has been shown directly to function as a Snf1-activating kinase in yeast cells lacking the three Snf-activating kinases, Pak1, Tos3, and Elm1 (29Hong S.-P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The protein products of the CaMKK gene family, CaMKKα and CaMKKβ, show significant homology to LKB1 and to the three aforementioned yeast kinases (3Hong S.P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 4Sutherland C.M. Hawley S.A. McCartney R.R. Leech A. Stark M.J. Schmidt M.C. Hardie D.G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 5Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar). In the present study, we have investigated the possibility that one or both CaMKKs might serve as AMPKKs to regulate AMPK in cell lines lacking expression of LKB1. Cell Culture and Incubations—Panc-1, AsPC-1, and COS cells were purchased from ATCC. Mouse embryo fibroblasts (MEFs) from LKB1+/+ and LKB1-/- mice and HeLa cells were kindly provided by Reuben Shaw (Harvard University). These cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. A549 cells (ATCC) were grown in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were incubated in 6-well plates after various additions (see the figure legends) prior to extraction for immunoblotting and analysis of AMPK activity. Preparation of Cell Extracts—Cell extracts were prepared by three different methods. For analysis of AMPK activity, digitonin lysis followed by ammonium sulfate precipitation was employed, as in Ref. 11Witters L.A. Kemp B.E. J. Biol. Chem. 1992; 267: 2864-2867Abstract Full Text PDF PubMed Google Scholar. For immunoblotting of total cellular protein, cells were lysed either in a Triton X-100-containing buffer, as in Ref. 12Hamilton S.R. O'Donnell Jr., J.B. Hammet A. Stapleton D. Habinowski S.A. Means A.R. Kemp B.E. Witters L.A. Biochem. Biophys. Res. Commun. 2002; 293: 892-898Crossref PubMed Scopus (55) Google Scholar, or in an SDS-containing buffer. For the latter, cells were rinsed with phosphate-buffered saline (2×) and lysed directly on the plate with boiling SDS buffer (1% SDS, 100 mm NaCl, 10 mm Tris-HCl, pH 7.5). These extracts were then sheared with a 25-gauge needle and boiled for 5 min. Protein concentration in all extracts was determined with a BCA assay (Pierce), according to the manufacturer's protocol. Enzyme Activities and Immunoblotting—AMPK activity against the SAMS peptide was determined at a saturating concentration of AMP, as in Ref. 11Witters L.A. Kemp B.E. J. Biol. Chem. 1992; 267: 2864-2867Abstract Full Text PDF PubMed Google Scholar. AMPKK activity in Triton X-100 cell lysates was determined by phosphorylation of a recombinant AMPKα protein, as in Ref. 12Hamilton S.R. O'Donnell Jr., J.B. Hammet A. Stapleton D. Habinowski S.A. Means A.R. Kemp B.E. Witters L.A. Biochem. Biophys. Res. Commun. 2002; 293: 892-898Crossref PubMed Scopus (55) Google Scholar. Cell extracts were examined by immunoblotting, as in Ref. 12Hamilton S.R. O'Donnell Jr., J.B. Hammet A. Stapleton D. Habinowski S.A. Means A.R. Kemp B.E. Witters L.A. Biochem. Biophys. Res. Commun. 2002; 293: 892-898Crossref PubMed Scopus (55) Google Scholar, employing a panel of different antibodies/reagents. These included anti-AMPK total α (reactive against α and α2), anti-AMPKαT172p, anti-ACCS79p, streptavidin-HRP (13Hamilton S.R. Stapleton D. O'Donnell Jr., J.B. Kung J.T. Dalal S.R. Kemp B.E. Witters L.A. FEBS Lett. 2001; 500: 163-168Crossref PubMed Scopus (96) Google Scholar), anti-CaMKKα/β (C-terminal; BD Transduction Laboratories catalog number 610544), and anti-LKB1 (a kind gift from Reuben Shaw and Ronald DePhino (Harvard University)) (7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). RNA Interference—siRNA oligonucleotides against a scrambled, non-targeting sequence as a negative control (D-001206-13-20) and CaMKKβ (human CaMKK2, SiGENOME SMARTpool reagent M-004842-00-0050, accession number NM_006549) were obtained from Dharmacon Research (Lafeyette, CO). siRNA oligonucleotides designed against CaMKKα (siRNA Gene Silencers, human) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HeLa cells were plated at a density of 5 × 105 cells/well in a 6-well plate and allowed to adhere overnight. Cells were transfected with a non-targeting control siRNA pool (200 nm), CaMKKβ siRNA pool (100 nm), or CaMKKα siRNA (200 nm). Preliminary studies established that the latter two were employed at their maximally effective concentrations (data not shown). Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 48 h after transfection, cells were either harvested or treated with AMPK activators and then harvested. Preparation of LKB1/STRAD/Mo25 Complex—COS cells were triply transfected with cDNAs expressing glutathione S-transferase-tagged LKB1 and FLAG-tagged STRAD and Mo25 (generous gifts from Reuben Shaw (Harvard University) and Dario Alessi (University of Dundee)) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were lysed in buffer containing 1% Triton X-100, as described above, 48 h after transfection. Cleared supernatants were aliquoted into microcentrifuge tubes (1 ml/tube) and incubated with 100 μl of 50% glutathione bead slurry (Sigma) for 2 h at 4 °C. Lysates were spun down briefly (20 to The was and were with buffer containing 1% Triton X-100 and then subsequently two with assay buffer were then in assay buffer containing mm glutathione (Sigma) to the enzyme a on are spun down for at to and the containing the purified LKB1/STRAD/Mo25 was and used in the in vitro kinase kinase analysis of data were by a with using the significant by the In an to AMPKKs from LKB1, two human cell lines that are deficient in LKB1 were for study, the HeLa and the A549 LKB1 is not in these two lines in LKB1-/- mouse embryo fibroblasts by immunoblotting We expression of LKB1 in other human lines and AsPC-1, the latter to be S. S. A. 2003; PubMed Google Scholar). HeLa and A549 cells were with a variety of known to AMPK by phosphorylation of and with ionomycin, the latter to 2-deoxyglucose and ionomycin, but not AICAR, both AMPK activity and phosphorylation in HeLa In A549 cells, and 2-deoxyglucose but not AICAR (data not AMPK activity and phosphorylation The of AICAR to AMPK in HeLa cells and in LKB1-/- mouse embryo fibroblasts has been (5Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). The indicate the presence of in HeLa and A549 cells from LKB1. other mammalian protein kinases have a significant homology to mammalian LKB1 and to the LKB1 in S. cerevisiae, Ca2+/CaM-dependent protein kinase kinase α and Ca2+/CaM-dependent protein kinase kinase β M.A. Goldstein E.G. Means A.R. Edelman A.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. Selbert M.A. Stapleton D. Goldstein E.G. Means A.R. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Means R.L. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Means A.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, J.T. Chem. 2001; 101: PubMed Scopus Google Scholar, Means A.R. Edelman A.M. Selbert M.A. Stapleton D. Goldstein E.G. Kemp B.E. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). CaMKK from pig brain prior to the that were two of the has been shown to and AMPK in this phosphorylation is by the of to the AMPK (10Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). both CaMKKα and CaMKKβ are in HeLa cells not in murine L. K. A. J. M. A. R. and L. A. were for study of the roles for these M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). the that one or both CaMKKs in HeLa cells as an AMPKK, HeLa cells were incubated or were with mannitol, 2-DG, or ionomycin in the presence of the CaMKK inhibitor, STO-609 M. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). At a concentration of STO-609 AMPK activation and phosphorylation in response to all three in HeLa cells The of of STO-609 was in the ionomycin-stimulated cells STO-609 totally the activation and phosphorylation of AMPK in response to and 2-DG, this might that these cells residual LKB1 by or STO-609 inhibited AMPK activation in response to and in A549 cells activation by of AMPK phosphorylation and phosphorylation of a downstream of AMPK, acetyl-CoA carboxylase were inhibited by STO-609 in HeLa cells at concentrations of mm The IC50 for STO-609 of phosphorylation in HeLa cells was μg/ml (data not shown). In other (data not we have that STO-609 the in vitro activity of purified AMPK against the SAMS the IC50 is than that for the we the possibility that of the STO-609 of phosphorylation in intact cells is to the of AMPK, the concentrations we employed in these intact cells studies was is not an AMPK effects of STO-609 be for by of CaMKKα activity is dependent upon and CaM, CaMKKβ has activity in its it be by a in M.A. Goldstein E.G. Means A.R. Edelman A.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. Selbert M.A. Stapleton D. Goldstein E.G. Means A.R. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Means R.L. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Means A.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an of and a R. Biochem. J. PubMed Scopus Google Scholar). In HeLa cells, the in AMPK activity and phosphorylation in response to ionomycin might be to a of either of the In of this STO-609 the of ionomycin to these in AMPK AMPKK activity, measured in HeLa cell lysates against a recombinant AMPKα in was inhibited by STO-609 The IC50 for this in vitro STO-609 was μg/ml (data not shown). IC50 is comparable with that for the of recombinant CaMKKα and CaMKKβ phosphorylation of a kinase protein in vitro M. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of STO-609 against several other protein kinases kinases and kinase, protein kinase protein kinase, and 10 its and as a CaMKK M. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we have that at these STO-609 not the activity of a LKB1/STRAD/Mo25 against the recombinant AMPKα in vitro the of this in the roles of the CaMKKs and LKB1 as AMPKKs in various tissues or cell specific for the the two CaMKK isoforms, CaMKKα and HeLa cells have been to both CaMKKα and CaMKKβ M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). The CaMKKβ gene several of as a of of the M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar, G.D. J.Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the of the protein products kinase activity, and have not been In HeLa cells, the and are two CaMKK and have been M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). with a that a sequence to CaMKKα and CaMKKβ, HeLa cell extracts two to a CaMKKα and at the of and M. R. FEBS Lett. 2003; PubMed Scopus Google Scholar). the possibility that the effects of STO-609 to AMPK activity were not by specific of the we employed RNA as an to their activity. with transfected non-targeting siRNA, siRNA substantially and AMPK activity and phosphorylation, siRNA but still effects on these AMPK significant effects of the on phosphorylation are and this is by the residual AMPK activity We have that the phosphorylation of in other is to small in AMPK activity S. M. Witters L.A. Kemp B.E. 2003; PubMed Scopus Google Scholar). The of both CaMKK both and AMPK activity and and phosphorylation and reduced ionomycin-stimulated AMPK activity siRNA the and the on immunoblotting, siRNA the that the in HeLa extracts is a of and CaMKKα and that the is We have been to the of a CaMKKα (Santa Cruz Biotechnology, to more the of the The have effects on either or total total AMPKα is in the presence of both The all of the CaMKK with a of AMPK activity and phosphorylation of CaMKKs AMPK activation by HeLa cells were transfected with against a non-targeting CaMKKα as or CaMKKβ as and incubated for 48 Cells were then incubated in the presence or of ionomycin for 5 and harvested by digitonin were against the SAMS peptide as described to AMPK kinase activity. These 4 lysates at are as of AMPK activity as of into the SAMS peptide of activity, and ionomycin-stimulated activity. determined by the of AMPK activity by both is significant at and that of the of the by ionomycin by siRNA is significant at For the data for the effects of the roles of LKB1 and the CaMKKs in a cell in which all three kinases are LKB1+/+ were with ionomycin and in the presence and of STO-609. and AMPK activity was then with that in LKB1-/- cells We and have that AICAR, and to AMPK in LKB1-/- (5Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28-37Crossref PubMed Google Scholar, 7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar). of AMPK and phosphorylation in response to ionomycin and is not impaired in LKB1-/- as with LKB1+/+ the response to ionomycin to be in the of STO-609 partially the response to ionomycin in both cell its to AMPK activation is in the LKB1-/- These data revealed that AMPK activation is one or both the response may be to by either LKB1 or the In the cells, of CaMKKs might be to that of LKB1. We of the of AMPKKs in either cell The data evidence that both CaMKKs function as AMPKKs in the cell lines for non-LKB1 AMPKKs in other tissues and cell Although and from both CaMKKα and CaMKKβ have a expression in rodent tissues A.M. Selbert M.A. Stapleton D. Goldstein E.G. Means A.R. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Means R.L. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Means A.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. M. N. N. Res. Mol. Res. 2003; PubMed Scopus Google Scholar), important roles for in the regulation of AMPK activity in vivo. The data indicate that both cellular and may or roles in the regulation of AMPK activity. the of three mammalian AMPKKs, be to in their to AMPK regulation in other tissues and cell Although LKB1 may be the AMPKK regulating AMPK to and in murine K. and D. R. Scholar), it expression of these AMPKKs, that may be in the regulation of AMPK both and dependent on the of the In data from yeast indicate that the different β of the kinase for Pak1, Tos3, and Elm1 upstream kinases R.R. Schmidt M.C. Curr. 2005; PubMed Scopus Google Scholar). it might be that different AMPK of of different catalytic α and and and might be by the three mammalian AMPKKs, the of regulation of AMPK activity in vivo. We the and the of from Reuben Shaw and Ronald DePinho (Harvard and Dario Alessi (University of with the with

Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1(E861A), where E861A denotes Glu(861)→Ala(861)). Adar1(E861A/E861A) embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)-sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3' untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1(E861A/E861A) were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

Cancer genome and other sequencing initiatives are generating extensive data on non-synonymous single nucleotide polymorphisms (nsSNPs) in human and other genomes. In order to understand the impacts of nsSNPs on the structure and function of the proteome, as well as to guide protein engineering, accurate in silicomethodologies are required to study and predict their effects on protein stability. Despite the diversity of available computational methods in the literature, none has proven accurate and dependable on its own under all scenarios where mutation analysis is required. Here we present DUET, a web server for an integrated computational approach to study missense mutations in proteins. DUET consolidates two complementary approaches (mCSM and SDM) in a consensus prediction, obtained by combining the results of the separate methods in an optimized predictor using Support Vector Machines (SVM). We demonstrate that the proposed method improves overall accuracy of the predictions in comparison with either method individually and performs as well as or better than similar methods. The DUET web server is freely and openly available at http://structure.bioc.cam.ac.uk/duet.

Although interleukin-6 (IL-6) has been associated with insulin resistance, little is known regarding the effects of IL-6 on insulin sensitivity in humans in vivo. Here, we show that IL-6 infusion increases glucose disposal without affecting the complete suppression of endogenous glucose production during a hyperinsulinemic-euglycemic clamp in healthy humans. Because skeletal muscle accounts for most of the insulin-stimulated glucose disposal in vivo, we examined the mechanism(s) by which IL-6 may affect muscle metabolism using L6 myotubes. IL-6 treatment increased fatty acid oxidation, basal and insulin-stimulated glucose uptake, and translocation of GLUT4 to the plasma membrane. Furthermore, IL-6 rapidly and markedly increased AMP-activated protein kinase (AMPK). To determine whether the activation of AMPK mediated cellular metabolic events, we conducted experiments using L6 myotubes infected with dominant-negative AMPK alpha-subunit. The effects described above were abrogated in AMPK dominant-negative-infected cells. Our results demonstrate that acute IL-6 treatment enhances insulin-stimulated glucose disposal in humans in vivo, while the effects of IL-6 on glucose and fatty acid metabolism in vitro appear to be mediated by AMPK.

Metformin is one of the most widely prescribed therapeutics for type 2 diabetes. But exactly how it works is still unclear. Gregory Steinberg and colleagues now show that it does so by activation of the enzyme AMP-activated protein kinase (Ampk) and Ampk's obligate targeting of two key enzymes involved in lipid homeostasis. The obesity epidemic has led to an increased incidence of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP-activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed type 2 diabetes drug metformin1,2. Ampk phosphorylates mouse acetyl-CoA carboxylase 1 (Acc1; refs. 3,4) at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl-CoA to malonyl-CoA. The latter metabolite is a precursor in fatty acid synthesis5 and an allosteric inhibitor of fatty acid transport into mitochondria for oxidation6. To test the physiological impact of these phosphorylation events, we generated mice with alanine knock-in mutations in both Acc1 (at Ser79) and Acc2 (at Ser212) (Acc double knock-in, AccDKI). Compared to wild-type mice, these mice have elevated lipogenesis and lower fatty acid oxidation, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Notably, AccDKI mice made obese by high-fat feeding are refractory to the lipid-lowering and insulin-sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism and, in the setting of obesity, for metformin-induced improvements in insulin action.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

<ns4:p>Single-cell RNA sequencing (scRNA-seq) is widely used to profile the transcriptome of individual cells. This provides biological resolution that cannot be matched by bulk RNA sequencing, at the cost of increased technical noise and data complexity. The differences between scRNA-seq and bulk RNA-seq data mean that the analysis of the former cannot be performed by recycling bioinformatics pipelines for the latter. Rather, dedicated single-cell methods are required at various steps to exploit the cellular resolution while accounting for technical noise. This article describes a computational workflow for low-level analyses of scRNA-seq data, based primarily on software packages from the open-source Bioconductor project. It covers basic steps including quality control, data exploration and normalization, as well as more complex procedures such as cell cycle phase assignment, identification of highly variable and correlated genes, clustering into subpopulations and marker gene detection. Analyses were demonstrated on gene-level count data from several publicly available data sets involving haematopoietic stem cells, brain-derived cells, T-helper cells and mouse embryonic stem cells. This will provide a range of usage scenarios from which readers can construct their own analysis pipelines.</ns4:p>

Single-cell RNA sequencing (scRNA-seq) has broad applications across biomedical research. One of the key challenges is to ensure that only single, live cells are included in downstream analysis, as the inclusion of compromised cells inevitably affects data interpretation. Here, we present a generic approach for processing scRNA-seq data and detecting low quality cells, using a curated set of over 20 biological and technical features. Our approach improves classification accuracy by over 30 % compared to traditional methods when tested on over 5,000 cells, including CD4+ T cells, bone marrow dendritic cells, and mouse embryonic stem cells.