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.

I. Introduction II. Role of Osteoblasts/Stromal Cells in Osteoclast Differentiation and Function A. Origin of osteoclasts B. Stimulation of osteoclast differentiation by osteoblasts/stromal cells C. Stimulation of osteoclast function by osteoblasts/stromal cells III. New Members of the Tumor Necrosis Factor (TNF) Receptor and Ligand Families A. Osteoprotegerin (OPG) B. Osteoclast differentiation factor (ODF) and stromal osteoclast-forming activity (SOFA) IV. Regulatory Mechanism in Osteoclast Development and Function A. Regulatory mechanism of osteoclast differentiation by RANKL B. Regulatory mechanism of RANKL action on osteoclast function C. Signals induced by interleukin-1 (IL-1) and RANKL in osteoclasts V. Regulation of Human Osteoclast Development VI. Summary and Conclusion OSTEOCLASTS, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. During the past decade, several new approaches have been developed to investigate osteoclast biology. A coculture system of mouse osteoblasts/stromal cells and hemopoietic cells for osteoclast formation has established the concept that osteoblasts/stromal cells are crucially involved in osteoclast development. Cell-to-cell contact between cells of the osteoblast lineage and hemopoietic cells is necessary for inducing differentiation of osteoclasts. It has been proposed that osteoblasts/stromal cells express osteoclast differentiation factor (ODF) or stromal osteoclast forming activity (SOFA) as a membrane-associated factor in response to several osteotropic factors such as 1α,25-dihydroxyvitamin D3[ 1α,25(OH)2D3], PTH, and interleukin 11 (IL-11). Osteoclast precursors of the monocyte-macrophage lineage recognize ODF/SOFA through cell-to-cell interaction with osteoblasts/stromal cells, and then differentiate into osteoclasts. Osteoblasts/stromal cells also play an essential role in the activation of osteoclast function. We emphasize that the term“ osteoblasts/stromal cells” is an operational one, used for convenience to describe those cells of the osteoblast lineage that have been shown convincingly in vitro to determine osteoclast formation. It is not certain in vivo which members of the lineage cells are responsible. In vitro data suggest that the osteoblast property is progressively lost with maturation of the osteoblast lineage cells, and in vivo, it is not at all likely that mature, synthesizing osteoblasts make any contribution to osteoclast formation. Nor are osteocytes likely to do so, but likely potential contributors are lining cells and early members of the osteoblast lineage that are situated close to the endosteal surface. Ultimately, the process of osteoclast formation is dependent on hemopoietic precursors being presented to the appropriate osteoblasts/stromal cells in an environment that provides appropriate stimulatory factors. Recently, three laboratories independently cloned cDNAs encoding the identical proteins, giving it the names osteoprotegerin (OPG), osteoclastogenesis inhibitory factor (OCIF), and tumor necrosis factor (TNF) receptor-like molecule 1 (TR1). This protein inhibits osteoclast development in vitro and in vivo. In an attempt to adopt a uniform nomenclature for this important biological activity, we propose that the name of choice be “osteoprotegerin.” OPG is a member of the TNF receptor family, but it does not have a transmembrane domain and possesses a signal sequence, suggesting that OPG functions as a secreted factor. Since OPG has the capacity to limit osteoclast formation, the ligand for this receptor was proposed to be the long-sought-after ODF/SOFA. Indeed, this hypothesis dictated the experiments carried out by the groups who subsequently identified a membrane-bound TNF-like ligand with the capacity to differentiate hematopoietic cells into functional osteoclasts. cDNA libraries from cell lines, which expressed specific binding sites for OPG, were screened by expression cloning approaches. As expected, the binding molecule of OPG was a membrane-associated protein of the TNF ligand family, which satisfied all the criteria of ODF/SOFA. In addition, ODF/SOFA was also able to maintain osteoclasts that had been induced by osteoblasts/stromal cells in an activated state. The discovery of this differentiation factor now opens a new era to investigate the molecular mechanism of osteoclast development and function. This review article describes the role of osteoblasts/stromal cells in osteoclast development and function at a molecular level, especially focusing on the central role of members of the TNF receptor and ligand superfamilies. Because discoveries in this area have originated from several directions and by different research groups, nomenclature has rapidly become confusing; thus, we propose an approach to overcome this. Development of osteoclasts proceeds within the local microenvironment of bone (1–4). This process can be replicated ex vivo using the coculture of mouse calvarial osteoblasts and spleen cells (5–9). Multinucleated cells formed in such cocultures satisfy the major criteria of osteoclasts such as tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) activity, calcitonin receptors, p60c-src, vitronectin receptors (αvβ3), and the ability to form resorption pits on bone and dentine slices. Some mouse stromal cell lines such as MC3T3-G2/PA6 and ST2 resemble calvarial osteoblasts and support osteoclastogenesis in coculture with mouse spleen cells (10). Experiments on the osteopetrotic op/op mouse model have established that an osteoblast/stromal cell product, macrophage colony-stimulating factor (M-CSF, also called CSF-1), is crucial for osteoclast formation. The M-CSF gene of op/op mice cannot code functionally active M-CSF protein due to an extra thymidine insertion in the coding region of the M-CSF gene (11, 12). Administration of recombinant human M-CSF restored impaired bone resorption in op/op mice (13, 14). Calvarial osteoblasts obtained from op/op mice failed to support osteoclast development in cocultures with normal spleen cells, but the addition of M-CSF to cocultures induced osteoclast formation in response to 1α,25(OH)2D3 (15–17). These findings indicate that M-CSF produced by osteoblasts/stromal cells plays an essential role in osteoclast development. After identification of the hemopoietic origin of osteoclasts, much attention has been focused on the cell lineages of osteoclast progenitors. Using ST2 cells as a stromal supportive cell line, it was shown that, in addition to spleen cells and bone marrow cells, peripheral blood mononuclear cells and alveolar macrophages acted as a source of osteoclast precursors (18). Chambers et al. (19) have established cell lines that express macrophage from et al. have also established the cell from a These cell lines into osteoclasts were with stromal cells in the of that osteoclasts are from cells of the et al. have shown that osteoclasts formed from of and macrophage the in human marrow et al. have that mouse cells also form osteoclasts in the These indicate that osteoclast precursors are from cells in the monocyte-macrophage with as the the of the gene and gene in mice the origin of osteoclasts. of the to osteopetrotic in bone by a in osteoclast of normal bone marrow cells into mice the In addition, the of and macrophages was in of suggesting that the of the gene a lineage between osteoclast and macrophage differentiation In cocultures of osteoblasts and spleen cells, spleen cells failed to differentiate into osteoclasts. is a and and mice were to be osteopetrotic The development of osteoclasts and macrophages was in The osteopetrotic of mice was by of normal bone marrow cells into the The of macrophages and osteoclasts in mice that this factor the of These support the that osteoclasts are from cells of the the mechanism by which osteoclast and the is not at In the coculture cell-to-cell contact between osteoblasts/stromal cells and hemopoietic cells was to be for osteoclast development the osteoblasts/stromal cells have been identified as the cells for osteotropic and M-CSF to osteoclast development. activity a cell receptor that of a receptor and a but protein A which transmembrane and was to the through in response to recombinant induced osteoclast formation in the but osteoclasts were formed in response to in the of This that a by is involved in osteoclast development. such as and inhibitory which through also induced osteoclast formation in coculture experiments in mice human the expression of in osteoblasts was shown to be for of osteoclast osteoblasts obtained from human mice were with normal spleen cells, osteoclast formation was induced in response to human human This that that as a signal on osteoblasts/stromal cells but not on osteoclast to osteoclast formation. are present in mice of the of as of the major used to osteoclast formation is with the for this being The cells of in inducing osteoclasts are also osteoblasts/stromal cells but not osteoclast in the of the human cell were established to human protein receptor a cell lines, and which expressed functional recombinant osteoclast formation in response to in the coculture with mouse spleen cells, the cells not of the for to be expressed on the osteoblast was using cocultures established between osteoblasts and spleen cells from normal and mice It was shown that osteoclasts were formed in response to in cocultures of spleen cells obtained from mice and normal calvarial osteoblasts These indicate that the expression of in osteoblasts/stromal cells is for osteoclast formation in the The used for osteoclast is that by and have in 1α,25(OH)2D3 receptor mice by of the obtained from mice failed to support osteoclast development in cocultures with normal spleen cells in response to 1α,25(OH)2D3 but in response to In spleen cells from mice into osteoclasts in coculture with normal osteoblasts in response to These suggest that the by and are also into osteoblasts/stromal cells to osteoclast formation in the The normal osteoclast formation in mice is by the the induced by all factors are in osteoblasts/stromal cells to osteoclast formation this in we have proposed the hypothesis that osteoblasts/stromal cells express which is a membrane-bound factor to differentiation of osteoclast into osteoclasts through a mechanism cell-to-cell contact (5–9). Chambers et al. (19) also proposed that expressed by osteoblasts/stromal cells is involved in osteoclast and are the to that were to be and the concept of of osteoclast formation. A concept of osteoclast factors such as PTH, and osteoclast formation in cocultures of osteoblasts/stromal cells and hemopoietic cells for factors are osteoblasts/stromal different by and or stromal osteoclast forming activity (SOFA) as a membrane-associated factor in osteoblasts/stromal Osteoclast of the monocyte-macrophage lineage recognize ODF/SOFA in osteoblasts/stromal cells through cell-to-cell and then differentiate into osteoclasts. M-CSF produced by osteoblasts/stromal cells is a for and differentiation of osteoclast progenitors. to the of osteoclast function is that to to formed on are to by with or or We have developed a of mouse bone marrow cells and osteoblasts to functionally active osteoclasts The of osteoclasts in this was only with osteoblasts the osteoclast this osteoclast to be a source by which to a resorption system using dentine This established a to determine the ability of osteoclasts by the property of bone or In we were able to osteoclasts by the osteoclast on a osteoclasts for on dentine failed to form resorption of osteoclasts was restored calvarial osteoblasts were to the osteoclasts Some stromal cell lines such as and ST2 also activity of osteoclasts. cell-to-cell contact between osteoblasts/stromal cells and osteoclasts was osteoclasts failed to form resorption osteoblasts/stromal cells to play an essential role not only in inducing osteoclast formation from but also in to osteoclasts to are cell-to-cell In has that cells are formed from human and hemopoietic in the of osteoblasts/stromal et al. have also used to mononuclear or osteoclasts from cocultures of mouse bone marrow cells and osteoblasts These cells expressed of the of osteoclasts such as calcitonin receptors, and vitronectin only resorb bone cells and 1α,25(OH)2D3 were These support the hypothesis that cells of the osteoblast lineage osteoclast function. In et al. the discovery (OPG) that bone OPG of acid was a member of the TNF receptor family, all members of the family, a transmembrane domain and a secreted TNF expression of OPG in mice in an et al. independently the protein osteoclastogenesis inhibitory factor as a protein from the of human and that cDNA was identical to that of OPG OPG osteoclast formation induced by PTH, or in the In vivo of OPG in an in bone and bone with a of active osteoclast in normal and was also by OPG into et al. also identified a new member of the TNF receptor receptor-like molecule from a of an expressed data was to be identical to OPG and osteoclast formation in the coculture formation by osteoclasts, and bone resorption in of mouse and to a uniform nomenclature we propose the (OPG) be for the that and A of the and receptor of the new TNF involved in osteoclast formation. for the are We to propose that and OPG be as the names of the signal and receptor for the new TNF family, members of the TNF receptor OPG In addition, OPG had domain by a with a and with of TNF receptor and which of human OPG have been using The of OPG was to osteoclast formation in the the of the protein that was to of that of the A was in and the for not with the OPG can as a or as a of formation of a using present in of OPG was not necessary for biological activity of OPG the of the to not the inhibitory activity of In addition, the transmembrane domain of was between and and the protein was expressed in the human cell was induced in the cells the role of and of OPG is not the are active in A of functional of Human OPG is of acid in domain in is for formation of OPG is also of acid with identical to human The of OPG have been in mice produced by of the gene mice were and but by osteoclast formation and function. of and of bone with an in the of osteoclasts were in of The and of were that the osteoblast as as the osteoclast were in mice mice also developed of the and These indicate that OPG is a of bone resorption bone It also that OPG of is a ligand that binding to receptors, and et al. that OPG to and of also that the activity of OPG in the These indicate a potential mechanism OPG and which also in the of bone As OPG was a member of the TNF receptor family, a likely for ODF/SOFA be a membrane-bound ligand for this Since ODF/SOFA be expressed on the of ST2 cells 1α,25(OH)2D3 and this cell was for the ability of OPG to to ST2 cells with 1α,25(OH)2D3 and cloning of the ligand for OPG was using a cDNA of ST2 A cDNA with an encoding acid was The molecule was a transmembrane protein of the TNF ligand cells with the molecule expression were with and with mouse spleen cells in the of osteoclasts were formed on the This that the molecule cell-to-cell for A form of the protein with M-CSF induced osteoclast formation from spleen cells in the of osteoblasts/stromal cells, which was by OPG of calvarial osteoblasts with the of osteoclast formation, PTH, or expression of of this molecule it was that the molecule was which an essential signal to osteoclast for differentiation into osteoclasts. the molecule was called In to the stimulatory of 1α,25(OH)2D3 and on OPG were suggesting that the of OPG is also for osteoclastogenesis induced by osteotropic factors. A of the of and the of on osteoclast formation in mouse spleen cell is of acid The transmembrane between and The domain in are and in the TNF ligand the of which is to the of to the used in this spleen cells cells were in in the or of and After for the of multinucleated cells three or were as osteoclasts. are expressed as the of three of mouse with a form of also the of from the bone which was by OPG OPG, bone resorption in induced by not only but also by PTH, and These indicate that bone resorption induced by osteotropic factors is by et al. also in the molecular cloning of a ligand for OPG from an expression of the cell The OPG ligand was identical to expressed by human in membrane-associated and is that the form of (ODF) is present in the microenvironment of A recombinant form of osteoclast development in bone marrow in the of and it induced formation by M-CSF in an of bone marrow activity of osteoclasts from was also by was into mice a for was induced the of osteoclasts was identical to those of mice These indicate that not only osteoclast differentiation but also osteoclast function. Recently, et al. have in mice with of bone marrow within endosteal mice osteoclasts but have normal osteoclast that can differentiate into functionally active osteoclasts with normal osteoblasts/stromal In addition, mice and have a in These suggest that is an for osteoclast and it plays an important role in cell differentiation as The molecular cloning of that this molecule was identical to and receptor of factor which were independently identified by groups as a member of the TNF ligand was cloned a for in cell that and expressed of A recombinant form of induced activation of in The receptor was on cells by the receptor for to be dependent on TNF factor was impaired in from mice the of mouse and human cells in vitro with of expression The in the of cells induced by was by an in cell in a of and ligand et al. cloned a new member of the TNF receptor from a cDNA of human The mouse was also from the mouse cDNA The mouse cDNA a transmembrane protein of acid OPG, this protein of human expression of with in the and failed to members of the TNF ligand such as or In for the binding molecule of a ligand was cloned from a cDNA of cells and to be identical to A form of RANKL the ability of cells to cell in a and the of et al. that and with at the acid of in human cells and factor the necessary for the binding was the receptor was of activity but not This that interaction with is necessary for activation but not for the activation of the et al. that was also with the of the domain in addition to the in the receptor of and receptor activation in the cells These suggest that differentiation and activation of osteoclasts through by These findings to be to the that the was impaired in from mice are for the of The and receptor of the new TNF members are in and RANKL are the molecule important for development and function of cells and cells as as osteoclasts. to be the receptor for is a receptor for and to function as a These and receptors and have a of functions and on cells osteoclasts and In the of and the nomenclature of to be is only the and we that this name be it be that an identical molecule that function has been proposed as the receptor The ligand for has been as and and in RANKL as the nomenclature for this molecule we into and The of that biological are specific to such a name be to the bone it is that be by action on and cells, and functions in tissues. of indicate that the molecule biological by binding to OPG, but OPG also to OPG to function as a receptor as have The from suggesting that this molecule is expressed only activation of is for expression of this which be We have several for that RANKL be the this molecule is to the only ligand identified for the membrane-bound it does not a or it describes the of the is giving rapidly to of this the of we the names of OPG, and subsequently in this review article M-CSF and RANKL are the essential factors for inducing osteoclasts from mouse hemopoietic had colony-stimulating activity in a of bone marrow cells, and it not the formation. This that RANKL is not a factor but a differentiation factor of osteoclast progenitors. Using and OPG, the process of osteoclast differentiation was in In the coculture the can be into the in which of osteoclast and the in which differentiation into osteoclasts is was to the coculture for the cells on in the of In to the cocultures the of osteoclast but not differentiation into osteoclasts in response to the of M-CSF in osteoclast normal spleen cells were with osteoblasts from op/op mice M-CSF was the coculture osteoclasts were formed in response to the of M-CSF for the or for the failed to in osteoclast formation. These that M-CSF is for the and the differentiation of osteoclast development et al. that M-CSF plays important in and differentiation of osteoclast in mouse bone marrow The differentiation of osteoclast into functionally active osteoclasts and the for of the Recently, we osteoclast precursors obtained from cocultures osteoclast precursors expressed of the such as and and into osteoclasts cell in the of osteoclast precursors were with and into multinucleated cells within in the of These also that M-CSF and RANKL are involved in the differentiation of osteoclast precursors into osteoclasts osteoblasts/stromal cells were from the osteoclasts rapidly within by several and and M-CSF the of osteoclasts the of and function of osteoclasts by and were using from cocultures of mouse cells and bone marrow within and M-CSF and of through receptors The of on were by the of receptor but not by a M-CSF receptor The on but not resorption activity of on dentine was induced by in the of osteoblasts/stromal M-CSF failed to formation in the on dentine slices. As osteoclasts from the coculture failed to form resorption activity of osteoclasts was by but not by in the of osteoblasts/stromal it is that and M-CSF and of but only to osteoclasts that are active in resorption These suggest that play a role as a of osteoclast activation in bone of osteoclasts was also by of osteoclasts with OPG the of osteoclasts by but not that by or and the and of In addition, induced the activity of These indicate that RANKL and to osteoclast function in the of osteoblasts/stromal cells This was by the experiments using osteoclast in which osteoblasts were marrow cells were on in the of and M-CSF but in the of osteoblasts/stromal formed were by osteoclasts were on dentine rapidly forming resorption and all the of those osteoclasts, but only and induced the osteoblasts were to osteoclasts, resorption pits were factors such as PTH, and formation only in the of osteoblasts from op/op mice also induced activity of osteoclasts, which was by These support the hypothesis that osteoblasts/stromal cells osteoclast function through RANKL as a membrane-associated factor. RANKL can be with to and activation of osteoclasts. not support differentiation of osteoclast precursors into in the of osteoblasts/stromal cells were These also suggest that RANKL is involved in bone is involved in bone resorption such as and RANKL has been shown to resorption by osteoclasts the of activation of osteoclasts is by using a system in which osteoclast formation We of osteoclasts by such a to determine that the of RANKL in osteoclasts from of cell as as a on osteoclast and activation of osteoclasts cannot be in vivo in the expressed by osteoblasts/stromal cells, out the of osteoclasts differentiation and that activated in the of osteoclasts, and the activation at addition formed in the cocultures have receptors The of which a with and the in the with the activation of The that a of was from the into all of the of the multinucleated osteoclasts. of osteoclasts with or to and of the of osteoclasts by These indicate that the of osteoclasts through activation that osteoclasts formed in the cocultures expressed of osteoclasts with activated within which was by the of and also activated within in the osteoclasts. These suggest that the activation of and in osteoclasts by and in of osteoclast activation expression in the osteoclasts with suggesting that a is involved in the of osteoclasts. Signals induced by and RANKL in osteoclasts. formed in the cocultures express receptor and and RANKL the and activation of osteoclasts in the of osteoblasts/stromal and RANKL and through receptor and OPG and Recently, et al. and et al. independently mice in and of The mice developed of a in osteoclast The osteopetrotic was by bone marrow that the osteoclast were were but the of macrophages was in bone from the These suggest that and can be with in formation with in osteoclast RANKL has been to in the cells and we have also this in the osteoclast These suggest that the activation of in osteoclast also plays a crucial role in differentiation into osteoclasts. It is also that factors are by in osteoclast precursors and osteoclasts. As RANKL and M-CSF are essential factors for mouse osteoclast formation. findings indicate that the mechanism of human osteoclast formation is to that of mouse osteoclast formation. et al. that cells and ST2 cells human osteoclast formation in coculture with human peripheral blood mononuclear cells in the of 1α,25(OH)2D3 and In addition of human M-CSF to the coculture was essential to human osteoclasts and ST2 cells and mouse which do not to human M-CSF receptors This also that and mouse RANKL can on human cells as We also that the human cell line, which expressed functionally active receptors, human osteoclast formation in response to and in coculture with human peripheral blood mononuclear cells M-CSF mouse and human osteoclast formation in coculture with These are with the of and who a role of M-CSF in human osteoclast formation as of human with mouse and human M-CSF with induced human osteoclasts OPG osteoclast formation from human that was by cells or by human induced expression of RANKL by cells, and this was not by These suggest that cells in human an inhibitory human of which is by osteoclasts were from human that had been on a and M-CSF were the for colony-stimulating factor has been shown to be an important factor for osteoclast formation in human bone marrow as in the of mouse osteoclast formation, human osteoclast formation induced by and M-CSF and This that of osteoclast but inhibits differentiation into osteoclasts. These also indicate that of human osteoclast formation are the as those of mouse osteoclast formation. Regulation of human osteoclast formation and function can be from the findings obtained from the mouse with human human osteoclast formation. Human were in cells in the or of human mouse human OPG, human and After for multinucleated cells three or were as osteoclasts. Human were in cells with or human M-CSF and mouse in the of After for cells were for Human were in cells in which a dentine had been were with human M-CSF and mouse in the of After for resorption pits formed on the were with Osteoblasts/stromal cells are involved in osteoclast differentiation and function through cell-to-cell contact have been to the mechanism of the by osteoblasts/stromal it has an OPG and binding molecule were The discovery of the new members of the TNF members has the that osteoclast differentiation and function are by osteoblasts/stromal which has also been called or is a member of the TNF ligand of RANKL in osteoblasts/stromal cells is by osteotropic factors such as PTH, and Osteoclast precursors express a TNF receptor recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into in the of RANKL is also involved in the and of and activation of osteoclasts. OPG, which has also been called or is a receptor for RANKL and as a receptor in the system A of osteoclast differentiation and function by osteoblasts/stromal In osteoblasts/stromal cells are involved in all of the of osteoclast such as and activation of osteoclasts Osteoblasts/stromal cells can now be with RANKL and M-CSF in with the of osteoclasts. and OPG are three that osteoclast and function. on the molecular mechanism of the of bone This of new to several bone by osteoclast and functions such as bone and bone We and of and and of for of the and

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.

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 AMPKalphaThr-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-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ 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, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. 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.

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.

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.

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.