Wellcome/MRC Cambridge Stem Cell Institute

Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.

RNA-sequencing (RNA-seq) has a wide variety of applications, but no single analysis pipeline can be used in all cases. We review all of the major steps in RNA-seq data analysis, including experimental design, quality control, read alignment, quantification of gene and transcript levels, visualization, differential gene expression, alternative splicing, functional analysis, gene fusion detection and eQTL mapping. We highlight the challenges associated with each step. We discuss the analysis of small RNAs and the integration of RNA-seq with other functional genomics techniques. Finally, we discuss the outlook for novel technologies that are changing the state of the art in transcriptomics.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.

Single-cell RNA-seq quantifies biological heterogeneity across both discrete cell types and continuous cell transitions. Partition-based graph abstraction (PAGA) provides an interpretable graph-like map of the arising data manifold, based on estimating connectivity of manifold partitions ( https://github.com/theislab/paga ). PAGA maps preserve the global topology of data, allow analyzing data at different resolutions, and result in much higher computational efficiency of the typical exploratory data analysis workflow. We demonstrate the method by inferring structure-rich cell maps with consistent topology across four hematopoietic datasets, adult planaria and the zebrafish embryo and benchmark computational performance on one million neurons.

Abstract Over the past few decades, neuroimaging has become a ubiquitous tool in basic research and clinical studies of the human brain. However, no reference standards currently exist to quantify individual differences in neuroimaging metrics over time, in contrast to growth charts for anthropometric traits such as height and weight 1 . Here we assemble an interactive open resource to benchmark brain morphology derived from any current or future sample of MRI data ( http://www.brainchart.io/ ). With the goal of basing these reference charts on the largest and most inclusive dataset available, acknowledging limitations due to known biases of MRI studies relative to the diversity of the global population, we aggregated 123,984 MRI scans, across more than 100 primary studies, from 101,457 human participants between 115 days post-conception to 100 years of age. MRI metrics were quantified by centile scores, relative to non-linear trajectories 2 of brain structural changes, and rates of change, over the lifespan. Brain charts identified previously unreported neurodevelopmental milestones 3 , showed high stability of individuals across longitudinal assessments, and demonstrated robustness to technical and methodological differences between primary studies. Centile scores showed increased heritability compared with non-centiled MRI phenotypes, and provided a standardized measure of atypical brain structure that revealed patterns of neuroanatomical variation across neurological and psychiatric disorders. In summary, brain charts are an essential step towards robust quantification of individual variation benchmarked to normative trajectories in multiple, commonly used neuroimaging phenotypes.

The dentate gyrus (DG) of the mammalian hippocampus is hypothesized to mediate pattern separation-the formation of distinct and orthogonal representations of mnemonic information-and also undergoes neurogenesis throughout life. How neurogenesis contributes to hippocampal function is largely unknown. Using adult mice in which hippocampal neurogenesis was ablated, we found specific impairments in spatial discrimination with two behavioral assays: (i) a spatial navigation radial arm maze task and (ii) a spatial, but non-navigable, task in the mouse touch screen. Mice with ablated neurogenesis were impaired when stimuli were presented with little spatial separation, but not when stimuli were more widely separated in space. Thus, newborn neurons may be necessary for normal pattern separation function in the DG of adult mice.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

squamous cell carcinoma antigen 1 or 2 α1 antitrypsin (α1 proteinase inhibitor) α2 antiplasmin amyloid-β α1 antichymotrypsin antithrombin III monocyte-neutrophil elastase inhibitor ovalbumin plasminogen activator inhibitor type 1 or 2 pigment epithelium-derived factor vascular endothelial growth factor reactive site loop tissue plasminogen activator urokinase plasminogen activator The serpins (serineproteinase inhibitors) are a superfamily of proteins (350–500 amino acids in size) that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. The serpins were last reviewed in 1994 (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). More recent studies show: 1) an expanded distribution within the kingdoms of metazoa and plantae, as well as certain viruses, 2) a surprising effect on the covalently bound target proteinase, and 3) novel biochemical and biological functions. Most serpins inhibit serine proteinases of the chymotrypsin family. However, cross-class inhibitors have been identified. The viral serpin CrmA and, to a lesser extent, PI9 (SERPINB9) inhibit the cysteine proteinase, caspase 1 (2Komiyama T. Ray C.A. Pickup D.J. Howard A.D. Thornberry N.A. Peterson E.P. Salvesen G. J. Biol. Chem. 1994; 269: 19331-19337Abstract Full Text PDF PubMed Google Scholar), and SCCA11 (SERPINB3) neutralizes the potent papain-like cysteine proteinases, cathepsins L, K, and S (3Schick C. Pemberton P.A. Shi G.-P. Kamachi Y. Cataltepe S. Bartuski A.J. Gornstein E.R. Bromme D. Chapman H.A. Silverman G.A. Biochemistry. 1998; 37: 5258-5266Crossref PubMed Scopus (259) Google Scholar). In addition, several members no longer function as proteinase inhibitors but perform other roles such as hormone transport (thyroid-binding globulin (SERPINA6), corticosteroid-binding globulin (SERPINA7)), and blood pressure regulation (angiotensinogen (SERPINA8)) (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). Data base searching provides evidence for ∼500 serpins, with full-length coding sequences known or predicted for about one-half of those (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (523) Google Scholar). A phylogenetic analysis divides serpins into 16 clades (see Supplemental Data, Table A) and 10 highly diverged “orphans” (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (523) Google Scholar). These data facilitate the construction of a consistent expandable nomenclature (see Supplemental Data for Serpin Nomenclature Guidelines, Table B). The completed DNA sequences of several organisms have yielded insight into the complexity of the family. The Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thalianagenomes encode for ∼20,000, 13,000, and 25,000 genes, respectively. However, these three species harbor ∼9, 32, and 13 serpin genes, respectively. The nonlinear relationship among the number of serpin genes, relative to the total gene number, suggests that at least a subset of serpins has evolved divergent functions despite a striking degree of sequence and structural conservation. Serpins adopt a metastable conformation that is required for their inhibitory activity (5Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (398) Google Scholar). This conformation consists of a conserved secondary structure comprised of β-sheets A, B, and C and at least 7 α-helices (most typically have 9, lettered A–I; Fig.1 A). The RSL, which contains the proteinase recognition site, is an exposed, flexible stretch of ∼17 residues tethered between β-sheets A and C. Serpins can undergo major structural rearrangements that involve alternative conformations for the RSL, β-sheet A, and the attached strand 1 of β-sheet C. Considering only intramolecular structural changes, serpins can convert to the more stable latent form (Fig. 1 B). The RSL inserts into the middle of β-sheet A to give a fully antiparallel β-sheet, and s1C is extracted from β-sheet C to provide an exposed “return” from the bottom of the serpin. Serpins in the latent conformation are noninhibitory but can be converted back to the active state by denaturation and refolding. The Tm for unfolding of latent PAI1 (SERPINE1) is 17 °C higher than that for the native state (reviewed in Ref. 6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). The most stable state for inhibitory serpins is the RSL-cleaved form, in which the RSL has fully inserted into β-sheet A, as in the latent conformation, but without the need to extract s1C from β-sheet C (Fig. 1 C). Estimates of the Tm for unfolding of such conformations are >120 °C, compared with ∼60 °C for the native state (7Kaslik G. Kardos J. Szabo E. Szilagyi L. Zavodszky P. Westler W.M. Markley J.L. Graf L. Biochemistry. 1997; 36: 5455-5464Crossref PubMed Scopus (104) Google Scholar). The most informative serpin structures, from a mechanistic viewpoint, are those of a Michaelis complex between Serpin 1 and trypsin (Fig.1 D) and of a covalent complex between α1AT (SERPINA1) and trypsin (8Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (970) Google Scholar) (Fig. 1 E). This latter structure represents the proteinase after it has been kinetically trapped in the acyl-enzyme intermediate that forms normally along the peptide bond cleavage pathway. Whereas the bound serpin is almost indistinguishable from that of the RSL-cleaved form (Fig. 1 C), the proteinase is grossly distorted (see below). Serpins inhibit serine proteinases by an irreversible suicide substrate mechanism when the interaction proceeds down the inhibitory arm of a branched pathway (Fig. 2) (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). In the inhibitory pathway, the proteinase initially forms a noncovalent Michaelis-like complex (Fig. 1 D) through interactions with residues flanking the scissile bond (P1–P1′). Attack of the active site serine on the scissile bond leads to a covalent ester linkage between Ser-195 of the proteinase and the backbone carbonyl of the P1 residue and cleavage of the peptide bond (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). It is likely that only at this stage, with removal of the restraint, does the RSL start to insert into β-sheet A and transport the covalently bound proteinase with it. Upon complete loop insertion the proteinase is translocated by over 70 Å, and its active site is distorted (Fig. 1 E). The alignment of the active site catalytic triad is altered by as much as 3 Å, and the P1 side chain is removed from the S1 pocket. Also, 40% of the body of the proteinase shows no traceable electron density. Proteinase distortion and hence inactivation results from compression of the proteinase against the base of the serpin as a consequence of the inserted RSL being just the right length. The energy needed to effect the distortion may come from the much greater stability of the cleaved loop-inserted conformation compared with the native-like conformation. The net result of this conformational rearrangement is kinetic trapping of the acyl intermediate due to slowing of the deacylation steps of the normal substrate reaction by 6–8 orders of magnitude (k5 in Fig. 2). Because of the small values for k5 (complex t12≅ hours to weeks), serpin-proteinase complexes in vivowould bind to their receptors and be cleared (complext12 ≅ minutes) long before significant complex decay could occur. The point in transit where the enzyme activity is reduced sufficiently to commit the intermediate to the kinetic trap is not known but in part contributes to the branched nature of the pathway and the ultimate fate of the complex. If, for example, RSL movement is impeded, the enzyme may successfully complete the deacylation step and escape before it is irreversibly trapped. This noninhibitory pathway yields an active proteinase and a cleaved, inactive serpin. The ratio of serpin products (complex versuscleaved) thus reflects a competition between the rate of ester hydrolysis (k3 in Fig. 2) and that of loop insertion (k4 in Fig. 2) to the point of proteinase distortion. This ratio is signified also by the stoichiometry of inhibition, which is defined as (k3 +k4)/k4, i.e.the number of moles of serpin needed to inhibit 1 mol of proteinase as a kinetically trapped complex. This mechanism accounts for the requirements for effective inhibition by serpins, which include a critical RSL length, appropriate residues within the loop that are compatible with rapid and favorable burial into β-sheet A, and the presence of Ser in the proteinase active site (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). Such a mechanism is adaptable to the inhibition of cysteine proteinases by serpins, with the difference being that the kinetically trapped intermediate is a thiol ester rather than an oxy ester. The detection of CrmA, a serpin that inhibits cysteine proteinases of the caspase family, in the loop-inserted cleaved conformation supports the feasibility of a common inhibitory mechanism (9Renatus M. Zhou Q. Stennicke H.R. Snipas S.J. Turk D. Bankston L.A. Liddington R.C. Salvesen G.S. Struct. Fold. Des. 2000; 8: 789-797Abstract Full Text Full Text PDF Scopus (55) Google Scholar), whereas the detection of an SDS-stable complex between SCCA1 and cathepsin S (a cysteine proteinase of the papain family) provides evidence for the formation of a stable, covalent thiol ester-type linkage (3Schick C. Pemberton P.A. Shi G.-P. Kamachi Y. Cataltepe S. Bartuski A.J. Gornstein E.R. Bromme D. Chapman H.A. Silverman G.A. Biochemistry. 1998; 37: 5258-5266Crossref PubMed Scopus (259) Google Scholar). The few convincing reports of reversible inhibition, such as of single-chain uPA by PCI (SERPINA5) (10Schwartz B.S. Espana F. J. Biol. Chem. 1999; 274: 15278-15283Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) or of chymotrypsin by α2AP (SERPINF2) (11Shieh B.H. Potempa J. Travis J. J. Biol. Chem. 1989; 264: 13420-13423Abstract Full Text PDF PubMed Google Scholar) may represent special cases in which unusual stabilization of the initial noncovalent Michaelis-like complex blocks progression to the substrate reaction. A negative consequence of the need for a metastable conformation in the active state is that natural mutations, either alone or in combination with environmental factors, can promote inappropriate loop insertion. When this occurs between the RSL of one molecule and the β-sheet of another, dimers and higher order oligomers can result. Either through depletion of active serpin or through pathological effects of the polymers themselves, such aggregate formation can lead to disease. The best characterized examples are the emphysema (serpin depletion) and cirrhosis (intracellular inclusions) associated with loop-sheet polymers of the Z or S variants of α1AT (12Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (249) Google Scholar) (see Supplemental Data, Fig. A) and the dementia associated with neuroserpin (SERPINI1) inclusion bodies (see below). Understanding the biologic function of serpins remains an ongoing challenge. For example, the biologic functions for many of the human serpins involved in the clotting and fibrinolytic cascades are well documented. However the role of human serpins in some other types of biologic processes awaits further validation (Fig.3). In 1993 amino acid similarities among chicken ovalbumin (ov), PAI2 (SERPINB2), and MNEI (SERPINB1) led to the identification of a subgroup of the serpin superfamily (13Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (217) Google Scholar). The N and C termini of the ov-serpins are shorter than the prototypical serpin α1AT, and they also lack a classical secretory signal peptide. At present, there are 13 human ov-serpins (see Supplemental Data, Table B). They map to 6p25 and 18q21 and fall into two classes based on a single difference in gene structure (14Scott F.L. Eyre H.J. Lioumi M. Ragoussis J. Irving J.A. Sutherland G.A. Bird P.I. Genomics. 1999; 62: 490-499Crossref PubMed Scopus (37) Google Scholar). Like ovalbumin, many of the 18q21 serpin genes have an exon encoding a polypeptide loop between helices C and D (CD loop) that may contribute to accessory functions. Unlike ovalbumin itself, most ov-serpins reside intracellularly with a cytoplasmic or nucleocytoplasmic distribution. However, several ov-serpins (PAI2, megsin (SERPINB7), MNEI, maspin (SERPINB5), and the SCCAs (SERPINB3 and -4)) may function extracellularly as they are released from cells under certain conditions. Release may be facilitated by an embedded, noncleaved hydrophobic N-terminal signal sequence and appears to involve both conventional and non-endoplasmic reticulum-Golgi secretory pathways (15Belin D. Thromb. Haemostasis. 1993; 70: 144-147Crossref PubMed Scopus (40) Google Scholar). Regardless of how ov-serpins are released from cells, those with RSL cysteine or methionine residues are susceptible to oxidative inactivation and are likely to have a limited half-life in the extracellular milieu. With the possible exception of maspin, all human ov-serpins are functional, competitive inhibitors of serine or cysteine proteinases. Several members of the group inhibit more than one proteinase, and dual reactive sites (utilization of more than one P1 residue) have been described for PI6 (SERPINB6), PI8 (SERPINB8), PI9, SCCA1, SCCA2, and MNEI (for example see Ref. 16Riewald M. Schleef R.R. J. Biol. Chem. 1996; 271: 14526-14532Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, the CD loops of the ov-serpins have the potential to interact with other proteins. For example, the CD loop of PAI2 is required for its cell survival function (17Dickinson J.L. Bates E.J. Ferrante A. Antalis T.M. J. Biol. Chem. 1995; 270: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and is a target for transglutamination (18Jensen P.H. Schuler E. Woodrow G. Richardson M. Goss N. Hojrup P. Petersen T.E. Rasmussen L.K. J. Biol. Chem. 1994; 269: 15394-15398Abstract Full Text PDF PubMed Google Scholar). Bomapin (SERPINB10; like the chicken ov-serpin, MENT, see below) carries a nuclear localization signal in its CD loop that presumably interacts with a nuclear importin (19Chuang T.L. Schleef R.R. J. Biol. Chem. 1999; 274: 11194-11198Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The physiological functions of ov-serpins are still emerging. PAI2 may play a role in the regulation of extracellular matrix remodeling through the inhibition of uPA, as high PAI2 and low uPA levels correlate with a positive prognosis in breast cancer (20Duggan C. Kennedy S. Kramer M.D. Barnes C. Elvin P. McDermott E. O'Higgins N. Duffy M.J. Br. J. Cancer. 1997; 76: 622-627Crossref PubMed Scopus (66) Google Scholar). Also, PAI2 may have a structural role inside some cells (perhaps keratinocytes) as suggested by its ability to spontaneously polymerize and undergo transglutamination (21Mikus P. Ny T. J. Biol. Chem. 1996; 271: 10048-10053Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Many ov-serpins reside in proteinase-secreting cells (22Bird P.I. Immunol. Cell Biol. 1999; 77: 47-57Crossref PubMed Scopus (58) Google Scholar). For example, PI9, a potent inhibitor of granzyme B, is also present in cytotoxic lymphocytes. Because PI9 can protect cells against granzyme B-mediated apoptosis, it probably protects cytotoxic lymphocytes from autodestruction due to misdirected granzyme B. A similar cytoprotective role can be envisaged for PI6, PI8, MNEI, PAI2, and the SCCAs. In addition, endogenous or exogenous ov-serpins may protect bystander cells and tissue from proteolytic damage. Studies in rats show that recombinant MNEI delivered to the airways prevents lung injury by neutrophil proteinases and point to its potential in treating inflammatory lung disease (23Rees D.D. Rogers R.A. Cooley J. Mandle R.J. Kenney D.M. Remold-O'Donnell E. Am. J. Respir. Cell Mol. Biol. 1999; 20: 69-78Crossref PubMed Scopus (43) Google Scholar). The ability of many ov-serpins to inhibit more than one proteinase and their presence in epithelial cells suggest that they play a role in barrier function or host defense against microbial or viral proteinases. For example, PI9 inhibits Bacillussubtilisin, and PI8 inhibits furin, a subtilisin-related enzyme (24Dahlen J.R. Foster D.C. Kisiel W. Biochem. Biophys. Res. Commun. 1997; 238: 329-333Crossref PubMed Scopus (19) Google Scholar,25Dahlen J.R. Jean F. Thomas G. Foster D.C. Kisiel W. J. Biol. Chem. 1998; 273: 1851Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Additional functions of ov-serpins include the regulation of: 1) cell growth or differentiation, as exemplified by the role of megsin in megakaryocyte differentiation (26Tsujimoto M. Tsuruoka N. Ishida N. Kurihara T. Iwasa F. Yamashiro K. Rogi T. Kodama S. Katsuragi N. Adachi M. Katayama T. Nakao M. Yamaichi K. Hashino J. Haruyama M. Miura K. Nakanishi T. Nakazato H. Teramura M. Mizoguchi H. Yamaguchi N. J. Biol. Chem. 1997; 272: 15373-15380Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), 2) tumor cell invasiveness and motility, as shown by the inhibitory role of maspin in breast and prostate tumors (27Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (842) Google Scholar), and 3) angiogenesis (see below). Grigoryev et al. (28Grigoryev S.A. Bednar J. Woodcock C.L. J. Biol. Chem. 1999; 274: 5626-5636Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) isolated a novel serpin, MENT, from the nuclei of terminally differentiated chicken hematopoietic cells. MENT is an ov-serpin with a CD loop that contains a nuclear localization signal, a lamin-like chromatin binding domain, and an A-T hook DNA binding motif. The molecule has a relatively high pI (9 versus 5–6.5 for that of other serpins) with the majority of positive charges clustering near the CD loop. Thus, MENT appears to utilize the CD loop to bind tightly to nucleosomes with an apparent stoichiometry of 2:1. MENT is the major non-histone chromatin protein in differentiated nuclei and is concentrated in the heterochromatin. MENT induces higher order chromatin compaction when it is expressed ectopically in cells or added to isolated nuclei in vitro. Although MENT contains a viable RSL, target proteinases have yet to be identified. which inhibits uPA, and growth factor in is from and cells G.A. S. E.P. R. S. M. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). may play a role in the from In a neuroserpin in within the and of the protein reduced the by and the number of cells by M. M. E. D.A. 2000; PubMed Google Scholar). In a form of dementia and neuroserpin Molecular analysis in two and in the C. D. P. J. F. D. M. D.A. B. P.R. Carrell R.W. Lomas D.A. Nature. 1999; PubMed Google Scholar). These are similar to that in α1AT in which an of β-sheet A and the formation of loop-sheet In these polymers and in the normal function is and, to a lesser extent, other serpins are within the of from with one of the most common forms of dementia (reviewed in Ref. S. 1998; 20: PubMed Scopus Google Scholar). Although the of this is the extracellular of may be by binding to low receptors and with appears to facilitate formation by as a for the The peptide inserts into A and C of in which it a conformation. Upon RSL is released into the extracellular in which the peptide is more to is a noninhibitory serpin that isolated from pigment epithelial cells but is also in and (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). This factor the survival and differentiation of and et al. P. H. W. Science. 1999; PubMed Scopus Google Scholar) show that inhibits of the and endothelial cell vitro. In the cell as potent as other angiogenesis inhibitors such as and the effects of the angiogenesis growth growth and in the with the of Thus, and to blood growth in the by and angiogenesis and respectively. maspin, and RSL-cleaved have been shown to with angiogenesis in M. Shi N. Nat. 2000; PubMed Scopus Google S. E. G.A. D.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google S. J. Science. 1999; PubMed Scopus Google Scholar). However, it has yet to be of these are involved in the or regulation of blood A of function in serpin leads to the and of the E. C. D. M. J.A. Science. 1999; PubMed Scopus Google Scholar). of the pathway proteolytic cleavage of the In leads to an in both and the pathway. appears to in a negative loop by proteinases that Thus, the of and the to be secondary to proteolytic The function of these proteins remains Several studies show that serpins are of serine proteinase H. Rasmussen J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, with the exception of a proteinase in conventional serine proteinase are in of the sequence of trypsin as a to classical In studies by et al. K. Y. R.J. B. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) show that of the with the of the to and on these a role for serpins in host Several of human serpins have been by in cells. of these have to an whereas show and structural as well as (see Supplemental Data, Table C). Serpins are within a number of within the of the and the (see Supplemental Data, Table of the serpins are required for growth in cell the and three highly conserved serpins, and types of proteinases The of the also three serpins, the contains serpin genes and lack serpin of encode serpins with P1 and the and all have a with a P1 residue the have a serpin with a at the P1 For more on serpins see Supplemental The serpins are a superfamily of genes that are the metazoa and Serpin members are by a conserved structure and a unique suicide substrate-like inhibitory mechanism. Serpins reside both intracellularly and extracellularly and are involved in a of biologic functions that the ability of these to irreversibly inhibit target proteinases. The of serpin function biological such as the Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the role that these play in and host

Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.

Abstract The SARS-CoV-2 Omicron BA.1 variant emerged in 2021 1 and has multiple mutations in its spike protein 2 . Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron’s evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2 , and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways 3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

The stromal microenvironment of tumors, which is a mixture of hematopoietic and mesenchymal cells, suppresses immune control of tumor growth. A stromal cell type that was first identified in human cancers expresses fibroblast activation protein-α (FAP). We created a transgenic mouse in which FAP-expressing cells can be ablated. Depletion of FAP-expressing cells, which made up only 2% of all tumor cells in established Lewis lung carcinomas, caused rapid hypoxic necrosis of both cancer and stromal cells in immunogenic tumors by a process involving interferon-γ and tumor necrosis factor-α. Depleting FAP-expressing cells in a subcutaneous model of pancreatic ductal adenocarcinoma also permitted immunological control of growth. Therefore, FAP-expressing cells are a nonredundant, immune-suppressive component of the tumor microenvironment.

Pluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming.

Despite the clinical and genetic heterogeneity of autism, bulk gene expression studies show that changes in the neocortex of autism patients converge on common genes and pathways. However, direct assessment of specific cell types in the brain affected by autism has not been feasible until recently. We used single-nucleus RNA sequencing of cortical tissue from patients with autism to identify autism-associated transcriptomic changes in specific cell types. We found that synaptic signaling of upper-layer excitatory neurons and the molecular state of microglia are preferentially affected in autism. Moreover, our results show that dysregulation of specific groups of genes in cortico-cortical projection neurons correlates with clinical severity of autism. These findings suggest that molecular changes in upper-layer cortical circuits are linked to behavioral manifestations of autism.

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

Despite decades of research, brain tumours remain among the deadliest of all forms of cancer. The ability of these tumours to resist almost all conventional and novel treatments relates, in part, to the unique cell-intrinsic and microenvironmental properties of neural tissues. In an attempt to encourage progress in our understanding and ability to successfully treat patients with brain tumours, Cancer Research UK convened an international panel of clinicians and laboratory-based scientists to identify challenges that must be overcome if we are to cure all patients with a brain tumour. The seven key challenges summarized in this Position Paper are intended to serve as foci for future research and investment.

Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

Mouse embryonic stem (ES) cells grown in serum exhibit greater heterogeneity in morphology and expression of pluripotency factors than ES cells cultured in defined medium with inhibitors of two kinases (Mek and GSK3), a condition known as "2i" postulated to establish a naive ground state. We show that the transcriptome and epigenome profiles of serum- and 2i-grown ES cells are distinct. 2i-treated cells exhibit lower expression of lineage-affiliated genes, reduced prevalence at promoters of the repressive histone modification H3K27me3, and fewer bivalent domains, which are thought to mark genes poised for either up- or downregulation. Nonetheless, serum- and 2i-grown ES cells have similar differentiation potential. Precocious transcription of developmental genes in 2i is restrained by RNA polymerase II promoter-proximal pausing. These findings suggest that transcriptional potentiation and a permissive chromatin context characterize the ground state and that exit from it may not require a metastable intermediate or multilineage priming.