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Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell’s clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence. Lineage tracing by barcoding of individual cells using a lentivirus library shows that cycling and non-cycling drug-tolerant persister cells in cancer arise from different lineages with distinct transcriptional and metabolic programs.

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Calt;supgt;2+lt;/supgt; signals by an as-yet unidentified high-affinity Calt;supgt;2+lt;/supgt; sensorlt;supgt;1,2lt;/supgt;. Here we identify synaptotagmin-3 (SYT3)lt;supgt;3,4lt;/supgt; as that presynaptic high-affinity Calt;supgt;2+lt;/supgt; sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Calt;supgt;2+lt;/supgt;. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref.lt;supgt;5lt;/supgt;). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Calt;supgt;2+lt;/supgt;-dependent vesicle docking.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

Saccharomyces cerevisiae bearing engineered alginate and mannitol catabolic pathways can ferment sugars from brown macroalgae to produce ethanol, potentially allowing the use of brown macroalgae as a viable feedstock for the production of biofuels and renewable chemicals. Brown macroalgae are seen as a viable feedstock for the production of biofuels, with the advantage that they can be farmed in coastal waters without using valuable arable land. However, the most abundant sugars in brown macroalgae are alginate, mannitol and glucan, and the full potential of this feedstock cannot be realized without extensive re-engineering of the alginate and mannitol catabolic pathways in Saccharomyces cerevisiae. In this paper the authors identify a 4-deoxy-L-erythro-5-hexoseulose uronate transporter in Asteromyces cruciatus brown algae and use it to develop a S. cerevisiae strain that can use the unique sugars in brown macroalgae for high-efficiency ethanol fermentation. With appropriate genetic modifications, this synthetic biology platform can be used to produce many other biofuels and renewable chemicals. The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways1,2,3 in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-l-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus4. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l−1) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Pathogenic aggregation of α-synuclein (αSyn) is implicated in Parkinson's disease and related disorders. αSyn has long been regarded as a natively unfolded monomer that acquires secondary structure only when it binds to its target. This model is the basis for a number of published studies, but Bartels et al. report that endogenous αSyn isolated under entirely non-denaturing conditions from brain tissue, cell lines and living human cells occurs principally as a folded tetramer. This finding suggests that destabilization of the αSyn tetramer precedes misfolding and aggregation in synucleinopathies, and that agents that stabilize the normal tetramer may reduce αSyn pathogenicity. Parkinson’s disease is the second most common neurodegenerative disorder1,2. Growing evidence indicates a causative role of misfolded forms of the protein α-synuclein in the pathogenesis of Parkinson’s disease3,4. Intraneuronal aggregates of α-synuclein occur in Lewy bodies and Lewy neurites5, the cytopathological hallmarks of Parkinson’s disease and related disorders called synucleinopathies4. α-Synuclein has long been defined as a ‘natively unfolded’ monomer of about 14 kDa (ref. 6) that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles7. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous α-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58 kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived α-synuclein showed α-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant α-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson’s disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α-synuclein pathogenicity.

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are approved for cholesterol reduction, may also be beneficial in the treatment of inflammatory diseases. Atorvastatin (Lipitor) was tested in chronic and relapsing experimental autoimmune encephalomyelitis, a CD4(+) Th1-mediated central nervous system (CNS) demyelinating disease model of multiple sclerosis. Here we show that oral atorvastatin prevented or reversed chronic and relapsing paralysis. Atorvastatin induced STAT6 phosphorylation and secretion of Th2 cytokines (interleukin (IL)-4, IL-5 and IL-10) and transforming growth factor (TGF)-beta. Conversely, STAT4 phosphorylation was inhibited and secretion of Th1 cytokines (IL-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha) was suppressed. Atorvastatin promoted differentiation of Th0 cells into Th2 cells. In adoptive transfer, these Th2 cells protected recipient mice from EAE induction. Atorvastatin reduced CNS infiltration and major histocompatibility complex (MHC) class II expression. Treatment of microglia inhibited IFN-gamma-inducible transcription at multiple MHC class II transactivator (CIITA) promoters and suppressed class II upregulation. Atorvastatin suppressed IFN-gamma-inducible expression of CD40, CD80 and CD86 co-stimulatory molecules. l-Mevalonate, the product of HMG-CoA reductase, reversed atorvastatin's effects on antigen-presenting cells (APC) and T cells. Atorvastatin treatment of either APC or T cells suppressed antigen-specific T-cell activation. Thus, atorvastatin has pleiotropic immunomodulatory effects involving both APC and T-cell compartments. Statins may be beneficial for multiple sclerosis and other Th1-mediated autoimmune diseases.

Although extensive data support a central pathogenic role for amyloid β protein (Aβ) in Alzheimer's disease1, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Aβ and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Aβ are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Aβ monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Aβ species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Aβ monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Aβ oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with γ-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Aβ oligomers can be targeted therapeutically.

During vertebrate development, the specification of distinct cell types is thought to be controlled by inductive signals acting at different concentration thresholds1. The degree of receptor activation in response to these signals is a known determinant of cell fate2, but the later steps at which graded signals are converted into all-or-none distinctions in cell identity remain poorly resolved. In the ventral neural tube, motor neuron and interneuron generation depends on the graded activity of the signalling protein Sonic hedgehog (Shh)3,4,5. These neuronal subtypes derive from distinct progenitor cell populations that express the homeodomain proteins Nkx2.2 or Pax6 in response to graded Shh signalling6,7. In mice lacking Pax6, progenitor cells generate neurons characteristic of exposure to greater Shh activity6,7. However, Nkx2.2 expression expands dosally in Pax6 mutants6, raising the possibility that Pax6 controls neuronal pattern indirectly. Here we provide evidence that Nkx2.2 has a primary role in ventral neuronal patterning. In Nkx2.2 mutants, Pax6 expression is unchanged but cells undergo a ventral-to-dorsal transformation in fate and generate motor neurons rather than interneurons. Thus, Nkx2.2 has an essential role in interpreting graded Shh signals and selecting neuronal identity.

It is problematic that geochemical estimates of new production - that fraction of total primary production in surface waters fueled by externally supplied nutrients - in oligotrophic waters of the open ocean surpass that which can be sustained by the traditionally accepted mechanisms of nutrient supply. In the case of the Sargasso Sea, for example, these mechanisms account for less than half of the annual nutrient requirement indicated by new production estimates based on three independent transient-tracer techniques. Specifically, approximately one-quarter to one-third of the annual nutrient requirement can be supplied by entrainment into the mixed layer during wintertime convection, with minor contributions from mixing in the thermocline and wind-driven transport (the potentially important role of nitrogen fixation - for which estimates vary by an order of magnitude in this region - is excluded from this budget). Here we present four lines of evidence - eddy-resolving model simulations, high-resolution observations from moored instrumentation, shipboard surveys, and satellite data - which suggest that the vertical flux of nutrients induced by the dynamics of mesoscale eddies is sufficient to balance the nutrient budget in the Sargasso Sea. Additional information is contained in the original extended abstract.

X-ray analyses have defined the three-dimensional structures of crystals of mouse and human renins complexed with peptide inhibitors at resolutions of 1.9 and 2.8 A, respectively. The exquisite specificity of renin arises partly from ordered loop regions at the periphery of the binding cleft. Although the pattern of main-chain hydrogen bonding in other aspartic proteinase inhibitor complexes is conserved in renins, differences in the positions of secondary structure elements (particularly helices) also lead to improved specificity in renins for angiotensinogen substrates.

THE nearly landlocked waters of the Black Sea support a valuable fishery, but are also particularly vulnerable to anthropogenic disturbance. Here we use dissolved barium and radium-226 as tracers, to investigate the biogeochemical health of the sea. Both elements are brought to surface waters by vertical mixing of deeper, enriched waters1,2, and by rivers3–8; these inputs should ordinarily be balanced by outflow of surface waters at the Bosporus, and by biologically mediated removal of 226Ra-bearing barite. We show, however, that surface-water inventories have been substantially depleted over the past few decades: recent (1988–89) barium concentrations were 1.6 times lower than in 19759; and recent 226Ra concentrations were 3.5 times lower than in 195810 and 196711. These observations suggest that steady-state cycling of these elements has been perturbed by increased primary productivity, presumably fuelled by nutrients from industry and agricultural runoff, and to a lesser extent by decreased fluvial sediment loads owing to extensive impoundment of rivers in the region.

replying to J. Burre . 10.1038/nature12125 (2013) In disagreeing with our report that native α-synuclein occurs physiologically as an α-helically folded tetramer in neural and erythroid cells1, Burre et al.2 conclude instead that ‘native brain α-synuclein’ consists of a largely unstructured monomer. They make two implications about our paper that are inaccurate: (1) that our findings pertained only to erythrocyte α-synuclein (we reported multiple experiments on neural cells); and (2) that we concluded that cellular α-synuclein is a stable tetramer under all conditions (we did not use the term ‘stable’, and we observed monomers and some other oligomers in normal cells (e.g., Fig. 1d of ref. 1)). Indeed, we emphasized the need to discover “compounds that … could kinetically stabilize native tetramers and prevent pathogenic α-synuclein aggregation”. Although the data in our report suggest that tetramers are the predominant native species, tetramers and other oligomers arise from monomers, so there must be an equilibrium between monomeric and oligomeric forms in cells. Pathogenic events (e.g., mutations) could alter this equilibrium, and some therapeutic compounds could potentially re-establish it, as we explicitly suggested1.

Human diseases characterized by insoluble extracellular deposits of proteins have been recognized for almost two centuries. Such amyloidoses were once thought to represent arcane secondary phenomena of questionable pathogenic significance. But it is has now become clear that many different proteins can misfold and form extracellular or intracellular aggregates that initiate profound cellular dysfunction. Particularly challenging examples of such disorders occur in the post-mitotic environment of the neuron and include Alzheimer's and Parkinson's diseases. Understanding some of the principles of protein folding has helped to explain how such diseases arise, with attendant therapeutic insights.

The spectrum of inflammatory diseases of the central nervous system has been steadily expanding from classical autoimmune disorders such as multiple sclerosis to far more diverse diseases. Evidence now suggests that syndromes such as Alzheimer's disease and stroke have important inflammatory and immune components and may be amenable to treatment by anti-inflammatory and immunotherapeutic approaches. The notion of 'vaccinating' individuals against a neurodegenerative disorder such as Alzheimer's disease is a marked departure from classical thinking about mechanism and treatment, and yet therapeutic vaccines for both Alzheimer's disease and multiple sclerosis have been validated in animal models and are in the clinic. Such approaches, however, have the potential to induce unwanted inflammatory responses as well as to provide benefit.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.

CEREBRAL deposition of the β-amyloid peptide (Aβ) is an invariant feature of Alzheimer's disease. Since the original isola-tion and characterization of αβ (ref. 1) and the subsequent cloning of its precursor protein2–5, no direct evidence for the actual production of discrete Aβ has been reported6–11. Here we investigate whether Aβ is present in human biological fluids using antibodies specific for an epitope within Aβ that spans the site of normal constitutive cleavage12,13. These antibodies were used to construct a sandwich type enzyme-linked immunosorbent assay that detects Aβ in cerebrospinal fluid, plasma and conditioned medium of human mixed-brain cells grown in vitro (see also ref. 14). By affinity chromatography, we have purified and sequenced Aβ and a novel Aβ fragment from human cerebrospinal fluid and conditioned medium of human mixed-brain cell cultures. These findings demonstrate that Aβ is produced and released both in vivo and in vitro. These observations offer new opportunities for developing diagnostic tests for Alzheimer's disease and therapeutic strategies aimed at reducing the cerebral deposition of Aβ.

Studies of the molecular basis of Alzheimer's disease exemplify the increasingly blurred distinction between basic and applied biomedical research. The four genes so far implicated in familial Alzheimer's disease have each been shown to elevate brain levels of the self-aggregating amyloid-beta protein, leading gradually to profound neuronal and glial alteration, synaptic loss and dementia. Progress in understanding this cascade has helped to identify specific therapeutic targets and provides a model for elucidating other neurodegenerative disorders.

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.

The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. Here we show that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human papilloma virus oncoprotein E7. We find that Rb can recruit histone deacetylase to E2F and that Rb cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated repression of a chromosomally integrated E2F-regulated promoter. Our results indicate that histone deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb may involve the modification of chromatin structure.