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Gāndhī Nagar, India

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The formation of new blood vessels through angiogenesis is crucial to meet the metabolic demands of organs1, 2. Accumulating evidence indicates that ECs regulate organ homeostasis and repair through the production of angiocrine factors in an angiogenesis-independent manner (Box 1). The Greek philosopher and scientist Aristotle, who is widely considered to be the founder of classical biology, proposed that blood vessels direct the configuration of organs8. On pathophysiological stress (exposure to ionizing radiation, chemical injury or hypoxic conditions, for example) or loss of tissue mass, defined angiocrine factors emanate from activated ECs (Table 1). The activated ECs relay inflammatory and injury-induced angiocrine signals to quiescent tissue-specific stem cells, which drives regeneration and enforces developmental set points to re-establish homeostatic conditions. Microvascular ECs therefore fulfil the criteria for professional niche cells that choreograph tissue regeneration by cradling and nurturing stem cells with physiological levels and proper stoichiometry of angiocrine factors. The contribution of the endothelial niche to mediating stem-cell homeostasis and function has been studied in depth in neural stem cells (NSCs), spermatogonial stem cells and haematopoietic stem and progenitor cells (HSPCs). The adult brain contains two regions in which NSCs undergo neurogenesis: the ventricular subventricular zone (V-SVZ) and the subgranular zone (SGZ). In the V-SVZ, type B1 quiescent and activated NSCs give rise to type C transit amplifying cells and type A mature neuroblastic cell progenies, which are positioned in the proximity of capillary ECs9, 10, 11, 12 (Fig. 2a). Similarly, in the SGZ, which is located in the dentate gyrus of the hippocampus, NSCs and their progenies reside near capillaries13. Brain capillaries are lined with ECs that are positive for VEGF receptor (VEGFR)-2 and vascular endothelial (VE)-cadherin, positive or negative for the CD133 antigen, negative for or express only low levels of thrombomodulin, and that display zones of variable permeability4, 10, 12 (Figs 1d, 2a). Subsets of V-SVZ and SGZ blood vessels have a specialized planar morphology in which NSCs extend their endfeet to contact ECs. This close proximity supports the possibility that angiocrine factors regulate neurogenesis. In vitro studies of neuronal cells that were co-cultured with heterotypic-derived ECs support a model in which ECs regulate NSC homeostasis and differentiation. Primary human umbilical vein ECs have been shown to produce brain-derived nerve growth factor (BDNF), which fosters the expansion of neuroblasts14. Bovine pulmonary artery ECs and polyoma-middle-T-immortalized mouse brain capillary ECs, but not smooth-muscle cells, trigger Notch signalling by secreting soluble factors that increase the self-renewal of NSCs and drive neurogenesis9. Follow-up studies showed that pigment epithelium-derived factor (PEDF) was one of the secreted angiocrine factors that stimulates Notch-dependent self-renewing symmetric divisions of NSCs15. Subsequent in vivo experiments demonstrated that angiocrine factors derived from brain ECs regulate the homeostasis and regeneration of NSCs both through direct cellular contact and in a paracrine manner13, 16, 17, 18. Under steady-state conditions, angiocrine expression of the membrane-bound proteins EphrinB2 and Jagged-1 (refs 19, 20) sustains the dormancy of quiescent NSCs. Direct contact of EC-derived EphrinB2 and Jagged-1 with the endfeet of these cells suppresses their entry into the cell cycle and keeps them in an undifferentiated state. Moreover, neurotrophin-3 (NT-3), which is selectively produced by ECs in the brain and choroid plexus, maintains NSC quiescence, in part, through the induction of endothelial nitric-oxide synthase and the production of nitric oxide21, 22. Although NSCs could also supply endothelial nitric-oxide synthase, the angiocrine release of NT-3 in the V-SVZ and cerebrospinal fluid dictates nitric oxide production that sustains stem-cell quiescence. Conditional deletion of NT-3 in adult mouse brain ECs depletes NT-3 in both cerebrospinal fluid and the V-SVZ, which leads to an increase in dividing activated NSCs that express glial fibrillary acidic protein (GFAP) and accelerates the exhaustion of the NSC pool. Thus, angiocrine factors actively enforce the quiescence that is crucial for the long-term maintenance of the NSC population. During regenerative processes, irrigation of the V-SVZ by soluble angiocrine factors such as BDNF14, PEDF23, betacellulin24 and placental growth factor-2 (PlGF-2)25, and of the SGZ by VEGF-C26, 27, orchestrates proliferation and differentiation of both quiescent and activated NSCs into transit amplifying cells and neuroblasts. Notably, graded angiocrine deposition of SDF-1 (ref. 28) and BDNF29 by blood vessels that run along the rostral migratory stream in the mouse brain guides the proliferation of transit amplifying cells and their migration to the olfactory bulb30. Therefore, brain capillary ECs not only supply the V-SVZ and SGZ with region-specific regenerative and path-finding cues, but also secrete angiocrine factors into cerebrospinal fluid to potentially modulate neuronal homeostasis throughout the brain. Crosstalk between neuronal cells and angiogenic ECs allows the endothelial niche to adapt to regenerative neurogenesis (Fig. 1e). During vascular sprouting, cross-activation of ECs by neuronal-derived angiogenic factors regulates the differential production of angiocrine factors (Fig. 2a). After hypoxic injury, upregulation of VEGF-A through the activation of VEGFR-2 enhances the production of nitric oxide, which induces BDNF in brain capillary ECs to drive the expansion and maturation of transit amplifying cells16. Growth differentiation factor (GDF)-11 also enhances neurogenesis by remodelling the blood vessels31. Thus, endothelial niche cells in the brain possess a remarkable angiocrine plasticity that can adapt to the physiological demands of NSCs to initiate, execute and finalize neurogenic programmes. Undifferentiated type A spermatogonial stem cells from mice reside in the vicinity of interstitial capillaries within the seminiferous tubules of the testes32. After perturbation of the testicular microenvironment, transplanted donor-derived spermatogonial stem cells localize to zones that are enriched in capillaries. In vitro studies have shown that spermatogonial stem cells that express the G-protein-coupled receptor GPR125 can directly convert to multipotent progenitor cells. Incubation of such spermatogonial stem cells with vascular-like stromal cells that carry the CD34 antigen is essential for the conversion of spermatogonial stem cells to pluripotent stem cells33, 34, 35, and indicates that angiocrine factors play an important part in regulating the maintenance and self-renewal of spermatogonial stem cells. Indeed, transcriptional analysis of testicular endothelium suggests that ECs could be a rich source of glial-cell-line-derived neurotrophic factor (GDNF)4. Further analysis of the phenotypic and functional properties of testicular ECs is necessary to determine the degree to which ECs influence spermatogonial stem-cell homeostasis by deploying angiocrine factors and depositing peritubular extracellular matrix components. The first evidence that ECs establish an instructive niche for haematopoietic cells (Fig. 2b) was the demonstration that homotypic human bone-marrow-derived ECs expand human umbilical cord blood-derived CD34+ cells ex vivo36, 37. Furthermore, heterotypic primary ECs isolated from brain, heart and fetal tissues have since been shown to promote the proliferation of mouse38, 39, human40, 41 and non-human primate HSPCs42. However, these co-culture studies were performed in media supplemented by serum that contained supraphysiological doses of growth factors and under ambient oxygen tension, which masked the full potential of ECs to regulate the function of the cells. The development of techniques for serum-free and xenobiotic-free culture of primary human or mouse homotypic EC monolayers (Box 2) has facilitated the identification of angiocrine factors that support the self-renewal and differentiation of HSPCs in such co-culture studies43, 44. Co-culture studies have also been used to demonstrate that bone-marrow sinusoidal ECs that are positive for VEGFR-3, VEGFR-2, VE-cadherin and CD31 stimulate the self-renewal of HSPCs by expressing soluble and membrane-bound angiocrine factors45, 46, 47, including bone morphogenic protein (BMP)2 and BMP4, insulin growth factor binding protein (IGFBP)2, SDF-1, Desert hedgehog (Dhh) protein, Notch ligands, Wingless-type MMTV integration site (Wnt)5a, and Kit ligand (Fig. 2b). Bone-marrow sinusoidal ECs also drive the lineage-specific differentiation of HSPCs by producing granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, granulocyte colony-stimulating factor (G-CSF), IL-1, tumour necrosis factor (TNF), chemokines and metalloproteinases45. Notably, ECs that are transitioning through various activation states also produce inhibitory factors, such as transforming growth factor (TGF)-β1 (ref. 48), dickkopf-related protein (DKK)1 and DKK3, which block WNT signalling, and Noggin, which interferes with BMP signalling45 (Fig. 2b, Table 1). Thus, ECs express inhibitory and stimulatory angiocrine factors that regulate the quiescence and proliferation of HSPCs. ECs cultured under serum-free conditions were shown to supply angiocrine factors at physiological levels that increase the self-renewal of repopulating authentic mouse haematopoietic stem cells by 150-fold46 and of human cord blood severe combined immunodeficiency repopulating cells by 8-fold49. Direct contact between haematopoietic cells and ECs is essential for the self-renewal and differentiation of HSPCs45, 46, 47. Compared with mesenchymal cells, ECs are more efficient at expanding umbilical cord blood-derived HSPCs50. Other angiocrine factors, such as prostaglandin E2 (PGE2) (refs 51, 52), pleiotrophin53 and epidermal growth factor (EGF)54, drive haematopoietic reconstitution, which establishes ECs as a physiological repository of HSPC-supportive factors. The first in vivo evidence to support the role of the endothelial niche in haematopoiesis came from a study of mice that are unable to produce soluble Kit ligand, an essential regulator of haematopoietic stem-cell biology55. It demonstrated that compartmentalized — yet interactive — stromal and endothelial niche cells regulate the regeneration of HSPCs. In response to physiological stress, the activation of matrix metalloproteinase (MMP)-9 leads to the release of soluble Kit ligand from cells in the niche, which stimulates the regeneration and proper transportation of HSPCs. Follow-up studies showed that phenotypically marked stem cells reside in close proximity to the endothelial niche56. Further evidence indicated that haematopoietic regeneration and thrombopoiesis after chemotherapy or irradiation is impaired by the conditional deletion of VEGFR-2 in ECs of adult mice47 and by the targeting of VE-cadherin to disrupt reconstitution of the endothelial niche57, 58. Therefore, the endothelial niche is essential not only for sustaining the self-renewal of haematopoietic stem cells, but also for multi-lineage reconstitution (Fig. 2b, Table 1). Haematopoietic regeneration is orchestrated by the differential production of angiocrine factors that are induced by signalling pathways activated within ECs45(Fig. 2b). After myeloablative stress, angiogenic factors such as VEGF-A, VEGF-C, FGF-2 and the angiopoietins upregulate other angiocrine factors, including Jagged-1, through activation of AKT (also known as protein kinase B). Conditional deletion of Jagged-1 in ECs impairs haematopoietic recovery59, which suggests that Notch activation prevents the exhaustion of HSPCs. During the angiogenic phase of regeneration, AKT phosphorylation is accompanied by the activation of p42/p44 mitogen-activated protein kinase (MAPK). This triggers the secretion of G-CSF, macrophage colony-stimulating factor (M-CSF), GM-CSF and IL-6 to expand populations of myeloid, megakaryocytic and lymphoid progenitor cells and aid haematopoietic reconstitution45 (Fig. 2b). In turn, maturing haematopoietic cells produce inhibitory angiogenic factors that prevent excessive sprouting of regenerating sinusoidal vessels. For example, mature megakaryocytes produce TSP-1, which decelerates angiogenesis and shuts off the production of activating angiocrine factors to restore homeostasis4, 60. Notably, AKT-activated bone marrow ECs, which emulate some of the functions of in vivo angiogenic ECs, expand long-term repopulating haematopoietic stem cells under serum-free culture conditions, whereas bone-marrow-derived stromal cells direct stem-cell attrition44. Moreover, protection of the haematopoietic microenvironment through transplantation of AKT-activated bone marrow ECs, but not mesenchymal ones, accelerates haematopoietic recovery after lethal irradiation44. Therefore, the equilibrium between AKT and MAPK activation regulates multi-lineage haematopoietic recovery. The contribution of the endothelial niche to steady-state haematopoiesis was unravelled by studies in which selective deletion in ECs of SDF-1, Kit ligand or Jagged-1 impaired the maintenance of HSPCs44, 61, 62, 63, 64. Several studies have also scrutinized the relative contribution of bone marrow perivascular cells to the homeostasis of HSPCs65, 66, 67. Because the functions and structural stability of endothelial and non-vascular cells is mutually dependent, the deletion of factors in one niche has the potential to perturb the constituents of the neighbouring one. Therefore, genetic manipulations within the intimately associated endothelial niche and accompanying perivascular cells could have off-target effects, which must be taken into consideration. Nonetheless, the findings of these in vivo and reductionist in vitro studies suggest that, irrespective of the localization of HSPCs, angiocrine factors that are presented by either arteriolar or sinusoidal endothelial niches have executive functions and serve as 'rheostats' that choreograph haematopoietic stem-cell self-renewal and differentiation during homeostasis and recovery after haematopoietic suppression. Furthermore, these studies demonstrate that some, but not all, heterotypic ECs can support HSPC expansion, which confirms that each organotypic vascular bed is endowed with unique angiocrine attributes that are suitable for stem-cell homeostasis and reconstitution4, 44, 46, 47. During fetal development, inductive signals from ECs68 specify the development of haemogenic ECs69, 70. Thus, an endothelial niche could induce the direct conversion of all ECs into haemogenic ones, which give rise to definitive haematopoietic stem cells. Notably, endothelial niche-derived angiocrine signals are essential for the direct conversion of adult ECs into haematopoietic cells. In this approach, adult ECs were transduced with the transcription factors FosB, Gfi1, Runx1 and Spi1 (collectively termed FGRS). However, FGRS-transduced ECs failed to convert to engraftable haematopoietic cells unless they were co-cultured in direct contact with ECs71. Moreover, co-culture of haematopoietic cells that were derived from mouse and non-human primate pluripotent stem cells and an endothelial niche enhanced the engraftment of putative haematopoietic cells, in part through the deployment of Notch ligands72, 73. Thus, angiocrine signals from ECs participate in the specification, development, homeostasis, self-renewal and differentiation of haematopoietic stem cells.

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Now scientists at the Department of Energy's Pacific Northwest National Laboratory have made a "vitamin mimic" - a molecule that looks and acts just like the natural vitamin to bacteria, but can be tracked and measured by scientists in live cells. The research offers a new window into the inner workings of living microbes that are crucial to the world's energy future, wielding great influence in the planet's carbon and nutrient cycle and serving as actors in the creation of new fuels. Vitamins are a powerful currency for researchers seeking to compel microbes to give up their secrets. "We have a lot to learn about how microbes accumulate and use nutrients that are necessary for their survival and growth. This provides a window for doing so," said chemist Aaron Wright, the corresponding author of the study published in ACS Chemical Biology. "Perhaps we will be able to make a microbial community do what we want, by controlling its access to a specific nutrient," Wright added. To control the bacteria via vitamins, Wright and his team have to know what other proteins in the cell the vitamins are consorting with, and where and when. Think of a planner analyzing emergency services for a large city. Knowing that an ambulance enters the city occasionally and transports some people somewhere, for instance, is not nearly as useful as knowing the precise address of the caller, the identity of the injured, and the location of the nearest hospital. It's the same for scientists trying to understand microbial cells. While a cell is infinitesimally small, the activity within resembles the hustle and bustle of a large city, with many functions within carried out by thousands of entities. Knowing precisely which vitamins aid which proteins, under what circumstances, to keep things running is a must if scientists are to maximize microorganisms for energy production and other processes. "Microbial communities are organized based on their ability to get the resources they need to survive and grow," said Wright. "We need to understand how the availability of nutrients, like vitamins, helps determine the structure of a microbial community as a step toward controlling that community in ways we would like to be able to do." Wright's team studied the bacterium Chloroflexus aurantiacus J-10-fl, which is a common member of microbial mats - gloopy natural structures (think pond scum) where layers containing different groups of microbes band together. In these collections, C. aurantiacus often plays the role of anchor, helping to hold together an assortment of microbes. The bacteria, which resemble strands of string under the microscope, are usually found in hot springs, since they enjoy temperatures above 100 degrees Fahrenheit. Wright's team performed a series of synthetic chemical steps to alter three vitamins that C. aurantiacus needs to survive: vitamin B1 (thiamine), vitamin B2 (riboflavin), and vitamin B7 (biotin). While the bacteria recognized the substances as normal vitamins, the researchers can monitor the mimics much more easily than their natural counterparts. Wright's team used the mimics to relay a treasure trove of information about how vitamins enter the cell and interact within the cell, by analyzing the precise location of the molecules' activity in living cells. Through a system called affinity-based protein profiling, Wright's group effectively tagged these molecules where they're active, then used techniques such as mass spectrometry to sort and measure proteins of interest. One of the team's findings suggests multiple vitamins may share the same molecular machinery to gain entry into the cell. The team is still investigating these data. These findings can provide a road map for scientists like Wright who are trying to direct microbes as part of broad efforts to create clean, renewable fuels and reduce the effects of climate change. Explore further: Vitamin water: Measuring essential nutrients in the ocean More information: Lindsey N. Anderson et al. Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions, ACS Chemical Biology (2015). DOI: 10.1021/acschembio.5b00918

Venkat Ratnam M.,National Atmospheric Research Laboratory NARL | Krishna Murthy B.V.,B1 | Jayaraman A.,National Atmospheric Research Laboratory NARL
Geophysical Research Letters

Indian Summer Monsoon (ISM) is mainly characterized by seasonal wind reversal in low level jet stream and tropical easterly jet (TEJ) among several other elements of monsoon systems. TEJ is observed in general between 100 and 150 hPa during June-September over the Indian region and its strength is directly related to the monsoon rainfall. In the context of changing climate, large reduction in its extent and weakening of its strength were reported. Using high resolution measurements, we report here the observation of a sharp strengthening of the TEJ during the recent warmest decade (2001-2010), reaching its 1970s value. We also show that this change is reflected in the tropical cyclone systems and finally on the precipitation patterns over the Indian region as they are interlinked. We attribute this unusual change partly to the change in the circulation due to the tropospheric warming and lower stratospheric ozone recovery. Key Points Showed that Indian Summer Monsoon circulation has changed in recent past Its effect on number of cyclones over Bay of Bengal is also going to change ISM modelers/forecasters need to consider these issues in future modeling ©2013. American Geophysical Union. All Rights Reserved. Source

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The automated body needs 6 hours rest per 24, completed in 1-2 intervals of horizontal interface during which nutrition and hydration are accomplished via oral-esophageal connection duct. Liquified grain-legume meal provides 1500-1900 calories with 2-3 liters water and simultaneously delivers pharmaceutical support, quantities variant with unit size. Excretion is accomplished during interface via anal duct and throughout labor activity via catheter. Program revisions and medical and/or hardware diagnostics and repair may be performed as necessary during this time. Interface terminates with antibacterial flush. When Energy Debt (ED) is nonrecuperable and/or violation is grave (see: PENALTIES level 6-9, severe and/or permanent energy depletion via productivity suppression or damage to persons/property, inadequate productivity (long-term), excessive resource intensivity (long-term), recidivism, etc.) the unit is fully automated for maximum efficiency and productivity. Full automation permits maximal communication that is not vulnerable to connectivity failures/interruptions. Hardware connections established via the sphenoid and magnum foramina allow control of sensorimotor and semi-autonomous systems. Operating systems integrate primarily with parietal and occipital lobes of the cerebrum and with the cerebellum. Activity is surgically suppressed in the frontal and temporal lobes and the limbic system. Full automation is permanent and irreversible. Fully automated units (FAUs) will experience catastrophic functional failure in disconnection. When significant functional lifetime remains such that average potential productivity exceeds ED (see: PENALTIES level 3-5, accidental or minor energy depletion via productivity suppression or damage of persons/property, inadequate productivity (short term), excessive resource intensivity (short term) etc.) semi-automation permits reintegration when ED recuperation is complete. In semi-automated units (SAUs) frontal cortical and limbic activity is pharmaceutically suppressed and hardware connectivity established with the parietal lobe. SAUs may retain partial awareness. Pharmaceutical support ensures that post-reintegration subjects retain few if any specific memories of incidents during automation. (see: GUIDELINES Ethics 5c.2 and PENALTIES section 2g) Damage and resulting infection shorten unit longevity. FAUs will not react to external or sensory stimuli and require regular visual/manual assessments for autonomic reactions and/or symptoms of damage or illness. SAUs may experience some voluntary and reflex reactions detectable by electronic systems. Evaluate damage according to degree of efficiency impairment and/or threat of product contamination. Non-contaminating injuries and illness (e.g. minor bone fractures, internal and non-ulcerating tumors, etc. See MAINTENANCE B1) and those that do not impede efficiency may not require treatment. Conditions affecting production should receive appropriate medical intervention, with the exception of analgesic/anaesthetic medication obviated by systematic neural suppression. System connectivity failures may result in brief interruptions of control. Such interruptions typically last 0.5-1 second before correction and do not significantly impede unit efficiency. More frequent and/or longer interruptions due to internal hardware malfunction or displacement impede efficiency and may result in equipment damage. SAUs in temporary or malfunctional disconnections experience disorientation and anxiety with unpredictable reactions and behaviors. Sedation may be required to facilitate repair. Increase ED to compensate for additional support. If semi-automation proves ineffective/inefficient due to continued or increased connectivity failure frequency, ED will increase and full automation may be required. Major hardware corrections should be attempted no more than once. Minor corrections should be administered if feasible and if the disconnected unit will submit to it. Average longevity of FAUs is 6-8 years. Expiration is determined based on frequency of damage or illness. Remains are cremated and added to growth medium. Average ED-recuperation period for SAUs is 1-3 years. Post-reintegration neural scarring and minor damage resulting in diminished mental capacity are expected. Former SAUs receive manual labor assignments appropriate to remaining mental capacity although individual outcomes differ widely, variant with automation time and neural recovery. Post-reintegrative compliance rates are high. Recidivism rates vary according to initial violation but average near 7%. Data excludes non-reporting former SAUs. Non-reports are presumed deceased or departed from the cities. This attrition rate is negligible. Disconnect and rise from the half-lit rows of the underground, emerge, proceed in line through the glow of the dome at sundown. ASSIGNED Rinse station 12. Stand by for supply. Pick spinach at night, once recovered from heat and daylight wilting. Leaves carry remnants of growth medium. The thin flesh so easily bruised. Three rinses, packaging in plastic; throughout, hands remain light. Finger prickles: dark eyes, long skinny legs. A crawl, a pinch along the index. The son, remember ERROR A spider. Nine hours, a cool damp dream of spinach, until next interface. ASSIGNED Tomatoes require staking, removal of extraneous growth. Wrong stems, too many flowers shrink existing fruit. Favor the hard bulbs, the green infant fists of future food. Fertilization requires bees. Floating gold vibrations spiral through the mist of solar light, tense and constant between blossoms. Can't stop moving just like his dad, stupid kid ERROR Hands turn green with sap, red with stings. Late shoots scatter the brown sponge of growth medium. So stubborn even as a baby ERROR stiffens and screams and won't ever sleep through ERROR Nine hours. Interface. ASSIGNED Enlarged fingers lose leaves in sinks, seek again, try to grasp, failed hand splashing in cold water FUNCTION FAILURE your fucking fault can't you ever ERROR so swing a fist for control UNIT FAILURE ALERT wires light across the cerebral cortex, thalamus to cerebellum and down through spine. Dendrites flare. Body crashes, crooks. Struggles to right REASSIGNED: Diagnostics. See what keeps blippin’ in this guy— fluorescent void and jolt shadow above, fingers disappearing in the dark half of empty other eye Okay, the central sulcus, can you enlarge and something rips open, something Here’s our problem— choking and the arms jolt up, find fingers in a soft mouth the wet the hard teeth Hey now look, can you— Push to upright, pull the tubes like tearing out a throatful of fire, flaming through the muscle like veins. Flat grayface figures with hands held out All right, guy, just calm Shove aside, out and past, gotta get away get alone and remove all this, layers of rubber, thighbag of piss, the filth and the smell— What’s his name, anyway, is he— halls curve like tubes around the floor. Like looking through rain. Clear plastic blurs rows of bodies into one body, one movement, one machine. And run. Like a rat between cages but there’s gotta be a way out, across the floor between rows under the crushing weight of smell: unwashed shit and untreated infection, rot and ferment and death. Bodies unperceiving, unperceived even as they crash. But go—through the next tube, lose the guard, toward the doorway, just go Okay, Eastman—Joseph Eastman, listen— and on the door, a reflection—the boy. you shouldn’t be here, are you really here? Joe how— but it’s the same as always, squared off and screaming full-grown boy can’t act his age, can’t respect his own father, brought you into this world, teach you how to act— and stop and the impact, body to body to floor okay hold him still, get his arms the same fist, the same lesson been teaching all this time, the blow that always falls, that cannot fall again starting sedation in the chest port now— eyes like a mirror to recognize mine, my face to defeat, my fault he can’t submit to it Fallen, failed again, lost eye filling with the same blood that he gets from me eyelid Interface. Elegant patterns of hands flow independent of slack-faced figures, gathered under the dome and the shortening night. The spinach swells emerald as the bees sleep, hives dormant in the dark. With the light they wake, merge into light, the condensed warmth of morning sun. And yes, it will come up again. And yes, I will remember.

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The proteasome, a ~2.5-MDa molecular machine, is the focal point for global regulation of ubiquitin pathway output10. Among its major regulators is a deubiquitinating enzyme known in mammals as USP14 (in yeast, Ubp6). USP14 negatively regulates proteasome activity by ubiquitin chain disassembly as well as by a noncatalytic mechanism1, 2, 3, 4, 5, 6. USP14 inhibitors produce selective effects on the turnover of proteasome substrates3, 8, suggesting that the rate at which USP14 disassembles proteasome-bound ubiquitin chains may depend on the nature of the substrate. We test this hypothesis in the present work. USP14 is thought to progressively trim monoubiquitin groups from the substrate-distal tip of a chain9 (Fig. 1a). To assess this model, we employed a canonical substrate of USP14, ubiquitinated cyclin B1 (refs 1, 3). When cyclin B1 is modified by the ubiquitin ligase anaphase-promoting-complex/cyclosome (APC/C) in the presence of E2 enzyme UbcH10, the resulting conjugates typically carry multiple short ubiquitin chains of varied length11, 12. We refer to these as supernumerary chains, insofar as only one chain is in principle required for substrate degradation. The flexible, 88-residue amino-terminal element of cyclin B1 (NCB1) is enriched in lysine residues, nine of which are competent for ubiquitination11. The N-terminal element is necessary for cyclin B1 degradation, and contains an APC/C recognition motif, the destruction box11, 12, 13. Ubiquitinated NCB1 was rapidly degraded by proteasomes lacking USP14, but in the presence of USP14 deubiquitinated species were observed. These were not fully deubiquitinated, but rather carried 2–4 ubiquitin groups and were resistant to both degradation and further deubiquitination (Fig. 1b). This strong stop to USP14-dependent processing might be assumed to reflect that these conjugates retain too few ubiquitin moieties for efficient proteasome binding. However, studies below show that USP14 activity depends on the architecture of ubiquitin chains on the substrate and not simply substrate binding to the proteasome. The behaviour of NCB1 was comparable to that of full-length cyclin B1 (data not shown)3, 12. We used the experimental system described above to test whether the architecture of ubiquitin chains on NCB1 was relevant to its rapid deubiquitination. Cyclin B1 is modified by APC/C and UbcH10 to form mixed-linkage chains11 primarily via residues K11, K48, and K63. Thus, a lysine-free mutant of ubiquitin12 will modify NCB1 via multiple mono-ubiquitination events (Fig. 1c). USP14 deubiquitinated this form of NCB1 at a rate comparable to that of wild-type conjugates, indicating that USP14 does not act obligatorily on ubiquitin–ubiquitin linkages (Fig. 1c). In a complementary experiment, we eliminated lysines from NCB1 rather than ubiquitin, leaving behind only K64 of NCB1 to allow for the synthesis of a single ubiquitin chain on NCB1. This mutant is competent for degradation (Fig. 1d)12. Although single-chain conjugates are canonical proteasome substrates14, USP14 showed no detectable activity on this substrate (Fig. 1d). The lack of deubiquitinating activity on the single-chain substrate was accompanied by a failure to inhibit its degradation. Similar results were obtained with NCB1 ubiquitinated with Ubc4 in place of UbcH10 (Extended Data Fig. 1a, b). A caveat to the experiment above is that, because NCB1 is degraded quickly by the proteasome, USP14 has little time to act. However, the proteasome must be present in the assay because it is required to activate USP14. When we quenched substrate degradation by replacing ATP with ADP (Extended Data Fig. 2), USP14-dependent deubiquitination of WT NCB1 conjugates was preserved (Fig. 1e). In contrast, no deubiquitination was detected with single-chain conjugates (Fig. 1f). Thus, USP14 shows marked specificity for NCB1 conjugates bearing multiple ubiquitin chains, whereas the proteasome does not effectively distinguish between these two classes of substrate in the absence of USP14. We next considered whether the resistance of single-chain conjugates to USP14 was idiosyncratic to the particular modified lysine in NCB1. On the contrary, conjugates generated using a K36-only form of NCB1 behaved equivalently to the K64-anchored conjugates (Extended Data Fig. 1c). The supernumerary chain effect was seen with the substrate PY-Sic1 as well as NCB1 (Extended Data Fig. 1d, e). We identified a third preferred substrate of USP14, securin, which is likewise modified at multiple lysines (Extended Data Fig. 1f)13, 15. Ubp6, the Saccharomyces cerevisiae orthologue of USP14, showed the same preference for supernumerary chains, indicating that this property is evolutionarily conserved (Extended Data Fig. 3). It has been hypothesized that USP14 or Ubp6 may preferentially cleave K63-linked chains16, 17. This model could account for our data if single-chain NCB1 conjugates were devoid of K63 linkages. To test this model, we prepared single-chain NCB1 with homogeneous K63 linkages. These conjugates were also cleaved slowly if at all by USP14 (Extended Data Fig. 4), regardless of length. One explanation for the specificity of USP14 for substrates modified at multiple sites is that cleavage of one chain is promoted allosterically by binding of a second chain to USP14 or another site on the proteasome. However, adding free chains over a wide concentration range failed to stimulate deubiquitination of single-chain NCB1 (Extended Data Fig. 5). Another measure that failed to stimulate removal of singleton chains was phosphomimetic substitution of Ser432, a site subject to modification by Akt18 (Extended Data Fig. 6). If singleton chains are as a rule poor substrates for USP14, then all free ubiquitin chains—those not attached to an acceptor protein—must be poor USP14 substrates, regardless of length or linkage type. Extended Data Fig. 7 shows that free chains of diverse length and linkage type are all cleaved minimally by proteasome-activated USP14, even upon long incubation. By ubiquitinating NCB1 with preformed tetraubiquitin chains, we prepared conjugates carrying multiple chains of homogeneous linkage type and length. When these conjugates were incubated with USP14, proteasomes, and either ATP or ADP, a limit digest product was formed consisting of NCB1 carrying four ubiquitin groups (Fig. 2a, b). Thus, USP14 seemed to completely remove each supernumerary chain, and stop when a single chain remained. Accordingly, ubiquitin was released from this substrate in the form of tetraubiquitin chains (Fig. 2c). Using mass spectrometry, we confirmed that K48 linkages were not consumed in this reaction (Fig. 2d). Studies with other linkage types (Extended Data Fig. 8) further indicated that USP14 removes chains preferentially en bloc (Fig. 2e), rather than trimming them from the distal tip, as previously thought. Why does USP14 discriminate against cleavage within chains? The presumptive active form of Ubp6 sits with its catalytic domain directly contacting the OB domain of subunit Rpt1 (refs 5,6). Modelling studies with diubiquitin as substrate indicated that, regardless of chain linkage type, the proximal ubiquitin may have restricted access to the active site of the enzyme (Fig. 2f, Extended Data Fig. 9). The coiled-coils of Rpt1 and Rpt2 are responsible for this occlusion (Fig. 2f). This effect may account for the dominant en bloc cleavage mode of Ubp6 and USP14. Cleavage within ubiquitin chains by free forms of USP14 and Ubp6, as observed by many researchers, may reflect that free USP14 is not subject to occlusion from proteasome subunits. In a recent study, we established a single-molecule TIRF (total internal reflection fluorescence) microscopy method to monitor deubiquitination events mediated by the en bloc zinc-dependent activity of RPN1119, 20, using USP14-free proteasomes15. Deubiquitination was identified as a stepwise reduction of signals from fluorescently-labelled ubiquitin on substrates (Fig. 3a, Extended Data Fig. 10). To study USP14-mediated deubiquitination, we inhibited RPN11 activity (as well as substrate degradation) using ADP and a zinc chelator15. ‘Stepped’ events representing deubiquitination increased sevenfold upon USP14 addition (Fig. 3a). Single-molecule traces with USP14-reconstituted proteasome in the presence of ADP showed remarkable similarity to those produced by RPN11 (Fig. 3b, Extended Data Fig. 10). The step size of ubiquitin removal by RPN11 and USP14 followed a similar distribution, supporting en bloc cleavage by USP14 (Fig. 3c). USP14-mediated deubiquitination reduced the dwell time of ubiquitin conjugates on the proteasome, suggesting that rapid deubiquitination can suppress degradation of the substrate by contracting the duration of its encounter with the proteasome (Fig. 3d). This dwell time effect suggests that, to suppress degradation, USP14 must remove chains at least as rapidly as the substrate is degraded in the absence of USP14. To test this scenario we used quench-flow methods, which allow reactions to be followed on a millisecond time scale. An excess of proteasome over substrate was employed to approximate single-turnover conditions. USP14-free proteasomes degraded NCB1 conjugates efficiently, with most degradation occurring between 9 and 30 s (Fig. 4a, b). USP14 delayed degradation (Fig. 4b), as high molecular weight conjugates were instead converted into partially deubiquitinated species. The production of lower molecular weight species through deubiquitination was initiated before degradation was evident with USP14-free proteasomes (Fig. 4a–c). Thus, USP14 is sufficiently fast that deubiquitination can be accomplished before the proteasome would otherwise degrade NCB1. Similar results were obtained using full-length cyclin B1 (data not shown), and we confirmed that deubiquitination was mediated by proteasome-bound rather than free USP14 (Extended Data Fig. 6b). The ability of USP14 to prevail over the proteasome in a kinetic competition may explain the suppression of protein degradation by USP14’s deubiquitinating activity3. Thus, USP14-loaded proteasomes may discriminate against substrates bearing multiple ubiquitin chains, whereas USP14-free proteasomes do not. Because USP14 and Ubp6 are modulated by growth factors and stress conditions2, 18, the ability of the proteasome to discriminate between single-chain and multi-chain substrates is likely to be under biological control. We expect that the supernumerary chain rule should bias USP14 action towards the products of ubiquitin ligases that are weak in chain-extending activity, thus adding ubiquitin to multiple sites on a target protein. Many ubiquitinated proteins can be modified at multiple lysines11, 12, 21, 22, as originally described for cell surface receptors23. In most cases however, it is unclear whether simultaneous modification of lysines occurs on endogenous substrates, because proteomics methods for analysis of ubiquitination generally rely on proteolytic digestion. Therefore, the development of new methodologies may be critical for a better understanding of how USP14 regulates proteasome function. We suggest that the last ubiquitin chain resists cleavage by USP14 because its site of attachment to substrate is not docked in proximity to USP14, regardless of the ubiquitin receptor to which the chain is bound. If the chain that is spared by USP14 is short, degradation will be suppressed; if it is long, then proteasome–substrate interaction will be preserved and substrate degradation will proceed. This chain will be removed by RPN11 once substrate translocation is initiated. Because RPN11 also cleaves chains proximally19, 20, our data reveal an unexpected point of similarity between these enzymes, which may act sequentially. However, RPN11 activity differs from that of USP14 in requiring ATP hydrolysis19, 20, presumably to drive translocation of the conjugate to its active site24, 25. That RPN11 promotes substrate degradation whereas USP14 suppresses degradation may reflect that RPN11 removes chains following the commitment step in the degradation pathway. It will be interesting to determine whether supernumerary chain removal is the principal role of USP14 in cells, or whether other specificity rules exist for this enzyme. Substrate flexibility, a feature of the cyclin B and securin degrons26, but not of ubiquitin itself, may be important for USP14 to act on these substrates. Indeed, the occlusion that we propose to impair docking of proximal ubiquitin in a free chain near the USP14 active site may apply as well to many folded globular domains that are ubiquitinated, which would be required to access the same site. This substrate docking constraint may further limit the substrate range of USP14. In contrast to USP14, RPN11 is expected to have a robust activity against chains on domains that are normally folded, because such chains may be presented to RPN11 only after the domain has been unfolded by mechanical force applied by the proteasomal ATPases.

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