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Category : Protein Folding

Stress increases in exopher-mediated neuronal extrusion require lipid biosynthesis, FGF, and EGF RAS/MAPK signaling –

Proteostasis is critical for the function, maintenance, and long-term survival of all cells (1). Successful maintenance of the proteome entails efficacious balancing of protein synthesis and degradation, with coordinated actions of regulated gene expression and translation, the chaperone-folding network, the ubiquitin proteasome system, and autophagy all serving critical functions in overall protein quality control. Neurons are particularly susceptible to proteostasis disruption, and aggregates are a striking and common feature of neuropathology in most human neurodegenerative diseases (2). Recent studies have shown that in diseases characterized by distinctive aggregates, including Alzheimers disease, Parkinsons disease, and Huntingtons disease, aggregates can spread from neurons to neighboring cells, inducing deleterious consequences (3). The mechanisms operative for aggregate spread are unclear and remain a matter of considerable research attention.

We discovered that adult Caenorhabditis elegans neurons can select and then extrude aggregated proteins, such as expanded polyglutamine HTTQ128-CFP (related to neurotoxic Huntingtons disease protein) and an aggregation-prone, high-expression mCherry reporter (4) (see Fig. 1A). Aggregates are sent out of the neuron in strikingly large, membrane-surrounded vesicles [4-m average diameter; for comparison, the neuron soma is 6 m; this vesicle is two orders of magnitude larger than exosomes that form by a distinct mechanism (5)]. We refer to these massive cell extrusions as exophers (exo = outside and pher = carry away). In the case of the C. elegans touch receptor neurons that mediate sensitivity to gentle touch stimuli (6), exophers are passed into the surrounding hypodermis, which can partially degrade their contents. Baseline production of exophers under nonstress growth conditions is low, detected in 5 to 20% of ALMR touch neurons, and occurs at lower rates for other touch neurons. However, we found that exacerbated proteostress (for example, expressing the human A142 fragment implicated in Alzheimers disease pathology, disrupting specific autophagy genes via RNA interference [RNAi] or introducing the MG132 proteasome inhibitor) can significantly increase exopher production (4). Animals expressing toxic HTTQ128-CFP in touch neurons that extruded aggregates in exophers retained better touch sensitivity than transgenic HTTQ128-CFP animals in which the neurons did not produce exophers (4), suggesting that exopher production that helps clear the neuron of toxic aggregates is neuroprotective, at least in the short term.

A key mystery in the biology of exophers is the precise nature of the cellular conditions and stresses that induce or elevate exophergenesis. To address this question, we systematically tested external, physiological stresses for the capacity to influence the expulsion of cellular trash. Our data 1) show a clear link between specific, environmental stresses and neuronal exopher production (namely food withdrawal, osmotic stress, and oxidative stress); 2) emphasize that there exists a permissive window for exopher production in early adult life during which external stresses can elevate exopher levels; and 3) identify a previously unrecognized limit to the level of stress that can induce exophers, such that, beyond this upper stress limit, exophergenesis is not observed. We linked the fasting-induced increase in exophers (as much as 10-fold elevation in exophers) to activities of the intestinal peptide transporter PEPT-1, the transcription factors MDT-15 and SBP-1/SREBF2, and their target FASN-1/fatty acid synthase, as well as to epidermal growth factor (EGF) and fibroblast growth factor (FGF)/RAS/MAPK signaling pathways. Our data reveal how stress conditions might promote the spread of aggregates from neurons to their neighbors and suggest pathways that might be targeted to regulate analogous processes, with implications for addressing human neurodegenerative disease.

A common stress that C. elegans encounter in their natural environment is variable food type and/or food abundance, which can have a significant impact on C. elegans gene expression, development, metabolism, and longevity (79). In the laboratory, C. elegans eat a diet of Escherichia coli spread on agar plates, and food sources can be easily manipulated. We first asked whether neuronal exophergenesis levels are sensitive to food source, quantitating ALMR exophers produced by neurons expressing an mCherry protein (strain ZB4065 bzIs166[Pmec-4mCherry1]), which is avidly expelled as exopher cargo (10) (see Fig. 1 for an example). For claritys sake we refer to assay strain ZB4065 as mCherryAg2 in the text hereafter. Exophers are typically produced by young adult animals, peaking at days 2 to 3 of adulthood and returning close to baseline by adult day 4 (Ad4) (4, 11). We therefore measured exophers produced each day, Ad1 to Ad4, to compare both daily relative levels and temporal profiles.

We quantitated exopher production when mCherryAg2 animals were fed four different E. coli strains: E. coli OP50, which is the standard C. elegans food source (12); HT115, a strain that lacks RNAaseIII and is used in RNAi studies (13); HB101, a food source that promotes larger body size and faster development (14); and NA22, a strain that fosters enhanced growth in liquid culture (15). We noted no major differences among the different E. coli strains in common laboratory use, all of which support the basic pattern of peak exopher production around Ad2 that returns to the baseline around Ad4 (SI Appendix, Fig. S1A).

Complete food withdrawal markedly induces exophers. We reared mCherryAg2 animals at 20 C and at Ad1 (onset of egg laying) moved animals to unseeded plates at 20 C for either 3 or 6 h, counting ALMR exophers shortly thereafter. Continuously fed control mCherryAg2 animals generate exophers at levels that range from 5 to 20% of animals, and in this study, baseline was close to 5% on Ad1. Subsequent to a 3-h fast, we found exophers in more than 20% ALMRs; a 6-h fast increased average exopher numbers 5- to 10-fold to nearly 50% of ALMRs examined (Fig. 1B). Fasting also causes an increase in multiple-exopher events, in which more than one exopher is generated (SI Appendix, Fig. S1B). We conclude that food withdrawal can have a rapid and dramatic impact on the extrusion of exophers in animals expressing a noxious mCherry reporter.

At 20 C, in the presence of food, the young adult wave of exopher production falls narrowly within the first 4 d of adulthood (SI Appendix, Fig. S1A). We were therefore curious as to whether the fasting-induced elevation of exophers was restricted to the Ad1 to Ad4 timeframe or, alternatively, whether food withdrawal experienced at any time point could be effective for inducing exopher production. We subjected animals from a single, synchronized population to 6-h fasting regimens as L4 larvae as well as on Ad1 to Ad10; we measured exopher levels immediately after the 6-h fast on the experimental fasting day (Fig. 1C). Our analysis of mCherryAg2 ALMR exophers in this study revealed the following: 1) a 6-h fasting protocol does not induce exopher production during the L4 stage; 2) the 6-h fasting protocol elevates exopher production to peak levels at Ad1 when administered on that day; 3) fasting efficacy is slightly lower when delivered at Ad2 and lower still when delivered at Ad3; and 4) by Ad4, a fasting-induced exopher increase is no longer observed. We conclude that the impact of food withdrawal on enhancing exopher production is limited to a window of time that covers Ad1 to Ad3. The flanking of the permissive phase by exopher-recalcitrant periods suggests the existence of a licensed period, in which the expulsion of significant amounts of neuronal material is biologically feasible and during which exophergenesis can be modulated by environmental stress signals. We observed the 6-h fastinginduced elevation of exophers in strains that expressed GFP (Fig. 1D) or expanded polyglutamine (Fig. 1F) mNG:HttQ74 in touch neurons or expressed GFP in dopaminergic neurons (Fig. 1E), indicating that the fasting induction of exophers was not reporter- or cell typespecific.

Temperature is an environmental factor that dramatically influences the C. elegans reproductive life cycle time, lifespan (16), and proteostress (17). We therefore asked whether culture temperature might influence the timing or levels of adult exopher production. We reared animals continuously at 15, 20, and 25 C from the egg stage and measured exopher levels over the first 4 d of adult life (SI Appendix, Fig. S2A). Somewhat unexpectedly, exopher levels did not vary substantially with culture temperature. In an alternative experimental design, we reared synchronized populations of animals at 20 C and then split cultures at the L4 adolescent stage into three parallel cultures that were thereafter maintained at 15, 20, and 25 C (SI Appendix, Fig. S2B). We found that, in animals shifted to 25 C, the peak of exopher production occurred earlier (Ad1) than 20 C animals, but exopher levels were not significantly elevated, relative to 20 C. Our data suggest that the experience of a temperature shift, rather than continuous noxious temperature exposure, might exert the strongest impact on levels of exopher production (SI Appendix, Fig. S2C). That said, the approximate doubling of exopher scores after an L4 shift from 20 to 25 C is modest in comparison to the 5 to 10 exopher elevation measured for food withdrawal. We conclude that, within the normal confines of C. elegans laboratory culture, the temperature at which animals are grown exerts a relatively minor influence on levels of exopher production.

Hypoxia can induce protein aggregation in C. elegans models (18), raising the possibility that hypoxia might enhance exopher production. We tested the exposure to 0.1% oxygen using an adjustable hypoxia C-174 chamber (Biospherix) to generate exposures to a controlled, hypoxic environment. We subjected Ad1 to Ad5 mCherryAg2 animals to 0.1% oxygen for 6 h and monitored ALMR exophers after the removal of animals from the chamber.

We failed to find differences in exophers produced under these hypoxic conditions on any day (SI Appendix, Fig. S2D). We also tested anoxic conditions induced by replacing oxygen with nitrogen in a sealed anoxia chamber (19), measuring ALMR exophers after 6-h exposure to anoxia (SI Appendix, Fig. S2D, dark purple line). Exposure to anoxia failed to markedly increase exopher production on any exposure day. While it is possible that longer exposures or different recovery times might alter outcomes, we conclude that hypoxia/anoxia treatments, as delivered, do not elevate exopher levels.

Osmotic stress introduces proteostasis challenges (20), and thus, we were curious as to how exopher production might respond to osmotic stress conditions. We first made agar plates designed to introduce osmotic stresses based on standard C. elegans conditions, testing 250 mM concentrations of sucrose, glucose, sorbitol, and NaCl. At Ad1, we introduced mCherryAg2 animals to these osmotic stresses for 6 h and scored ALMR exophers thereafter (Fig. 2A). We found that a 6-h exposure to osmotic stress increased exopher production 4 above baseline. Since each solute-induced stress resulted in a similar elevation of exophers, the exopher response is likely grounded in osmotic stress itself, rather than the chemical nature of the specific osmolytes.

Osmotic stress and oxidative stress can increase exopher production, but extreme stress levels can decrease exophergenesis. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. (A) Transient osmotic stress significantly elevates ALMR exophers on Ad1. We exposed mCherryAg2 animals to 250 mM sucrose, glucose, sorbitol, or NaCl on Ad1 and scored for exophers 6 h later: 5 trials and 50 animals per trial. (B) Animals chronically exposed to osmotic stress from Ad1 into adulthood produce elevated exophers with early onset peak production. We grew mCherryAg2 animals to the Ad1 stage under standard growth conditions and shifted to 250 mM sucrose, glucose, sorbitol, or NaCl, measuring ALMR exophers in these populations on Ad1 to Ad4: 4 trials and 50 animals per time point. CMH compares osmotic stress media to normal growth media. (C) Osmotic stress at 250 mM enhances exopher production more than at 500 mM. We exposed strain mCherryAg2 to 250 and 500 mM sucrose, glucose, sorbitol, and NaCl for 6 h on Ad1 and measured exophers shortly thereafter: 5 trials and 50 animals per trial. (DF) Increasing oxidative stress enhances exopher production to a limit, after which exopher production is suppressed. We grew mCherryAg2 animals to Ad1 at 20 C, and then, transferred animals to plates housing increasing concentrations of juglone, rotenone (rot), or paraquat (PQ) for 6 h, as indicated, measuring ALMR exophers shortly thereafter (black asterisks compare to control): 7 trials and 50 animals per trial. (G) Combined food withdrawal and osmotic stress can suppress exopher induction under conditions that individually induce high-exopher levels. We fasted Ad1 mCherryAg2 animals for 6 h on plates containing the solutes indicated (20 C) and scored for ALMR exophers shortly thereafter: 3 trials and 50 animals per trial (asterisks compare two indicated conditions at bar ends). (H) Combined food withdrawal with oxidative stress can suppress exopher induction under conditions that individually induce high-exopher levels. We fasted Ad1 mCherryAg2 animals for 6 h on juglone, rotenone, and paraquat at the concentrations indicated, scoring for ALMR exophers shortly thereafter: 5 trials and 50 animals per trial (asterisks compare two indicated conditions). (I) While anoxia does not increase exophers, combining food withdrawal and anoxic stress can suppress fasting-associated exopher induction. We fasted Ad1 mCherryAg2 animals for 6 h under anoxic conditions, scoring for ALMR exophers shortly thereafter: 4 trials and 50 animals per trial. Note that our anoxia protocol is not effective in limiting fasting-induced exopher elevation (SI Appendix, Fig. S2 D and H).

We also quantitated exopher levels under conditions of long-term, hyperosmotic stress during adult life (Fig. 2B). We raised animals under standard growth conditions until the L4 stage and then moved animals to 250 mM sucrose, glucose, sorbitol, or NaCl plates for adult life. We first measured exopher levels 24 h after initiating osmotic stress and tested the same population on Ad1 to Ad4 thereafter. For all solutes tested, osmotic stress resulted in an 5 increase in exophers over the baseline at peak and shifted the measured peak of exopher onset forward 1 d. Notably, exopher levels returned to baseline at Ad4, even in the presence of osmotic stressors. We conclude that both transient and extended exposure to hyperosmotic stress can elevate exopher production substantially in early adult life and infer that hyperosmotic conditions rapidly generate a trigger that elevates exophers.

Oxidative stress is a central player in aging biology and proteostasis (21). We tested whether well-characterized chemical inducers of mitochondrial oxidative stress can influence exopher production in ALMR neurons. We exposed Ad1 mCherryAg2 animals to 6 h of increasing concentrations of juglone (juglone is a plant-derived compound that can induce superoxide production when incorporated into nematode growth medium [NGM]); rotenone, which interacts with mitochondrial electron transport complex I to elevate reactive oxygen species (ROS); and paraquat, which increases mitochondrial superoxide production (Fig. 2 DF). For all mitochondrial ROS generators, we observed a dose-dependent increase in exophers at Ad1 (to a point, discussed in Excessive Stress Decreases Exopher Production) that ranged from 4 to 6 over untreated controls. We conclude that exposure to mitochondrial ROS induction can enhance exopher production.

We also tested conditions of continuous ROS exposure during adult life. We raised animals under standard growth conditions until the L4 stage and shifted cultures to paraquat plates, measuring ALMR exophers on Ad1 to Ad8 (SI Appendix, Fig. S2E). We find that continuous paraquat exposure increases exopher levels. Interestingly, exopher levels in the 2-mM paraquat treatment do not begin to fall at Ad3, as is typical for other stresses, but instead remain higher than control levels through Ad6, returning to baseline at Ad7. Thus, paraquat-mediated oxidative stress can extend the time period of permissive exophergenesis by 2 d, raising the possibility that paraquat might induce signals that normally promote exopher production. Overall, data from three mitochondrial ROS-generating compounds indicate that chemically-induced oxidative stress can increase exopher formation.

While initially testing the ability of stressors to modulate exopher production, we often utilized a doseresponse approach. Our studies revealed a striking commonality regardless of stressor type: excessive stress suppresses exophergenesis. For example, 6-h exposure of 240 M juglone (Fig. 2D), 25 M rotenone (Fig. 2E), or 25 mM paraquat (Fig. 2F) reduces exopher levels, even though lower levels of these ROS stressors enhance exopher production. The same pattern emerges under osmotic stress conditions; 6-h exposure to 500 mM concentrations of sucrose, glucose, sorbitol, and NaCl suppresses exopher levels, compared to 250 mM concentrations of each of these solutes (Fig. 2C). Although at Ad1 ALMR exophers modestly increase with 6-h exposures to increasing temperatures up to 30 C, 6 h at 37 C causes a collapse in exophergenesis (SI Appendix, Fig. S2F).

We generated additional evidence in support of the idea that excessive stress can inhibit exopher formation by exposing Ad1 mCherryAg2 animals to combined two-stress conditions that, by themselves, individually enhance exopher production. For example, whereas a 6-h fast elevates exophers (Figs. 1 BF and 2G), cointroducing osmotic stress with fasting (which also normally also elevates exophers; Fig. 2 A and B) suppresses exopher levels (Fig. 2G). We also found a combined inhibitory effect for fasting + oxidative stress (Fig. 2H).

Furthermore, temperature (SI Appendix, Fig. S2G) and anoxia (Fig. 2I and SI Appendix, Fig. S2H), stresses that did not affect exophergenesis on their own (SI Appendix, Fig. S2 A, B, and D), could suppress the effects of fasting on exopher production. Data suggest that under extreme stress neurons either cannot meet molecular requirements for exopher production or might enact mechanisms that actively suppress exophergenesis (see Discussion).

In summary, food withdrawal, osmotic stress, and oxidative stress can enhance exopher production, although above specific threshold levels of these stresses (including when two individual, exopher-promoting stresses are combined), exopher production can be suppressed. Thus, in addition to a temporal constraint on when environmental stresses can elevate exopher levels, we demonstrate that there is a limit to the severity of environmental stress capable of inducing the exopher production response.

With a goal of defining molecular mechanisms by which stresses elevate neuronal exopher production, we sought to define genetic requirements for the fasting-dependent induction response. We elected to focus on the fasting response because of the robust and highly reproducible level of induction associated with 6 h food withdrawal (Fig. 1 B and C), combining genetic mutant and RNAi strategies. To probe the mechanism by which fasting elevates neuronal exopher production, we first tested mutants for well-characterized, stress-activated transcription factors: heat shock factor 1 hsf-1/HSF1, required for transcription of heat shock chaperones and proteostasis (22); hypoxia inducible factor hif-1/HIF1, required for hypoxia stress responses (23); hlh-30/TFEB, required for starvation resistance and lysosomal integration with metabolism (24); skn-1/NRF2, which promotes response to oxidative stress and xenobiotic challenge (25, 26); and daf-16/FOXO, which is activated by low insulin pathway signaling and functions in a range of stress-protective responses, including proteostasis (27).

We constructed mCherryAg2 strains with viable mutant alleles of each transcription factor and subjected mutants to 6 h food deprivation on Ad1, measuring ALMR exophers thereafter (Fig. 3A). We observed the significant elevation of exophergenesis in hsf-1, hif-1, hlh-30, and skn-1 backgrounds in response to food withdrawal, indicating that these transcription factors are not critical for the induction of exophers in response to fasting. In contrast, the daf-16 null mutant exhibited a partial defect in the food withdrawal response, with exopher levels clearly increasing over baseline in the absence of food (P < 0.001) but never reaching the levels observed in wild-type (WT) animals (P < 0.001) (Fig. 3A). The partial effect in the daf-16 null mutant background is consistent with a model in which a daf-16dependent process mediates one component of the fasting response but that a daf-16independent process works in parallel to elevate exophers when food is withdrawn.

Lipid synthesis and FGF-activated RAS/ERK signaling are required for the induction of neuronal exophers in response to fasting. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. Feeding RNAi was initiated at the L4 larval stage and continued until Ad2. (A) The daf-16 null mutant has diminished exopher production in response to fasting. We tested mutants defective in major stress-responsive transcription factors (daf-16/FOXO, hsf-1/HSF-1, hif-1/HIF1, hlh-30/TFEB and skn-1/NRF2) in the mCherryA2 background, Ad2: exopher counts 6 trials, 50 animals per trial, and CMH difference between WT control and daf-16 deletion mutant ***P < 0.001. (B) Lipid synthesis is implicated in the fasting-induced boost in exophergenesis. RNAi knockdown of pept-1, mdt-15, sbp-1, and fasn-1 beginning at L4 in the mCherryAg2 strain, 6 h fast followed by exopher assay on Ad2, control is empty vector RNAi. We compared WT fed versus fasted P < 0.001 CMH, the others are not significant: 3 trials and 50 animals per trial. (C) Schematic of C. elegans MAPK signaling pathways targeted in genetic tests for fasting-induced exopher elevation: p38 MAPK signaling (purple), JUN/FOS MAP kinase signaling (orange), EGF-mediated MAPK signaling (green), and FGF-mediated MAPK signaling (blue). Black boxes highlight a common function in both EGF and FGF MAPK signaling. (D) RNAi knockdown of most components of the p38 and JUN/FOS signaling cascades does not impair fasting-induced exopher increases. The strain was mCherryAg2; RNAi was initiated at the L4 stage; and 6 h fast was followed by exopher counts at Ad2: 3 trials and 50 animals per trial. mek-1(RNAi) stood out as exceptional in failing to induce exophers. (E) Loss-of-function mutants for JUN/FOS signaling do not suppress exopher production. kgb-1; mCherryAg2 and jun-1; mCherryAg2 mutants were fasted for 6 h at Ad2 before exopher counts: 5 trials and 50 animals per trial. (F) An egl-17/FGFmediated MAPK signaling cascade is necessary for a fasting-induced increase in exopher production. RNAi for the indicated genes was initiated at the L4 stage on strain mCherryAg2, and at Ad2, animals were fasted 6 h and then scored for exophers. RNAi knockdown of let-23, ksr-2, and FGF ligand let-756 did not disrupt the fasting exopher response 3 trials and 50 animals per trial.

We also tested whether autophagy, a pathway activated by food limitation, might be critical for the fasting-induced increase in exophers. We used RNAi approaches to knockdown autophagy genes lgg-1, atg-7, and bec-1 in mCherryAg2 animals and scored for exophers on Ad2 (SI Appendix, Fig. S3A). Since all three disruptions in the autophagy pathway failed to suppress fasting-induced exopher elevation, we infer that engagement of autophagy functions is not required for fasting-induced exopher increase. Pharmacological inhibition of autophagy with 1 mM spautin or of the proteasome with 10 mM MG132 (SI Appendix, Fig. S3B) did not block the fasting-induced elevation of exophers, in further support that autophagy and proteasome contributions are not critical for fasting-induced exopher increase.

To better characterize the pathways involved in fasting-induced exopher production, we compiled a list of known genes implicated in starvation and feeding in the C. elegans literature and screened for fasting-induced exopher elevation when the candidate genes were knocked down using RNAi (28) (see SI Appendix, Table S1 for a list of candidates tested). Note that the strain we targeted with feeding RNAi, mCherryAg2, should permit efficient RNAi knockdown in all tissues except neurons, because neurons do not express a double-stranded RNAi transporter, sid-1, required for the efficacious knockdown in feeding RNAi (29). Utilizing this experimental design, we expected to identify genes operative in the nonautonomous initial events in the sensation and signaling of fasting stress to the touch neurons, rather than genes involved in neuron-intrinsic exophergenesis.

We tested positive clones from the first round of the RNAi screen in triplicate to identify intestinal peptide transporter pept-1, lipid synthesis-implicated Mediator complex factor mdt-15, MDT-15 binding partner sbp-1/SREBF2, and fatty acid synthase fasn-1, as required for robust exopher elevation in response to fasting (Fig. 3B).

pept-1 encodes a conserved intestinal di-/triamino acid transporter implicated in C. elegans nutrient sensing (30). Loss of the pept-1 function increases the intestinal absorption of free fatty acids from ingested bacteria, such that short- and medium-chain fatty acids are highly increased in the mutant, and de novo synthesis of long-chain and polyunsaturated fatty acids is greatly decreased (31). Our data suggest that sudden withdrawal of food causes a metabolic reconfiguration that signals for enhanced exopher production and that the sensing of food limitation requires PEPT-1 transporter activity.

MDT-15 has been shown to promote health and longevity by orchestrating many of the metabolic changes that occur in response to short-term fasting (32). MDT-15 encodes a subunit of the transcriptional coregulator Mediator complex that is required to express fatty acid metabolism genes and fasting-induced transcripts (3235), heavy metal and xenobiotic detoxification genes (32, 36), and oxidative stress genes (37). SBP-1, the homolog of the mammalian sterol regulatory element-binding protein (SREBF2) transcription activator that regulates fatty acid homeostasis, is a known partner of MDT-15, and together, MDT-15 and SBP-1 promote the expression of lipid synthesis genes (32). FASN-1 encodes the sole C. elegans fatty acid synthase, and its expression is regulated by MDT-15/SBP-1 (38, 39). Together, MDT-15, SBP-1, and FASN-1 may act to promote the synthesis of a lipid-based factor that signals for, or is otherwise required for, neuronal exopher production under fasting stress.

Expression of lipid synthesis genes in multiple tissues may contribute to fasting-induced exopher elevation. To address where the lipid synthesis gene group required for fasting-induced exopher elevation acts, we took a tissue-specific RNAi knockdown approach. We worked with strains that expressed mCherryAg2 in touch neurons but were defective in either the double-stranded RNA (dsRNA) transporter sid-1 (neurons, muscle, pharynx, intestine, and hypodermis) or RISC complex factor rde-1 (germline and vulva), and we reintroduced sid-1 or rde-1 using tissue-specific promoters to drive expression and restore RNAi knockdown capability only in the rescued tissue. The promoters we used to restore expression (and therefore RNAi targeting) in specific tissues were the following: pan-neuronal rgef-1, muscle myo-3, pharynx myo-2, intestine vha-6, hypodermis hyp7 semo-1, vulva lin-31, and germline sun-1. This test set of seven tissue-specific RNAi lines enabled us to target most cells of the animal. We fasted animals to ask whether RNAi disruption in individual tissues is sufficient to disrupt the exopher induction response, which would indicate that expression in that targeted tissue is necessary in the response. We found that although whole body knockdown of pept-1, mdt-15, sbp-1, and fasn-1 could disrupt the fasting-induced exopher elevation (Fig. 3B), no tissue-specific knockdown was effective in blocking this response (SI Appendix, Fig. S4A). Data are consistent with a model in which multiple tissues can contribute the required lipid biosynthesis, although we cannot rule out that RNAi targeting was ineffective or missed necessary cells.

Food limitation stresses engage multiple signaling pathways that can activate animal defenses, including RAS/MAPK pathways that transduce developmental and stress responses to activate specific transcription programs (40, 41). We tested members of three canonical, well-characterized C. elegans MAPK signaling pathwaysthe PMK-1/p38 pathway that functions in some innate immunity and oxidative stress responses; the JNK pathway, which, among other activities, functions in intermittent fasting programs (42); and the RAS/ERK pathway, which, among other things, affects vulval precursor fate and vulval development response to starvation stress (43) (pathways summarized in Fig. 3C).

The core MAP kinases in the p38 pathway are nsy-1/MAPKKK and pmk-1/MAPK (40). RNAi directed against nsy-1 and pmk-1 genes was ineffective in blocking the fasting induction of neuronal exophers (Fig. 3D), and thus, we do not find evidence supporting the engagement of the pmk-1/p38 stress pathway in fasting-induced exopher elevation.

In C. elegans, an adult intermittent fasting protocol of 2 d without food followed by 2 d of food extends lifespan via an MLK-1, MEK-1, and KGB-1 JNK pathway that converges on AP-1 (JUN-1, FOS-1)mediated transcription (42). We find that kgb-1(RNAi), kgb-1, and jun-1 genetic mutations do not block the fasting-induced elevation of exopher production (Fig. 3 D and E). This result, coupled with published data examining a transcriptional time course following food withdrawal that revealed a distinct transcription pattern for 3 to 6 h after fasting, as compared to the more chronic starvation of 9 h and longer (44), suggests that the response to short-term food withdrawal that we characterize here does not operate via the characterized, intermittent fasting pathway. Still, the positive mek-1/MAPKK and partial mlk-1/MAPK outcome (mlk-1(RNAi) fasted versus WT fasted, not significant; mlk-1(RNAi) fasted versus mlk-1(RNAi) fed, P < 0.01) (Fig. 3D), suggest possible pathway involvement or cross-talk involving these kinases. As evidence for more definitive engagement of the RAS/ERK pathway was evident in our studies (Fig. 3F), we focused on examining pathway members in more detail.

RAS/ERK signaling in C. elegans (reviewed in refs. 40 and 41; see Fig. 3C) can involve the EGF or FGF activation of receptor tyrosine kinases that interact with adaptor proteins such as SEM-5/GRB2 or SOC-1/GAB1, which recruit guanine nucleotide exchange factor SOS-1 to activate small GTPase LET-60/RAS. LET-60/RAS-GTP activates MAPKKK LIN-45/RAF. Scaffold proteins KSR-1 and/or KSR-2 collect downstream members of the MAPK cascade, so LIN-45/MAPKKK activates MEK-2/MAPKK, which in turn phosphorylates and activates MPK-1/ERK. MPK-1 can then enter the nucleus to phosphorylate transcription factors that execute a transcriptional response. We found RNAi knockdown of multiple members of the conserved core RAS/ERK signaling, namely let-60/RAS, mek-2/MAPKK, mpk-1/ERK, sem-5, and ksr-1, disrupts the fasting-induced increase of touch neuron exophers (Fig. 3F). Given the implication of five core MAPK components in fasting-induced exopher elevation, we conclude that the RAS/ERK pathway plays a critical role in the mechanism by which neurons increase mCherry expulsion (and other cell contents) under food withdrawal stress.

The characterized C. elegans EGF and FGF signaling pathways share the aforementioned signaling components in the conserved RAS/ERK pathways but differ in ligands, receptors, KSR-type (EGFR uses both KSR-1 and KSR-2; FGFR exclusively uses KSR-1), and FGF pathway requirement for the adaptor SOC-1/Gab1 (41). We tested MAPK components in an effort to distinguish whether FGF, EGF, or both, contribute to exopher elevation in response to fasting.

We used RNAi approaches to test for a requirement of FGF ligands egl-17 and let-756 as well as FGF receptor egl-15 in fasting-induced exopher elevation. Interventions with ligand egl-17/FGF and receptor egl-15/FGFR, but not FGF ligand let-756, disrupted the capacity to increase exophers in response to 6-h fasting, implicating a specific FGF in exopher regulation biology (Fig. 3F). Moreover, we find that FGF pathway-specific soc-1 (RNAi) diminishes fasting-induced exopher production. Our data, which implicate eight FGF pathway genes (FGF ligand egl-17, FGF receptor egl-15, FGF pathway-specific soc-1 and sem-5, and core pathway components let-60, mek-2, ksr-1, and mpk-1) in the fasting induction of exophers, identify an FGF-activated ERK/MAPK pathway that acts in response to 6-h food withdrawal, as an essential mechanistic step in neuronal exopher increase.

To address the potential source of egl-17/FGF and identify the tissue via which FGFR acts to promote fasting-elevated exopher production, we used tissue-specific RNAi approaches to disrupt all identified components in all neurons, muscle, pharynx, intestine, hypodermis, vulva, or germline (SI Appendix, Fig. S4A).

We were unable to identify single-tissue sources that were required for FGF ligand EGL-17 or FGF receptor EGL-15 activity in fasting-induced exopher elevation, suggesting that either multiple tissues can execute effective FGF signaling, that cells that we were unable to target are responsible, or that unknown technical issues might apply. We did, however, find that knockdown of the most downstream pathway targets, mek-2 and mpk-1, only in the hypodermis or only in the germline (SI Appendix, Fig. S4B) could disrupt fasting induction. Our data thus suggest that mek-2 and mpk-1 are required in both the hypodermis and in the germline for fasting-induced exopher induction.

Importantly, mek-2 and mpk-1 are downstream kinases for both the FGF and the EGF MAPK pathways. Indeed, our RNAi perturbation screens of MAPK pathway members indicated that lin-3/EGF also acts in fasting-induced exopher elevation (Fig. 3F). Tissue-specific RNAi studies reveal that germline-specific knockdown of lin-3/EGF disrupted fasting-induced exopher elevation (while hypodermal knockdown did not) (SI Appendix, Fig. S4C), implicating the germline production of LIN-3/EGF in a transgenerational influence on neuronal exophergenesis. Knockdown of mek-2 and mpk-1 are also needed in the germline for fasting-induced exophergenesis (SI Appendix, Fig. S4B).

RNAi disruption of EGF receptor let-23 and EGF pathway-specific ksr-2 did not eliminate the exopher elevation in response to fasting in our original screen (Fig. 3F). This observation raised the possibility that the EGF-responsive pathway might function in neurons (which are generally not as susceptible to RNAi as other tissues). Because let-23(gf) alleles caused extensive reproductive system development consequences that complicated the interpretation of exopher production (not shown), we tested the available strains that expressed the EGF pathway-activating EGFR/let-23(gf) in specific tissues for elevated exopher levels in the presence of food. We found that the transgenic introduction of EGFR/let23(gf) in neurons (but not muscle or intestine) elevates exophers in the presence of abundant food (Fig. 4A), suggesting a neuronal-based EGF pathway in promoting fasting-induced exopher elevation.

Molecular pathways that influence fasting-induced exopher elevation. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. (A) let-23 (gain of function) increases exopher formation when expressed specifically in neurons. let23(sa62) is a gain-of-function allele that activates MAPK signaling. We measured the exopher levels in strains that expressed let-23(sa62gf) from neuronal (unc-119), intestinal (vha-6), or muscle (myo-3) promoters in the mCherryAg2 background at Ad2 in the presence of food: 3 replicates and 50 animals per trial. (B) let-60 (gain of function) increases exopher formation under abundant food conditions. let-60(n1046) is a gain-of-function RAS allele that activates MAPK signaling. We measured exopher levels in control mCherryAg2 and in let-60(n1046);mCherryAg2 strains at Ad2 in the presence of food: 3 replicates and 50 animals per trial. (C) Schematic of the logic of epistasis within the MAPK signaling context. The phenotype of a gain-of-function allele that continuously signals will not be affected by the knockdown of any effector upstream of it within the cascade. On the other hand, any actors downstream of a constitutively active component will affect the phenotype. FGF signaling is used as an example here. (D) Lipid synthesis genes act upstream of let-60(gf)/RAS to elevate neuronal exopher production. RNAi interventions were initiated at the L4 stage on gain-of-function let-60;mCherryAg2 animals. Exophers were scored at Ad2: 3 replicates and 50 animals per trial. Although mek-2 and mpk-1, known to act downstream of let-60(gf), are critical for exopher elevation, the known upstream genes egl-15, egl-17, and soc-1 are not, consistent with data that previously ordered the FGF pathway. pept-1, mdt-15, sbp-1, and fasn-1 do not suppress let-60(gf)-induced exophergenesis, indicating that the lipid synthesis genes likely act upstream of let-60/RAS. (E) Lipid synthesis genes and lin-3 act upstream of let-23(gf) activity when it is expressed in neurons. RNAi knockdown was initiated at the L4 developmental stage on gain-of-function let-23(sa62);mCherryAg2 animals. Exophers were scored at Ad2: 3 replicates and 50 animals per trial. Cascade components let-60, mek-2, and mpk-1 are known to act downstream of let-23 and effectively abrogate the MAPK signaling leading to exophers, while lin-3 works upstream of let-23 and therefore cannot suppress a downstream gain-of-function mutation. RNAi knockdown of pept-1, mdt-15, sbp-1, and fasn-1 do not suppress neuronal let-23(gf)-induced exophergenesis, indicating that the lipid synthesis genes likely act upstream of neuronal let-23(gf). (F) Model for signaling that elevates neuronal exopher formation in response to fasting. In the sudden absence of food or when dipeptide transporter PEPT-1 activity is low, a lipid-based signal is produced/released that depends upon MDT-15/SBP-1 transcriptional activity and fatty acid synthase FASN-1. The lipid signal may act on the neurons directly or may contribute to the activation of a required FGF pathway in a relay tissue (unlikely to be neurons) to promote transcription of an essential signal that acts on touch neurons to elevate exopher production. EGF pathway activation in neurons can elevate exopher production; it remains to be determined whether EGF signaling is required in touch neurons per se. In sum, transtissue stress signaling influences remote neuronal trash management through lipid, FGF, and EGF RAS/MAPK signaling. Note that we cannot distinguish between an inducible lipid signal and a static, essential lipid product required for exopher elevation.

let-60(n1046) is a well-characterized, gain-of-function RAS allele that constitutively activates MAPK signaling (45). Our RNAi data, identifying the FGF and EGF/RAS/MAPK pathways as required for fasting-induced exopher induction, predict that the let-60(gf) allele should induce higher exopher levels, even in the absence of starvation. To test this model, we constructed a let-60(n1046gf);mCherryAg2 strain and measured exopher levels at Ad2. We find that exophers are indeed elevated in the let-60(n1046gf) background in the absence of fasting, confirming that RAS activation enhances exopher production (Fig. 4B).

let-60(n1046gf) has been used extensively in epistasis pathway ordering (45), and thus we pursued an epistasis approach to clarify the relationship between the lipid synthesis gene group and the FGF/RAS/MAPK pathways (Fig. 4C). We reasoned that if the genes involved in lipid synthesis act downstream or in parallel to the RAS/MAPK pathway that elevates exophers, RNAi knockdown of the lipid synthesis genes in the let-60(gf) background should suppress constitutive exopher production. Alternatively, if the lipid synthesis branch acts upstream of LET-60/RAS activation, disruption of the upstream genes should not change the exopher elevation associated with let-60(gf) (Fig. 4C). We therefore repeated RNAi knockdowns of genes in the lipid synthesis and FGF/RAS/MAPK pathways in the let-60(gf);mCherryAg2 background, quantitating the impact on exopher formation. As expected for known downstream kinases in the RAS pathway, the RNAi disruption of mek-2/MAPKK and mpk-1/MAPK suppressed the let-60(gf) phenotype (Fig. 4D). In contrast, genes encoding egl-17/FGF, egl-15/FGFR, and soc-1 that act upstream of let-60/RAS did not suppress the let-60(gf)/RAS phenotype, consistent with the known action of these genes upstream of RAS in the signaling pathway.

Importantly, RNAi directed against pept-1, and the lipid biosynthesis genes mdt-15, sbp-1, and fasn-1 did not suppress elevated exopher levels in the let-60(gf) background (Fig. 4D). We conclude that the lipid biosynthesis branch is likely to act upstream of the FGF/RAS/MAPK to promote exopher elevation.

Lipid and FGF signaling may act upstream of EGF signaling. We also used an epistasis strategy to begin to address how lipid biosynthesis and FGF pathways might relate to the EGF pathway in fasting-induced exopher elevation. We performed the RNAi knockdown of required lipid synthesis genes and of upstream FGF pathway-specific genes egl-17/FGF, egl-15/FGFR, and soc-1 on the neuronal let-23(gf) strain in which exopher levels are elevated, reasoning that if a critical lipid or FGF pathway is normally activated downstream of neuronal EGFR activation, perturbation of lipid or FGF pathway-specific genes would block the let-23(gf) exopher elevation under well-fed conditions. Our RNAi-dependent disruptions of either pathway, however, did not suppress neuronal let-23(gf) high-exopher levels (Fig. 4E), suggesting that the essential lipid and FGF signaling required for fasting-induced exopher induction normally occurs upstream of neuronal EGF signaling. Although these studies constitute only the first rudimentary tests required to establish pathway details (caveats discussed in more detail in SI Appendix, Fig. S4), data suggest a basic framework for mechanistic evaluation (Fig. 4F).

Overall, we identify three pathways that are required for fasting-induced exopher elevation in stressed touch receptor neurons: lipid synthesis, an FGF/MAPK pathway, and an EGF/MAPK pathway that can act in neurons. Our data suggest a model for signaling that influences a dramatic expulsion of neuronal contents upon the introduction of fasting stress. Upon food withdrawal, di-/tripeptide transporter PEPT-1 plays a role in nutrient sensing and a lipid-based stress signal (the generation of which depends on MDT-15/SBP-1 transcriptional activity and fatty acid synthase FASN-1) is produced. The lipid-dependent process could act directly to nonautonomously influence exopher production in the touch neurons or could trigger/activate the required FGF/RAS/MAPK and/or EGF MAPK signaling and the likely consequent downstream transcription. Downstream, or in parallel, RAS/MAPK activity in hypodermis and germline contributes to the stress-sensing tissue network, establishing that that nonautonomous signaling directs exopher production in the touch neurons. FGFR signaling pathway genes are known to be highly expressed in the hypodermis (46). Notably, RNAi evidence supports the hypothesis that the same group of genes that mediate fasting-induced exopher increase is required for exopher elevation in response to osmotic stress (SI Appendix, Fig. S3C), indicating that general, rather than fasting-specific, mechanisms are engaged.

Although many details of this complex lipid-FGF-EGF signaling network remain to be further elucidated, our data provide documentation that aging and proteostasis-relevant stresses engage multiple pathways that can act over multiple tissues to influence a dramatic expulsion of neuronal contents. Conserved signaling molecules can modulate a process of fundamental interest in neuronal proteostasis, relevant to the understanding of neuronal degeneration.

Maintaining neuronal proteostasis is a critical goal for healthy brain aging and a fundamental challenge for diseased neurons in a range of neurodegenerative diseases (47). A recently identified facet of Alzheimers disease, Parkinsons disease, and other proteopathies is the transfer of aggregates to neighboring cells, which can seed aggregate spread and promote pathology (48, 49). In vivo dissection of the biology of protein aggregate spread is challenging to investigate in mammalian brain, but it is clear that the understanding of mechanisms that regulate autonomous and nonautonomous aggregate expulsion in relation to other neuroprotective strategies is of considerable importance in addressing potential treatment.

C. elegans touch neuron exopher production, which increases with high proteostress (4), models several aspects of aggregate/organelle transfer biology. We find that the production of neuronal exophers can be dramatically responsive to specific stress conditions, being enhanced by food withdrawal, oxidative stress, and osmotic stress but influenced relatively little by temperature or hypoxia. We also demonstrate the temporal restriction of stress-induced exopher production to the first 3 d of adult life, and we document a stress ceiling phenomenon, in which the highest levels of individual stress, or a combination of two distinct noninhibitory stresses, suspend exopher production. Finally, we show that fasting-induced exophergenesis is dependent on nonautonomous lipid biosynthesis, FGF-activated RAS/MAPK, and EGF-activated RAS/MAPK signaling pathways. Although details remain to be filled in regarding the complex interactions of the signaling steps, a major point is that environmental and genetic factors can be manipulated nonautonomously to regulate the expulsion of offensive aggregates from neurons. Given the importance of aggregate management in aging and neurodegenerative disease and the poorly understood biology of in vivo aggregate transfer, exopher-related mechanisms may suggest new strategies toward the manipulation of the analogous process in higher organisms.

Acute food withdrawal, oxidative stress, and hyperosmotic stress elevate exopher production, but temperature elevation and hypoxia/anoxia are relatively ineffective at provoking similar responses. Starvation (8, 50), oxidative stress (51), and osmotic stress (52) perturb proteostasis and share ROS elevation (53, 54). The future dissection of the intersection of the genetic and physiological conditions common to these three stresses should provide insight into molecular mechanisms that promote exophergenesis. Likewise, physiological differences in stress responses to temperature and hypoxia, which are not potent inducers of exophers, may help distinguish particular conditions that are specifically correlated with exopher induction.

Exopher production, comprising the release of a large, membrane-surrounded vesicle filled with cellular contents, has the appearance of an energetically costly incident that involves the dynamic loss of organelles and aggregates. Our working model posits that exophergenesis is invoked when the levels of damaged organelles and proteins surpass the neuronal capacity for internal degradation. Consistent with this idea, increasing oxidative challenge and increasing hyperosmotic exposure both increase exophergenesis. Interestingly, conditions of extreme osmotic and oxidative stress markedly suppress the formation of exophers. Moreover, combining two stresses, either of which is sufficient to promote exopher production when introduced alone, can result in exopher suppression. This combinatorial effect can also occur with stress stimuli that themselves do not significantly induce exophers, such as anoxia and modestly elevated temperature. Together, these observations reveal a molecular summing of stress signals that appear to flip the off switch for exophergenesis. The suppression of exopher production under conditions of extreme stress may be caused by energy exhaustion, a molecular repression mechanism, or grievous loss of homeostasis, leading to physiological dysregulation.

Exopher production follows a distinctive and reproducible temporal profile in early adult life (4, 11). In the Ag2mCherry strain, exophers are not produced in larval development but begin to be detected after animals reach reproductive maturity, typically peaking in numbers around Ad2 and returning to low-baseline detection by Ad4. Data included here underscore that the temporal pattern is generally maintained, despite the continued or introduced presence of stresses. In other words, stresses definitively elevate exopher production, but for the most part, these stresses do not extend the period of exopher production later into adult life. Our findings thus define a limited temporal window in which exopher production can be modulated by stresses and suggest the existence of physiological states permissive (or restrictive) for exopher production. A link to reproduction likely defines this permissive period. For some stimuli (paraquat, rotenone, osmotic stress, and a shift to 25 C), we do report a capacity to move the peak day of exopher production ahead to Ad1 or at least to markedly enhance Ad1 levels above nonstressed controls. We infer that these stimuli reach the molecular threshold for exopher triggering faster than other conditions.

The fasting-induced elevation of exopher levels does not require stress transcription factors HSF-1, HIF-1, HLH-30/TFEB, or SKN-1/NRF2 but does depend in part on DAF-16/FOXO, a conserved stress-responsive transcription factor that drives the expression of food-sensitive, oxidative stress resistance and proteostasis genes (27, 44, 55) and is known to exert autonomous and nonautonomous impacts on stress resistance and longevity (56). Our data implicate a FOXO family member in regulation of neuronal aggregate expulsion. Our data do not rule out whether HSF-1, HIF-1, HLH-30/TFEB, or SKN-1/NRF2 might function redundantly in the exopher response to fasting.

How DAF-16 interacts with fat biosynthesis pathways and RAS/ERK signaling in exopher induction remains to be clarified. DAF-16 can control the expression of mdt-15 and has been previously implicated in transtissue benefits by interactions with MDT-15 (57). DAF-16 also intersects with FGF, ERK, and lipid biogenesis pathways and vice versa (for example, refs. 5861). The future definition of how DAF-16 integrates with these signaling pathways and the identification of the transcription factor that mediates the DAF-16independent component of the fasting-induced exopher response will add molecular understanding to what appears to be a complex regulatory network.

Mediator complex subunit MDT-15 is a transcriptional coregulator involved in lipid metabolism (34), response to fasting (32, 62), and oxidative, stress-induced expression of detoxification genes associated with the exposure to reagents like paraquat (63). The requirement for mdt-15 in fasting-induced, neuronal exophergenesis adds a new facet to the MDT-15 integration of multiple, transcriptional regulatory pathways (32), expanding the known roles of MDT-15/SBP-1 to include the activation of extrusion of remote neuronal aggregates in exophers. SBP-1/SREBF2 acts with MDT-15 to promote the expression of lipid metabolism genes (33, 34), and fatty acid synthase fasn-1 can be regulated by these (38, 39, 62, 64, 65). The requirement for multiple genes involved in lipid synthesis in fasting-induced exopher increase suggests that a lipid-based signal may be issued to ultimately direct or modulate neuronal trash expulsion. An equally plausible model is that lipid-dependent machinery is required for upstream signaling that promotes exophergenesis.

Our findings identify FGF ligand egl-17 (but not FGF ligand let-756), FGF receptor egl-15, FGF pathway specific soc-1, and pathway components let-60, mek-2, ksr-1, and mpk-1 as required for the fasting induction of exophers. These data reveal a specific FGF/RAS/ERK signaling pathway that enhances neuronal exopher production when food is withdrawn. Since the candidate RNAi screen for the factors required for fasting-induced exopher elevation was conducted in a strain background that is not readily permissive for neuronal RNAi effects and since RNAi knockdown of pathway components (specifically in neurons) does not block fasting-induced exopher increase, FGF/ERK signaling likely takes place outside of the touch receptor neurons to exert a regulatory role on neuronal exopher production. An interesting potential site of FGFR action is the hypodermis, which is necessary for some MEK-2/MPK-1 MAPK signaling. Such signaling is permissive for fasting-induced exopher production and the hypodermis is a known site of expression of FGFR pathway components (SI Appendix, Fig. S4B). Detailed, cell-specific expression studies will be required to test this model.

The conserved FGF pathway executes numerous roles in mammalian development and homeostasis (66). Interestingly, mammalian FGF21 acts as a global starvation signal that, among other things, impacts lipid metabolism. Although most FGF21 studies feature extended starvation and mousehuman differences have been noted (67), FGF21 is one of the most up-regulated rat liver genes under the conditions of an 8-h fast (68). FGF21 can cross the bloodbrain barrier to change hypothalamic neuron gene expression (69). Overall, the implication of FGF/ERK pathways in the response of neurons to food limitation across diverse metazoans suggests a mechanism that may be ancient and raises the possibility that the FGF branch of these pathways might activate extracellular trash expulsion mechanisms within mammalian neurons. If so, FGF signaling might be considered as a target for the therapeutic elimination of stored neuronal aggregates.

EGF RAS/MAPK signaling is also engaged in fasting-induced exopher elevation. The knockdown of the sole C. elegans EGF ligand lin-3 in whole animal, or only in germline, impaired fasting-induced exopher elevation.

Although RNAi knockdown of the EGFR let-23 and EGF pathway-specific ksr-2 in whole animal or only neurons was not effective, expressing activated, gain-of-function EGFR receptor allele let-23(sa62) in neurons resulted in elevated exopher levels in the absence of fasting. Epistasis studies suggest that EGFR activation in neurons could occur as a downstream target of lipid biosynthesis and FGF signaling. Although definitive establishment of fasting-induced EGFR activation in neurons remains (experimental caveats in interpretation of data are discussed in detail in SI Appendix, Fig. S4C), data are consistent with a role for EGF signaling originating in the germline as an inducer of EGFR-activated responses in neurons that promote exopher production. Future studies will need to confirm neuron requirements and to address whether EGFR directly activates exophergenesis in touch neurons or engages additional neurons as intermediatory signaling centers.

It is important that the germline serves as an EGF source needed for fasting-induced exopher elevation. Indeed, in studies of exopher production under standard growth conditions, we have defined a role for germline in the production of young adult exophers. A key point here is that food-sensing, nonautonomous growth factor signaling across generations can influence seemingly extreme neuronal proteostasis activity.

EGF- and FGF-dependent processes cooperate in development. For example, EGF signaling activates FGF production and a downstream FGF pathway required in vulval epithelial fate specification (70). Starvation conditions can influence the signaling level for the EGF/RAS/ERK pathway that specifies C. elegans vulval cell fateseither starvation or pept-1(RNAi)can enhance RAS/MAPK signaling during vulval fate specification (43). Our documentation of FGF signaling in food limitation responses that elevate neuronal proteostasis outcomes identifies a second C. elegans EGF- and FGF-regulated signaling pathway that responds to food limitation. Why food limitation might induce neuronal trash elimination is unclear. One possibility is that exophergenesis [which we track in single neurons in our study but is likely to also occur in other neurons and cells (4)] might serve as a mechanism to discard superfluous, neuronal proteins and organelles for degradative recycling in neighboring cells as resources become limited.

Our study defines the basic framework by which metabolic stresses engage a distributed network that influences a significant neuronal expulsion phenomenon. Evidence is accumulating that exopher-like extrusion capabilities are not limited to stressed C. elegans neurons [mammalian examples in refs. 71 and 72)]. For example, a recent comprehensive study of mitochondrial expulsion by mouse cardiomyocytes revealed numerous analogies between C. elegans exophers and mouse mitochondrial expulsion models (72). Although elaborating details of molecular homologies remain for the future, that related biology is likely to be conserved holds significant implications of interest with regard to mammalian aging and neurodegeneration. 1) It is interesting that stresses, particularly associated with aging (i.e., oxidative stress) or proteostasis impairment (i.e., osmotic stress), are especially potent in inducing C. elegans exopher elevation; the disruption of exopher-related biology may contribute generally to the decline/dysfunction in aging neurons across phyla. 2) Likewise, the direct demonstration that extreme stress levels, or the summation of distinct, nonconsequential stresses, can effectively shut down the exopher response suggests a type of potential excessive stress impairment relevant to pathological mechanisms. 3) Our data establish that exopher production can be responsive to specific, conserved biochemical signaling, such that chemical strategies for inducing, or limiting, the expulsion of neurotoxic material by exploiting exopher-related mechanisms in mammals might be considered targets for therapeutic manipulation.

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From the journals: MCP – American Society for Biochemistry and Molecular Biology

Discovering drugs in deadly venom. Improving transplant outcomes with better storage. Linking mannose glycosylation to eye development. Read about papers on these topics recently published in the journal Molecular & Cellular Proteomics.

The purple cone snail, Conus purpurascens, hunts fish and uses venom to immobilize its prey. Cone snail venom contains diverse toxic peptides, or conopeptides, that are biologically active, target-specific and valuable for drug discovery. One of the most powerful known painkillers, ziconotide, marketed as Prialt, was derived from a conopeptide.

Alex Holt/NIST

A purple cone snail uses its harpoon to pierce through a latex-covered tube,allowing a researcher to collect its venom.

Conopeptides vary among the more than 800 cone snail species and among members of the same species. Even a single cone snail specimen can produce unique conopeptide cocktails, called cabals, specialized for predation or defense. Cone snails also can hypermodify conopeptides at the post-translational step, increasing the diversity of the toxins and extending their range of biological targets. The extreme diversity of conopeptides provides a rich source of biologically active molecules for drug discovery.

In a recent study in the journal Molecular & Cellular Proteomics, Meghan Grandal and colleagues at the National Institute of Standards and Technology collected and analyzed the injected venom from 27 specimens of the purple cone snail. Using high-resolution mass spectrometry techniques, they discovered 543 unique conopeptides derived from 33 base peptide sequences 21 of which were newly identified base peptides. An abundant and newly discovered conopeptide named PVIIIA illustrates the complexity and diversity of modifications to the base conopeptide that occur among various purple cone snail specimens. Building on previous studies, the researchers showed that the different snail specimens produce one of two unique venom cocktails. These two cocktails correspond to what are known as the lightning strike cabal that rapidly induces paralysis of the snails prey and the motor cabal that acts more slowly to induce irreversible paralysis.

Knowing which conopeptides are co-expressed within a specific cocktail will give the researchers important clues as to the possible neural targets of newly identified conopeptides. This will be a critical step in developing new conopeptides into neural probes or therapeutics.

The demand for kidney transplants exceeds the supply of available kidneys. Some donated kidneys go unused, however, due to the prolonged time between circulatory arrest and the start of cold storage. These kidney graftsoften fail or are slow to function. Repairing such kidneys before transplant could greatly increase the available supply.

An unusual type of protein glycosylation, C-mannosylation, involves attaching a single mannose sugar to the amino acid tryptophan by a carboncarbon bond. C-mannosylation, which regulates protein secretion, folding and function, occurs at a specific sequence of four amino acids that begins with the modified tryptophan. Even though about 18% of secreted or transmembrane proteins have this sequence, few studies have looked for the modification. Consequently, researchers know of few proteins that are C-mannosylated.

In a new study in the journal Molecular & Cellular Proteomics, Karsten Cirksena of the Institute of Clinical Biochemistry and a team of researchers in Germany found numerous proteins with altered secretion levels in cells lacking the C-mannosylation machinery. One of these potentially C-mannosylated proteins, a disintegrin and metalloprotease with thrombospondin motifs, or ADAMTS16, is essential during eye development and optic fissure closure. In Chinese hamster ovary cells and Japanese rice fish, the researchers demonstrated that ADAMTS16 can be C-mannosylated, that its secretion depends on C-mannosylation and that loss of a C-mannosylation enzyme causes a developmental eye defect known as a partial coloboma a gap in the eye tissue. Their findings suggest that C-mannosylation, an understudied protein modification, plays a critical role in eye development by regulating secretion of ADAMTS16.

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From the journals: MCP - American Society for Biochemistry and Molecular Biology

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PATHWAYS TO HEALING: Eating on the go | Pathways To Healing | – Lake Oconee Breeze

When it comes to traveling, planning ahead is always key to a successful excursion. This is especially true when it comes to eating. Whether youre traveling by car, boat or plane, a little forethought and preparation can help ensure you have what you need to remain comfortably fed without resorting to junk food.

Raw vegetables make a great travel snack, providing both nutrition and convenience. Vegetables such as celery sticks, baby carrots, sugar snap peas and cut up cucumber with a pinch of salt are all highly portable. There are also many simple recipes for salads in a jar. Simply prepare your jar salad in advance and pack a fork for a healthy and easy meal on the go.

Other simple snacks include crackers paired with meat sticks and pre-cubed cheese. Nuts are another high-protein option. You can make your own trail mix by mixing raw nuts and seeds in a sandwich bag or small reusable container. Fresh fruit, such as apples, grapes or oranges, is a great portable source of fiber. Small packets of nut butters are great with crackers or can pair nicely with a banana.

A simple travel smoothie recipe is another great tool to have in your arsenal. Whether you have an early morning flight or a red eye, it pays to always head to the airport prepared. Pack a shaker bottle and a serving of protein powder in a sandwich bag or just dry in the bottom of the bottle. Once at the airport, order cold or steamed milk from a coffee shop and blend it with the protein powder for a high protein latte. This is a foolproof way to balance your blood sugar, curb your appetite and keep you satiated, which makes you much less likely to reach for junk food later.

If youre headed out and want to pack your lunch, look no further than these healthy roll up recipes:

Pickle Roll Ups

Pickle roll ups are a tasty snack that only take 10 minutes to prepare and yields up to 30 servings! Gather an 8-ounce package of cream cheese (brought to room temperature), 16 ounces of whole dill pickles and pound thinly sliced deli ham. Grab a slice of ham, slather with cream cheese and place a pickle in the middle of the ham slice. Roll the pickle in the ham, and then slice evenly into bite-sized pieces. For variety, try using corned beef instead of ham or whipped cream cheese instead of a block. Some people like adding a couple tablespoons of dry ranch dressing mix to the cream cheese for added flavor.

Turkey Caprese Roll Ups

Turkey caprese roll ups are a low carb, high-protein snack option, with no bread or tortilla needed. These can be made in advance and are a great snack to store in the cooler. Gather thinly sliced deli turkey breast, pesto, fresh tomato slices and fresh mozzarella. Spread the pesto on the top of the turkey slices, layer with tomato and mozzarella. Roll up the turkey, slice and enjoy.

Turkey Club Roll Ups

These turkey club roll ups take just five minutes to prepare and have such a punch of flavor, you wont even miss the bread! Youll need romaine lettuce leaves, lunch meat of your choice, cooked bacon, avocado, and your favorite sandwich spread. Begin by laying parchment paper on a cutting board or large plate. Remove the stems from your romaine lettuce and lay the lettuce on top of the parchment paper, overlapping pieces until you form a 10 by 8 rectangle of lettuce. Drizzle your sandwich spread of choice (anything from mustard to ranch to mayonnaise or jalapenos). Next, layer your lunch meat, followed by the tomatoes, bacon, and avocado. Use the parchment paper to help roll the sandwich into a tight tube by folding in the ends as you roll. Slice the sandwich in half and fold down the parchment paper as you eat, with no mess!

With a little planning, its easy to healthy and well fed while traveling. on that next flight, road trip or day on the lake.

All and all you will want to pack snacks that do not require refrigeration and remain fresh at room temperature unless you have access to a cooler.

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PATHWAYS TO HEALING: Eating on the go | Pathways To Healing | - Lake Oconee Breeze

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Glycomics Market | By Solution Type, By Application Type, By Industry Type, By Brand,By Region and Forecast 2021-2027 UNLV The Rebel Yell – UNLV The…

Glycomics Market research report delivers a comprehensive study on production capacity, consumption, import and export for all major regions across the world. Report provides is a professional inclusive study on the current state for the market. Analysis and discussion of important industry like market trends, size, share, growth estimates are mentioned in the report.

Glycomics is an emerging field which aims to focus on the structure and function of the glycans in a cell, tissue or in an organism. Glycans are the chain like structures of the carbohydrates that are free or conjugated to macromolecules such as lipids or proteins. They contribute in a diverse selection of biological processes such as protein folding, cell signaling, and immune recognition. These are implicated in a number of diseases such as oncological, autoimmune, and others.

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Competitive Landscape Glycomics Market:

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To comprehend global Glycomics market dynamics in the world mainly, the worldwide market is analyzed across major global regions: North America (United States, Canada and Mexico), Europe (Germany, France, United Kingdom, Russia and Italy), Asia-Pacific (China, Japan, Korea, India, Southeast Asia and Australia), South America (Brazil, Argentina), Middle East & Africa (Saudi Arabia, UAE, Egypt and South Africa)

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Glycomics Market | By Solution Type, By Application Type, By Industry Type, By Brand,By Region and Forecast 2021-2027 UNLV The Rebel Yell - UNLV The...

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ASC Therapeutics Receives IND Clearance From the US Food and Drug Administration for ASC618 Second-Generation Gene Therapy for Hemophilia A | DNA RNA…

DetailsCategory: DNA RNA and CellsPublished on Thursday, 08 July 2021 09:39Hits: 355

MILPITAS, CA, USA I July 7, 2021 I ASC Therapeutics, a privately-held biopharmaceutical company pioneering the development of transformative in-vivo gene replacement, gene editing and allogeneic cell therapies for hematologic and other rare disorders today announced that the U.S. Food & Drug Administration (FDA) has cleared an Investigational New Drug (IND) application for ASC618, an investigational second-generation gene therapy for patients with severe and moderately severe hemophilia A. The transformational Adeno-Associated Virus (AAV) construct contains a proprietary B-domain deleted codon-optimized bioengineered chimeric Factor VIII (FVIII) gene and a minimal-length liver-specific promoter, shown in pre-clinical studies to produce therapeutic levels of FVIII protein at doses that are significantly lower than other constructs expressing the native human FVIII currently in clinical trials.

Ruhong Jiang, PhD, ASC Therapeutics CEO said, FDAs IND clearance of ASC618 is a significant endorsement of the discovery, pre-clinical, analytical, clinical, regulatory, quality, manufacturing, and overall capabilities of our organization. This milestone culminates our transformation into a clinical-stage gene and cell therapy biopharmaceutical company.

Steven Pipe, MD, PhD, professor of pediatrics and pathology and pediatric medical director of the hemophilia and coagulation disorders program at the University of Michigan, and the principal investigator of the ASC618 phase 1/2 clinical trial, stated, ASC618 design provides a novel perspective to the field of hemophilia gene therapy. For the first time we will be able to investigate in a clinical setting the relevance of a novel bioengineered construct that has been proven in pre-clinical studies to significantly improve the biosynthesis, protein folding and secretion of factor VIII.

About Hemophilia A

Hemophilia A accounts for most cases of hemophilia (~80%), affecting approximately 1 of every 5000 live-born males. Over a million people around the world are estimated to have hemophilia, including more than 30,000 in the United States (US)1.

Currently, patients with hemophilia A are managed with prophylactic or on-demand replacement therapy with recombinant FVIII or bypassing agents. The major challenges of current treatment regimens, such the short half-life of hemophilia therapeutics with need for frequent intravenous injections, justify ongoing focus on gene replacement therapies.

About ASC618

ASC618 is an AAV8-based gene therapy product incorporating a novel liver-specific promoter and a bioengineered, codon-optimized B domain-deleted FVIII variant; in preclinical studies, ASC618 exhibits at least a 10-fold increase in the biosynthesis and secretion of FVIII compared with native human FVIII bioengineered constructs. ASC618 has the potential to increase durability of clotting factor biosynthesis and secretion by minimizing cellular stress and induction of the unfolded protein response, which may lead to diminished FVIII production from liver cells. ASC618 was developed based on extensive work on a second-generation gene therapy for hemophilia A from an academic team at Emory University2. ASC Therapeutics has obtained exclusive global rights to ASC618 from Expression Therapeutics and has conducted IND-enabling studies in multiple animal models3.

ASC Therapeutics will conduct a phase 1/2 clinical trial to evaluate the safety, tolerability, and preliminary efficacy of ASC618. The program was granted Orphan Drug Designation by the FDA in 2020. The study design is available at http://www.clinicaltrials.gov4.

About ASC Therapeutics

ASC Therapeutics is a biopharmaceutical company pioneering the development of gene replacement therapies, in-vivo gene editing and allogeneic cell therapies for hematological and other rare diseases. Led by a management team of industry veterans with significant global experience in gene and cell therapy, ASC Therapeutics is developing multiple therapeutic programs based on three technology platforms: 1) In-vivo gene therapies that use hepatocytes as a protein biofactory, initially focusing on ASC618 for hemophilia A; 2) In-vivo gene editing, initially focusing on ASC518 for hemophilia A; and 3) Allogeneic cell therapy, initially focusing on Decidua Stromal Cell-based therapy for steroid-refractory acute Graft-versus-Host Disease in patients receiving bone marrow transplantation, mostly to treat hematological malignancies. To learn more please visit


1 Peyvandi F, Garagiola I, Young G. The past and future of haemophilia: diagnosis, treatments, and its complications. Lancet. 2016 Jul 9;388(10040):187-97 2 Brown HC, Wright JF, Zhou S, et al. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery. Mol Ther Methods Clin Dev. 2014;1:14036 3 Veselinovic M, Gilam A, Ross A, et al. Preclinical Development of ASC618, an Advanced Human Factor VIII Gene Therapy Vector for the Treatment of Hemophilia A: Results from FRG KO Humanized Liver Mice, C57Bl/6 Mice and Cynomolgus Monkeys. Molecular Therapy 2020;28/4:167-8 4 ASC618 Gene Therapy in Hemophilia A Patients.

SOURCE: ASC Therapeutics

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ASC Therapeutics Receives IND Clearance From the US Food and Drug Administration for ASC618 Second-Generation Gene Therapy for Hemophilia A | DNA RNA...

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Natural Human Molecule Identified That Blocks Toxic Forms of Parkinsons-Related Protein – SciTechDaily

Schematic representation of the human peptide LL37 binding the toxic oligomers of -synuclein, blocking its propagation and preventing its neurotoxicity. Credit: Irantzu Pallars, IBB-UAB

Researchers at the UAB and the UniZar have identified a human peptide found in the brain that blocks the -synuclein aggregates involved in Parkinsons disease and prevents their neurotoxicity. The study, published in Nature Communications, suggests that this could be one of the organisms natural mechanisms with which to fight aggregation. The discovery may help to develop new therapeutic and diagnosis strategies for Parkinsons disease and other synuclein pathologies.

The death of neurons specialized in the synthesis of dopamine, one of the brains main neurotransmitters, deteriorates the motor and cognitive capacities of those with Parkinsons disease. The loss of these neurons is related to alpha-synuclein aggregation. Recent studies show that oligomers, the initial aggregates of this protein, are the most pathogenic forms of -synuclein and are responsible for the spreading of the disease in the brain.

Therefore, one of the more promising approaches in fighting this disorder consists in neutralizing these oligomers and, thus, slow down the pathological progression. However, the fact that these aggregates do not present a defined structure and that they are transitory by nature makes it extremely difficult to identify molecules that bind with enough strength as to explore any clinical application.

A scientific collaboration between researchers from the Institute for Biotechnology and Biomedicine (IBB) at the Universitat Autnoma de Barcelona (UAB) and from the Instituto de Biocomputacin y Fsica de Sistemas Complejos (BIFI) of the Universidad de Zaragoza (UniZar) now has been able to identify a human endogenous peptide which strongly and specifically attaches to the -synuclein oligomers, thus avoiding their aggregation and blocking their neurotoxicity, two processes closely related to the neurodegenerative decline of Parkinsons disease. The identification and study of the peptide, called LL-37, was recently published in Nature Communications.

LL-37 interacts with the toxic alpha-synuclein oligomers in a selective manner and with a strength superior to that of any peptide previously described, equivalent to the strength exhibited by antibodies. It inhibits aggregation at very low concentrations and protects neuronal cells from being damaged, researchers point out.

They add that, LL-37 is found naturally in the human organism, both in the brain and in the intestine, organs in which -synuclein aggregation takes place in Parkinsons disease. This suggests that LL-37s activity might respond to a mechanism developed by the body itself as a means to naturally fight this disease.

Encouraged by this idea, researchers now want to study how its expression can be regulated and if this strategy can become a safe therapy with the potential of influencing the course of the disease. There is a possibility that a therapy for Parkinsons disease already lies in our interior and that it only needs to be activated correctly, states Salvador Ventura, researcher at the IBB and coordinator of the study.

The identification of LL-37 was conducted under the framework of research analyzing the structure and characteristics of pathogenic oligomers with the aim of neutralizing them in a specific manner. The analyses conducted demonstrate that helical peptides with a hydrophobic side and another positively charged side are ideal for this type of activity. The trials allowed researchers to identify three molecules with anti-aggregation activity: in addition to the human molecule, a second peptide present in bacteria and a third artificially made molecule were identified.

In addition to representing a possible therapeutic route for Parkinsons disease and other synuclein pathologies, the molecules identified in the study are promising tools for its diagnosis, given that they discriminate between functional and toxic -synuclein species.

Until now there were no molecules capable of selectively and efficiently identifying toxic -synuclein aggregates; the peptides we present on these issues are unique and, therefore, have great potential as diagnostic and prognostic tools, says study co-coordinator Nunilo Cremades, researcher at BIFI-UniZar.

In the study, over 25,000 human peptides were computationally analyzed, and single-molecule spectroscopy methods, as well as protein engineering, were applied, in addition to cell cultures in vitro using toxic oligomers.

Reference: -Helical peptidic scaffolds to target -synuclein toxic species with nanomolar affinity by Jaime Santos, Pablo Gracia, Susanna Navarro, Samuel Pea-Daz, Jordi Pujols, Nunilo Cremades, Irantzu Pallars and Salvador Ventura, 18 June 2021, Nature Communications.DOI: 10.1038/s41467-021-24039-2

Participating in the study were researchers from the IBB-UAB and the Department of Biochemistry and Molecular Biology at the UAB Jaime Santos (first author of the article), Irantzu Pallars and Salvador Ventura (co-coordinators of the study), members of the Protein Folding and Conformational Diseases group; and BIFI-UniZar researchers Pablo Gracia (second author of the article) and Nunilo Cremades (co-coordinator of the study, predoctoral researcher and lead researcher, respectively, of the Amyloid Protein Misfolding and Aggregation NEUROMOL group from the BIFI-Unizar.

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AlphaZero to analyse no-castling match of the champions – Chessbase News

Press release

For the first time, the internationally renowned chess tournament Dortmund Chess Days will host a special match between legends Vladimir Kramnik and Viswanathan Anand playing the No-Castlingchess format. The rich, creative possibilities of this chess variant were recently explored by the ground-breaking artificial intelligence system AlphaZero, created by world-leading AI company DeepMind. Online audiences will now get to experience novel AlphaZero insights first hand in the post-match commentary of the no castling tournament, as part of DeepMinds support for Dortmund Chess Days.

Demis Hassabis [pictured], DeepMind Founder and CEO, says:

Its been incredibly exciting to see world-class players like Vladimir Kramnik use AlphaZero to explore new possibilities for the game of chess. Im looking forward to seeing two former world champions play the no castling variant in Dortmund and hope the games - and AlphaZeros insights - inspire chess players everywhere.

Event director Carsten Hensel said:

The Dortmund Chess Days has found a growing audience online, which we would like to develop in the coming years. We hope the inclusion of the no-castling variant and the inclusion of cutting-edge AI analysis from AlphaZero will further reinforce Dortmund as a ground-breaking tournament and will attract people from around the world to watch what will surely be a remarkable and historic match.

The tournament will see Vladimir Kramnik and Viswanathan Anand play four games against each other with classical thinking time, but with no option of castling. This tiny rule change will force the players to deviate from memorized opening lines, encouraging new creative play without deviating from the games familiar rules and patterns.

Plans for the 2022 event, which will be sponsored by DeepMind, are already underway with the hope that the no-castling format will become a regular fixture and an audience favourite, thanks to dynamic and entertaining play.

Master Class Vol. 12: Viswanathan Anand

This DVD allows you to learn from the example of one of the best players in the history of chess and from the explanations of the authors how to successfully organise your games strategically, consequently how to keep your opponent permanently under press

Anand and Kramnik playing the World Chess Championship in 2008

DeepMind is a multidisciplinary team of scientists, engineers, machine learning experts and more, working together to research and build safe AI systems that learn how to solve problems and advance scientific discovery for all.

Having developed AlphaGo, the first program to beat a world champion at the complex game of Go, DeepMind has published over 1000 research papers including more than a dozen in the journals Nature and Science and achieved breakthrough results in many challenging AI domains from StarCraft II to protein folding.

DeepMind was founded in London in 2010, and joined forces with Google in 2014 to accelerate its work. Since then, its community has expanded to include teams in Alberta, Montreal, Paris, New York and Mountain View in California.

The purpose of the association founded in the year 2019 is the promotion of chess. IPS is realizing this goal by the organization of chess events in the fields of sport, art, science, education, cultural and chess history. The outstanding project of the IPS is the Sparkassen Chess Trophy International Dortmund Chess Days, with its famous history since 1973. TheIPS is developing a modern concept and pay considerable attention to the digital requirements of today, especially regarding the topic of chess and its modern development.

Master Class Vol.11: Vladimir Kramnik

This DVD allows you to learn from the example of one of the best players in the history of chess and from the explanations of the authors (Pelletier, Marin, Mller and Reeh) how to successfully organise your games strategically, consequently how to keep y

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AlphaZero to analyse no-castling match of the champions - Chessbase News

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Joey Chestnut is commemorated with bobblehead as S.I. competitive eaters can gear up for contests –

STATEN ISLAND, N.Y. Nathans Famous competitive eating champ Joey Chestnut now can claim fame on another level. The National Bobblehead Hall of Fame and Museum presented a new bobblehead this week to commemorate the 14-time winner of Coney Islands annual hot dog downing contest.

Chestnut helped design the limited edition bobblehead, each numbered to 2,020. They are available for purchase through the Milwaukee, Wisconsin-based, National Bobblehead Hall of Fame and Museums online store. Each sells for $30 plus a flat-rate shipping charge of $8 per order.

The Joey Chestnuts bobblehead (Courtesy of the National Bobblehead Museum)

The figurine features a slender Chestnut holding a tray of franks while standing on a hot dog-shaped base with a built-in counter. The chomping champ nicknamed Jaws wears a t-shirt that says, Hot Dog Eating Champion.

This year on the Fourth of July, the 37-year-old Chestnut had consumed 76 hot dogs and buns in the annual 10-minute contest. He beat his own record established over a six-year tenure as the reigning Mustard Belt holder. He took the top title in Nathans contest six years ago from another world famous food scoffer, Takeru Tsunami Kobayashi.

According to the HOF and Museum, Chestnut holds over 50 world eating records that include speed consumption of foods that range from lobsters to tamales to Twinkies. The Museum said that he prepares for gorges by expanding the stomach with milk, water and protein supplements, then fasts a few days prior to competition. Last year, Chestnut told CNBC he trains for three months prior to the Coney contest with weekend practice runs and recovery days to follow.

Museum co-founder and CEO Phil Sklar said, Were excited to unveil this unique bobblehead of the greatest eater of all time. By claiming top honors in the Nathans Hot Dog Eating Contest year after year, Joeys victories have become a Fourth-of-July tradition and his legacy as the worlds greatest eater will be hard to top.

Bill LaCurtis of Eltingville celebrates his hot dog eating contest win with a hot dog at the Richmond County Fair Sunday, September 4, 2016 at Historic Richmond Town. (Staten Island Advance File Photo)Staff-Shot


Staten Islands competitive eating contests will happen at the Richmond County Fair this September. Details are forthcoming from the Historical Society that runs the showdowns but, generally speaking, the edibles will involve pies and hot dogs. Former champions have included Eltingville resident Bill LaCurtis, a perennial competitor who tackles the bun and dog separately. He first soaks the bread in water which eases it down the esophagus.

In 2019, Empire Outlets sponsored the boroughs first cannoli-eating competition. Nine competitors from around the country came to the St. George shopping complex to gobble down the goods for gift cards and a gym membership. Eaters bellied up to folding tables for a three-minute session. The winner that year was Gentleman Joe Menchetti of Cheshire, Ct. who shoveled down 22 red-white-and-blue sprinkled cannoli crafted by Marks Bake Shoppe in Richmond.

The annual San Gennaro Feast also holds a contest with cannoli as the centerpiece. Staten Islanders have been in the spotlight at this presentation in Little Italy in years past. Former winners have included Joe Gavone Rose and Krazy Kevin Lipsitz. Lipsitz announced his retirement from competitive eating at the 2008 Feast.

Pamela Silvestri is Advance Food Editor. She can be reached at

Joey Chestnut is commemorated with bobblehead as S.I. competitive eaters can gear up for contests -

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FROM THE LABS: Cells arrange their chromosomes following one of two designs – Baylor College of Medicine News

A team of biologists studying the tree of life has unveiled a new classification system for cell nuclei according to how chromosomes, which can be several meters long, fold up to fit inside the nuclei. They also discovered a method for transmuting one type of cell nucleus into another.

The study, which appears in the journal Science, emerged from several once-separate efforts. One effort is centered on the DNA Zoo, an international consortium spanning dozens of institutions including Baylor College of Medicine, the National Science Foundation-supportedCenter for Theoretical Biological Physics (CTBP) at Rice University, the University of Western Australia and SeaWorld. The other effort emerged from an independent team in theNetherlands Cancer Institute.

Whether we were looking at worms or urchins, sea squirts or coral, we kept seeing the same folding patterns coming up, saidDr. Olga Dudchenko, co-first author of the new study and a member of theCenter for Genome Architecture at Baylor and CTBP.

Eventually, the team realized it was just seeing variants on two overall nuclear designs. In some species, chromosomes are organized like the pages of a printed newspaper, with the outer margins on one side and the folded middle at the other, explained Dudchenko, who also is co-director of DNA Zoo. And then in other species, each chromosome is crumpled into a little ball.

So we had a puzzle, saidDr. Erez Lieberman Aiden, an associate professor ofmolecular and human geneticsand Emeritus McNair Scholar at Baylor, co-director of the DNA Zoo and senior author on the new study. The data implied that over the course of evolution, species can switch back and forth from one type to the other. We wondered: What is the controlling mechanism? Might it be possible to change one type of nucleus into another in the lab? Aiden also is director of the Center for Genome Architecture and a senior investigator at CTBP.

Meanwhile, an independent team in the Netherlands had discovered something unexpected. I was doing experiments on a protein called condensin II, which we knew plays a role in how cells divide, said Claire Hoencamp, co-first author of the study and a member of the laboratory ofDr. Benjamin Rowlandat theNetherlands Cancer Institute. But we observed the strangest thing: When we mutated the protein in human cells, the chromosomes would totally rearrange. It was baffling!

The two teams met at a conference in the Austrian mountains, where Rowland presented his labs latest work. They soon realized that Hoencamp had hit on a way to convert human cells from one nuclear type to another.

When we looked at the genomes being studied at the DNA Zoo, we discovered that evolution had already done our experiment many, many times! When mutations in a species break condensin II, they usually flip the whole architecture of the nucleus, said Rowland, senior author on the study.

Its always a little disappointing to get scooped on an experiment, but evolution had a very long head start.

The team decided to work together to confirm condensin IIs role. But then the COVID-19 pandemic struck, and much of the world shut down.

Without access to our laboratories, we were left with only one way to establish what condensin II was doing, Hoencamp said. We needed to create a computer program that could simulate the effects of condensin II on the chain of hundreds of millions of genetic letters that comprise each human chromosome.

The team turned toDr. Jos Onuchic, the Harry C. and Olga K. Wiess Chair of Physics at Rice. Our simulations showed that by destroying condensin II, you could make a human nucleus reorganize to resemble a fly nucleus, said Onuchic, co-director of CTBP, which includes collaborators at Rice, Baylor, Northeastern University and other institutions in Houston and Boston.

The simulations were performed by a team within Onuchics lab at CTBP, led by postdoctoral fellow and co-first author Dr. Sumitabha Brahmachari, working with Dr. Vinicius Contessoto, a former postdoc at CTBP, and Dr. Michele Di Pierro, a CTBP senior investigator and currently an assistant professor at Northeastern University.

We began with an incredibly broad survey of 2 billion years of nuclear evolution, Brahmachari said.

We found that much boils down to one simple mechanism, that we can simulate as well as recapitulate, on our own, in a test tube.

Its an exciting step on the road to a new kind of genome engineering in 3D!

Work at Rice, Baylor and Northeastern was supported by the National Science Foundation (NSF), the Welch Institute, the National Institutes of Health, the NSF-supported Behavioral Plasticity Research Institute, IBM, the Pawsey Supercomputing Center and Illumina Inc.

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FROM THE LABS: Cells arrange their chromosomes following one of two designs - Baylor College of Medicine News

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Six faculty elected to National Academy of Sciences – Stanford Today – Stanford University News

Six Stanford University researchers are among the 120 newly elected members of the National Academy of Sciences. Scientists are elected to the NAS by their peers.

The six Stanford faculty members newly elected to the National Academy of Sciences. (Image credit: Andrew Brodhead)

The new members from Stanford are Savas Dimopoulos, the Hamamoto Family Professor and professor of physics in the School of Humanities and Sciences; Daniel Freedman, a visiting professor at theStanford Institute for Theoretical Physics (SITP) and professor of applied mathematics and theoretical physics, emeritus, at MIT; Judith Frydman, professor of biology and the Donald Kennedy Chair in the School of Humanities and Sciences, and professor of genetics in the Stanford School of Medicine; Kathryn A. Kam Moler, vice provost and dean of research, and the Marvin Chodorow Professor and professor of applied physics and of physics in the School of Humanities and Sciences; Tirin Moore, professor of neurobiology in the Stanford School of Medicine; and John Rickford, professor of linguistics and the J.E. Wallace Sterling Professor in the Humanities, emeritus, in the School of Humanities and Sciences.

Savas Dimopoulos collaborates on a number of experiments that use the dramatic advances in atom interferometry to do fundamental physics. These include testing Einsteins theory of general relativity to fifteen decimal precision, atom neutrality to thirty decimals, and looking for modifications of quantum mechanics. He is also designing an atom-interferometric gravity-wave detector that will allow us to look at the universe with gravity waves instead of light.

Daniel Freedmans research is in quantum field theory, quantum gravity and string theory with an emphasis on the role of supersymmetry. Freedman, along with physicists Sergio Ferrara and Peter van Nieuwenhuizen, developed the theory of supergravity. A combination of the principles of supersymmetry and general relatively, supergravity is a deeply influential blueprint for unifying all of natures fundamental interactions.

Judith Frydman uses a multidisciplinary approach to address fundamental questions about protein folding and degradation, and molecular chaperones, which help facilitate protein folding. In addition, this work aims to define how impairment of cellular folding and quality control are linked to disease, including cancer and neurodegenerative diseases, and examine whether reengineering chaperone networks can provide therapeutic strategies.

Kam Molers research involves developing new tools to measure magnetic properties of quantum materials and devices on micron length-scales. These tools can then be used to investigate fundamental materials physics, superconducting devices and exotic Josephson effects a phenomenon in superconductors that shows promise for quantum computing.

Tirin Moore studies the activity of single neurons and populations of neurons in areas of the brain that relate to visual and motor functions. His lab explores the consequences of changes in that activity and aims to develop innovative approaches to fundamental problems in systems and circuit-level neuroscience.

John Rickfords research and teaching are focused on sociolinguistics the relation between linguistic variation and change and social structure. He is especially interested in the relation between language and ethnicity, social class and style, language variation and change, pidgin and creole languages, African American Vernacular English, and the applications of linguistics to educational problems.

The academy is a private, nonprofit institution that was created in 1863 to advise the nation on issues related to science and technology. Scholars are elected in recognition of their outstanding contributions to research. This years election brings the total of active academy members to 2,461.

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