The C. elegans truncated insulin receptor DAF-2B regulates survival of L1 arrested larvae.

We have previously characterized a truncated isoform of the C. elegans insulin-like receptor, DAF-2B, which retains the ligand binding domain but cannot transduce a signal due to the absence of the intracellular signaling domain. DAF-2B modifies insulin / insulin-like growth factor signaling-dependent processes, such as dauer formation and lifespan, by sequestering insulin-like peptides (ILP) and preventing signaling through full length DAF-2 receptors. Here we show that DAF-2B is also important for starvation resistance, as genetic loss of daf-2b reduces survival in arrested first stage larvae (L1). Under fed conditions, we observe daf-2b splicing capacity in both the intestine and the hypodermis, but in starved L1s this becomes predominantly hypodermal. Using a novel splicing reporter system, we observe an increase in the ratio of truncated to full length insulin receptor splicing capacity in starved L1 larvae compared with fed, that may indicate a decrease in whole body insulin responsiveness. Consistent with this, overexpression of DAF-2B from the hypodermis, but not the intestine, confers increased survival to L1 animals under starvation conditions. Our findings demonstrate that the truncated insulin receptor DAF-2B is involved in the response to L1 starvation and promotes survival when expressed from the hypodermis.

Intragenic DNA inversions expand bacterial coding capacity.

Bacterial populations that originate from a single bacterium are not strictly clonal. Often, they contain subgroups with distinct phenotypes. Bacteria can generate heterogeneity through phase variation: a preprogrammed, reversible mechanism that alters gene expression levels across a population. One well studied type of phase variation involves enzyme-mediated inversion of specific intergenic regions of genomic DNA. Frequently, these DNA inversions flip the orientation of promoters, turning ON or OFF adjacent coding regions within otherwise isogenic populations. Through this mechanism, inversion can affect fitness, survival, or group dynamics. Here, we develop and apply bioinformatic approaches to discover thousands of previously undescribed phase-variable regions in prokaryotes using long-read datasets. We identify 'intragenic invertons', a surprising new class of invertible elements found entirely within genes, in bacteria and archaea. To date, inversions within single genes have not been described. Intragenic invertons allow a gene to encode two or more versions of a protein by flipping a DNA sequence within the coding region, thereby increasing coding capacity without increasing genome size. We experimentally characterize specific intragenic invertons in the gut commensal Bacteroides thetaiotaomicron, presenting a 'roadmap' for investigating this new gene-diversifying phenomenon.

The SR protein RSP-2 influences expression of the truncated insulin receptor DAF-2B in Caenorhabditis elegans.

The alternatively spliced daf-2b transcript in Caenorhabditis elegans encodes a truncated isoform of the nematode insulin receptor that retains the extracellular ligand binding domain but lacks the intracellular signaling domain and is therefore unable to transduce a signal. To identify factors that influence expression of daf-2b, we performed a targeted RNA interference screen of rsp genes, which encode splicing factors from the serine/arginine protein family. Loss of rsp-2 significantly increased the expression of a fluorescent daf-2b splicing reporter, as well as increasing expression of endogenous daf-2b transcripts. Correspondingly, rsp-2 mutants exhibited similar phenotypes to those previously observed with DAF-2B overexpression, namely suppression of pheromone-induced dauer formation, enhancement of dauer entry in insulin signaling mutants, inhibition of dauer recovery, and increased lifespan. However, the epistatic relationship between rsp-2 and daf-2b varied according to the experimental context. Increased dauer entry and delayed dauer exit of rsp-2 mutants in an insulin signaling mutant background were partially dependent on daf-2b. Conversely, suppression of pheromone-induced dauer formation and increased lifespan in rsp-2 mutants were independent of daf-2b. These data demonstrate that C. elegans RSP-2, an ortholog of human splicing factor protein SRSF5/SRp40, is involved in regulating the expression of the truncated DAF-2B isoform. However, we also find that RSP-2 can influence dauer formation and lifespan independently of DAF-2B.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

F Plasmids Are the Major Carriers of Antibiotic Resistance Genes in Human-Associated Commensal Escherichia coli.

The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic.IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.

An alternatively spliced, non-signaling insulin receptor modulates insulin sensitivity via insulin peptide sequestration in C. elegans.

In the nematode C. elegans, insulin signaling regulates development and aging in response to the secretion of numerous insulin peptides. Here, we describe a novel, non-signaling isoform of the nematode insulin receptor (IR), DAF-2B, that modulates insulin signaling by sequestration of insulin peptides. DAF-2B arises via alternative splicing and retains the extracellular ligand binding domain but lacks the intracellular signaling domain. A daf-2b splicing reporter revealed active regulation of this transcript through development, particularly in the dauer larva, a diapause stage associated with longevity. CRISPR knock-in of mScarlet into the daf-2b genomic locus confirmed that DAF-2B is expressed in vivo and is likely secreted. Genetic studies indicate that DAF-2B influences dauer entry, dauer recovery and adult lifespan by altering insulin sensitivity according to the prevailing insulin milieu. Thus, in C. elegans alternative splicing at the daf-2 locus generates a truncated IR that fine-tunes insulin signaling in response to the environment.

Genetic behavioral screen identifies an orphan anti-opioid system.

Opioids target the µ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/ß protein.

Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe â†’ Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates oxidative DNA damage induced aging.

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

Synthesis of Cyclic Peptide Mimetics by the Successive Ring Expansion of Lactams.

A successive ring-expansion protocol is reported that enables the controlled insertion of natural and non-natural amino acid fragments into lactams. Amino acids can be installed into macrocycles via an operationally simple and scalable iterative procedure, without the need for high dilution. This method is expected to be of broad utility, especially for the synthesis of medicinally important cyclic peptide mimetics.

A genome-wide screen of bacterial mutants that enhance dauer formation in C. elegans.

Molecular pathways involved in dauer formation, an alternate larval stage that allows Caenorhabditis elegans to survive adverse environmental conditions during development, also modulate longevity and metabolism. The decision to proceed with reproductive development or undergo diapause depends on food abundance, population density, and temperature. In recent years, the chemical identities of pheromone signals that modulate dauer entry have been characterized. However, signals derived from bacteria, the major source of nutrients for C. elegans, remain poorly characterized. To systematically identify bacterial components that influence dauer formation and aging in C. elegans, we utilized the individual gene deletion mutants in E. coli (K12). We identified 56 diverse E. coli deletion mutants that enhance dauer formation in an insulin-like receptor mutant (daf-2) background. We describe the mechanism of action of a bacterial mutant cyaA, that is defective in the production of cyclic AMP, which extends lifespan and enhances dauer formation through the modulation of TGF-ß (daf-7) signaling in C. elegans. Our results demonstrate the importance of bacterial components in influencing developmental decisions and lifespan in C. elegans. Furthermore, we demonstrate that C. elegans is a useful model to study bacterial-host interactions.

Barriers to the Preclinical Development of Therapeutics that Target Aging Mechanisms.

Through the progress of basic science research, fundamental mechanisms that contribute to age-related decline are being described with increasing depth and detail. Although these efforts have identified new drug targets and compounds that extend life span in model organisms, clinical trials of therapeutics that target aging processes remain scarce. Progress in aging research is hindered by barriers associated with the translation of basic science discoveries into the clinic. This report summarizes discussions held at a 2014 Geroscience Network retreat focused on identifying hurdles that currently impede the preclinical development of drugs targeting fundamental aging processes. From these discussions, it was evident that aging researchers have varied perceptions of the ideal preclinical pipeline. To forge a clear and cohesive path forward, several areas of controversy must first be resolved and new tools developed. Here, we focus on five key issues in preclinical drug development (drug discovery, lead compound development, translational preclinical biomarkers, funding, and integration between researchers and clinicians), expanding upon discussions held at the Geroscience Retreat and suggesting areas for further research. By bringing these findings to the attention of the aging research community, we hope to lay the foundation for a concerted preclinical drug development pipeline.

Synthetic Ligands of Cannabinoid Receptors Affect Dauer Formation in the Nematode Caenorhabditis elegans.

Under adverse environmental conditions the nematode Caenorhabditis elegans can enter an alternate developmental stage called the dauer larva. To identify lipophilic signaling molecules that influence this process, we screened a library of bioactive lipids and found that AM251, an antagonist of the human cannabinoid (CB) receptor, suppresses dauer entry in daf-2 insulin receptor mutants. AM251 acted synergistically with glucose supplementation indicating that the metabolic status of the animal influenced the activity of this compound. Similarly, loss of function mutations in the energy-sensing AMP-activated kinase subunit, aak-2, enhanced the dauer-suppressing effects of AM251, while constitutive activation of aak-2 in neurons was sufficient to inhibit AM251 activity. Chemical epistasis experiments indicated that AM251 acts via G-protein signaling and requires the TGF-ß ligand DAF-7, the insulin peptides DAF-28 and INS-6, and a functional ASI neuron to promote reproductive growth. AM251 also required the presence of the SER-5 serotonin receptor, but in vitro experiments suggest that this may not be via a direct interaction. Interestingly, we found that other antagonists of mammalian CB receptors also suppress dauer entry, while the nonselective CB receptor agonist, O-2545, not only inhibited the activity of AM251, but also was able to promote dauer entry when administered alone. Since worms do not have obvious orthologs of CB receptors, the effects of synthetic CBs on neuroendocrine signaling in C. elegans are likely to be mediated via another, as yet unknown, receptor mechanism. However, we cannot exclude the existence of a noncanonical CB receptor in C. elegans.

Chemical activation of a food deprivation signal extends lifespan.

Model organisms subject to dietary restriction (DR) generally live longer. Accompanying this lifespan extension are improvements in overall health, based on multiple metrics. This indicates that pharmacological treatments that mimic the effects of DR could improve health in humans. To find new chemical structures that extend lifespan, we screened 30 000 synthetic, diverse drug-like chemicals in Caenorhabditis elegans and identified several structurally related compounds that acted through DR mechanisms. The most potent of these NP1 impinges upon a food perception pathway by promoting glutamate signaling in the pharynx. This results in the overriding of a GPCR pathway involved in the perception of food and which normally acts to decrease glutamate signals. Our results describe the activation of a dietary restriction response through the pharmacological masking of a novel sensory pathway that signals the presence of food. This suggests that primary sensory pathways may represent novel targets for human pharmacology.

Mitigation of Acetylcholine Esterase Activity in the 1,7-Diazacarbazole Series of Inhibitors of Checkpoint Kinase 1.

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.

Characterization of N-acyl phosphatidylethanolamine-specific phospholipase-D isoforms in the nematode Caenorhabditis elegans.

N-acylethanolamines are an important class of lipid signaling molecules found in many species, including the nematode Caenorhabditis elegans (C. elegans) where they are involved in development and adult lifespan. In mammals, the relative activity of the biosynthetic enzyme N-acyl phosphatidylethanolamine-specific phospholipase-D and the hydrolytic enzyme fatty acid amide hydrolase determine N-acylethanolamine levels. C. elegans has two N-acyl phosphatidylethanolamine-specific phospholipase-D orthologs, nape-1 and nape-2, that are likely to have arisen from a gene duplication event. Here, we find that recombinant C. elegans NAPE-1 and NAPE-2 are capable of generating N-acylethanolamines in vitro, confirming their functional conservation. In vivo, they exhibit overlapping expression in the pharynx and the nervous system, but are also expressed discretely in these and other tissues, suggesting divergent roles. Indeed, nape-1 over-expression results in delayed growth and shortened lifespan only at 25°C, while nape-2 over-expression results in significant larval arrest and increased adult lifespan at 15°C. Interestingly, deletion of the N-acylethanolamine degradation enzyme faah-1 exacerbates nape-1 over-expression phenotypes, but suppresses the larval arrest phenotype of nape-2 over-expression, suggesting that faah-1 is coupled to nape-2, but not nape-1, in a negative feedback loop. We also find that over-expression of either nape-1 or nape-2 significantly enhances recovery from the dauer larval stage in the insulin signaling mutant daf-2(e1368), but only nape-1 over-expression reduces daf-2 adult lifespan, consistent with increased levels of the N-acylethanolamine eicosapentaenoyl ethanolamine. These results provide evidence that N-acylethanolamine biosynthetic enzymes in C. elegans have conserved function and suggest a temperature-dependent, functional divergence between the two isoforms.

Aberrant autolysosomal regulation is linked to the induction of embryonic senescence: differential roles of Beclin 1 and p53 in vertebrate Spns1 deficiency.

Spinster (Spin) in Drosophila or Spinster homolog 1 (Spns1) in vertebrates is a putative lysosomal H+-carbohydrate transporter, which functions at a late stage of autophagy. The Spin/Spns1 defect induces aberrant autolysosome formation that leads to embryonic senescence and accelerated aging symptoms, but little is known about the mechanisms leading to the pathogenesis in vivo. Beclin 1 and p53 are two pivotal tumor suppressors that are critically involved in the autophagic process and its regulation. Using zebrafish as a genetic model, we show that Beclin 1 suppression ameliorates Spns1 loss-mediated senescence as well as autophagic impairment, whereas unexpectedly p53 deficit exacerbates both of these characteristics. We demonstrate that 'basal p53' activity plays a certain protective role(s) against the Spns1 defect-induced senescence via suppressing autophagy, lysosomal biogenesis, and subsequent autolysosomal formation and maturation, and that p53 loss can counteract the effect of Beclin 1 suppression to rescue the Spns1 defect. By contrast, in response to DNA damage, 'activated p53' showed an apparent enhancement of the Spns1-deficient phenotype, by inducing both autophagy and apoptosis. Moreover, we found that a chemical and genetic blockage of lysosomal acidification and biogenesis mediated by the vacuolar-type H+-ATPase, as well as of subsequent autophagosome-lysosome fusion, prevents the appearance of the hallmarks caused by the Spns1 deficiency, irrespective of the basal p53 state. Thus, these results provide evidence that Spns1 operates during autophagy and senescence differentially with Beclin 1 and p53.

Effects of elevated oxygen and carbon dioxide partial pressures on respiratory function and cognitive performance.

Hyperoxia during diving has been suggested to exacerbate hypercapnic narcosis and promote unconsciousness. We tested this hypothesis in male volunteers (12 at rest, 10 at 75 W cycle ergometer exercise) breathing each of four gases in a hyperbaric chamber. Inspired Po2 (PiO2 ) was 0.21 and 1.3 atmospheres (atm) without or with an individual subject's maximum tolerable inspired CO2 (PiO2 = 0.055-0.085 atm). Measurements included end-tidal CO2 partial pressure (PetCO2 ), rating of perceived discomfort (RPD), expired minute ventilation (V̇e), and cognitive function assessed by auditory n-back test. The most prominent finding was, irrespective of PetCO2 , that minute ventilation was 8-9 l/min greater for rest or exercise with a PiO2 of 1.3 atm compared with 0.21 atm (P < 0.0001). For hyperoxic gases, PetCO2 was consistently less than for normoxic gases (P < 0.01). For hyperoxic hypercapnic gases, n-back scores were higher than for normoxic gases (P < 0.01), and RPD was lower for exercise but not rest (P < 0.02). Subjects completed 66 hyperoxic hypercapnic trials without incident, but five stopped prematurely because of serious symptoms (tunnel vision, vision loss, dizziness, panic, exhaustion, or near syncope) during 69 normoxic hypercapnic trials (P = 0.0582). Serious symptoms during hypercapnic trials occurred only during normoxia. We conclude serious symptoms with hyperoxic hypercapnia were absent because of decreased PetCO2 consequent to increased ventilation.

Bacterial fatty acids enhance recovery from the dauer larva in Caenorhabditis elegans.

The dauer larva is a specialized dispersal stage in the nematode Caenorhabditis elegans that allows the animal to survive starvation for an extended period of time. The dauer does not feed, but uses chemosensation to identify new food sources and to determine whether to resume reproductive growth. Bacteria produce food signals that promote recovery of the dauer larva, but the chemical identities of these signals remain poorly defined. We find that bacterial fatty acids in the environment augment recovery from the dauer stage under permissive conditions. The effect of increased fatty acids on different dauer constitutive mutants indicates a role for insulin peptide secretion in coordinating recovery from the dauer stage in response to fatty acids. These data suggest that worms can sense the presence of fatty acids in the environment and that elevated levels can promote recovery from dauer arrest. This may be important in the natural environment where the dauer larva needs to determine whether the environment is appropriate to support reproductive growth following dauer exit.