Germline stem cell integrity and quiescence are controlled by an AMPK-dependent neuronal trafficking pathway.

During periods of energetic stress, Caenorhabditis elegans can execute a developmentally quiescent stage called "dauer", during which all germline stem cells undergo a G2 cell cycle arrest. In animals that lack AMP-activated protein kinase (AMPK) signalling, the germ cells fail to arrest, undergo uncontrolled proliferation, and lose their reproductive capacity upon recovery from this quiescent stage. These germline defects are accompanied by, and likely result from, an altered chromatin landscape and gene expression program. Through genetic analysis we identified an allele of tbc-7, a predicted RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is modulated by tbc-7 and show that the activity of RAB-7 is critical for the maintenance of germ cell integrity during the dauer stage. We reveal that TBC-7 is regulated by AMPK through two mechanisms when the animals enter the dauer stage. Acutely, the AMPK-mediated phosphorylation of TBC-7 reduces its activity, potentially by autoinhibition, thereby preventing the inactivation of RAB-7. In the more long term, AMPK regulates the miRNAs mir-1 and mir-44 to attenuate tbc-7 expression. Consistent with this, animals lacking mir-1 and mir-44 are post-dauer sterile, phenocopying the germline defects of AMPK mutants. Altogether, we have uncovered an AMPK-dependent and microRNA-regulated cellular trafficking pathway that is initiated in the neurons, and is critical to control germline gene expression cell non-autonomously in response to adverse environmental conditions.

Unscheduled epigenetic modifications cause genome instability and sterility through aberrant R-loops following starvation.

During starvation, organisms modify both gene expression and metabolism to adjust to the energy stress. We previously reported that Caenorhabditis elegans lacing AMP-activated protein kinase (AMPK) exhibit transgenerational reproductive defects associated with abnormally elevated trimethylated histone H3 at lysine 4 (H3K4me3) levels in the germ line following recovery from acute starvation. Here, we show that these H3K4me3 marks are significantly increased at promoters, driving aberrant transcription elongation resulting in the accumulation of R-loops in starved AMPK mutants. DNA-RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq) analysis demonstrated that a significant proportion of the genome was affected by R-loop formation. This was most pronounced in the promoter-transcription start site regions of genes, in which the chromatin was modified by H3K4me3. Like H3K4me3, the R-loops were also found to be heritable, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK mutant germ lines show considerably more RAD-51 (the RecA recombinase) foci at sites of R-loop formation, potentially sequestering them from their roles at meiotic breaks or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation. The downstream effects of R-loops on DNA damage sensitivity and germline stem cell integrity may account for inappropriate epigenetic modification that occurs in numerous human disorders, including various cancers.

Developmental plasticity and the response to nutrient stress in Caenorhabditis elegans.

Developmental plasticity refers the ability of an organism to adapt to various environmental stressors, one of which is nutritional stress. Caenorhabditis elegans require various nutrients to successfully progress through all the larval stages to become a reproductive adult. If nutritional criteria are not satisfied, development can slow or completely arrest. In poor growth conditions, the animal can enter various diapause stages, depending on its developmental progress. In C. elegans, there are three well-characterized diapauses: the L1 arrest, the dauer diapause, and adult reproductive diapause, each associated with drastic changes in metabolism and germline development. At the centre of these changes is AMP-activated protein kinase (AMPK). AMPK is a metabolic regulator that maintains energy homeostasis, particularly during times of nutrient stress. Without AMPK, metabolism is disrupted during dauer, leading to the rapid consumption of lipid stores as well as misregulation of metabolic enzymes, leading to reduced survival. During the L1 arrest and dauer diapause, AMPK is responsible for ensuring germline quiescence by modifying the germline chromatin landscape to maintain germ cell integrity until conditions improve. Similar to classic hormonal signalling, small RNAs also play a critical role in regulating development and behaviour in a cell non-autonomous fashion. Thus, during the challenges associated with developmental plasticity, AMPK summons an army of signalling pathways to work collectively to preserve reproductive fitness during these periods of unprecedented uncertainty.

AMPK regulates germline stem cell quiescence and integrity through an endogenous small RNA pathway.

During suboptimal growth conditions, Caenorhabditis elegans larvae undergo a global developmental arrest called "dauer." During this stage, the germline stem cells (GSCs) become quiescent in an AMP-activated Protein Kinase (AMPK)-dependent manner, and in the absence of AMPK, the GSCs overproliferate and lose their reproductive capacity, leading to sterility when mutant animals resume normal growth. These defects correlate with the altered abundance and distribution of a number of chromatin modifications, all of which can be corrected by disabling components of the endogenous small RNA pathway, suggesting that AMPK regulates germ cell integrity by targeting an RNA interference (RNAi)-like pathway during dauer. The expression of AMPK in somatic cells restores all the germline defects, potentially through the transmission of small RNAs. Our findings place AMPK at a pivotal position linking energy stress detected in the soma to a consequent endogenous small RNA-mediated adaptation in germline gene expression, thereby challenging the "permeability" of the Weismann barrier.

Spaceborne Cloud and Precipitation Radars: Status, Challenges, and Ways Forward.

Spaceborne radars offer a unique three-dimensional view of the atmospheric components of the Earth's hydrological cycle. Existing and planned spaceborne radar missions provide cloud and precipitation information over the oceans and land difficult to access in remote areas. A careful look into their measurement capabilities indicates considerable gaps that hinder our ability to detect and probe key cloud and precipitation processes. The international community is currently debating how the next generation of spaceborne radars shall enhance current capabilities and address remaining gaps. Part of the discussion is focused on how to best take advantage of recent advancements in radar and space platform technologies while addressing outstanding limitations. First, the observing capabilities and measurement highlights of existing and planned spaceborne radar missions including TRMM, CloudSat, GPM, RainCube, and EarthCARE are reviewed. Then, the limitations of current spaceborne observing systems, with respect to observations of low-level clouds, midlatitude and high-latitude precipitation, and convective motions, are thoroughly analyzed. Finally, the review proposes potential solutions and future research avenues to be explored. Promising paths forward include collecting observations across a gamut of frequency bands tailored to specific scientific objectives, collecting observations using mixtures of pulse lengths to overcome trade-offs in sensitivity and resolution, and flying constellations of miniaturized radars to capture rapidly evolving weather phenomena. This work aims to increase the awareness about existing limitations and gaps in spaceborne radar measurements and to increase the level of engagement of the international community in the discussions for the next generation of spaceborne radar systems.

Surviving Starvation: AMPK Protects Germ Cell Integrity by Targeting Multiple Epigenetic Effectors.

Acute starvation can have long-term consequences that are mediated through epigenetic change. Some of these changes are affected by the activity of AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis. In Caenorhabditis elegans, the absence of AMPK during a period of starvation in an early larval stage results in developmental defects following their recovery on food, while many of them become sterile. Moreover, the loss of AMPK during this quiescent period results in transgenerational phenotypes that can become progressively worse with each successive generation. Our recent data describe a chromatin-based mechanism of how AMPK mediates adjustment to acute starvation in the germ cells, however, the heritable aspect of this AMPK mutant phenotype remains unresolved. Here, we explore how AMPK might affect this process and speculate how the initial transcription that occurs in the germ cells may adversely affect subsequent germline gene expression and/or genomic integrity.

AMPK blocks starvation-inducible transgenerational defects in Caenorhabditis elegans.

Life history events, such as traumatic stress, illness, or starvation, can influence us through molecular changes that are recorded in a pattern of characteristic chromatin modifications. These modifications are often associated with adaptive adjustments in gene expression that can persist throughout the lifetime of the organism, or even span multiple generations. Although these adaptations may confer some selective advantage, if they are not appropriately regulated they can also be maladaptive in a context-dependent manner. We show here that during periods of acute starvation in Caenorhabditis elegans larvae, the master metabolic regulator AMP-activated protein kinase (AMPK) plays a critical role in blocking modifications to the chromatin landscape. This ensures that gene expression remains inactive in the germ-line precursors during adverse conditions. In its absence, critical chromatin modifications occur in the primordial germ cells (PGCs) of emergent starved L1 larvae that correlate with compromised reproductive fitness of the generation that experienced the stress, but also in the subsequent generations that never experienced the initial event. Our findings suggest that AMPK regulates the activity of the chromatin modifying COMPASS complex (complex proteins associated with Set1) to ensure that chromatin marks are not established until nutrient/energy contingencies are satisfied. Our study provides molecular insight that links metabolic adaptation to transgenerational epigenetic modification in response to acute periods of starvation.

AMPK Regulates Developmental Plasticity through an Endogenous Small RNA Pathway in Caenorhabditis elegans.

Caenorhabditis elegans larvae can undergo developmental arrest upon entry into the dauer stage in response to suboptimal growth conditions. Dauer larvae can exit this stage in replete conditions with no reproductive consequence. During this diapause stage, the metabolic regulator AMP-activated protein kinase (AMPK) ensures that the germ line becomes quiescent to maintain germ cell integrity. Animals that lack all AMPK signalling undergo germline hyperplasia upon entering dauer, while those that recover from this stage become sterile. Neuronal AMPK expression in otherwise AMPK-deficient animals is sufficient for germline quiescence and germ cell integrity and its effects are likely mediated through an endogenous small RNA pathway. Upon impairing small RNA biosynthesis, the post-dauer fertility is restored in AMPK mutants. These data suggest that AMPK may function in neurons to relay a message through small RNAs to the germ cells to alter their quiescence in the dauer stage, thus challenging the permeability of the Weismann barrier.

Correction: The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1005284.].

Genome-wide surveys reveal polarity and cytoskeletal regulators mediate LKB1-associated germline stem cell quiescence.

BACKGROUND: Caenorhabditis elegans can endure long periods of environmental stress by altering their development to execute a quiescent state called "dauer". Previous work has implicated LKB1 - the causative gene in the autosomal dominant, cancer pre-disposing disease called Peutz-Jeghers Syndrome (PJS), and its downstream target AMPK, in the establishment of germline stem cell (GSC) quiescence during the dauer stage. Loss of function mutations in both LKB1/par-4 and AMPK/aak(0) result in untimely GSC proliferation during the onset of the dauer stage, although the molecular mechanism through which these factors regulate quiescence remains unclear. Curiously, the hyperplasia observed in par-4 mutants is more severe than AMPK-compromised dauer larvae, suggesting that par-4 has alternative downstream targets in addition to AMPK to regulate germline quiescence. RESULTS: We conducted three genome-wide RNAi screens to identify potential downstream targets of the protein kinases PAR-4 and AMPK that mediate dauer-dependent GSC quiescence. First, we screened to identify genes that phenocopy the par-4-dependent hyperplasia when compromised by RNAi. Two additional RNAi screens were performed to identify genes that suppressed the germline hyperplasia in par-4 and aak(0) dauer larvae, respectively. Interestingly, a subset of the candidates we identified are involved in the regulation of cell polarity and cytoskeletal function downstream of par-4, in an AMPK-independent manner. Moreover, we show that par-4 temporally regulates actin cytoskeletal organization within the dauer germ line at the rachis-adjacent membrane, in an AMPK-independent manner. CONCLUSION: Our data suggest that the regulation of the cytoskeleton and cell polarity may contribute significantly to the tumour suppressor function of LKB1/par-4.

The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans.

AMP-activated kinase (AMPK) is a key regulator of many cellular mechanisms required for adjustment to various stresses induced by the changing environment. In C. elegans dauer larvae AMPK-null mutants expire prematurely due to hyperactive Adipose Triglyceride Lipase (ATGL-1) followed by rapid depletion of triglyceride stores. We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers. We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure. Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs). Our data reveal a novel structural role of CGI-58 in maintaining lipid droplet homeostasis through its effects on droplet composition, morphology and lipid hydrolysis; a conserved function that may account for some of the ATGL-1-independent features unique to Chanarin-Dorfman Syndrome.

Two-Element Mixture of Bose and Fermi Superfluids.

We report on the production of a stable mixture of bosonic and fermionic superfluids composed of the elements ^{174}Yb and ^{6}Li which feature a strong mismatch in mass and distinct electronic properties. We demonstrate elastic coupling between the superfluids by observing the shift in dipole oscillation frequency of the bosonic component due to the presence of the fermions. The measured magnitude of the shift is consistent with a mean-field model and its direction determines the previously unknown sign of the interspecies scattering length to be positive. We also observe the exchange of angular momentum between the superfluids from the excitation of a scissors mode in the bosonic component through interspecies interactions. We explain this observation using an analytical model based on superfluid hydrodynamics.

Mechanisms of animal diapause: recent developments from nematodes, crustaceans, insects, and fish.

Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages.

An empirical test of convergent evolution in rhodopsins.

Rhodopsins are photochemically reactive membrane proteins that covalently bind retinal chromophores. Type I rhodopsins are found in both prokaryotes and eukaryotic microbes, whereas type II rhodopsins function as photoactivated G-protein coupled receptors (GPCRs) in animal vision. Both rhodopsin families share the seven transmembrane α-helix GPCR fold and a Schiff base linkage from a conserved lysine to retinal in helix G. Nevertheless, rhodopsins are widely cited as a striking example of evolutionary convergence, largely because the two families lack detectable sequence similarity and differ in many structural and mechanistic details. Convergence entails that the shared rhodopsin fold is so especially suited to photosensitive function that proteins from separate origins were selected for this architecture twice. Here we show, however, that the rhodopsin fold is not required for photosensitive activity. We engineered functional bacteriorhodopsin variants with novel folds, including radical noncircular permutations of the α-helices, circular permutations of an eight-helix construct, and retinal linkages relocated to other helices. These results contradict a key prediction of convergence and thereby provide an experimental attack on one of the most intractable problems in molecular evolution: how to establish structural homology for proteins devoid of discernible sequence similarity.

Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survival.

Many organisms can enter a dormant state or diapause to survive harsh environmental conditions for extended durations. When Caenorhabditis elegans larvae enter dauer they arrest feeding but remain active and motile, yet become stress-resistant, extremely long-lived and non-ageing. Entry into dauer is associated with a reduction in insulin-like signalling, the accumulation of nutritive resources and a concomitant global change in metabolism, yet the precise molecular and physiological processes that enable long-term survival in the absence of caloric intake remain largely unknown. We show here that C. elegans larvae that lack LKB1/AMPK (AMP-activated protein kinase) signalling enter dauer normally, but then rapidly consume their stored energy and prematurely expire following vital organ failure. We found that this signalling pathway acts in adipose-like tissues to downregulate triglyceride hydrolysis so that these lipid reserves are rationed to last the entire duration of the arrest. Indeed, the downregulation of adipose triglyceride lipase (ATGL-1) activity suppresses both the rapid depletion of stored lipids and reduced life span of AMPK mutant dauers, while AMPK directly phosphorylates ATGL-1. Finally, we show that the slow release of energy during dauer is critical for appropriate long-term osmoregulation, which fails as triglyceride resources become depleted. These mechanisms may be essential for survival through diapause, hibernation, or long-term fasting in diverse organisms and may also underlie AMPK-dependent life span extension.

Ultracold heteronuclear mixture of ground and excited state atoms.

We report on the realization of an ultracold mixture of lithium atoms in the ground state and ytterbium atoms in an excited metastable (3P2) state. Such a mixture can support broad magnetic Feshbach resonances which may be utilized for the production of ultracold molecules with an electronic spin degree of freedom, as well as novel Efimov trimers. We investigate the interaction properties of the mixture in the presence of an external magnetic field and find an upper limit for the background interspecies two-body inelastic decay coefficient of K2'<3×10(-12) cm3/s for the 3P2 mJ=-1 substate. We calculate the dynamic polarizabilities of the Yb(3P2) magnetic substates for a range of wavelengths, and find good agreement with our measurements at 1064 nm. Our calculations also allow the identification of magic frequencies where Yb ground and metastable states are identically trapped and the determination of the interspecies van der Waals coefficients.

Differential requirements for STRAD in LKB1-dependent functions in C. elegans.

The protein kinase LKB1 is a crucial regulator of cell growth/proliferation and cell polarity and is the causative gene in the cancer-predisposing disease Peutz-Jeghers syndrome (PJS). The activity of LKB1 is greatly enhanced following its association with the Ste20-like adapter protein STRAD. Unlike LKB1 however, mutations in STRAD have not been identified in PJS patients and thus, the key tumour suppressive role(s) of LKB1 might be STRAD independent. Here, we report that Caenorhabditis elegans strd-1/STRAD mutants recapitulate many phenotypes typical of par-4/LKB1 loss of function, showing defects during early embryonic and dauer development. Interestingly, although the growth/proliferation defects in severe par-4 and strd-1 mutant dauers are comparable, strd-1 mutant embryos do not share the polarity defects of par-4 embryos. We demonstrate that most of par-4-dependent regulation of germline stem cell (GSC) quiescence occurs through AMPK, whereby PAR-4 requires STRD-1 to phosphorylate and activate AMPK. Consistent with this, even though AMPK plays a major role in the regulation of cell proliferation, like strd-1 it does not affect embryonic polarity. Instead, we found that the PAR-4-mediated phosphorylation of polarity regulators such as PAR-1 and MEX-5 in the early embryo occurs in the absence of STRD-1. Thus, PAR-4 requires STRD-1 to phosphorylate AMPK to regulate cell growth/proliferation under reduced insulin signalling conditions, whereas PAR-4 can promote phosphorylation of key proteins, including PAR-1 and MEX-5, to specify early embryonic polarity independently of STRD-1. Our results therefore identify a key strd-1/STRAD-independent function of par-4/LKB1 in polarity establishment that is likely to be important for tumour suppression in humans.

C. elegans STRADalpha and SAD cooperatively regulate neuronal polarity and synaptic organization.

Neurons are polarized cells with morphologically and functionally distinct axons and dendrites. The SAD kinases are crucial for establishing the axon-dendrite identity across species. Previous studies suggest that a tumour suppressor kinase, LKB1, in the presence of a pseudokinase, STRADalpha, initiates axonal differentiation and growth through activating the SAD kinases in vertebrate neurons. STRADalpha was implicated in the localization, stabilization and activation of LKB1 in various cell culture studies. Its in vivo functions, however, have not been examined. In our present study, we analyzed the neuronal phenotypes of the first loss-of-function mutants for STRADalpha and examined their genetic interactions with LKB1 and SAD in C. elegans. Unexpectedly, only the C. elegans STRADalpha, STRD-1, functions exclusively through the SAD kinase, SAD-1, to regulate neuronal polarity and synaptic organization. Moreover, STRD-1 tightly associates with SAD-1 to coordinate its synaptic localizations. By contrast, the C. elegans LKB1, PAR-4, also functions in an additional genetic pathway independently of SAD-1 and STRD-1 to regulate neuronal polarity. We propose that STRD-1 establishes neuronal polarity and organizes synaptic proteins in a complex with the SAD-1 kinase. Our findings suggest that instead of a single, linear genetic pathway, STRADalpha and LKB1 regulate neuronal development through multiple effectors that are shared in some cellular contexts but distinct in others.

Comparison of paraspinal cutaneous temperature measurements between subjects with and without chronic low back pain.

OBJECTIVE: The purpose of this study was to evaluate the effects of chiropractic manipulative treatment on paraspinal cutaneous temperature (PCT) for subjects with chronic low back pain and compare these PCT findings to subjects without chronic low back pain. METHODS: Two groups were created, a symptomatic treatment group (subjects with chronic low back pain, n = 11, 7 males, 4 females) and an asymptomatic, nontreatment group (asymptomatic subjects, n = 10, 6 males, 4 females). Outcomes included the modified Oswestry questionnaire and PCT measurements in the prone position after an 8-minute acclimation period. The treatment group received 9 chiropractic spinal instrument-based manipulative treatments over 2 weeks. Reevaluation was done 2 weeks after the initial evaluation for both groups. RESULTS: The preintervention Oswestry results (29.8% ± 11.8%) for the treatment group were higher than the asymptomatic group (10.2% ± 10.6%). The postintervention Oswestry results for the treatment group were 14.20 % ± 11.5%. The resulting Cohen's effect size of the spinal manipulation on the Oswestry evaluation is 0.58. The preintervention PCT showed higher temperature for the nontreatment group compared with the treatment group. Comparing the levels associated with low back pain, the nontreatment group PCT was stable, varying from 0.01°C to 0.02°C, whereas the treatment group PCT varied from 0.10°C to 0.18°C. The treatment group postintervention PCT showed an increase in temperature after the 9 visits; however, this did not reach the values of the asymptomatic group. CONCLUSION: The PCT readings for subjects with chronic low back pain were lower than the asymptomatic, nontreatment group. The PCT temperature of the treatment group increased after 9 treatments.

Cell cycle regulators control centrosome elimination during oogenesis in Caenorhabditis elegans.

In many animals, the bipolar spindle of the first zygotic division is established after the contribution of centrioles by the sperm at fertilization. To avoid the formation of a multipolar spindle in the zygote, centrosomes are eliminated during oogenesis in most organisms, although the mechanism of this selective elimination is poorly understood. We show that cki-2, a Caenorhabditis elegans cyclin-dependent kinase (Cdk) inhibitor, is required for their appropriate elimination during oogenesis. In the absence of cki-2, embryos have supernumerary centrosomes and form multipolar spindles that result in severe aneuploidy after anaphase of the first division. Moreover, we demonstrate that this defect can be suppressed by reducing cyclin E or Cdk2 levels. This implies that the proper regulation of a cyclin E-Cdk complex by cki-2 is required for the elimination of the centrosome that occurs before or during oogenesis to ensure the assembly of a bipolar spindle in the C. elegans zygote.