Dehydrated Caenorhabditis elegans Stocks Are Resistant to Multiple Freeze-Thaw Cycles.

Ultracold preservation is widely used for storage of genetic stocks of Caenorhabditis elegans Current cryopreservation protocols are vulnerable to refrigeration failures, which can result in the loss of stock viability due to damage during re-freezing. Here we present a method for preserving worms in a dehydrated and frozen form that retains viability after multiple freeze-thaw cycles. After dehydration in the presence of trehalose or glycerol, C. elegans stocks can be frozen and thawed multiple times while maintaining viability. While both dauer and non-dauer larvae survive desiccation and freezing, the dauer defective mutant daf-16 does not survive desiccation. Our technique is useful for storing stocks in a manner robust to freezer failures, and potentially for shipping strains between laboratories.

Antagonistic Serotonergic and Octopaminergic Neural Circuits Mediate Food-Dependent Locomotory Behavior in Caenorhabditis elegans.

Biogenic amines are conserved signaling molecules that link food cues to behavior and metabolism in a wide variety of organisms. In the nematode Caenorhabditis elegans, the biogenic amines serotonin (5-HT) and octopamine regulate a number of food-related behaviors. Using a novel method for long-term quantitative behavioral imaging, we show that 5-HT and octopamine jointly influence locomotor activity and quiescence in feeding and fasting hermaphrodites, and we define the neural circuits through which this modulation occurs. We show that 5-HT produced by the ADF neurons acts via the SER-5 receptor in muscles and neurons to suppress quiescent behavior and promote roaming in fasting worms, whereas 5-HT produced by the NSM neurons acts on the MOD-1 receptor in AIY neurons to promote low-amplitude locomotor behavior characteristic of well fed animals. Octopamine, produced by the RIC neurons, acts via SER-3 and SER-6 receptors in SIA neurons to promote roaming behaviors characteristic of fasting animals. We find that 5-HT signaling is required for animals to assume food-appropriate behavior, whereas octopamine signaling is required for animals to assume fasting-appropriate behavior. The requirement for both neurotransmitters in both the feeding and fasting states enables increased behavioral adaptability. Our results define the molecular and neural pathways through which parallel biogenic amine signaling tunes behavior appropriately to nutrient conditions.SIGNIFICANCE STATEMENT Animals adjust behavior in response to environmental changes, such as fluctuations in food abundance, to maximize survival and reproduction. Biogenic amines, such as like serotonin, are conserved neurotransmitters that regulate behavior and metabolism in relation to energy status. Disruptions of biogenic amine signaling contribute to human neurological diseases of mood, appetite, and movement. In this study, we investigated the roles of the biogenic amines serotonin and octopamine in regulating locomotion behaviors associated with feeding and fasting in the roundworm Caenorhabditis elegans We identified neural circuits through which these signals work to govern behavior. Understanding the molecular pathways through which biogenic amines function in model organisms may improve our understanding of dysfunctions of appetite and behavior found in mammals, including humans.

Food responsiveness regulates episodic behavioral states in Caenorhabditis elegans.

Animals optimize survival and reproduction in part through control of behavioral states, which depend on an organism's internal and external environments. In the nematode Caenorhabditis elegans a variety of behavioral states have been described, including roaming, dwelling, quiescence, and episodic swimming. These states have been considered in isolation under varied experimental conditions, making it difficult to establish a unified picture of how they are regulated. Using long-term imaging, we examined C. elegans episodic behavioral states under varied mechanical and nutritional environments. We found that animals alternate between high-activity (active) and low-activity (sedentary) episodes in any mechanical environment, while the incidence of episodes and their behavioral composition depend on food levels. During active episodes, worms primarily roam, as characterized by continuous whole body movement. During sedentary episodes, animals exhibit dwelling (slower movements confined to the anterior half of the body) and quiescence (a complete lack of movement). Roaming, dwelling, and quiescent states are manifest not only through locomotory characteristics but also in pharyngeal pumping (feeding) and in egg-laying behaviors. Next, we analyzed the genetic basis of behavioral states. We found that modulation of behavioral states depends on neuropeptides and insulin-like signaling in the nervous system. Sensory neurons and the Foraging homolog EGL-4 regulate behavior through control of active/sedentary episodes. Optogenetic stimulation of dopaminergic and serotonergic neurons induced dwelling, implicating dopamine as a dwell-promoting neurotransmitter. Our findings provide a more unified description of behavioral states and suggest that perception of nutrition is a conserved mechanism for regulating animal behavior.NEW & NOTEWORTHY One strategy by which animals adapt to their internal states and external environments is by adopting behavioral states. The roundworm Caenorhabditis elegans is an attractive model for investigating how behavioral states are genetically and neuronally controlled. Here we describe the hierarchical organization of behavioral states characterized by locomotory activity, feeding, and egg-laying. We show that decisions to engage in these behaviors are controlled by the nervous system through insulin-like signaling and the perception of food.

Sleep and cargo reorganization: A hypothesis.

Several molecules that act in the nervous system to regulate sleep and wake were first identified based on their transport effects in pigmented cells. I compiled a list of such molecules like melatonin, melanin-concentrating hormone, and pigment dispersing factor, etc. Molecules that induce pigment aggregation promote sleep whereas molecules that induce pigment dispersal promote wake. I call these Sleep and PIgment Regulating Factors SPIRFs. SPIRFs regulate organelle trafficking in both pigmentary models and neurons. I propose that cargo transport fulfills necessary sleep functions such as remodeling synapses and restoring homeostasis in the distribution of cell components. I put forth the hypothesis that sleep-promoting SPIRFs induce states of increased cargo movement towards the cell body, and propose that this function is a critical neuron maintenance task for which animals must sleep.

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity.

FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.

Deubiquitylation machinery is required for embryonic polarity in Caenorhabditis elegans.

The Caenorhabditis elegans one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another C. elegans DUB gene, usp-46, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in Schizosaccharomyces pombe are also required in C. elegans raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.

Efficacy of a comprehensive program for reducing stress in women: a prospective, randomized trial.

OBJECTIVE: This study evaluated the efficacy of a comprehensive stress management program in reducing perceived stress among women who reported moderate-to-high stress levels. METHODS: A total of 562 highly motivated females, aged 25-45, with moderate to high stress levels, were enrolled in a 14-week study. Participants were randomized into one of three groups: Group 1 included Internet-based coaching focusing on behavior modification, daily use of proprietary olfactive-based personal care products, and periodic feedback reports; Group 2 consisted of only online coaching; and Group 3 had no active stress management program. Participants in the three groups filled out validated psychometric assessments at baseline and throughout the study period. Several outcomes including Perceived Stress Scale (PSS), Profile of Mood States (POMS), St Mary's Hospital Sleep Questionnaire (SMS), the Trier Inventory of Chronic Stress (TICS), Spielberger State-Trait Anxiety Inventory (STAI), Short-Form-36 (SF-36) and the Work Productivity and Activity Impairment (WPAI), were measured periodically to assess changes in subject-perceived stress, stress-related comorbidities, and sleep quality and to evaluate overall program efficacy. Ethical approval of protocols was conducted by the Allendale Investigational Review Board (AIRB). Voluntary informed consent was obtained from each subject. RESULTS: At the end of the 14-week study period, subjects in Group 1 had statistically significant improvement in the PSS score vs. Group 3 (p < 0.01). There were statistically significant improvements in other efficacy outcomes such as POMS total mood disturbance, TICS work overload and social responsibility subscales, STAI and in the number of night awakenings, assessed by the SMS questionnaire (p < 0.05). Self-reported program efficacy was also significantly higher for Group 1 (p < 0.001). CONCLUSION: Despite study limitations, including reduction of stress in Group 3, this study demonstrates that this comprehensive stress management program is effective in reducing stress among women with moderate to high stress levels.

Creating therapeutic proteins from bioengineered systems.

If scientific and social obstacles can be overcome, the use of plants and animals as living production facilities to effect protein expression could be the key to reducing R&D costs - and ultimately, the cost of therapies that will benefit millions of people.