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[
Lab Chip,
2013]
Direct observation of developmental and physiological changes in certain model organisms over time has been technically challenging. In the model organism Caenorhabditis elegans, these studies require frequent or continuous imaging at physiologically benign conditions. However, standard methods use anaesthetics, glue, or microbeads, which prevent animals from feeding during the experiment. Thus, the animals' normal physiological function may be affected over time. Here we present a platform designed for dynamic studies of C. elegans. The system is capable of immobilizing only the animals' bodies under benign conditions and without physical deformation. Simultaneously, the animals' heads remain free to move and feed for the duration of the experiment. This allows for high-resolution and high-magnification fluorescent imaging of immobilized and feeding animals. The system is very easy to fabricate, set up, and operate, and should be widely applicable to many problems in developmental and physiological studies.
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[
Lab Chip,
2010]
Developmental studies in multicellular model organisms such as Caernohabditis elegans rely extensively on the ability to cultivate and image animals repeatedly at the cell or subcellular level. However, standard high-resolution imaging techniques require the use of anaesthetics for immobilization, and may have undesirable side effects on development. Thus such techniques are not ideal in allowing the same animals to grow and be imaged throughout development to observe specific developmental processes. In this paper, we present a microfluidic system designed to overcome these difficulties. The system allows for long-term culture of C. elegans starting at L1 larval stage and repeated high-resolution imaging at physiological temperatures without using anaesthetics. We use a commercially available biocompatible polymer, Pluronic F127 for immobilization; this polymer is capable of a reversible thermo-sensitive sol-gel transition within approximately 2 degrees C, which is well-controlled in the microfluidic chip. The gel phase is sufficient to immobilize the animals. While animals are not imaged, they are cultured in individual chambers in media containing nutrients required for development. We show here that this method facilitates time-lapse studies of single animals at high-resolution and lends itself to live imaging experiments on developmental processes and dynamic events.
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[
Biochemistry,
1987]
The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].
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Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP
[
Nature,
2015]
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.
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[
Mol Immunol,
1999]
Invertebrate cells lack the
p53 recombination checkpoint but contain mobile DNA sequences that transpose by a mechanism in part shared with excision of the V(D)J recombination signal sequences (RSS). In this work, inversion, deletion, and duplication of sequences associated with an invertebrate C. elegans Tc6 element is described. The structure of this C. elegans sequence and other dispersed Tc6 elements suggests that covalently closed 'hairpin' structures are not unique to excision of the V(D)J RSS by the RAG proteins, but rather can be generated by transposases at transposon termini leading to characteristic inversion and duplication events. Comparative analysis of recombination events at invertebrate sequences resembling the vertebrate V(D)J RSS may be useful in understanding V(D)J recombination-mediated recombination events in malignant vertebrate cells or genetic diseases such as ataxia telangectasia, in which the
p53 recombination checkpoint is defective.
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[
Phytother Res,
2008]
A bioassay-guided fractionation of Juniperus procera berries yielded antiparasitic, nematicidal and antifouling constituents, including a wide range of known abietane, pimarane and labdane diterpenes. Among these, abieta-7,13-diene (1) demonstrated in vitro antimalarial activity against Plasmodium falciparum D6 and W2 strains (IC(50) = 1.9 and 2.0 microg/mL, respectively), while totarol (6), ferruginol (7) and 7beta-hydroxyabieta-8,13-diene-11,12-dione (8) inhibited Leishmania donovani promastigotes with IC(50) values of 3.5-4.6 microg/mL. In addition, totarol demonstrated nematicidal and antifouling activities against Caenorhabditis elegans and Artemia salina at a concentration of 80 microg/mL and 1 microg/mL, respectively. The resinous exudate of J. virginiana afforded known antibacterial E-communic acid (4) and 4-epi-abietic acid (5), while the volatile oil from its trunk wood revealed large quantities of cedrol (9). Using GC/MS, the two known abietanes totarol (6) and ferruginol (7) were identified from the berries of J. procera, J. excelsa and J. phoenicea.
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[
Aging Cell,
2017]
Protein aggregation is enhanced upon exposure to various stress conditions and aging, which suggests that the quality control machinery regulating protein homeostasis could exhibit varied capacities in different stages of organismal lifespan. Recently, an efficient metazoan disaggregase activity was identified invitro, which requires the Hsp70 chaperone and Hsp110 nucleotide exchange factor, together with single or cooperating J-protein co-chaperones of classes A and B. Here, we describe how the orthologous Hsp70s and J-protein of Caenorhabditis elegans work together to resolve protein aggregates both invivo and invitro to benefit organismal health. Using an RNAi knockdown approach, we show that class A and B J-proteins cooperate to form an interactive flexible network that relocalizes to protein aggregates upon heat shock and preferentially recruits constitutive Hsc70 to disaggregate heat-induced protein aggregates and polyQ aggregates that form in an age-dependent manner. Cooperation between class A and B J-proteins is also required for organismal health and promotes thermotolerance, maintenance of fecundity, and extended viability after heat stress. This disaggregase function of J-proteins and Hsc70 therefore constitutes a powerful regulatory network that is key to Hsc70-based protein quality control mechanisms in metazoa with a central role in the clearance of aggregates, stress recovery, and organismal fitness in aging.
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[
Lab Chip,
2014]
This paper describes a novel selective immobilization technique based on optical control of the sol-gel transition of thermoreversible Pluronic gel, which provides a simple, versatile, and biocompatible approach for high-resolution imaging and microinjection of Caenorhabditis elegans.
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[
MicroPubl Biol,
2018]
Numerous microfluidic systems have been developed and used for live imaging of Caenorhabditis nematodes (Allen et al., 2008; Zhang et al., 2008; Krajniak and Lu, 2010; Krajniak et al., 2013; Cornaglia et al., 2015). These systems can be costly, complex to set up, or require high-maintenance between uses. In addition, microfluidic rigs can be thick, preventing live imaging of worms from strains expressing low fluorescence fusion proteins. In the absence of elaborate microfluidic rigs, most live imaging protocols utilize flat agarose pads along with anesthetics and/or microbeads to immobilize the nematodes (Kim et al., 2013). Since this method does not allow the user to maintain the nematode straight and does not prevent small movements that disturb live imaging, a higher number of worms need to be mounted to ensure that a some settle in an optimal position. This is especially problematic when trying to image nematodes genotypes that are scarce, since there is a very small number of nematodes with the desired genotype in a plate making it challenging to find enough animals to image. Here is a protocol, modified from Zhang, M. et al., 2008, to make grooved agarose pads utilizing a 12-inch vinyl Long Play (LP) record as a mold for agar pads in which nematodes can be positioned and immobilized for live imaging. This method is simple, effective, and allows long-term time-lapse imaging of young adult and adult hermaphrodites, and males expressing low fluorescence fusion proteins.
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[
Nat Genet,
2002]
Mice that are homozygous with respect to a mutation (ax(J)) in the ataxia (ax) gene develop severe tremors by 2-3 weeks of age followed by hindlimb paralysis and death by 6-10 weeks of age. Here we show that ax encodes ubiquitin-specific protease 14 (Usp14). Ubiquitin proteases are a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein in vitro, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates seen in Parkinson disease, spinocerebellar ataxia type 1 (SCA1; ref. 4) and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a mono-ubiquitin side chain, the removal of which would regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in ax(J)/ax(J) mice as a result of the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In contrast to other neurodegenerative disorders such as Parkinson disease and SCA1 in humans and GAD in mice, neither ubiquitin-positive protein aggregates nor neuronal cell loss is detectable in the central nervous system (CNS) of ax(J) mice. Instead, ax(J) mice have defects in synaptic transmission in both the central and peripheral nervous systems. These results suggest that ubiquitin proteases are important in regulating synaptic activity in mammals.