Hwang SW (1970) Nematologica "Freezing and storage of nematodes in liquid nitrogen."

Hwang SW

[Nematologica, 1970]

Free-living nematodes of the genera Aphelenchoides, Panagrellus, Turbatrix, and Caenorhabditis were successfully frozen in heat-sealed glass ampoules using dimethyl sulfoxide as a protectant. After 6 months storage in liquid nitrogen refrigerator (below -160C), living nematodes were recovered after thawing. The nematodes were slowly cooled to -22 +/- 1C and immediately transferred to liquid nitrogen at -196C.

Pandey T et al. (2020) Exp Gerontol "Swertiamarin, a secoiridoid glycoside modulates nAChR and AChE activity."

Smita SS, Mishra A, Sammi SR, Pandey R, Pandey T

[Exp Gerontol, 2020]

The ailments related to a malfunction in cholinergic functioning currently employ the use of inhibitors for acetylcholinesterase (AChE) and N-methyl-d-aspartate (NMDA) receptors. The present study was designed to elucidate the potential of swertiamarin (SW), a secoiridoidal glycoside isolated from Enicostemma littorale in curtailing the cholinergic dysfunction. Using Caenorhabditis elegans as a model, SW was found to enhance neurotransmission by modulating AChE and nicotinic acetylcholine receptor (nAChR) activity; being orchestrated through up-regulation of unc-17 and unc-50. SW exhibited AChE inhibition both in vivo and cell-free system. The in silico molecular docking of SW and human AChE (hAChE) displayed good binding energy of -6.02. Interestingly, the increase in aldicarb and levamisole sensitivity post SW treatment was curtailed to a significant level in daf-16 and skn-1 mutants. SW raised the level of the endogenous antioxidant enzymes through up-regulation of sod-3 and gst-4 that act downstream to DAF-16 and SKN-1, imparting protection against neurodegeneration. The outcome of our study displays SW as a potential natural molecule for the amelioration of cholinergic dysfunction. Moreover, the study also indicates that SW elicits antioxidant response via up-modulation of daf-16 possibly through unc-17 upregulation. Further research on SW pertaining to the underlying mechanism and potential is expected to significantly advance the current understanding and design of possible ameliorative or near ameliorative regimens for cholinergic dysfunction.

Bo S et al. (2023) Front Genet "A novel approach to analyze the association characteristics between post-spliced introns and their corresponding mRNA."

Ning P, Sun Q, Bo S, Wang H, Weng Y, Zhao X, Liang Y, Lu Z, Yuan N, Li Z

[Front Genet, 2023]

Studies have shown that post-spliced introns promote cell survival when nutrients are scarce, and intron loss/gain can influence many stages of mRNA metabolism. However, few approaches are currently available to study the correlation between intron sequences and their corresponding mature mRNA sequences. Here, based on the results of the improved Smith-Waterman local alignment-based algorithm method (SW method) and binding free energy weighted local alignment algorithm method (BFE method), the optimal matched segments between introns and their corresponding mature mRNAs in Caenorhabditis elegans (C.elegans) and their relative matching frequency (RF) distributions were obtained. The results showed that although the distributions of relative matching frequencies on mRNAs obtained by the BFE method were similar to the SW method, the interaction intensity in 5'and 3'untranslated regions (UTRs) regions was weaker than the SW method. The RF distributions in the exon-exon junction regions were comparable, the effects of long and short introns on mRNA and on the five functional sites with BFE method were similar to the SW method. However, the interaction intensity in 5'and 3'UTR regions with BFE method was weaker than with SW method. Although the matching rate and length distribution shape of the optimal matched fragment were consistent with the SW method, an increase in length was observed. The matching rates and the length of the optimal matched fragments were mainly in the range of 60%-80% and 20-30bp, respectively. Although we found that there were still matching preferences in the 5'and 3'UTR regions of the mRNAs with BFE, the matching intensities were significantly lower than the matching intensities between introns and their corresponding mRNAs with SW method. Overall, our findings suggest that the interaction between introns and mRNAs results from synergism among different types of sequences during the evolutionary process.

Davis, Kristen et al. (2015) International Worm Meeting "The appetite control circuit in C. elegans."

You, Young-jai, Davis, Kristen

[International Worm Meeting, 2015]

Satiety quiescence is a behavioral state defined as a lack of movement or feeding following satiation. ASI is important for worms to enter satiety quiescence. We found the nutrients directly activate ASI. The nutrient rich stimulus we use in the lab is Luria broth (LB) and we are working to identify the active component by testing individual components of LB. Feeding is stimulated or suppressed depending not only on metabolic needs but also on environmental cues. To understand how feeding is controlled by environmental cues, we treated worms with nutrients mixed with noxious stimuli such as a high concentration of NaCl (4 M). 1 We found that these noxious stimuli override the ASI activation by nutrients, suggesting feeding, a potential result after sensing nutrients via ASI, can be suppressed in the presence of noxious stimuli. Furthermore, ASI suppression by noxious stimuli is entirely dependent on ASH function, a pair of neurons required for sensation of noxious stimuli. In an ASH genetic ablation background, ASI continue to respond to LB in the presence or absence of noxious stimuli. We believe there is a circuit between ASH, ASI, and an interneuron pair, AIA, which regulates ASI activation to nutrients. Our behavioral data show in an AIA genetic ablation background and an ASH genetic ablation background that satiety quiescence is enhanced when compared to concurrent controls, suggesting the signals from these neuron pairs inhibit ASI and satiety quiescence when they are intact. Recent work by others has identified a potential circuit between ASH and ASI that included three other neuron pairs RIM/RIC and ADF, although this circuit was not tested in the context of feeding or nutrient availability. 21. Chatzigeorgiou M, Bang S, Hwang SW, Schafer WR. tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans. Nature. 2013;494(7435):95-9. doi:10.1038/nature11845.2. Guo M, Wu T-H, Song Y-X, et al. Reciprocal inhibition between sensory ASH and ASI neurons modulates nociception and avoidance in Caenorhabditis elegans. Nat Commun. 2015;6:1-13. doi:10.1038/ncomms6655.

Chatzigeorgiou M et al. (2013) Nature "tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans."

Hwang SW, Chatzigeorgiou M, Bang S, Schafer WR

[Nature, 2013]

Transmembrane channel-like (TMC) genes encode a broadly conserved family of multipass integral membrane proteins in animals. Human TMC1 and TMC2 genes are linked to human deafness and required for hair-cell mechanotransduction; however, the molecular functions of these and other TMC proteins have not been determined. Here we show that the Caenorhabditis elegans tmc-1 gene encodes a sodium sensor that functions specifically in salt taste chemosensation. tmc-1 is expressed in the ASH polymodal avoidance neurons, where it is required for salt-evoked neuronal activity and behavioural avoidance of high concentrations of NaCl. However, tmc-1 has no effect on responses to other stimuli sensed by the ASH neurons including high osmolarity and chemical repellents, indicating a specific role in salt sensation. When expressed in mammalian cell culture, C. elegans TMC-1 generates a predominantly cationic conductance activated by high extracellular sodium but not by other cations or uncharged small molecules. Thus, TMC-1 is both necessary for salt sensation in vivo and sufficient to generate a sodium-sensitive channel in vitro, identifying it as a probable ionotropic sensory receptor.

Balasubramaniam M et al. (2024) iScience "Alzheimer's-specific brain amyloid interactome: Neural-network analysis of intra-aggregate crosslinking identifies novel drug targets."

Ayyadevara S, Pahal S, Shmookler Reis RJ, Mainali N, Balasubramaniam M, Ganne A

[iScience, 2024]

Alzheimer's disease (AD) is characterized by peri-neuronal amyloid plaque and intra-neuronal neurofibrillary tangles. These aggregates are identified by the immunodetection of "seed" proteins (A&#x3b2;_1-42 and hyperphosphorylated tau, respectively), but include many other proteins incorporated nonrandomly. Using click-chemistry intra-aggregate crosslinking, we previously modeled amyloid "contactomes" in SY5Y-APP_Sw neuroblastoma cells, revealing that aspirin impedes aggregate growth and complexity. By an analogous strategy, we now construct amyloid-specific aggregate interactomes of AD and age-matched-control&#xa0;hippocampi. Comparing these interactomes reveals AD-specific interactions, from which neural-network (NN) analyses predict proteins with the highest impact on pathogenic aggregate formation and/or stability. RNAi knockdowns of implicated proteins, in <i>C.&#xa0;elegans</i> and human-cell-culture models of AD, validated those predictions. Gene-Ontology meta-analysis of AD-enriched influential proteins highlighted the involvement of mitochondrial and cytoplasmic compartments in AD-specific aggregation. This approach derives dynamic consensus models of aggregate growth and architecture, implicating highly influential proteins as new targets to disrupt amyloid accrual in the AD brain.

Chatzigeorgiou M et al. (2010) Nat Neurosci "Specific roles for DEG/ENaC and TRP channels in touch and thermosensation in C. elegans nociceptors."

Chatzigeorgiou M, Spencer WC, Miller III DM, Hwang SW, Watson JD, Driscoll M, Kindt KS, Treinin M, Schafer WR, Lee WH, Yoo S

[Nat Neurosci, 2010]

Polymodal nociceptors detect noxious stimuli, including harsh touch, toxic chemicals and extremes of heat and cold. The molecular mechanisms by which nociceptors are able to sense multiple qualitatively distinct stimuli are not well understood. We found that the C. elegans PVD neurons are mulitidendritic nociceptors that respond to harsh touch and cold temperatures. The harsh touch modality specifically required the DEG/ENaC proteins MEC-10 and DEGT-1, which represent putative components of a harsh touch mechanotransduction complex. In contrast, responses to cold required the TRPA-1 channel and were MEC-10 and DEGT-1 independent. Heterologous expression of C. elegans TRPA-1 conferred cold responsiveness to other C. elegans neurons and to mammalian cells, indicating that TRPA-1 is a cold sensor. Our results suggest that C. elegans nociceptors respond to thermal and mechanical stimuli using distinct sets of molecules and identify DEG/ENaC channels as potential receptors for mechanical pain.

Balasubramaniam M et al. (2018) Sci Rep "Structural insights into pro-aggregation effects of C. elegans CRAM-1 and its human ortholog SERF2."

Ayyadevara S, Balasubramaniam M, Shmookler Reis RJ

[Sci Rep, 2018]

Toxic protein aggregates are key features of progressive neurodegenerative diseases. In addition to &quot;seed&quot; proteins diagnostic for each neuropathy (e.g., A_1-42 and tau in Alzheimer's disease), aggregates contain numerous other proteins, many of which are common to aggregates from diverse diseases. We reported that CRAM-1, discovered in insoluble aggregates of C. elegans expressing Q40::YFP, blocks proteasomal degradation of ubiquitinated proteins and thus promotes aggregation. We now show that CRAM-1 contains three -helical segments forming a UBA-like domain, structurally similar to those of mammalian adaptor proteins (e.g. RAD23, SQSTM1/p62) that shuttle ubiquitinated cargos to proteasomes or autophagosomes for degradation. Molecular modeling indicates that CRAM-1, through this UBA-like domain, can form tight complexes with mono- and di-ubiquitin and may thus prevent tagged proteins from interacting with adaptor/shuttle proteins required for degradation. A human ortholog of CRAM-1, SERF2 (also largely disordered), promotes aggregation in SH-SY5Y-APP_Sw human neuroblastoma cells, since SERF2 knockdown protects these cells from amyloid formation. Atomistic molecular-dynamic simulations predict spontaneous unfolding of SERF2, and computational large-scale protein-protein interactions predict its stable binding to ubiquitins. SERF2 is also predicted to bind to most proteins screened at random, although with lower average stability than to ubiquitins, suggesting roles in aggregation initiation and/or progression.

Huan Wang et al. (2003) International Worm Meeting "Y110A2AL.14 is a glycosyltransferase that is important for proteoglycan tetrasaccharide linker biosynthesis."

Huan Wang, Mark Sullivan, Fred Hagen

[International Worm Meeting, 2003]

Galactosyltransferases modify carbohydrate chains that are attached to glycoproteins, proteoglycans, and glycolipids. Seven putative C. elegans genes have sequence motifs indicative of -1,3-galactosyltransferase function. Through an RNAi screen, one of these genes, Y110A2AL.14, was found to be required for early embryonic development. Sequence similarities using the BLAST program suggest that Y110A2AL.14 is a C. elegans homolog to human Galactosyltransferase II (GalT2) in the proteoglycan biosynthesis pathway. This cDNA encodes a type II membrane protein of 330 amino acids, shows 57 % amino acid similarity to human GalT2 and contains a DXD sequence motif that is potentially critical for enzymatic activity. A recombinant GalT2 fusion protein was expressed in insect cells and was purified to homogeneity. In vitro enzyme assays are consistent with this protein having GalT2 function. Glycosyltransferase activity was specific for galactose, utilizing the sugar donor UDP-galactose, and the acceptor, a terminal -linked galactose residue. We conclude that C. elegans Y110A2AL.14 encodes the enzyme likely act to attach the third sugar in the tetrasaccharide linker (GlcA--1,3-Gal--1,3-Gal--1,4-Xyl--1-O-Serine) for glycosaminoglaycan attachment. Glycosaminoglycan chains like heparan sulfate, chondroitin sulfate and dermatin sulfate inpact many properties of cells and require this tetrasaccharide for attachment. Therefore, reduction of C. elegans GalT II levels using RNAi, indicate that embryonic development depends on the expression of glycosaminoglycans. The RNAi knock-down phenotype is stronger in the F2 generation, suggesting that GalT2 is a maternal protein stored in oocytes at the time of fertilization. High yields of purified recombinant GalT2 has allowed us to produce single-chain anti-GalT2 antibodies to localize GalT2 in developing oocytes, embryos and larva. Y110A2AL.14 has recently been identified as sqv-2 by Hwang, Olson, Brown, Esko and Horvitz (JBC, in press)

H Hwang et al. (1999) International C. elegans Meeting "Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis"

H Hwang, HR Horvitz

[International C. elegans Meeting, 1999]

Eight sqv ( sq uashed v ulva) genes ( sqv-1 to 8 ) have a mutant vulval morphogenetic phenotype that is defined by reduced separation between the anterior and posterior halves of the vulva in the L4 (1). SQV-3 and SQV-8 are glycosyltransferase-like proteins, and SQV-7 is similar to a putative nucleotide-sugar transporter (2). We have previously reported the cloning of sqv-1 and sqv-4 (3). Both SQV-1 and SQV-4 are similar to enzymes involved in nucleotide-sugar metabolism. These molecular identities suggest that glycosylation is important in shaping the vulva during development. We have now determined biochemically that SQV-4 is a UDP-glucose dehydrogenase. Recombinant SQV-4 expressed in E. coli can reduce NAD in the presence of UDP-glucose, suggesting that UPD-glucose is being converted to UDP-glucuronate. Like known UDP-glucose dehydrogenases, SQV-4 appears to act specifically on UDP-glucose, as it fails to reduce NAD using any of the many other nucleotide-sugars tested. Mutant recombinant SQV-4 derived from either of the two genetically identified mutant alleles does not show detectable activity. Preliminary antibody staining using a rabbit polyclonal antibody directed against a GST::SQV-4 indicates that SQV-4 is localized to the vulval cells and several other tissues, including seam cells. This result agrees with the expression of SQV-4::GFP fusion protein and supports a model in which SQV-4 acts in the vulval cells during vulval development. We also are trying to determine the functions of the other four cloned sqv genes using biochemical assays and heterologous complementation. Lastly, we are mapping and cloning the three remaining sqv genes. 1. Herman, T. et al. (1999). PNAS 96 : 968-973. 2. Herman, T. and Horvitz, H.R. (1999). PNAS 96 : 974-979. 3. Hwang, H. and Horvitz, H.R. (1998) East Coast C. elegans meeting, p. 103.