PIEZO mediates a protective mechanism for nematode Caenorhabditis elegans in response to nanoplastics caused dopaminergic neurotoxicity at environmentally relevant concentrations.

Studies have probed nanoplastic toxicity on environmental organisms, but the regulatory role of animal PIEZO-type mechanosensitive ion channel component (PIEZO) remains unclear. Herein, we identified the sole PIEZO in Caenorhabditis elegans (C. elegans), utilizing amino acid homology analysis and Trans-Membrane prediction using Hidden Markov Models (TMHMM). In C. elegans, RNAi knockdown of pezo-1 had no impact on lifespan, body length, lethality, locomotion behaviors, or oxidative response (P > 0.05). However, exposure to 15 µg/L nanopolystyrene in the pezo-1 RNAi group resulted in severe locomotion changes: head trashes (P < 0.01), body bends (P < 0.05), forward turns (P < 0.05), backward turns (P < 0.01), and impaired sensory perception, including abnormal chemotaxis to NaCl (P < 0.01) and diacetyl (P < 0.01), as well as aversive responses (P < 0.05) to nanopolystyrene compared to the wild-type group. Dopaminergic neuron damage explains these behaviors, with GST-4 (P < 0.01) and SKN-1/Nrf2 (P < 0.01) activation mitigating nanoplastic-induced damage. Our results emphasize that even at the environmentally relevant concentrations (ERC), nanoplastics can impact neurotoxicity-related endpoints, with PIEZO mediating the regulation of oxidative and antioxidative systems in response to these effects. PIEZO may be applied for assessing the neurotoxicity or oxidative stress induced by other environmental toxicants besides nanoplastics.

Polystyrene nanoparticles strengthen high glucose toxicity associated with alteration in insulin signaling pathway in C. elegans.

Using Caenorhabditis elegans as animal model, we investigated the effect of exposure to polystyrene nanoparticles (PS-NPs) in the range of µg/L on high glucose toxicity induction. With lifespan and locomotion behavior as endpoints, we observed that PS-NP (10 and 100 µg/L) enhanced toxicity in 50 mM glucose treated animals. In insulin signaling pathway, expressions of genes encoding insulin receptor (daf-2), kinases (age-1 and akt-1/2), and insulin peptides (ins-9, ins-6, and daf-28) were increased, and expressions of daf-16 and its target of sod-3 were decreased in high glucose treated nematodes followed by PS-NP exposure. Toxicity enhancement in high glucose treated nematodes by PS-NP exposure was inhibited by RNAi of daf-2, age-1, akt-2, akt-1, and 3 insulin peptides genes, but increased by RNAi of daf-16 and sod-3. The resistance of animals with RNAi of daf-2 to toxicity in high glucose treated nematodes followed by PS-NP exposure could be suppressed by RNAi of daf-16. Moreover, in high glucose treated animals followed by PS-NP exposure, daf-2 expression was inhibited by RNAi of ins-6, ins-9, and daf-28. Our data demonstrated the risk of PS-NP exposure in enhancing the high glucose toxicity. More importantly, alteration in expression of genes in insulin signaling pathway was associated with the toxicity enhancement in high glucose treated nematodes followed by PS-NP exposure.

Polystyrene nanoplastics at predicted environmental concentrations enhance the toxicity of copper on Caenorhabditis elegans.

Excessive nanoplastics not only pose a direct threat to the environment but also have the propensity to adsorb and interact with other pollutants, exacerbating their impact. The coexistence of nanoplastics and heavy metals in soils is a prevalent phenomenon. However, limited research existed about the joint effects of the two contaminants on soil organisms. In this paper, we ascertained the combined toxicity of polystyrene nanoplastics (PS-NPs) and copper (Cu2+) on soil organisms (Caenorhabditis elegans) at quantities that were present in the environment, further exploring whether the two toxicants were synergistic or antagonistic. The outcomes manifested that single exposure to low-dose PS-NPs (1 µg/L) would not cause significant damage to nematodes. After treatment with PS-NPs and Cu2+, the locomotion ability of nematode was impaired, accompanied by an elevation in reactive oxygen species (ROS) level and a biphasic response in antioxidant enzyme activity. Moreover, combined exposure to PS-NPs and Cu2+ induced the mRNA up-regulation of vit-6, cyp-35a2, hsp-16.2, age-1, and cep-1, both of which were stress-related genes. The comparative analysis between groups (with or without PS-NPs) revealed that the combined exposure group resulted in significantly greater toxic effects on nematodes compared with Cu2+ exposure alone. Furthermore, the addition of PS-NPs influenced the metabolic profiles of Caenorhabditis elegans under Cu2+ stress, with numerous differential metabolites associated with oxidative damage or defense mechanism. Overall, these findings manifested that PS-NPs at the expected environmental concentration elevated Cu2+ toxicity on nematodes.

Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Induction of transgenerational toxicity is associated with the activated germline insulin signals in nematodes exposed to nanoplastic at predicted environmental concentrations.

Exposure to nanoplastics can induce toxicity on organisms at both parental generation (P0-G) and the offspring. However, the underlying mechanism remains unknown. Using Caenorhabditis elegans as a model organism, exposure to 20-nm polystyrene nanoparticle (PS-NP) (1-100 µg/L) upregulated the expressions of insulin ligands (INS-39, INS-3, and DAF-28), and this increase could be further detected in the offspring after PS-NP exposure. Germline ins-39, ins-3, and daf-28 RNAi induced resistance to transgenerational toxicity of PS-NP, indicating that increase in expression of these three insulin ligands mediated induction of transgenerational toxicity. These three insulin ligands transgenerationally activated function of insulin receptor DAF-2 to control transgenerational toxicity of PS-NP. Exposure to 1-100 µg/L PS-NP further upregulated DAF-2, AGE-1, and AKT-1 expressions and downregulated DAF-16 expression. During transgenerational toxicity control, DAF-16/AKT-1/AGE-1 was identified as downstream signaling cascade of DAF-2. Moreover, transcriptional factor DAF-16 activated two downstream targets of HSP-6 (a mitochondrial UPR marker) and SOD-3 (a mitochondrial SOD) to modulate transgenerational toxicity of PS-NP. Our findings indicate a crucial link between activation of insulin signaling and induction of transgenerational toxicity of nanoplastics at low concentrations in organisms.

Neuronal Gα subunits required for the control of response to polystyrene nanoparticles in the range of µg/L in C. elegans.

The aim of this study was to identify Gα proteins mediating function of neuronal G protein-coupled receptors (GPCRs) in controlling the response to polystyrene nanoparticles (PS-NPs). Caenorhabditis elegans was used as an animal model, and both gene expression and functional analysis were performed to identify the Gα proteins in controlling PS-NPs toxicity. In nematodes, exposure to PS-NPs (1-100 µg/L) significantly altered transcriptional expressions of some neuronal Gα genes, including gpa-5, gpa-10, gpa-11, gpa-15 gsa-1, egl-30, and goa-1. Among these 7 Gα genes, only neuronal RNAi knockdown of gsa-1, gpa-10, and goa-1 affected toxicity of PS-NPs in inducing ROS production and in decreasing locomotion behavior. Some neuronal GPCRs (such as GTR-1, DCAR-1, DOP-2, NPR-8, NPR-12, NPR-9, and DAF-37) functioned upstream of GOA-1, some neuronal GPCRs (such as DCAR-1, DOP-2, NPR-9, NPR-8, and DAF-37) functioned upstream of GSA-1, and some neuronal GPCRs (such as DOP-2, NPR-8, DAF-37, and DCAR-1) functioned upstream of GPA-10 to regulate the toxicity of PS-NPs. Moreover, GOA-1 acted upstream of MPK-1/ERK MAPK, JNK-1/JNK MAPK, DBL-1/TGF-ß, and DAF-7/ TGF-ß, GSA-1 functioned upstream of MPK-1/ERK MAPK, JNK-1/JNK MAPK, and DBL-1/TGF-ß, and GPA-10 functioned upstream of GLB-1/Globin and DBL-1/TGF-ß to control the PS-NPs toxicity. Therefore, neuronal Gα proteins of GOA-1, GSA-1, and GPA-10 functioned to transduce signals of multiple GPCRs to different downstream signaling pathways during the control of PS-NPs toxicity in nematodes. Our results provide clues for understanding the important function of GPCRs-Gα signaling cascade in the neurons in controlling response to nanoplastics in organisms.

Polystyrene nanoparticles induced neurodevelopmental toxicity in Caenorhabditis elegans through regulation of dpy-5 and rol-6.

Micro- and nano- polystyrene particles have been widely detected in environment, posing potential threats to human health. This study was designed to evaluate the neurodevelopmental toxicity of polystyrene nanoparticles (NPs) in Caenorhabditis elegans (C. elegans), to screen crucial genes and investigate the underlying mechanism. In wild-type C. elegans, polystyrene NPs (diameter 50 nm) could concentration-dependently induce significant inhibition in body length, survival rate, head thrashes, and body bending, accompanying with increase of reactive oxygen species (ROS) production, lipofuscin accumulation, and apoptosis and decrease of dopamine (DA) contents. Moreover, pink-1 mutant was demonstrated to alleviate the locomotion disorders and oxidative damage induced by polystyrene NPs, indicating involvement of pink-1 in the polystyrene NPs-induced neurotoxicity. RNA sequencing results revealed 89 up-regulated and 56 down-regulated differently expressed genes (DEGs) response to polystyrene NPs (100 µg/L) exposure. Gene Ontology (GO) enrichment analysis revealed that predominant enriched DEGs were correlated with biological function of cuticle development and molting cycle. Furthermore, mutant strains test showed that the neurodevelopmental toxicity and oxidative stress responses induced by 50 nm polystyrene NPs were regulated by dpy-5 and rol-6. In general, polystyrene NPs induced obvious neurodevelopmental toxicity in C. elegans through oxidative damage and dopamine reduction. Crucial genes dpy-5 and rol-6 might participate in polystyrene NPs-induced neurodevelopmental toxicity through regulation on synthesis and deposition of cuticle collagen.

Response of tyramine and glutamate related signals to nanoplastic exposure in Caenorhabditis elegans.

Neurotransmission related signals are involved in the control of response to toxicants. We here focused on the tyramine and the glutamate related signals to determine their roles in regulating nanoplastic toxicity in Caenorhabditis elegans. In the range of µg/L, exposure to nanopolystyrene (100 nm) increased the expression of tdc-1 encoding a tyrosine decarboxylase required for synthesis of tyramine, and decreased the expression of eat-4 encoding a glutamate transporter. Both TDC-1 and EAT-4 could act in the neurons to regulate the nanopolystyrene toxicity. Meanwhile, neuronal RNAi knockdown of tdc-1 induced a susceptibility to nanopolystyrene toxicity, and neuronal RNAi knockdown of eat-4 induced a resistance to nanopolystyrene toxicity. In the neurons, TYRA-2 functioned as the corresponding receptor of tyramine and acted upstream of MPK-1 signaling to regulate the nanopolystyrene toxicity. Moreover, during the control of nanopolystyrene toxicity, GLR-4 and GLR-8 were identified as the corresponding glutamate receptors, and acted upstream of JNK-1 signaling and DBL-1 signaling, respectively. Our results demonstrated the crucial roles of tyramine and glutamate related signals in regulating the toxicity of nanoplastics in organisms.

Dysregulated mir-76 mediated a protective response to nanopolystyrene by modulating heme homeostasis related molecular signaling in nematode Caenorhabditis elegans.

The underlying mechanisms of microRNAs (miRNAs) in regulating nanoplastic toxicity are still largely unclear in organisms. In nanopolystyrene (NPS) exposed Caenorhabditis elegans, the expression of mir-76 (a neuronal miRNA) was significantly decreased, and the mir-76 mutant was resistant to the toxicity of NPS. The aim of this study was to determine the molecular basis of mir-76 in controlling NPS toxicity in nematodes. The mir-76 mutation increased expression of glb-10 encoding a globin protein in NPS (1 µg/L) exposed nematodes. Exposure to NPS (1-100 µg/L) increased the glb-10 expression, and the glb-10(RNAi) worm was susceptible to NPS toxicity in inducing reactive oxygen species (ROS) production and in decreasing locomotion behavior. Using ROS production and locomotion behavior as endpoints, mutation of glb-10 inhibited resistance of mir-76 mutant to NPS toxicity, and neuronal overexpression of mir-76 inhibited the resistance to NPS toxicity in nematodes overexpressing neuronal glb-10 containing 3' untranslated region (3'UTR). Thus, GLB-10 functioned as a target of mir-76 in the neurons to regulate the NPS toxicity. Moreover, a signaling cascade of HRG-7-HRG-5 required for the control of heme homeostasis was identified to function downstream of neuronal GLB-10 to regulate the NPS toxicity. In this signaling cascade, the neuronal HRG-7 regulated the NPS toxicity by antagonizing function of intestinal HRG-5. Furthermore, in the intestine, HRG-5 controlled NPS toxicity by inhibiting functions of hypoxia-inducible transcriptional factor HIF-1 and transcriptional factor ELT-2. Our results highlight the crucial function of heme homeostasis related signaling in regulating the NPS toxicity in organisms.

Molecular cloning and biochemical characterization of the phospholipid scramblase SCRM-1 from Caenorhabditis elegans.

In this study, the SCRM-1 gene from Caenorhabditis elegans was cloned and overexpressed in E. coli to study the biochemical properties of scramblase. This is the first report showing that this scramblase from C. elegans possesses a Ca2+-dependent and head group-independent scramblase activity. The SCRM-1 of C.elegans possesses functional domains including a single EF-hand-like Ca2+ binding domain, as human scramblases do. A point mutation in the EF-hand-like Ca2+ binding motif results in loss of scramblase activity. Other biochemical assays like carbocyanine staining, Tb3+ luminescence, Tryptophan fluorescence, and CD spectroscopy strongly proved the role of the EF-hand motif for functional activity. The increase in protein size in solution upon incubating with Ca2+ shows ligand-dependent oligomerization and conformational changes.

Epigenetic response to nanopolystyrene in germline of nematode Caenorhabditis elegans.

microRNAs (miRNAs) provide an epigenetic regulation mechanism for the response to environmental toxicants. mir-38, a germline miRNA, was increased by exposure to nanopolystyrene (100 nm). In this study, we further found that germline overexpression of mir-38 decreased expressions of nhl-2 encoding a miRISC cofactor, ndk-1 encoding a homolog of NM23-H1, and wrt-3 encoding a homolog of PPIL-2. Meanwhile, germline-specific RNAi knockdown of nhl-2, ndk-1, or wrt-3 caused the resistance to nanopolystyrene toxicity. Additionally, mir-38 overexpression suppressed the resistance of nematodes overexpressing germline nhl-2, ndk-1, or wrt-3 containing 3'UTR, suggesting the role of NHL-2, NDK-1, and WRT-3 as the targets of germline mir-38 in regulating the response to nanopolystyrene. Moreover, during the control of response to nanopolystyrene, EKL-1, a Tudor domain protein, was identified as the downstream target of germline NHL-2, kinase suppressors of Ras (KSR-1 and KSR-2) were identified as the downstream targets of germline NDK-1, and ASP-2, a homolog of BACE1, was identified as the downstream target of germline WRT-3. Our results raised a mir-38-mediated molecular network in the germline in response to nanopolystyrene in nematodes. Our data provided an important basis for our understanding the response of germline of organisms to nanoplastic exposure.

Intestinal mir-794 responds to nanopolystyrene by linking insulin and p38 MAPK signaling pathways in nematode Caenorhabditis elegans.

Caenorhabditis elegans is sensitive to toxicity of environmental pollutants. The alteration in expression of mir-794, a microRNA (miRNA) molecule, mediated a protective response to nanopolystyene (100 nm) at predicted environmental concentration (1 µg/L) in nematodes. However, the underlying molecular basis for mir-794 function in regulating the response to nanopolystyrene remains largely unclear. In this study, we found that intestinal overexpression of mir-794 caused the susceptibility to nanopolystyrene toxicity, suggesting that mir-794 acted in the intestine to regulate the response to nanopolystyrene. Intestinal overexpression of mir-794 further decreased the expressions of daf-16 encoding a FOXO transcriptional factor in insulin signaling pathway, skn-1 encoding a Nrf transcriptional factor in p38 MAPK signaling pathway, and mdt-15 encoding a lipid metabolic sensor acting downstream of SKN-1 in nanopolystyrene exposed nematodes. Meanwhile, intestinal overexpression of mir-794 could suppress the resistance of nematodes overexpressing intestinal daf-16, skn-1, or mdt-15 containing the corresponding 3' untranslated region (3' UTR) to nanopolystyrene toxicity. Therefore, DAF-16, SKN-1, and MDT-15 acted as the downstream targets of intestinal mir-794 to regulate the response to nanopolystyrene. In the intestine, DAF-16 functioned synergistically with SKN-1 or MDT-15 to regulate the response to nanopolystyrene. Our results suggested that the intestinal mir-794 provided an important epigenetic regulation mechanism to control the response to nanopolystyrene by linking insulin and p38 MAPK signaling pathways in nematodes.

Preparative Separation of Flavonoids from Goji Berries by Mixed-Mode Macroporous Adsorption Resins and Effect on Aß-Expressing and Anti-Aging Genes.

Flavonoids are the main constituents of Goji berries and have good biological and pharmacological activities. The mixed-mode macroporous adsorption resins (MARs) for purification of flavonoids from Goji berries through computer-assisted calculation of the molecular size of flavonoids and the precise matching of MAR physical and chemical properties was firstly developed in the present study. Ten varieties of MARs with suitable molecular dimensions and polarities were used for investigating the adsorption/desorption behaviors of the flavonoids. Both AUKJ-1 and BWKX-1 showed higher separation efficiency than other MARs and then were mixed in different ratios to constitute a mixed-mode macroporous adsorption resin to obtain the optimal adsorption phase. Under optimal conditions, total flavonoid content of purified flavonoid (p-FLA) extract increased from 0.97% to 36.88% after one purification. The p-FLA extract from Goji berries significantly improved the expression of six genes with anti-aging effects and played an important role in aging-related Alzheimer's disease by down-regulating Aß expression.

Dentin dysplasia type I-A dental disease with genetic heterogeneity.

Hereditary dentin disorders include dentinogenesis imperfecta (DGI) and dentin dysplasia (DD), which are autosomal dominant diseases characterized by altered dentin structure such as abnormality in dentin mineralization and the absence of root dentin. Shields classified DGI into three subgroups and DD into two subtypes. Although they are all hereditary dentin diseases, they do not share the same causative genes. To date, the pathogenic genes of DGI type I, which is considered a clinical manifestation of syndrome osteogenesis imperfecta, include COL1A1 and COL1A2. Mutations of the DSPP gene, which encodes the dentin sialophosphoprotein, a major non-collagenous protein, are responsible for three isolated dentinal diseases: DGI-II, DGI-III, and DD-II. However, DD-I appears to be special in that researchers have found three pathogenicity genes-VPS4B, SSUH2, and SMOC2-in three affected families from different countries. It is believed that DD-I is a genetically heterogeneous disease and is distinguished from other types of dentin disorders. This review summarizes the DD-I literature in the context of clinical appearances, radiographic characteristics, and functions of its pathogenic genes and aims to serve clinicians in further understanding and diagnosing this disease.

Dysregulation of let-7 by PEG modified graphene oxide in nematodes with deficit in epidermal barrier.

In nematode Caenorhabditis elegans, epidermal RNA interference (RNAi) knockdown of bli-1 encoding a cuticular collagen caused the toxicity induction of GO-PEG (PEG surface modified graphene oxide). In this study, we further found that epidermal RNAi knockdown of bli-1 increased expression of a microRNA let-7, and let-7 mutation suppressed the susceptibility of bli-1(RNAi) nematodes to GO-PEG toxicity. let-7 regulated the toxicity induction of GO-PEG by suppressing expression and function of its direct targets (HBL-1 and LIN-41). Like the nematodes with epidermal RNAi knockdown of bli-1, epidermal RNAi knockdown of hbl-1 or lin-41 also induced functional abnormality in epidermal barrier. Therefore, a signaling cascade of BLI-1-let-7-HBL-1/LIN-41 was raised to be involved in GO-PEG toxicity induction. Our data imply the dysregulation of let-7-mediated molecular machinery for developmental timing control by GO-PEG in nematodes with deficit in epidermal barrier caused by bli-1(RNAi).

Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance.

A stem cell's immediate microenvironment creates an essential "niche" to maintain stem cell self-renewal. Many niches and their intercellular signaling pathways are known, but for the most part, the key downstream targets of niche signaling remain elusive. Here, we report the discovery of two GLP-1/Notch target genes, lst-1 (lateral signaling target) and sygl-1 (synthetic Glp), that function redundantly to maintain germ-line stem cells (GSCs) in the nematode Caenorhabditis elegans. Whereas lst-1 and sygl-1 single mutants appear normal, lst-1 sygl-1 double mutants are phenotypically indistinguishable from glp-1/Notch mutants. Multiple lines of evidence demonstrate that GLP-1/Notch signaling activates lst-1 and sygl-1 expression in GSCs within the niche. Therefore, these two genes fully account for the role of GLP-1/Notch signaling in GSC maintenance. Importantly, lst-1 and sygl-1 are not required for GLP-1/Notch signaling per se. We conclude that lst-1 and sygl-1 forge a critical link between Notch signaling and GSC maintenance.

Membrane-associated collagens with interrupted triple-helices (MACITs): evolution from a bilaterian common ancestor and functional conservation in C. elegans.

BACKGROUND: Collagens provide structural support and guidance cues within the extracellular matrix of metazoans. Mammalian collagens XIII, XXIII and XXV form a unique subgroup of type II transmembrane proteins, each comprising a short N-terminal cytosolic domain, a transmembrane domain and a largely collagenous ectodomain. We name these collagens as MACITs (Membrane-Associated Collagens with Interrupted Triple-helices), and here investigate their evolution and conserved properties. To date, these collagens have been studied only in mammals. Knowledge of the representation of MACITs in other extant metazoans is lacking. This question is of interest for understanding structural/functional relationships in the MACIT family and also for insight into the evolution of MACITs in relation to the secreted, fibrillar collagens that are present throughout the metazoa. RESULTS: MACITs are restricted to bilaterians and are represented in the Ecdysozoa, Hemichordata, Urochordata and Vertebrata (Gnathostomata). They were not identified in available early-diverging metazoans, Lophotrochozoa, Echinodermata, Cephalochordata or Vertebrata (Cyclostomata). Whereas invertebrates encode a single MACIT, collagens XIII/XXIII/XXV of jawed vertebrates are paralogues that originated from the two rounds of en-bloc genome duplication occurring early in vertebrate evolution. MACITs have conserved domain architecture in which a juxta-membrane furin-cleavage site and the C-terminal 34 residues are especially highly conserved, whereas the cytoplasmic domains are weakly conserved. To study protein expression and function in a metazoan with a single MACIT gene, we focused on Caenorhabditis elegans and its col-99 gene. A col-99 cDNA was cloned and expressed as protein in mammalian CHO cells, two antibodies against COL-99 protein were generated, and a col-99-bearing fosmid gene construct col-99::egfp::flag was used to generate transgenic C. elegans lines. The encoded COL-99 polypeptide is 85 kDa in size and forms a trimeric protein. COL-99 is plasma membrane-associated and undergoes furin-dependent ectodomain cleavage and shedding. COL-99 is detected in mouth, pharynx, body wall and the tail, mostly in motor neurons and muscle systems and is enriched at neuromuscular junctions. CONCLUSIONS: Through identification of MACITs in multiple metazoan phyla we developed a model for the evolution of MACITs. The experimental data demonstrate conservation of MACIT molecular and cellular properties and tissue localisations in the invertebrate, C. elegans.

Mitochondrial oxidative stress alters a pathway in Caenorhabditis elegans strongly resembling that of bile acid biosynthesis and secretion in vertebrates.

Mammalian bile acids (BAs) are oxidized metabolites of cholesterol whose amphiphilic properties serve in lipid and cholesterol uptake. BAs also act as hormone-like substances that regulate metabolism. The Caenorhabditis elegans clk-1 mutants sustain elevated mitochondrial oxidative stress and display a slow defecation phenotype that is sensitive to the level of dietary cholesterol. We found that: 1) The defecation phenotype of clk-1 mutants is suppressed by mutations in tat-2 identified in a previous unbiased screen for suppressors of clk-1. TAT-2 is homologous to ATP8B1, a flippase required for normal BA secretion in mammals. 2) The phenotype is suppressed by cholestyramine, a resin that binds BAs. 3) The phenotype is suppressed by the knock-down of C. elegans homologues of BA-biosynthetic enzymes. 4) The phenotype is enhanced by treatment with BAs. 5) Lipid extracts from C. elegans contain an activity that mimics the effect of BAs on clk-1, and the activity is more abundant in clk-1 extracts. 6) clk-1 and clk-1;tat-2 double mutants show altered cholesterol content. 7) The clk-1 phenotype is enhanced by high dietary cholesterol and this requires TAT-2. 8) Suppression of clk-1 by tat-2 is rescued by BAs, and this requires dietary cholesterol. 9) The clk-1 phenotype, including the level of activity in lipid extracts, is suppressed by antioxidants and enhanced by depletion of mitochondrial superoxide dismutases. These observations suggest that C. elegans synthesizes and secretes molecules with properties and functions resembling those of BAs. These molecules act in cholesterol uptake, and their level of synthesis is up-regulated by mitochondrial oxidative stress. Future investigations should reveal whether these molecules are in fact BAs, which would suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome might participate in disease processes by affecting the regulation of metabolism by BAs.

Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region.

XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.