Gomez-Saladin E et al. (1994) Cell Mol Neurobiol "Isolation and in situ localization of a cDNA encoding a Kex2-like prohormone convertase in the nematode Caenorhabditis elegans."

Dickerson IM, Wilson DL, Gomez-Saladin E

[Cell Mol Neurobiol, 1994]

1. A cDNA that encodes a Kex2-like prohormone convertase (PC) containing an active site similar to that of mammalian PC2 has been isolated from C. elegans. Total RNA was isolated from a mixed population of strain BA713 worms. After poly-(A)-selection and reverse transcription, degenerate/nested polymerase chain reactions (PCR) were performed using primers based on conserved regions within the active sites of the known vertebrate and invertebrate endoproteases. 2. Two distinct 300-bp PCR products that shared homologies with the active sites of known Kex2-like endoproteases were isolated. These two PCR products were used to screen a C. elegans cDNA library. 3. The complete cDNA for a Kex2-like endoprotease, designated CELPC2, was isolated and determined to be 2527 bp in length. This size was confirmed by northern analysis. The deduced amino acid sequence for the CELPC2 cDNA is very similar to the known Kex2-like endoproteases, especially at conserved regions within the active sites, but not identical to any one of them. The strongest structural homology was to vertebrate and invertebrate PC2 sequences. 4. In situ hybridization suggests that CELPC2 is synthesized primarily in cells associated with the circumpharyngeal nerve ring and the dorsorectal ganglion.

Lindberg I et al. (1998) DNA Cell Biol "Cloning and functional analysis of C. elegans 7B2."

Muller L, Lindberg I, Tu B, Dickerson IM

[DNA Cell Biol, 1998]

The neuroendocrine protein 7B2 is a binding protein for the prohormone convertase 2 (PC2) and is required for the intracellular conversion of proPC2 to active PC2. Both full-length 7B2 and its carboxy-terminal 31-residue peptide (CT peptide) are capable of potent inhibition of PC2; the 7B2 protein thus regulates both the biosynthesis and the activity of PC2. Vertebrate 7B2s are highly conserved (92%-97% homology), and thus, species comparison has not been informative in assessing the crucial protein domains responsible for bioactivity. We here report the cloning of the Caenorhabditis elegans 7B2 protein. Although weakly conserved with the vertebrate sequences (23% similarity with mouse 7B2), C. elegans 7B2 contains the signature PPNPCP motif as well as a highly conserved heptapeptide within the CT peptide. In in vitro assays, C. elegans 7B2 possessed significant inhibitory activity against recombinant vertebrate PC2 (IC50 130 nM), and in two functional tests, the amino-terminal domain of C. elegans 7B2 facilitated the activation of proPC2. We conclude that despite low amino acid conservation overall, both functional domains within 7B2 have been conserved between the C. elegans and the vertebrate proteins.

Gomez-Saladin E et al. (1997) DNA Cell Biol "Isolation of a cDNA encoding a Kex2-like endoprotease with homology to furin from the nematode Caenorhabditis elegans."

Wilson DL, Luebke AE, Gomez-Saladin E, Dickerson IM

[DNA Cell Biol, 1997]

A cDNA was isolated from the nematode Caenorhabditis elegans that encodes an endoprotease which is a member of the Kex2 family of serine endoproteases. Degenerate oligonucleotide primers were designed based on conserved regions within the active sites of known Kex2-like endoproteases, and were used for reverse transcription-polymerase chain reaction (RT-PCR) of poly(A)+RNA isolated from C. elegans. A PCR product was isolated that had homology to the active sites of known furin endoproteases, and was used as a probe to screen a C. elegans cDNA library. A Kex2-like endoprotease (CelfurPC) which encoded a 692-amino-acid pre-proendoprotease, was identified. The deduced amino acid sequence for the catalytic domain of CelfurPC is homologous to the known Kex2-like endoproteases, with strongest structural homology to the furin/PACE4 family. However, all furins and PACE4 proteins contain a characteristic cysteine-rich domain, and all furins contain a transmembrane domain, neither of which is present in the CelfurPC protein. CelfurPC may thus represent a new class of Kex2-like endoprotease.

Zhao YG et al. (2017) Mol Cell "The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation."

Feng D, Chen Y, Liu N, Zhang H, Zhao H, Miao G, Liu P, Li D, Qu W, Wang Z, Li L, Chen S, Zhao YG

[Mol Cell, 2017]

During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.

Li BW et al. (2004) Filaria J "Antibody responses to Brugia malayi antigens induced by DNA vaccination."

Curtis KC, Zhang SR, Li BW, Weil GJ, Rush A

[Filaria J, 2004]

BACKGROUND: DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. METHODS: Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR trade mark 3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. RESULTS: Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. CONCLUSIONS: Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.

Asef CK et al. (2023) Anal Chem "Unknown Metabolite Identification Using Machine Learning Collision Cross-Section Prediction and Tandem Mass Spectrometry."

Rainey MA, Fernandez FM, McIntyre LM, Garcia BM, Gouveia GJ, Asef CK, Edison AS, Shaver AO, Leach FE, Morse AM

[Anal Chem, 2023]

Ion mobility (IM) spectrometry provides semiorthogonal data to mass spectrometry (MS), showing promise for identifying unknown metabolites in complex non-targeted metabolomics data sets. While current literature has showcased IM-MS for identifying unknowns under near ideal circumstances, less work has been conducted to evaluate the performance of this approach in metabolomics studies involving highly complex samples with difficult matrices. Here, we present a workflow incorporating de novo molecular formula annotation and MS/MS structure elucidation using SIRIUS 4 with experimental IM collision cross-section (CCS) measurements and machine learning CCS predictions to identify differential unknown metabolites in mutant strains of Caenorhabditis elegans. For many of those ion features, this workflow enabled the successful filtering of candidate structures generated by in silico MS/MS predictions, though in some cases, annotations were challenged by significant hurdles in instrumentation performance and data analysis. While for 37% of differential features we were able to successfully collect both MS/MS and CCS data, fewer than half of these features benefited from a reduction in the number of possible candidate structures using CCS filtering due to poor matching of the machine learning training sets, limited accuracy of experimental and predicted CCS values, and lack of candidate structures resulting from the MS/MS data. When using a CCS error cutoff of +/-3%, on average, 28% of candidate structures could be successfully filtered. Herein, we identify and describe the bottlenecks and limitations associated with the identification of unknowns in non-targeted metabolomics using IM-MS to focus and provide insights into areas requiring further improvement.

Li H et al. (2022) Sci Total Environ "Direct toxicity of environmentally persistent free radicals to nematode Caenorhabditis elegans after excluding the concomitant chemicals."

Liu Y, Li H, Steinberg C, Zuo N, Pan B, Lang D, Xing B

[Sci Total Environ, 2022]

Environmentally persistent free radicals (EPFRs) have attracted extensive attention due to their potential toxicity. However, EPFRs-containing particles always coexist with their parent organic contaminants and intermediate degradation products (IM), which may have hindered the toxicity assessment of EPFRs. In this study, the toxicity of EFFRs was specifically verified after comparing the systems without EPFRs, such as the immediate mixture of catechol (CT) and particles, solutions of CT only, IM extracted from the particles, as well as particles after EPFRs quenching. Caenorhabditis elegans (C. elegans) were used as model organisms. Our results showed that EPFRs-containing particles (Si-Al-CT) exhibited significant toxicity to C. elegans, but not for the parent chemical CT and IM on the particles. Higher levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the Si-Al-CT system were attributed to the mediated generation of -O₂^- and -OH via EPFRs. EPFRs could increase gene expressions related not only to oxidative stress and biotransformation in C. elegans, but also to indications of disturbances in energy homeostasis, survival, proliferation, cell and embryonic development. Overall, these results confirmed the direct toxicity of EPFRs and highlighted the key role of EPFRs which may be neglected in assessing the environmental risks of organic contaminants.

Dec Bronsvoort BM et al. (2008) Parasit Vectors "UMF-078: A modified flubendazole with potent macrofilaricidal activity against Onchocerca ochengi in African cattle."

Ekale D, Dec Bronsvoort BM, Tanya VN, Trees AJ, Makepeace BL, Fleckenstein L, Renz A

[Parasit Vectors, 2008]

UNLABELLED: Human onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is currently controlled using the microfilaricidal drug, ivermectin. However, ivermectin does not kill adult O. volvulus, and in areas with less than 65% ivermectin coverage of the population, there is no effect on transmission. Therefore, there is still a need for a macrofilaricidal drug. Using the bovine filarial nematode O. ochengi (found naturally in African cattle), the macrofilaricidal efficacy of the modified flubendazole, UMF-078, was investigated. METHODS: Groups of 3 cows were treated with one of the following regimens: (a) a single dose of UMF-078 at 150 mg/kg intramuscularly (im), (b) 50 mg/kg im, (c) 150 mg/kg intraabomasally (ia), (d) 50 mg/kg ia, or (e) not treated (controls). RESULTS: After treatment at 150 mg/kg im, nodule diameter, worm motility and worm viability (as measured by metabolic reduction of tetrazolium to formazan) declined significantly compared with pre-treatment values and concurrent controls. There was abrogation of embryogenesis and death of all adult worms by 24 weeks post-treatment (pt). Animals treated at 50 mg/kg im showed a decline in nodule diameter together with abrogated reproduction, reduced motility, and lower metabolic activity in isolated worms, culminating in approximately 50% worm mortality by 52 weeks pt. Worms removed from animals treated ia were not killed, but exhibited a temporary embryotoxic effect which had waned by 12 weeks pt in the 50 mg/kg ia group and by 24 weeks pt in the 150 mg/kg ia group. These differences could be explained by the different absorption rates and elimination half-lives for each dose and route of administration. CONCLUSION: Although we did not observe any signs of mammalian toxicity in this trial with a single dose, other studies have raised concerns regarding neuro- and genotoxicity. Consequently, further evaluation of this compound has been suspended. Nonetheless, these results validate the molecular target of the benzimidazoles as a promising lead for rational design of macrofilaricidal drugs.

Harrison RA et al. (2000) Parasite Immunol "DNA immunization with Onchocerca volvulus genes, Ov-tmy-1 and OvB20: serological and parasitological outcomes following intramuscular or GeneGun delivery in a mouse model of onchocerciasis."

Harrison RA, Bianco AE

[Parasite Immunol, 2000]

The objective of this study was to evaluate whether the distinct immune responses invoked by epidermal and intramuscular DNA immunization could be harnessed to improve upon the levels of protection to Onchocerca volvulus infective larvae achieved previously by recombinant protein immunization. Intramuscular (IM) and epidermal (GeneGun) routes of DNA immunization generally drive T helper1 and Th2 dominant responses, respectively. This dichotomy was used in an attempt to further define the nature of host-protective immunity in a mouse model of onchocerciasis. Mice were immunized with DNA plasmids expressing the O. volvulus antigens, Ov-TMY-1 (tropomyosin) and OvB20 (a nematode specific gene product). While, IM and GeneGun immunization of mice with Ov-tmy-1 induced expected Th1/Th2-associated IgG isotype profiles, mice responded to OvB20 immunization with a Th2 dominant response, irrespective of the delivery route. Despite inducing potent serological responses, neither DNA construct promoted statistically significant levels of protection to L3 challenge infection. We conclude that DNA immunization has good potential for induction of humoral responses against nematode infections and that serological responses alone do not predict vaccination efficacy under the conditions used here to measure host resistance to parasite challenge.

Raneri M et al. (2018) Sci Rep "Pseudomonas aeruginosa mutants defective in glucose uptake have pleiotropic phenotype and altered virulence in non-mammal infection models."

Briani F, Pinatel E, Jousson O, Bianconi I, Raneri M, Rampioni G, Peano C, Dalmasio C, Leoni L, Ferrante P

[Sci Rep, 2018]

Pseudomonas spp. are endowed with a complex pathway for glucose uptake that relies on multiple transporters. In this work we report the construction and characterization of Pseudomonas aeruginosa single and multiple mutants with unmarked deletions of genes encoding outer membrane (OM) and inner membrane (IM) proteins involved in glucose uptake. We found that a triple gltKGF gntP kguT mutant lacking all known IM transporters (named GUN for Glucose Uptake Null) is unable to grow on glucose as unique carbon source. More than 500 genes controlling both metabolic functions and virulence traits show differential expression in GUN relative to the parental strain. Consistent with transcriptomic data, the GUN mutant displays a pleiotropic phenotype. Notably, the genome-wide transcriptional profile and most phenotypic traits differ between the GUN mutant and the wild type strain irrespective of the presence of glucose, suggesting that the investigated genes may have additional roles besides glucose transport. Finally, mutants carrying single or multiple deletions in the glucose uptake genes showed attenuated virulence relative to the wild type strain in Galleria mellonella, but not in Caenorhabditis elegans infection model, supporting the notion that metabolic functions may deeply impact P. aeruginosa adaptation to specific environments found inside the host.