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[
Mol Biochem Parasitol,
2000]
The gene encoding the Wuchereria bancrofti orthologue of the Brugia malayi-derived diagnostic antigen SXP1 was identified from a W. bancrofti L3 cDNA library and characterized. The
Wb-sxp-1 cDNA encoded a basic protein with a calculated molecular mass of 20.8 kDa. Wb-SXP-1 was 85% identical to the SXP1 protein described from B. malayi (Bm-SXP-1). The Wb-SXP-1 sequence also showed significant identity with proteins described from B. pahangi, Onchocerca volvulus, Acanthochilonema vitea, Ascaris suum, Loa loa, Litomosoides sigmodontis and Caenorhabditis elegans. The presence of a number of invariant and conserved residues in all of these nematode-derived molecules suggests that Wb-SXP-1 is a member of a new protein family. A recombinant form of Wb-SXP-1 was produced and it was determined that the anti-Wb-SXP-1 antibody response in patients with W. bancrofti infections was restricted to the IgG4 subclass. An anti-Wb-SXP-1 IgG4 ELISA was developed and this assay was found to be 100% sensitive for patients with patent W. bancrofti infection. Sera from individuals experiencing chronic pathology, endemic normals or patients with non-filarial nematode infections had no detectable IgG4 against Wb-SXP-1. While patients with patent Onchocerca volvulus infections were uniformly negative in the Wb-SXP-1 assay, 40% of sera from patent Loa loa infections were positive. When Bm-SXP-1 was used as the antigen under identical conditions, the assay was 88% specific for patent W. bancrofti infections and the antigen was recognized by antibodies from both O. volvulus and L. loa infections. The results strongly suggested that, for certain diagnostic filarial antigens, the use of same-species molecules can enhance the specificity of diagnostic tests.
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[
Bioinformation,
2005]
GST (glutathione S-transferases) are a family of detoxification enzymes that catalyze the conjugation of reduced GSH (glutathione) to xenobiotic (endogenous electrophilic) compounds. GST from Wb (Wuchereria bancrofti) and Bm (Brugia malayi) are significantly different from human GST in sequence and structure. Thus, Wb-GST and Bm-GST are potential chemotherapeutic targets for anti-filarial treatment. Comparison of modeled Wb and Bm GST with human GST show structural difference between them. Analysis of the active site residues for the binding of electrophilic co-substrates provides insight towards the design of parasite specific GST inhibitors.
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[
Cancers (Basel),
2021]
Metastasis is a complex process that affects patient treatment and survival. To routinely monitor cancer plasticity and guide treatment strategies, it is highly desired to provide information about metastatic status in real-time. Here, we proposed a worm-based (WB) microfluidic biosensor to rapidly monitor biochemical cues related to metastasis in a well-defined environment. Compared to conventional biomarker-based methods, the WB biosensor allowed high throughput screening under low cost, requiring only visual quantification of outputs; Methods: Caenorhabditis elegans were placed in the WB biosensor and exposed to samples conditioned with cancer cell clusters. The chemotactic preference of these worms was observed under discontinuous imaging to minimize the impact on physiological activity; Results: A chemotaxis index (CI) was defined to standardize the quantitative assessment from the WB biosensor, where moderate (3.24-6.5) and high (>6.5) CI levels reflected increased metastasis risk and presence of metastasis, respectively. We demonstrated that the secreted metabolite glutamate was a chemorepellent, and larger clusters associated with increased metastatic potential also enhanced CI levels; Conclusions: Overall, this study provided a proof of concept for the WB biosensors in assessing metastasis status, with the potential to evaluate patient-derived cancer clusters for routine management.
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[
Database (Oxford),
2020]
Biological knowledgebases rely on expert biocuration of the research literature to maintain up-to-date collections of data organized in machine-readable form. To enter information into knowledgebases, curators need to follow three steps: (i) identify papers containing relevant data, a process called triaging; (ii) recognize named entities; and (iii) extract and curate data in accordance with the underlying data models. WormBase (WB), the authoritative repository for research data on Caenorhabditis elegans and other nematodes, uses text mining (TM) to semi-automate its curation pipeline. In addition, WB engages its community, via an Author First Pass (AFP) system, to help recognize entities and classify data types in their recently published papers. In this paper, we present a new WB AFP system that combines TM and AFP into a single application to enhance community curation. The system employs string-searching algorithms and statistical methods (e.g. support vector machines (SVMs)) to extract biological entities and classify data types, and it presents the results to authors in a web form where they validate the extracted information, rather than enter it de novo as the previous form required. With this new system, we lessen the burden for authors, while at the same time receive valuable feedback on the performance of our TM tools. The new user interface also links out to specific structured data submission forms, e.g. for phenotype or expression pattern data, giving the authors the opportunity to contribute a more detailed curation that can be incorporated into WB with minimal curator review. Our approach is generalizable and could be applied to additional knowledgebases that would like to engage their user community in assisting with the curation. In the five months succeeding the launch of the new system, the response rate has been comparable with that of the previous AFP version, but the quality and quantity of the data received has greatly improved.
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[
Chemosphere,
2012]
Copper pollutions are typical heavy metal contaminations, and their ability to move up food chains urges comprehensive studies on their effects through various pathways. Currently, four exposure pathways were prescribed as food-borne (FB), water-borne plus clean food (WCB), water-food-borne (WFB) and water-borne (WB). Caenorhabditis elegans was chosen as the model organism, and growth statuses, feeding abilities, the amounts of four antioxidant enzymes, and corresponding recovery effects under non-toxic conditions with food and without food were investigated. Based on analysis results, copper concentrations in exposure were significantly influenced by the presence of food and its uptake by C. elegans. Both exposure and recovery effects depended on exposure concentrations and food conditions. For exposure pathways with food, feeding abilities and growth statuses were generally WFB<WCBFB (p<0.05). The antioxidant activities were up-regulated in the same order. Meanwhile, the exposure pathway without food (WB) caused non-up-regulated antioxidant activities, and had the best growth statuses. For recoveries with food, growth statuses, feeding abilities and the inductions of the antioxidant enzymes were all WBWFB<WCB<FB (p<0.05). For recoveries without food, the order of growth statuses remained WB>FB>WCB>WFB (p<0.05), while the antioxidant activities were all inhibited in a concentration-dependent fashion. In conclusion, contaminated food was the primary exposure pathway, and various pathways caused different responses of C. elegans.
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[
Proc Natl Acad Sci U S A,
1998]
Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.
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Diomede L, Romeo M, Romanucci V, Galati C, Zarrelli A, Monaco I, Milardi D, Bongiorno C, Di Fabio G, Salmona M, Grasso G, Sciacca MFM, Spinella N, D'Urso L, La Rosa C, Lolicato F
[
ACS Chem Neurosci,
2017]
The self-assembling of the amyloid (A) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's Disease (AD). Based on the "amyloid hypothesis" much efforts have been devoted in designing molecules able to halt disease progression by inhibiting A self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS) and computational (all-atom Molecular Dynamics) techniques to investigate the capacity of four optically pure components of the natural product Silymarin (Silybin A, Silybin B, 2,3-Dehydrosilybin A, 2,3-Dehydrosilybin B), to inhibit A aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only Silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that Silybin B interacts mainly with the C-terminal hydrophobic segment 35MVGGVV40 of A40. Consequently to Silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast Silybin A interacts preferentially with the segments 17LVFF20 and 27NKGAII32 of A40 which shows a high tendency to form bend, turn and -sheet conformation in and around these two domains. Both 2,3-Dehydrosilybin enantiomers bind preferentially the segment 17LVFF20 but lead to the formation of different small-sized, ThT-positive A aggregates. Finally, in vivo studies in a transgenic C. elegans strain expressing human A, indicated that Silybin B is the most effective of the four compounds in counteracting A proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of Silybins and points to Sil B as a promising lead compound for further development in anti AD therapeutics.
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[
FEBS Lett,
2002]
6-Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6-double bond in the steroid nucleus, was used recently to identify cholesterol-binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42-49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725-1736], and to probe lipid-protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143-154]. To determine whether 6-photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.
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[
Microbiol Immunol,
2002]
Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.
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[
Diagn Microbiol Infect Dis,
2014]
Lymphatic filariasis is the second leading cause of permanent long-term disability globally and control of this disease needs efficient diagnostic methods. In this study, abundantly expressing microfilarial sheath protein (Shp-1) from Brugia malayi was characterized as a filarial diagnostic candidate using samples from different clinical population. Monoclonal antibodies were developed against E. coli expressed recombinant Shp-1 in order to assess its efficiency in filarial antigen detection assay system. Endemic Normal (EN, n = 170), asymptomatic microfilaeremics (MF, n = 65), symptomatic chronic pathology (CP, n = 45) and non endemic normal (NEN, n = 10) sera were analyzed by antigen capture enzyme-linked immunosorbent assay. Of the 290 individuals, all MF individuals (both brugian and bancroftian) were positive in this assay followed by CP and EN. When compared with SXP-1 and Og4C3 antigen assays, all assays detected Wb MF correctly, Bm MF was detected by Shp-1 and SXP-1 assays, and only Shp-1 was able to detect EN (12%) and CP (29%). Results showed that this assay may be useful for monitoring prior to mass drug administration.