Mining the “glycocode”–exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy
- FASEB journal : official publication of the Federation of American Societies for Experimental Biology
- Published over 4 years ago
Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.-Gunning, A. P., Kirby, A. R., Fuell, C., Pin, C., Tailford L. E., Juge, N. Mining the “glycocode”-exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy.
Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 2 years ago
Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant-containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.
Bioenergy production combined with phytoremediation has been suggested to help in solving two critical world problems: the gradual reduction of fossil fuels and soil contamination. The aim of this research was to investigate the potential for the use of Ricinus communis L. (castor oil plant) as an energy crop and plant species to remediate metal-polluted sites. This study was performed in mine tailings containing high concentrations of Cu, Zn, Mn, Pb and Cd. Physico-chemical characterization, total, DTPA-extractable and water-soluble metals in rhizospheric tailings heap samples were carried. Metal concentrations in plant tissues and translocation factors (TFs) were also determined. The Ricinus seed-oil content was high between 41 and 64%, seeds from San Francisco site 6 had the highest oil content, while these from site 7 had the lowest. No trend between oil yield vs seed origin site was observed. Seed-oil content was negatively correlated with root concentration of Cu, Zn, Pb and Cd, but no correlation was observed with the extractable-metals. According to its shoot metal concentrations and TFs, castor bean is not a metal accumulator plant. This primary colonizing plant is well suited to cope with the local toxic conditions and can be useful for the stabilization of these residues, and for then decreasing metal bioavailability, dispersion and human health risks on these barren tailings heaps and in the surrounding area. Our work is the first report regarding combined oil production and a phytostabilization role for Ricinus plants in metal mine tailings and may give a new value to suitable metal-polluted areas.
OBJECTIVE: In immunotherapy of HPV-16-associated cervical cancers, the E7 protein is considered as a prime candidate. However, it is a poor inducer of a cytotoxic T-cell response when used as a singular antigen in protein vaccination. Therefore, to design effective cancer vaccines, the best tumor antigens should be combined with the most effective immunogens or drug delivery tools to achieve positive clinical results. In this study, we fused HPV-16 E7 with the lectin subunit of ricin toxin (RTB) from castor plant as a vaccine adjuvant/carrier. MATERIALS AND METHODS: After reaching the soluble form of the recombinant protein, we designed 2 preventive and inhibition tumor models for investigation of the prevention and rejection of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-RTB or E7 without any adjuvant. RESULTS: We demonstrated that prophylactic immunization of E7-RTB protected mice against challenge from TC-1 cells. Also in the therapeutic model, E7-RTB could inhibit TC-1 tumor growth in the lung. The results were significant compared with the immunization of E7 singularly. CONCLUSIONS: We concluded that immunization with E7-RTB protein without any adjuvant could generate antitumor effects in mice challenged with TC-1 cells. This research verifies the clinical applications and the future prospects for development of HPV-16 E7 therapeutic vaccines fused to immunoadjuvants.
In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed storage proteins, toxins and allergens. Additionally, we have used an off-line Hydrophilic Interaction Chromatography (HILIC) as a step of peptide fractionation preceding the Reverse Phase nanoLC coupled with a LTQ Orbitrap. We were able to identify a total of 1875 proteins and from these 1748 could be mapped to extant castor gene models, expanding considerably the number proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism and catabolism of fatty acid and the pattern of deposition of reserve proteins, toxins and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of reserve proteins that are differentially expressed during seed development.
Context. Ingestions of the seed of the castor bean plant (Ricinus communis) carries the risk of toxicity from ricin, a potent inhibitor of protein synthesis. Objective. We sought to describe characteristics of castor bean seed exposures reported to a state-wide poison control system. Methods. This was an observational case series. A state-wide poison control system’s database was reviewed for exposures to castor bean plant seeds from 2001 to 2011. Case notes were reviewed and data collected, when available, included age, gender, circumstances surrounding exposure, number of castor beans consumed, whether beans were chewed or crushed, symptoms described, laboratory values (aspartate aminotransferase [AST], alanine aminotransferase [ALT], prothrombin time [PT] and international normalized ratio [INR]), duration of follow-up, treatment, and patient outcomes. Results. Eighty-four cases were identified. Ingestions were unintentional in 50 cases (59%) cases and intentional in 34 (40%) cases. A median of 10 seeds (range: 1-20) were ingested in intentional cases versus 1 seed (range: 1-40) in unintentional cases. In 49 (58%) of cases the seeds were reported to have been chewed or crushed. Gastrointestinal symptoms were the most commonly reported symptoms. Vomiting (n = 39), nausea (n = 24), diarrhea (n = 17), and abdominal pain (n = 16) predominated. One patient developed hematochezia and vomiting after reportedly ingesting and intravenously injecting castor bean seeds. Laboratory values were documented in 17 (20%) cases. Only one abnormality was noted; an asymptomatic patient one week following ingestion had AST/ALT of 93 U/L and 164 U/L, respectively. Ricinine was confirmed in the urine of two patients. Twenty-three (27%) cases received activated charcoal. Seventy-two (86%) of cases were calls from health care facilities or referred to health care facilities by the poison control center. Twenty-two (26%) cases were admitted for a median of 2 days (range: 1-10). Admitted cases ingested a median of 8.5 seeds (range: 1-20). Intentional ingestions were followed for median of 37.5 h (range: 0.5-285.5) while unintentional cases were followed for 14 h (range: 1-182). No delayed symptoms, serious outcomes, or deaths were reported. Discussion. Due to the presence of ricin, there is concern for serious outcomes after ingestions of the seeds of the castor bean plant. In this study GI symptoms were most commonly reported but serious morbidity or mortality was not present. The true risk of castor bean plant seed ingestions should continue to be re-evaluated. Conclusion. In this retrospective review, gastrointestinal symptoms were the most common symptoms described after reported exposures to castor bean seeds. These exposures were not associated with serious morbidity, mortality, or delayed symptoms.
This study aimed to evaluate the radiosensitivity of castor bean seeds after applications of different doses of Cobalt60 gamma radiation. Seeds were pre-soaked for 24 hours in distilled water and then irradiated with 50, 100, 150, and 200 Gy, except the control. Sowing was performed in trays, which contained soil as substrate and were maintained in a greenhouse. The electrical conductivity, emergence, emergence speed index, growth parameters and activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, and catalase) were evaluated in the leaves and roots of castor bean seedlings. Gamma radiation did not affect the electrical conductivity of the seeds; however, at a dose of 200 Gy, the emergence and emergence speed index of the seedlings was negatively affected. An analysis of the morphophysiological parameters revealed a reduction in seedling size as the radiation dose increased. There was a significant increase in superoxide dismutase and ascorbate peroxidase activities at higher radiation doses in the leaves, but not in roots. Thus, the analysis of all the variables suggests a response pattern as to the morphophysiological and biochemical changes of castor bean seedlings due to the increase of gamma radiation, which may serve as a tool for generating greater genetic variability.
For counter-terrorism purposes, a selective nano liquid chromatography-mass spectrometry (nanoLC-MS) platform was developed for detecting the highly lethal protein ricin from castor bean extract. Manual sample preparation steps were omitted by implementing a trypsin/Lys C enzyme-immobilized multi-channel reactor (MCR) consisting of 126 channels (8 µm inner diameter in all channels) that performed on-line digestion of proteins (5 minute reaction time, instead of 4-16 hours in previous in-solution methods). Reduction and alkylation steps were not required. The MCR allowed identification of ricin by signature peptides in all targeted mode injections performed, with a complete absence of carry-over in blank injections. The MCRs (interior volume ≈ 1 µL) have very low backpressure, allowing for trivial on-line coupling with commercial nanoLC-MS systems. The open tubular nature of the MCRs allowed for repeatable within/between-reactor preparation and performance.
Soils impacted by metallurgy activities pose serious risks to the health of exposed populations, whether by ingestion of soil or contaminated food and water. The municipality of Santo Amaro, Bahia state, presents the most important case of human lead contamination in Brazil. It occurred because of inadequate slag disposal. The aims of this research were to: (i) determine the environmentally available concentrations and the distribution of As, Cd, Pb, and Zn in soil fractions; (ii) estimate the non-carcinogenic and carcinogenic risks of these elements for children; and (iii) to evaluate the use of corn (Zea mays) and castor bean (Ricinus communis) either for phytoextraction induced by chelating agents or phytostabilization. Our data demonstrated that the environmentally available concentrations of As, Cd, Pb, and Zn in soils surrounding the Pb smelting plant are among the highest that have been reported. Apart from Cd, sequential extraction demonstrated that most metals are in recalcitrant forms in the soil. However, the daily exposure of children to Pb, Zn, Cd, and As exceeded the acceptable daily intake as established by the World Health Organization. Non-carcinogenic risk modeling indicated probable adverse health effects from chronic exposure to soil Pb. The mean estimated time for remediation of the area using phytoextraction was high, ranging from 76 to 259 years; therefore, this is not a viable alternative for remediating soils in the studied area. However, good development in the contaminated soil along with restriction of the metal(oid) translocation to shoots enables castor bean to phytostabilize metal(oid)s. Additionally, castor bean cultivation may be an alternative for an economic return because of biofuel production.
Ricinus communis L. (castor bean) as a potential candidate for revegetating industrial waste contaminated sites in peri-urban Greater Hyderabad: remarks on seed oil
- Environmental science and pollution research international
- Published 2 months ago
Ricinus communis L. (castor bean or castor oil plant) was found growing on metal-contaminated sites (4) of peri-urban Greater Hyderabad comprises of erstwhile industrial areas viz Bollaram, Patancheru, Bharatnagar, and Kattedan industrial areas. During 2013-2017, about 60 research papers have appeared focusing the role of castor bean in phytoremediation of co-contaminated soils, co-generation of biomaterials, and environmental cleanup, as bioenergy crop and sustainable development. The present study is focused on its use as a multipurpose phytoremediation crop for phytostabilization and revegetation of waste disposed peri-urban contaminated soils. To determine the plant tolerance level, metal accumulation, chlorophyll, protein, proline, lipid peroxidation, oil content, and soil properties were characterized. It was noticed that the castor plant and soils have high concentration of metals such as cadmium (Cd), lead (Pb), iron (Fe), manganese (Mn), and zinc (Zn). The soils have high phosphorous (P), adequate nitrogen (N), and low concentration of potassium (K). Iron (Fe) concentrations ranged from1672±50.91 to 2166±155.78 mg kg(-1) in the soil. The trend of metal accumulation Fe>Zn>Mn>Pb>Cd was found in different plant parts at polluted sites. The translocation of Cd and Pb showed values more than one in industrial areas viz Bollaram, Kattedan, and Bharatnagar indicating the plants resistance to metal toxicity. Chlorophyll and protein content reduced while proline and malondialdehyde increased due to its tolerance level under metal exposure. The content of ricinoleic acid was higher, and the fatty acids composition of polluted areas was almost similar to that of the control area. Thus, R. communis L. can be employed for reclamation of heavy metal contaminated soils.