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Microplastics in wastewater treatment plant (WWTP) effluent have been identified and quantified, but few studies have examined the microplastics in advanced treatment systems. A new method for isolating, quantifying, and determining the polymer type of microplastics was developed that included chemical digestion coupled with Raman microspectroscopy to investigate microplastics in the effluent of reverse osmosis nanofiltration and activated carbon filtration systems. This method allows for the removal of organics and the quantification and identification of all microplastics present in the sample. A large number of microplastics, the majority of which were smaller than 10 μm, were identified in the effluent of the advanced filtration systems with polyethylene the most common polymer identified. This study not only reports a new method for microplastic identification and quantification but also shows the importance of measuring the smallest fraction of microplastics, those smaller than 20 μm, which have previously been understudied., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

It has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor-bearing cells. We now report that DT388-GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF-receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I-GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388-GM-CSF at 4 x 10(-9) mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 x 10(-9) mol/L to 3 x 10(-13) mol/L. GM-CSF-receptor occupancy at 0.7 nmol/L GM-CSF-ligand concentration correlated with the protein synthesis IC50. Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL, in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388-GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.

We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC. The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence. Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 degrees C and, on incubation at 37 degrees C, distributed initially to endosomes and then to Golgi vesicles. Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing. Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured. Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells. Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells. These data support a model requiring a Golgi-dependent step in cell intoxication by ricin. The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.

Ricin intoxication of mammalian cells is initiated by B chain (RTB) binding to cell surface galactosides. Recombinant insect-derived RTB mutants with modifications in lectin-site subdomains 2 gamma, 1 alpha/2 gamma, and 1 alpha/1 beta/2 gamma were reassociated with plant RTA and tested for lethality in C57B1/6 6-8 weeks old mice. The LD50 of intraperitoneally injected castor bean ricin was 75 ng per 18 g mouse. The LD50 of single-site 2 gamma mutant heterodimer was 100 ng: the LD50 of the double-site 1 alpha/2 gamma mutant heterodimer was 500 ng, and the LD50 of the triple-site 1 alpha/1 beta,2 gamma mutant heterodimer was > 10 micrograms. Plant RTA alone had an LD50 of 300 micrograms. Animals died between 1 and 10 days post-injection. Histopathological examination of morbid animals receiving an LD50 dose of each toxin revealed only apoptosis in the thymus and spleen. The present data provide clear evidence for participation of three lectin sites in ricin in vivo toxicity. These results suggest an origin for some of the normal tissue toxicities observed with clinical trials of doubly blocked ricin conjugates and suggest modification of at least three RTB subdomains will be necessary in genetically engineered ricin fusion proteins.

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, consists of a cell binding lectin subunit (RTB) disulfide linked to an rRNA N-glycosidase protein synthesis inactivating subunit (RTA). While X-ray crystallography and equilibrium dialysis suggested two sugar-combining sites located in subdomains 1 alpha and 2 gamma, biochemical and mutational analyses suggested the existence of a third lectin site. We performed oligonucleotide-directed mutagenesis on RTB cDNA to create mutants with modifications in subdomains 1 alpha, 2 gamma, and either 1 beta or 2 alpha. The triple-site mutant RTBs were expressed in insect cells. Partially purified recombinant proteins obtained from infected cell extracts and cell supernatants were characterized for asialofetuin and cell binding, immunoreactivites, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Yields of both triple-site mutants were similar to the parent double-site mutant. Both mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. The triple-site mutant with modification of amino acid residues in subdomains 1 alpha, 2 alpha, and 2 gamma bound asialofetuin and cells similarly to the parent 1 alpha, 2 gamma, subdomain mutant. In contrast, the 1 alpha, 1 beta, 2 gamma subdomain triple-site mutant had a one and one-half log decrease in asialofetuin and cell binding relative to the parent double-site mutant. The 1 alpha, 2 alpha, 2 gamma triple-site mutant and 1 alpha, 2 gamma parent protein had sugar binding which was inhibited by 3-27-fold by lactose and asialofetuin. Both triple-site mutants reassociated well with RTA. The 1 alpha, 2 alpha, 2 gamma triple-site mutant-RTA was equally cytotoxic to mammalian cells as the double-site mutant-RTA heterodimer. In contrast, the 1 alpha, 1 beta, 2 gamma triple-site mutant-RTA was 25 times less toxic than the double mutant and 20 times more toxic than RTA alone. These data support a model for at least three lectin-binding subdomains in RTB.

Fusion toxins are hybrid proteins consisting of peptide ligands linked through amide bonds to polypeptide toxins. The ligand directs the molecule to the surface of target cells and the toxin enters the cytosol and induces cell death. Ricin is an excellent candidate for use in fusion toxins because of its extreme potency, the extensive knowledge of its atomic structure and the lack of prior immunological exposure in patients. We synthesized a baculovirus transfer vector with the polyhedrin promoter followed sequentially from the 5' end with DNA encoding the gp67A leader sequence, the tripeptide ADP, IL-2 (interleukin-2), another ADP tripeptide and RTB (ricin toxin B chain) with lectin-site mutations W37S and Y248H. Recombinant baculovirus was generated in Sf9 insect cells and used to infect Sf9 cells. Recombinant IL-2-RTB[W37S/Y248H] protein (fusion protein of IL-2 with modifications W37S and Y248H) was recovered at high yields from day 6 insect cell supernatants, partially purified by affinity chromatography and reassociated with RTA (ricin toxin A chain). The fusion toxin was soluble, immunoreactive with antibodies to RTB, IL-2 and RTA and had a molecular weight of 80 kDa by SDS-PAGE. The molecule reacted poorly with asialofetuin, but bound strongly to IL-2 receptor based on selective cytotoxicity to IL-2 receptor bearing cells. The specific cytotoxicity could be blocked with IL-2 but not lactose. Thus, we report a novel targeted fusion toxin protein with full biological activity.

Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactose-binding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens-mediated gene transfer. Yields of ricin in soluble cell extracts were 1 microg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10(-12) M for tobacco ricin and 1 x 10(-11) M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10(-13) and 2 x 10(-12) M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.

Three distinct double-site and two single-site ricin B chain (RTB) mutants were expressed in Spodoptera frugiperda insect cells and purified from infected cell supernatants. The yields of recombinant proteins were 0.01-0.2 mg/l. The purity after monoclonal antibody affinity chromatography was 1-20%. The mutant proteins were soluble, immunoreactive with monoclonal antibodies and polyclonal antibodies to RTB and demonstrated molecular weights of 32 kDa, similar to plant RTB. All three double-site and both single-site mutants bound asialofetuin and mammalian cell surfaces based on an asialofetuin ELISA and cell binding immunofluorescence assay. While one double-site mutant, W37S/Y248S, had a 1 log drop in sugar binding, the other two double-site mutants W37S/Y248H and D22E/D234E had 2 log reductions in sugar binding. Each mutant reassociated efficiently (25-75%) with plant ricin A chain (RTA) to form cytotoxic heterodimers. The concentration of protein required to reduce protein synthesis 50% (ID50) was 1 log higher than plant ricin for W37S/Y248S-RTA and the single-site mutant heterodimers, Q35N-RTA and D22E-RTA and 2 logs higher than plant ricin for the other two double-site mutant heterodimers. The results suggest amino acid residues in both the 1 alpha and 2 gamma subdomains of RTB participate in sugar binding. However, other subdomains must contribute to the avidity of ricin for cell surface oligosaccharides.

Fusion toxins consist of peptide ligands linked through amide bonds to polypeptide toxins. The ligand directs the molecule to the surface of target cells and the toxin enters the cytosol and induces cell death. Ricin toxin is an excellent candidate for use in fusion toxins because of its extreme potency, the extensive knowledge of its atomic structure, and the years of experience with RTA chemical conjugates in clinical trials. We synthesized a baculovirus transfer vector with the polyhedrin promoter followed sequentially from the 5' end with DNA encoding the gp67A leader sequence, the tripeptide ADP, IL2, another ADP tripeptide, and RTB. Recombinant baculovirus was generated in Sf9 insect cells and used to infect Sf9 cells. Recombinant IL2-RTB protein was recovered at high yields from day 5 insect cell supernatants, partially purified by affinity chromatography, and characterized. The recombinant product was soluble and immunoreactive with antibodies to RTB and IL2, bound asialofetuin and lactose, and reassociated with RTA. In the presence of lactose to block galactose-binding sites on RTB, the IL2-RTB-RTA heterodimer was selectively cytotoxic to IL2 receptor, bearing cells. Specific cytotoxicity could be blocked with IL2. Thus, we report a novel targeted plant toxin fusion protein with full biological activity.