Chain Of Ricin Research Articles

Computational design of fusion proteins against ErbB2-amplified tumors inspired by ricin toxin.

Although the anti-cancer activity of ricin is well-known, its non-specific targeting challenges the development of ricin-derived medicines. In the present study, novel potential ribosome-inactivating fusion proteins (RIPs) were computationally engineered by incorporation of an ErbB2-dependant penetrating peptide (KCCYSL, MARAKE, WYSWLL, MARSGL, MSRTMS, and WYAWML), a linker (either EAAAK or GGGGS) and chain A of ricin which is responsible for the ribosome inactivation. Molecular dynamics simulations assisted in making sure that the least change is made in conformation and dynamic behavior of ricin chain A in selected chimeric protein (CP). Moreover, the potential affinity of the selected CPs against the ligand-uptaking ErbB2 domain was explored by molecular docking. The results showed that two CPs (CP2 and 10) could bind the receptor with the greatest affinity.

Inactivation of ricin by constituents present in a skin decontamination lotion

Ricin is a proteinaceous toxin, listed on the schedules of both the chemical and biological weapons conventions. The ease of accessibility to the Ricinus communis plant and toxin extraction makes ricin a viable concern for use of intentional release and causal effects. The adverse effects following exposure to the toxin are caused by the bipartite molecular structure of ricin which allows binding to the mammalian cell surface, enter via endocytic uptake, and deliver the catalytically active polypeptide into the cell cytosol where it irreversibly inhibits protein synthesis, causing cell death. In the present study, the inactivation effectiveness of RSDL® (Reactive Skin Decontamination Lotion) and its individual inactivating constituents (Potassium 2,3-butanedione monoximate (KBDO) and 2,3-butanedione (DAM)) was evaluated for ricin using a number of read out systems including a cytotoxicity assay, quantitative sandwich ELISA test, and a mass spectrometry-based assay. The results demonstrate that RSDL is able to abolish ricin activity after an incubation time of 30 min as determined in the cytotoxicity assay, and after 2 min as determined in the ELISA assay. Mass spectrometric analysis provided evidence that RSDL is able to induce cleavage of the disulfide linkage between the A- and B- polypeptide chain of ricin which is crucial to the inactivation of the toxin, but this seems not the only mechanism of inactivation. Follow on studies would assist to elucidate the details of the toxin inactivation because it is possible that additional generic mechanisms are in place for denaturation with the RSDL lotion components. This may also provide a promise for testing and inactivation with RSDL of other protein toxins.

Characterizing Binding Interactions and Elucidating Structure of Aptamer‐Based Biosensors

Electrochemical biosensors based on the conformational dynamics of DNA aptamers have found success against a wide variety of proteins, toxins, antibodies, and heavy metals. However, the mechanistic underpinnings of the mechanism by which these surface‐bound DNA molecules change conformation upon target binding, thus changing the dynamics of an appended redox‐active tag and generating a measurable signal, is poorly understood. Our first target for investigation was a previously reported Ricin Chain A binding aptamer biosensor. Here, we have investigated this biosensor using a variety of nucleic acid assays, including PCR‐termination via basepair modification, fluorescence anisotropy, gel mobility shift assays, and FRET tagging to determine 3D orientation. These have allowed us to better characterize the basepair interactions involved in binding targets, as well as offer clues to the changing three dimensional folded structures of these biosensors. These results will help inform the field of biosensors and aptamers in general on strategies for future optimization.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B-chain.

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis.

Baicalin Inhibits the Lethality of Ricin in Mice by Inducing Protein Oligomerization

Toxic ribosome-inactivating proteins abolish cell viability by inhibiting protein synthesis. Ricin, a member of these lethal proteins, is a potential bioterrorism agent. Despite the grave challenge posed by these toxins to public health, post-exposure treatment for intoxication caused by these agents currently is unavailable. In this study, we report the identification of baicalin extracted from Chinese herbal medicine as a compound capable of inhibiting the activity of ricin. More importantly, post-exposure treatment with baicalin significantly increased the survival of mice poisoned by ricin. We determined the mechanism of action of baicalin by solving the crystal structure of its complex with the A chain of ricin (RTA) at 2.2 Å resolution, which revealed that baicalin interacts with two RTA molecules at a novel binding site by hydrogen bond networks and electrostatic force interactions, suggesting its role as molecular glue of the RTA. Further biochemical and biophysical analyses validated the amino acids directly involved in binding the inhibitor, which is consistent with the hypothesis that baicalin exerts its inhibitory effects by inducing RTA to form oligomers in solution, a mechanism that is distinctly different from previously reported inhibitors. This work offers promising leads for the development of therapeutics against ricin and probably other ribosome-inactivating proteins.

Antibody Interactions with Ricinus communis Agglutinins Studied by Biolayer Interferometry

Two related agglutinins are present in the seeds of Ricinus communis (castor): ricin, a dichain ribosome-inactivating protein and Ricinus communis agglutinin-1, a much less toxic tetrameric hemagglutinin. The immunochemical analysis of these agglutinins is of special interest because ricin toxicity has resulted in both accidental and intentional poisonings, while it has also provided a potential cancer chemotherapeutic in the form of an immunoconjugate. We previously characterized a panel of monoclonal antibodies (mAbs) for the analysis of potential contamination with ricin in several food matrices. In this study, an optical sensing technique, biolayer interferometry (BLI), was used to study the binding of two mAbs to the agglutinins. MAbs were immobilized on sensors with amine-reactive, Fc-binding, and streptavidin-coated tips to study the interactions with the agglutinins and with ricin A- and B-chains in solution. The kinetically determined equilibrium dissociation constants generally agreed with the relative binding observed in ELISA, although binding was less predictable for the isolated ricin chains. BLI analysis of kinetic constants for mAb 1797 was not affected by nonfat milk (0.5% by volume). BLI provides a useful method to characterize the binding of antibodies, with the potential for immunodiagnostic applications in food matrices.

A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate

Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD50 challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD50 challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 μg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD50 in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT.

Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection

A gold nanoparticle (GNP) probe-based assay (GNPA) modified from the bio-barcode assay (BCA) was developed for ultrasensitive detection of ricin, a potential biothreat agent. In the GNPA, a chain of ricin was captured by a GNP probe coated with polyclonal antibodies and single-stranded signal DNA. A magnetic microparticle (MMP) probe coated with ricin A chain monoclonal antibody was then added to form an immuno-complex. After being magnetically separated, the immuno-complex containing the single-stranded signal DNA was characterized by PCR and real-time PCR. A detection limit of 10−2 fg/ml was determined for the ricin A chain; this is eight orders of magnitude more sensitive than that achieved with an ELISA and two orders more sensitive than that obtained with the BCA. The coefficients of variation (CV) of the intra- and inter-assay values ranged from 3.82–6.46%. The results here show that this novel assay is an ultrasensitive method for detection of ricin proteins and may be suitable for the ultrasensitive detection of other proteins.

Immunosorbent analysis of ricin contamination in milk using colorimetric, chemiluminescent and electrochemiluminescent detection

Analytical methodology to detect active ricin in food matrices is important because of the potential use of foodborne ricin as a terrorist weapon. Monoclonal antibodies (mAbs) that bind ricin were used as both capture and detection ligands in sandwich enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescent (ECL) immunosorbent assays. ELISA employed two types of substrate for colorimetric or chemiluminescent detection. Although both fat content and protein content of samples influenced the recovery of ricin, the lower limit of detection (LOD) in ELISA and ECL systems permitted detection of 0.1 ng/mL for milk samples containing 0–4% fat. The assay systems detect pure ricin- or crude ricin-containing castor extract, but do not significantly respond to isolated ricin chains, heat-denatured ricin or the related agglutinin, Ricinus communis agglutinin 1 (RCA-1). Using the standard 96-well-plate formats, the assays detect less than 0.01% of an adult human lethal dose in a typical serving of milk.

Immunotoxins constructed with chimeric, short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T1/2 of 263 h, the mcRFB4s had T1/2s ranging from 39 to 106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.

A nanoparticle-based bio-barcode assay for ultrasensitive detection of ricin toxin

The ultrasensitive bio-barcode amplification assay (BCA) technique was developed for the specific detection of the A chain of ricin toxin. The target antigen A chain was first captured by gold nanoparticles (GNPs) coated with polyclonal antibodies. Magnetic microparticles (MMPs) coated with A chain monoclonal antibody were then added to form a sandwich immuno-complex. After the immuno-complex was formed, signal DNA annealed to DNA strands covalently bound to the GNPs were released by heating and characterized by PCR and real-time fluorescence PCR. A detection limit of 1fg/ml was measured for A chain, six orders of magnitude more sensitive than that of conventional antigen-capture ELISA. The coefficient of variation (CV) of intra-assay and inter-assay ranged from 3.39% to 6.84%. The BCA can detect the A chain in milk and water mimic samples. In the following work it is demonstrated that this assay is a highly sensitive method for the detection of ricin proteins that could be adapted to measure other proteins.

The conjugation of an AQP1-directed immunotoxin in the study of site-directed therapy within the CNS

The water channel, aquaporin (AQP)1, is highly specific to the choroid plexus (CP) epithelium within the brain. It is therefore a potential target through which therapeutic agents could be selectively directed to the CP. Here we describe the conjugation of a monoclonal antibody (mAb), raised against an extra-cellular domain of AQP1, to the A chain of ricin (RCA). This reagent should allow study of a highly specific chemical lesion of the CP. A published method was used to couple the anti-AQP1 mAb to the A chain of ricin using an SPDP cross-linker. The conjugate was purified by Superdex S-200 chromatography. Column fractions were analysed by SDS-PAGE and Western blotting. Fractions containing conjugate were assayed for cytotoxicity on rat CP cells in culture, using a WST-1 viability assay to assess cell death. SDS-PAGE and Western blotting demonstrated separation of conjugated anti-AQP1 mAb from unconjugated ricin by the Superdex column. Cultured rat CP cells were killed with an IC(50) of 1.81 μg/ml when exposed to the anti-AQP1 mAb-RCA conjugation reaction products. Selectivity of this reagent was demonstrated by the higher IC(50) exhibited by non-AQP1-expressing primary fibroblast cultures and cultures exposed to the reaction products from the non-specific IgG-RCA conjugation attempt. We have been able to demonstrate selective cytotoxicity in vitro of an anti-AQP1 mAb-RCA conjugate. We hope that this work will generate further interest in the use of this site-specific conjugate to target the CP in conditions such as hydrocephalus, tumours that arise from here, and other AQP1-expressing tumours.

Shiga toxin 1 and ricin A chain bind to human polymorphonuclear leucocytes through a common receptor

The main cause of acute renal failure in children is HUS (haemolytic uraemic syndrome), a consequence of intestinal infections with Escherichia coli strains producing Stx (Shiga toxins). Stx released in the gut by the non-invasive bacteria reach the bloodstream and are targeted to cerebral and renal endothelium triggering HUS. PMN (polymorphonuclear leucocytes) seem to be involved in Stx delivery through an unidentified membrane receptor (Kd=10⁻⁸ M; 2×10⁵ binding sites) which does not allow internalization. Some experts in the field have defined the Stx-PMN interaction as non-specific and of little biological significance. In the present study, we show that the A chain of ricin, the well-known plant RIP (ribosome-inactivating protein), interacts with PMN (Kd=10⁻⁹ M; 2×10⁵ binding sites) competing for the same receptor that recognizes Stx, whereas diphtheria toxin and several agonists of TLRs (Toll-like receptors) or the mannose receptor were ineffective. No toxic effects of ricin A chain on PMN were observed, as assessed by measuring protein synthesis and the rate of spontaneous apoptosis of leucocytes. Moreover, two single-chain RIPs (gelonin and saporin S6) had the same competing effect. Thus RIPs and Stx1 share structural similarities, the same enzymatic activity and a common receptor on PMN. These observations reveal that the Stx-PMN interaction is specific, confirming that PMN recognize molecular patterns common to different foreign molecules.

In vitro ruminal degradation of ricin and its effect on microbial growth

Ricin is a toxic protein found in castorseed ( Ricinus communis L.). We hypothesized that ruminal microbiota are capable of degrading ricin, and that the toxin inhibits ruminal microbial growth. Therefore, first we evaluated the in vitro ruminal degradation of ricin from solvent castorseed meal (SCM) by SDS-PAGE and densitometry analysis of culture medium (Experiment 1). Culture medium (three replicates) were collected after 0, 3, 6, 12, 24 and 48 h of incubation content initially 0, 61, 122 and 244 μg of ricin/mL or 122 μg of ricin/mL (without ruminal inoculum). No protein compounds were detected by SDS-PAGE in the culture medium without ricin, indicating an absence of interference from the ruminal inoculum. Ricin chains remained intact in the absence of rumen inoculum, but they were degraded at rates of 0.2725, 0.1504 and 0.0648 h −1 with ruminal inoculum, at initial ricin concentrations of 61, 122 and 244 μg/mL. Next, the effect of ricin denaturation on rumen microbial specific growth rate (SGR) (OD-600 nm) and the average ammonia concentration at the same time of incubation were investigated (Experiment 2). This experiment had a completely randomized design in a 3 × 3 factorial (three replicates) arrangement, with three sources of protein (trypticase-control; crude extract of soluble protein at pH 3.8 buffer of solvent castorseed meal (CEP) intact, containing 1.46 mg of ricin/mL; and denatured CEP with calcium oxide, containing 0.04 mg of ricin/mL) and three protein levels (0.42, 0.84, and 1.68 mg/mL). There was interaction (P=0.021) between protein level and protein source for SGR. A linear increase (P<0.001) of SGR was observed with increase of trypticase level, but there was a quadratic effect (P=0.023) with increase of intact CEP level, with a minimum value of SGR of −0.004 h −1 at a protein level of 1.45 mg/mL (210 μg of ricin/mL) of intact CEP. There was no effect (P=0.099) of denatured CEP level, but SGR increased (P<0.001) 3.2 times with denaturation of intact CEP. Ruminal microbial growth was inhibited by 50% with 89 μg of ricin/mL. Ammonia concentration was 91% lower (P<0.001) for the CEP source when compared to trypticase, but the denaturation of intact CEP had no effect (P=0.9560) on the ammonia concentration. Although ruminal microbiota was able to degrade ricin in in vitro conditions, the toxin inhibits ruminal microbial growth. Therefore, complete detoxification of CSM before using it to feed ruminants is recommended. The denatured CEP presents potential of use as modifier of rumen microbial fermentation.

Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

BackgroundRicin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.ResultsIn this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 ± 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity).ConclusionResults of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication.

Site-directed mutagenesis at amino terminus of recombinant ricin A chain

Successful immunotoxin therapy may depend upon reduction of the size of the components in order to decrease antigenicity and rate of clearance. In initial attempts to modify the A chain of ricin by deletion analyses within a prokaryotic expression system, coding sequences were modified by the insertion of unique restriction endonuclease sites and by the removal of 22 codons near the 5' terminus. The work presented here examines the expression, solubility, and activity of these mutant proteins and demonstrates that while amino acid residues may be altered in this region, the deletion of residues 19 through 40 yields an insoluble and inactive toxin molecule.

Ricin B Chain Targeted to the Endoplasmic Reticulum of Tobacco Protoplasts Is Degraded by a CDC48- and Vacuole-independent Mechanism*

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway.

Biochemical properties of ricin in immature castor seed

The biochemical properties of ricin at different stages of seed i.e. from immature to mature seed were studied. Hemagglutination, SDS-PAGE and UV-spectrometry studies showed total absence of RCA protein in the immature seed. Interestingly, ricin extract on SDS-PAGE showed only one protein band with a molecular weight of 29,000 dalton corresponding to the molecular weight of A chain of ricin. Our results have shown that at immature seed level only the toxic moiety of ricin (A chain) is being synthesized first and gradually the RCA and B chain of ricin.

The Catalytic Subunit of Shiga-like Toxin 1 Interacts with Ribosomal Stalk Proteins and is Inhibited by Their Conserved C-Terminal Domain

Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A1 domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A1 chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A1 chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A1 chain, whereas P0, lacking this common C terminus, still binds to the A1 domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A1 chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A1 chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A1 chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA.

Comparison of an electrochemiluminescence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain (RCA-B)

An electrochemiluminescence (ECL) assay for the detection of the B chain of ricin (RCA-B) in a 96-well plate format was developed in parallel with a colorimetric ELISA utilizing the same pair of antibodies. Sensitivity results were interpreted with the ANOVA and Tukey statistical tests, allowing a direct comparison between the two technologies, that can probably be extended to other protein antigens such as toxins. Reproducibility, repeatability and rapidity of the two techniques were also compared. The ELISA assay utilized an alkaline phosphatase conjugate for signal generation. After optimization, its limit of detection was 400 pg of RCA-B per ml buffer, with an intra-day standard deviation (SD) of 2.2% of the mean and an inter-day SD of 5.1%. The ECL assay utilized ruthenylated antibodies for detection. The ECL measurement was carried out using a Sector™ PR 400 plate reader. After optimization, its limit of detection was 50 pg of RCA-B per ml buffer, with an intra-day SD of 4.1% of the mean and an inter-day SD of 4.3%. Starting from a pre-coated plate, the ELISA assay was completed in 7 h and the ECL assay took 2.5 h. While reproducibility and repeatability of the two assays were equivalent, this ECL assay in plate format had an 8-fold better sensitivity for RCA-B detection than the colorimetric ELISA in buffer and in various matrices. The ECL assay was also three times faster, and retained the robustness and convenience of the 96-well plate format.