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Virus inactivation is a prerequisite for safe handling of high-risk infectious samples. ß-Propiolactone (BPL) is an established reagent with proven virucidal efficacy. BPL primarily reacts with DNA, RNA, and amino acids. The latter may modify antigenic protein epitopes interfering with binding properties of affinity reagents such as antibodies and aptamers used in affinity proteomic screens. We investigated (i) the impact of BPL treatment on the analysis of protein levels in plasma samples using the aptamer-based affinity proteomic platform SomaScan and (ii) effects on protein detection in conditioned medium samples using the proximity extension assay-based Olink Target platform. In the former setup, BPL-treated and native plasma samples from patients with ovarian cancer (n = 12) and benign diseases (n = 12) were analyzed using the SomaScan platform. In the latter, conditioned media samples collected from cultured T cells with (n = 3) or without (n = 3) anti-CD3 antibody stimulation were analyzed using the Olink Target platform. BPL-related changes in protein detection were evaluated comparing native and BPL-treated states, simulating virus inactivation, and impact on measurable group differences was assessed. While approximately one-third of SomaScan measurements were significantly changed by the BPL treatment, a majority of antigen/aptamer interactions remained unaffected. Interaction effects of BPL treatment and disease state, potentially altering detectability of group differences, were observable for less than one percent of targets (0.6%). BPL effects on protein detection with Olink Target were also limited, affecting 3.6% of detected proteins with no observable interaction effects. Thus, effects of BPL treatment only moderately interfere with affinity proteomic detectability of differential protein expression between different experimental groups. Overall, the results prove high-throughput affinity proteomics well suited for the analysis of high-risk samples inactivated using BPL.

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S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.

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During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.

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Although the copolymerizations of l-lactide (LA) with seven- or six-membered ring lactones have been extensively studied, the copolymerizations of LA with four-membered ring lactones have scarcely been reported. In this work, we studied the copolymerization of LA with ß-propiolactone (PL) and the properties of the obtained copolymers. The copolymerization of LA with PL was carried out using trifluoromethanesulfonic acid as a catalyst and methanol as an initiator to produce poly(LA-co-PL) with Mn of ~50,000 and PL-content of 6-67 mol %. The Tg values of the copolymers were rapidly lowered with increasing PL-contents. The Tm and ΔHm of the copolymers gradually decreased with increasing PL-contents, indicating their decreased crystallinity. Biodegradation test of the copolymers in compost demonstrated their improved biodegradability in comparison with the homopolymer of LA.

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We evaluated the effect of a ß-propiolactone (BPL)-inactivated coxsackievirus A16 (CA16) vaccine, using three immunogenicity evaluation and two animal challenge systems. A CA16 virus strain, named 419, was used as the production strain. Another CA16 strain, named 1131, was isolated and used as the challenge strain in intracerebral inoculation of neonatal mice for the calculation of median lethal dose (LD 50). In the passive and maternal antibody-protection challenge systems, all results indicated that the vaccine could protect mouse pups from lethal challenge with the CA16 virus. In the immunogenicity systems, three types of animal (mouse, rat, and cynomolgus monkey), were immunized with the 419/CA16 vaccine. The dose-effect relationship and the antibody-generation routine were described. The CA16 vaccine induced a more potent serum antibody effect in rat than in mouse. The serum antibody titer was detectable more than 63 days after the initial vaccination. We also identified tools to evaluate the effect of the BPL-inactivated CA16 vaccine.

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Studies have been initiated to find compounds that can trap direct-acting carcinogens within the lumen of the gastrointestinal tract and thus prevent these carcinogens from attacking tissues of the host. Sodium 4-mercaptobenzene sulfonate (4-MBSNa) is a potent nucleophile and was found to react rapidly in vitro with the direct-acting carcinogen beta-propiolactone (BPL). In further investigations 4-MBSNa was shown to inhibit mutagenesis resulting from exposure of Salmonella typhimurium strain TA-100 to BPL and a second direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine. Subsequent experiments were performed to determine if 4-MBSNa would inhibit BPL-induced carcinogenesis in vivo. In the first of these, 4-MBSNa was administered by p.o. intubation to female A/J mice 5 min before p.o. administration of BPL. Under these conditions inhibition of carcinogenesis of the forestomach occurred. In a second experiment, 4-MBSNa was given by rectal intubation 5 min before BPL also administered intrarectally. Administration of BPL intrarectally produced adenomatous polyps of the large intestine. The occurrence of these neoplasms was inhibited by the prior administration of 4-MBSNa. The data presented show that 4-MBSNa has the capacity to trap direct-acting carcinogens and to inhibit the occurrence of BPL-induced neoplasia.

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Three direct-acting carcinogens, beta-propiolactone (BPL), methylmethane sulfonate (MMS), and dimethylcarbamyl chloride (DMCC), were evaluated for their carcinogenic potencies in the nasal mucosa of rats and for their abilities to bind in vivo to rat nasal mucosal DNA. The relative carcinogenic potencies of BPL and MMS corresponded well with their overall levels of binding to nasal mucosal DNA. DMCC, however, the most potent carcinogen of the three compounds, produced the lowest level of binding to nasal mucosal DNA. These results indicate that the DNA adducts formed by DMCC in rat nasal mucosa DNA are more readily expressed as cancer than those formed by BPL or MMS.

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The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen beta-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0-7.5, 37 degrees C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N4-bis(2-carboxyethyl)deoxycytidine (3,N4-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N4-BCEdCyd were hydrolyzed (1.5 N HCl, 100 degrees C, 2 h) to 3-CEC and 3,N4-bis(2-carboxyethyl)cytosine (3,N4-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d27) tri-TMS derivative of 3-CEC. The structure of 3,N4-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. Ei and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMs derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N4-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N4-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)-adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)-thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0-5 degrees C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H2O at 37b degrees C and pH 7.0-7.5.

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In vitro reaction of beta-propiolactone (BPL) with calf thymus DNA and mouse liver DNA followed by acid (HCL) hydrolyses of the BPL-reacted DNA's resulted in the isolation of a new compound, 1-(2-carboxyethyl)-adenine (1-CEA). The structure was assigned on the basis of ultraviolet spectra at acidic, alkaline and neutral pH and electron impact and chemical ionization mass spectra as well as chemical synthesis of 1-CEA from BPL and 2'-adenosine-5'-monophosphoric acid. The only other compound previously isolated from the in vitro and in vivo reactions of BPL and DNA was 7-(2-carboxyethyl)guanine (7-CEG) which we also identified as a product of our in vitro reaction. Under the conditions used the main product of alkylation was 1-CEA and the ratios of the concentrations of 1-CEA to 7-CEG was approx 3 : 1. The possible effect of the formation of 1-CEA on the structure of DNA and its role in chemical carcinogenesis is discussed.

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