Unfolding Protein-Based Hapten Coupling via Thiol-Maleimide Click Chemistry: Enhanced Immunogenicity in Anti-Nicotine Vaccines Based on a Novel Conjugation Method and MPL/QS-21 Adjuvants.

Vaccines typically work by eliciting an immune response against larger antigens like polysaccharides or proteins. Small molecules like nicotine, on their own, usually cannot elicit a strong immune response. To overcome this, anti-nicotine vaccines often conjugate nicotine molecules to a carrier protein by carbodiimide crosslinking chemistry to make them polymeric and more immunogenic. The reaction is sensitive to conditions such as pH, temperature, and the concentration of reactants. Scaling up the reaction from laboratory to industrial scales while maintaining consistency and yield can be challenging. Despite various approaches, no licensed anti-nicotine vaccine has been approved so far due to the susboptimal antibody titers. Here, we report a novel approach to conjugate maleimide-modified nicotine hapten with a disulfide bond-reduced carrier protein in an organic solvent. It has two advantages compared with other approaches: (1) The protein was unfolded to make the peptide conformation more flexible and expose more conjugation sites; (2) thiol-maleimide "click" chemistry was utilized to conjugate the disulfide bond-reduced protein and maleimide-modified nicotine due to its availability, fast kinetics, and bio-orthogonality. Various nicotine conjugate vaccines were prepared via this strategy, and their immunology effects were investigated by using MPL and QS-21 as adjuvants. The in vivo study in mice showed that the nicotine-BSA conjugate vaccines induced high anti-nicotine IgG antibody titers, compared with vaccines prepared by using traditional condensation methods, indicating the success of the current strategy for further anti-nicotine or other small-molecule vaccine studies. The enhancement was more significant by using MPL and QS-21 than that of traditional aluminum adjuvants.

Environmental-related doses of afidopyropen induced toxicity effects in earthworms (Eisenia fetida).

Afidopyropen has high activity against pests. However, it poses potential risks to the soil ecology after entering the environment. The toxicity of afidopyropen to earthworms (Eisenia fetida) was studied for the first time in this study. The results showed that afidopyropen had low level of acute toxicity to E. fetida. Under the stimulation of chronic toxicity, the increase of reactive oxygen species (ROS) level activated the antioxidant and detoxification system, which led to the increase of superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. Lipid peroxidation and DNA damage were characterized by the increase of malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents. Meanwhile, the functional genes SOD, CAT, GST, heat shock protein 70 (HSP70), transcriptionally controlled tumor protein (TCTP), and annetocin (ANN) played a synergistic role in antioxidant defense. However, the comprehensive toxicity of high concentration still increased on the 28th day. In addition, strong histopathological damage in the body wall and intestine was observed, accompanied by weight loss, which indicated that afidopyropen inhibited the growth of E. fetida. The molecular docking revealed that afidopyrene combined with the surface structure of SOD and GST proteins, which made SOD and GST become sensitive biomarkers reflecting the toxicity of afidopyropen to E. fetida. Summing up, afidopyropen destroys the homeostasis of E. fetida through chronic toxic. These results provide theoretical data for evaluating the environmental risk of afidopyropen to soil ecosystem.

Fluopicolide-Induced Oxidative Stress and DNA Damage in the Earthworm Eisenia foetida.

Fluopicolide is a new benzamide fungicide with a unique mechanism of action and is toxic to some non-target organisms. However, there is a lack of research on the chronic toxicity of fluopicolide to earthworms. In this study, in order to evaluate the chronic toxicity of fluopicolide to earthworms, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and DNA oxidative damage (8-hyoxy-2-deoxyguanosine content) in earthworms were measured at 7, 14, 21, and 28 days after exposure to different concentrations (0, 0.1, 0.5, 1, 2.5, 5, and 10 mg/kg) of fluopicolide. In most treatment groups, the ROS levels increased significantly 7 days after exposure and then decreased gradually with an increase in exposure time, a certain dose-effect relationship. The antioxidant enzymes' activities (SOD and CAT) in most treatment groups were activated, showing an increasing trend at first and then a decreasing trend; however, the CAT activity in the high-concentration treatment group was inhibited 21 days after exposure. The GST activity and MDA content showed an increasing trend at first and then a decreasing trend, which was dependent on the dose. As a biomarker of DNA damage, the 8-OHdG content was positively correlated with the concentration of fluopicolide. The results showed that a low dose of fluopicolide could cause oxidative stress and DNA damage in earthworms.

Effect of different exposure times and doses of cyantraniliprole on oxidative stress and genotoxicity in earthworms (Eisenia fetida).

Cyantraniliprole, the second generation of diamide insecticides, is widely used to control various pests, which will certainly result in adverse effects on earthworms in soil. In this study, after exposure with six doses of cyantraniliprole (0, 0.5, 1, 2.5, 5, and 10 mg kg-1) by artificial soil method, six biomarkers, four functional genes, and histopathological changes of Eisenia fetida were measured on the 7th, 14th, 21st, and 28th days. The comprehensive toxicity was assessed by the IBR version 2 (IBRv2) method. The results showed that the reactive oxygen species (ROS) level was induced significantly. The superoxide dismutase (SOD) activity was activated in 7-28 days. The catalase (CAT) and glutathione S-transferases (GST) activities were also activated in the initial 14 days. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) contents in the high treatment increased until the late stage of exposure. On the 28th day, the metallothionein (MT) and calreticulin (CRT) genes were up-regulated, the transcriptionally controlled tumor protein (TCTP) gene was down-regulated. The SOD gene showed a good correlation with SOD activity. Extensive histopathological damage was found in the endoderm and ectoderm of E. fetida. The 5 and 10 mg kg-1 treatments showed higher comprehensive toxicity than the 0.5, 1, and 2.5 mg kg-1 treatments on the 28th day. These results suggest that cyantraniliprole exerted certain subchronic toxic effects of oxidative stress, DNA damage, and histopathological changes to E. fetida, which provided theoretical basis for rational use of cyantraniliprole and evaluation of its safety to soil environment.

Sensitivity Differences and Biochemical Characteristics of Laodelphax striatellus (Fallén) to Seven Insecticides in Different Areas of Shandong, China.

Laodelphax striatellus Fallén is one of the main pests that can severely harm rice, corn, and wheat. Insecticides acting on the nicotinic acetylcholine receptor (nAChR) are the main type of pesticides used for the control of L. striatellus in Shandong Province, a major grain-producing region in China. In this study, the rice seedling dipping method was used to determine the sensitivities of six field L. striatellus populations in Shandong to seven insecticides acting on nAChR. The results showed that all the field populations were sensitive to clothianidin, nitenpyram, and triflumezopyrim, and the Jiaxiang population exhibited the lowest resistance ratio (RR) to imidacloprid, dinotefuran, sulfoxaflor, and thiamethoxam. The Donggang population showed a medium-level resistance to imidacloprid, with the highest RR of 17.48-fold. The Yutai population showed low-level resistance to imidacloprid and thiamethoxam, with RRs of 7.23- and 7.02-fold, respectively. The contents of cytochrome P450 monooxygenase (P450s), carboxylesterase (CarE), and glutathione S-transferase (GST) were the highest in the Donggang population and the lowest in the Jiaxiang population. The P450 gene CYP314A1 and the CarE gene LsCarE12 were highly up-regulated in all populations. No mutations of V62I, R81T, and K265E in the nAChR ß1 subunit were found in any of the populations. These results provide valuable information for the strategies of resistance management of L. striatellus in the field.

Oxidative stress and DNA damage in earthworm (Eisenia fetida) induced by triflumezopyrim exposure.

Triflumezopyrim is an excellent pesticide for preventing and controlling rice planthoppers. It is widely used in the production of field rice and mainly through spraying with some inadvertent loss of pesticide to the soil. The future may bring development and utilization of seeds containing triflumezopyrim which will certainly impact earthworms. To evaluate the toxic effects of triflumezopyrim on earthworms, reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, malondialdehyde content (MDA), glutathione S-transferase (GST), and DNA oxidative damage (8-hydroxy-2'-deoxyguanosine, 8-OHdG) were measured after 7, 14, 21, and 28 d analyzing the effects of 6 concentrations (0, 0.5, 1, 2.5, 5 and 10 mg/kg) of triflumezopyrim, respectively. ROS content did not change significantly in the early stages but showed a significant dose-effect relationship in the late stages. Antioxidant enzymes were activated in most treatment groups, and catalase activity was the same as that in the control group at 28 d. GST activity showed a trend of increasing first and then decreasing whereas MDA content had no obvious change in trend. 8-OHdG showed significant positive correlation with the concentration of triflumezopyrim at 28 d, indicating that the degree of DNA oxidative damage increased accordingly. The results showed that chronic exposure of triflumezopyrim may cause both oxidative stress and DNA damage in earthworms and alter the activity of antioxidant enzymes.

Solvothermal synthesis of well-dispersed ZnSe microspheres.

Well-defined crystalline ZnSe microspheres have been fabricated successfully via a simple solvothermal route under mild conditions in the presence of ethylenediamine tetraacetic acid disodium salt (Na(2)EDTA) and octadecylamine (ODA). X-ray powder diffraction (XRD), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectrum (EDS), X-ray photoelectron spectra (XPS), Fourier transform-infrared spectroscopy (FT-IR), and photoluminescence spectra (PL) were used to characterize the phase structures, morphologies, and properties of the products. The result of XRD demonstrated that we obtained ZnSe products crystallized well with a stilleite structure. The influencing elements such as reaction time, sodium hydroxide (NaOH), addition of chelating reagent Na(2)EDTA, and the water/ethanol volume ratio were investigated, and the possible formation mechanism was also proposed. Furthermore, the experimental result indicated that ZnSe microspheres showed blue emission under ultraviolet excitation.