Simultaneously improving the activity and thermostability of hyperthermophillic pullulanase by modifying the active-site tunnel and surface lysine.

Pullulanases are important starch-debranching enzymes that mainly hydrolyze the α-1,6-glycosidic linkages in pullulan, starch, and oligosaccharides. Nevertheless, their practical applications are constrained because of their poor activity and low thermostability. Moreover, the trade-off between activity and thermostability makes it challenging to simultaneously improve them. In this study, an engineered pullulanase was developed through reshaping the active-site tunnel and engineering the surface lysine residues using the pullulanase from Pyrococcus yayanosii CH1 (PulPY2). The specific activity of the engineered pullulanase was increased 3.1-fold, and thermostability was enhanced 1.8-fold. Moreover, the engineered pullulanase exhibited 11.4-fold improvement in catalytic efficiency (kcat/Km). Molecular dynamics simulations demonstrated an anti-correlated movement around the entrance of active-site tunnel and stronger interactions between the surface residues in the engineered pullulanase, which would be beneficial to the activity and thermostability improvement, respectively. The strategies used in this study and dynamic evidence for insight into enzyme performance improvement may provide guidance for the activity and thermostability engineering of other enzymes.

Understanding the Role of Low-Dose Polystyrene Microplastic in Copper Toxicity to Rice Seed (Oryza sativa L.).

There is still much to learn with respect to the potential for microplastics (MPs) to interact with environmental toxins and biota. In the present study, we investigated the effect of MPs on the toxicity of copper (Cu) to rice seeds (Oryza sativa L.). The 7-day median effective concentration (EC50) value of MPs on rice seed germination was 864 mg/L (95% confidence interval [CI] 839 to 897 mg/L). We found that MPs slightly reduced Cu toxicity to rice seeds. The 7-day EC50 of Cu on rice seed germination increased from 7.29 mg/L (95% CI 7.10-7.52 mg/L) to 7.93 mg/L (95% CI 7.58-8.08 mg/L) in the presence of 20 mg/L MPs. We examined this toxicity reduction phenomenon by investigating the role of MPs in the process of Cu transport, Cu accumulation, and metabolic responses. Further investigation found that the MPs used in the present study hardly adsorbed Cu, but these MPs accumulated on the coats of rice seeds and significantly reduced Cu accumulation in rice seedlings. When Cu concentration was 10 mg/L, the presence of MPs reduced the accumulation of Cu in rice seedlings by 34%. We also found that, compared with only Cu present, the addition of MPs resulted in lower reactive oxygen species accumulation and higher catalase activity and glutathione levels in rice seedlings, which also contributed to Cu toxicity reduction. Collectively, the present study shows that polystyrene MPs have the potential to form associations with plant structures which can ultimately impact heavy metal bioaccessibility and therefore toxicity. Environ Toxicol Chem 2024;00:1-10. © 2024 SETAC.

Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

BACKGROUND: Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

Iron minerals: A frontline barrier against combined toxicity of microplastics and arsenic.

The coexistence of microplastics (MPs) and arsenic (As) in terrestrial ecosystems presents challenges to controlling soil pollution and performing environmental risk assessments. In this study, the interactions among As, polystyrene MPs, and goethite in porous media were investigated and the individual and combined toxicities of MPs and As on wheat germination were evaluated. An additional experiment was conducted to assess the mitigating effect of goethite on the toxicity of the two contaminants. The results showed that the presence of MPs reduced As accumulation in wheat and decreased the acute lethal toxicity of As pollutants (the half-lethal concentration of As during wheat germination increased by 68.21%). However, MPs exhibited inhibitory effects on wheat germination and served as carriers to promote the migration of As within the plant body. The addition of goethite mitigated both individual and combined toxicities and further increased the half-lethal concentration for the combined pollution of As and MPs by 39.48%. This was primarily attributed to the adsorption and immobilization of arsenate and MPs on the medium and root surfaces. In our study, goethite reduced soluble As by 48.29% under the combined pollution scenarios and formed iron plaques on wheat roots, effectively obstructing pollutant entry. Thus, iron minerals serve as pioneering barriers to combined toxicity. Our findings contribute to the understanding of the combined toxicity of MPs and As in crops and offer potential strategies for managing combined pollution.

Using multi-technology to characterize transboundary Hg pollution in the largest presently active Hg deposit in China.

Active Hg mines are primary sources of Hg contamination in the environment of mining districts and surrounding areas. Alleviation of Hg pollution requires knowledge of pollution sources, migration, and transform pathways across various environmental media. Accordingly, the Xunyang Hg-Sb mine, the largest active Hg deposit in China, presently was selected as the study area. GIS, TIMA, EPMA, µ-XRF, TEM-EDS, and Hg stable isotopes were adopted to investigate the spatial distribution, mineralogical characteristics, in situ microanalysis, and pollution sources of Hg in the environment medium at the macro- and micro-levels. The total Hg concentration in samples showed a regional distribution, with higher levels in areas close to the mining operations. The in situ distribution of Hg in soil was mainly associated with the mineralogical phases of quartz, and Hg was also correlated with Sb and S. Hg was also found to be rich mainly in quartz minerals in the sediment and showed different distributions of Sb. Hg hotspots had S abundances and contained no Sb and O. The contributions from the anthropogenic sources to soil Hg were estimated to be 55.35%, among which 45.97% from unroasted Hg ore and 9.38% from tailing. Natural input of soil Hg due to pedogenic processes accounted for 44.65%. Hg in corn grain was mainly derived from the atmosphere. This study will provide a scientific basis for assessing the current environmental quality in this area and minimizing further impacts that affect the nearby environmental medium.

Effects of arbuscular mycorrhizal fungi on frond antimony enrichment, morphology, and proteomics in Pteris cretica var. nervosa during antimony phytoremediation.

Pteris cretica var. nervosa is a dominant fern species found in antimony (Sb) mining areas, capable of forming symbiosis with arbuscular mycorrhizal fungi (AMF), especially with those members of the Glomus genus. Despite this fern's relevance and the potential contribution of mycorrhizal symbiosis to phytoremediation, the AMF's impact on P. var. nervosa phytoremediation of Sb remains unknown. Here, we exposed P. var. nervosa to different concentrations of Sb for 6 months. Our results showed that Sb reduced shoot biomass, enlarged the root/shoot ratio, and disrupted the fronds' intracellular structure. AMF inoculation, however, was able to moderate these phenotypic changes and increased the accumulation level of Sb in plants. From a proteomics analysis of this plant's fronds, a total of 283 proteins were identified. Notably, those proteins with catalytic function, carbon fixing and ATP metabolic function were highly enriched. K-means clustering demonstrated protein-changing patterns involved in multiple metabolic pathways during exposure to Sb. Further, these patterns can be moderated by AMF inoculation. Pearson correlations were used to assess the plant biomarkers-soil Sb relationships; This revealed a strong correlation between ribosome alteration and the root/shoot ratio when inoculated with AMF, and a positive correlation between photosynthesis proteins and chlorophyll (SPAD value). Our results indicate AMF could moderate the fronds impairment by maintaining the sufficient protein levels for ribosomal functioning, photosynthesis activity and to counter ROS production. We demonstrate the effective use of AMF associated with P. cretica var. nervosa for Sb phytoremediation and the potential of applying proteomics to better understand the mechanism behind this symbiotic plant physiological response.

Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

Ebola virus (EBOV) disease outbreaks have resulted in many fatalities, yet no licensed vaccines are available to prevent infection. Recombinant glycoprotein (GP) production may contribute to finding a cure for Ebola virus disease, which is the key candidate protein for vaccine preparation. To explore GP1,2 expression in BmN cells, EBOV-GP1,2 with its native signal peptide or the GP64 signal peptide was cloned and transferred into a normal or gp64 null Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid via transposition. The infectivity of the recombinant bacmids was investigated after transfection, expression and localization of EBOV-GP were investigated, and cell morphological changes were analyzed by TEM. The GP64 signal peptide, but not the GP1,2 native signal peptide, caused GP1,2 localization to the cell membrane, and the differentially localized GP1,2 proteins were cleaved into GP1 and GP2 fragments in BmN cells. GP1,2 expression resulted in dramatic morphological changes in BmN cells in the early stage of infection. However, GP1,2 expression did not rescue GP64 deficiency in BmNPV infection. This study provides a better understanding of GP expression and processing in BmN cells, which may lay a foundation for EBOV-GP expression using the BmNPV baculovirus expression system.