Divergent molecular assembly and catalytic mechanisms between bacterial and archaeal RNase P in pre-tRNA cleavage.

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single-molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from E. coli (Escherichia coli) and Mja (Methanocaldococcus jannaschii) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between E. coli and Mja at both the RNA and protein levels. Specifically, the RNA of E. coli RNase P (EcoRPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of E. coli RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, Mja RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of Mja RPR. Mutation experiments suggest that the functional deficiency of Mja RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for Mja RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms.

Characterizing the Specific Recognition of Xanthurenic Acid by GEP1 and GEP1-GCα Interactions in cGMP Signaling Pathway in Gametogenesis of Malaria Parasites.

Gametogenesis is an essential step for malaria parasite transmission and is activated in mosquito by signals including temperature drop, pH change, and mosquito-derived xanthurenic acid (XA). Recently, a membrane protein gametogenesis essential protein 1 (GEP1) was found to be responsible for sensing these signals and interacting with a giant guanylate cyclase α (GCα) to activate the cGMP-PKG-Ca2+ signaling pathway for malaria parasite gametogenesis. However, the molecular mechanisms for this process remain unclear. In this study, we used AlphaFold2 to predict the structure of GEP1 and found that it consists of a conserved N-terminal helical domain and a transmembrane domain that adopts a structure similar to that of cationic amino acid transporters. Molecular docking results showed that XA binds to GEP1 via a pocket similar to the ligand binding sites of known amino acid transporters. In addition, truncations of this N-terminal sequence significantly enhanced the expression, solubility, and stability of GEP1. In addition, we found that GEP1 interacts with GCα via its C-terminal region, which is interrupted by mutations of a few conserved residues. These findings provide further insights into the molecular mechanism for the XA recognition by GEP1 and the activation of the gametogenesis of malaria parasites through GEP1-GCα interaction.

Identification of a novel substrate motif of yeast separase and deciphering the recognition specificity using AlphaFold2 and molecular dynamics simulation.

Separase is a giant cysteine protease and has multiple crucial functions. The most well-known substrate of separase is the kleisin subunit of cohesin, the cleavage of which triggers chromosome segregation during cell division (Uhlmann et al., 1999; Kamenz and Hauf, 2016) [1,2]. Recently, separase has also been found to cleave MCL-1 or BCL-XL proteins to trigger apoptosis (Hellmuth and Stemmann, 2020) [3]. Although substrate recognition through a short sequence right upstream of the cleavage site is well established, recent studies suggested that sequence elements outside this minimum cleavage site are required for optimal cleavage activity and specificity (Rosen et al., 2019; Uhlmann et al., 2000) [4,5]. However, the sequences and their underlying mechanism are largely unknown. To further explore the substrate determinants and recognition mechanism, we carried out sequence alignments and found a conserved motif downstream of the cleavage site in budding yeast. Using Alphafold2 and molecular dynamics simulations, we found this motif is recognized by separase in a conserved cleft near the binding groove of its inhibitor securin. Their binding is mutually exclusive and requires conformation changes of separase. These findings provide deeper insights into substrate recognition and activation of separase, and paved the way for discovering more substrates of separase.

A Guided Vehicle under Fire Conditions Based on a Modified Ultrasonic Obstacle Avoidance Technology.

Low visibility and hot smoke environment under fire conditions can largely hamper the related fire rescue processes. Ultrasound obstacle avoidance technology is then useful for guidance. However, the biggest challenge of adopting ultrasound technology comes from accurate distance measurements under the disturbances of high temperature and soot particle concentration. It is critical to measure the propagation speed under the complicated fire conditions. Therefore, in this study, a baffle calibration method was proposed to improve the accuracy of distance measurement of an obstacle. The method is based on two ultrasound measurement systems, while one is used to calibrate the propagation speed of ultrasound based on the fixed distanced baffle and the other is for the dynamic measurement of obstacle distance based on the calibrated speed. The viability of this method on the guided vehicle was confirmed based on the experiments. From its comparison to those existing methods, such as constant speed and temperature compensation methods, it was known from that the proposed baffle calibration method provides the best prediction. It was obtained that the maximum errors based on the baffle calibration method are 2.75% and 2.62% under the two representative fire scenarios, respectively, which are much lower than those of constant speed (7.81% and 8.4%) and temperature compensation methods (10.4% and 5.12%).

[Content and Health Risks of Microplastics and Phthalate Esters in Bottled Water].

To study the content and health risks of microplastics （MPs） and phthalate esters （PAEs） in bottled water, a quantitative analysis of MPs was conducted by using Rose Bengal staining and stereomicroscopy. Seven PAEs were quantified by using gas chromatography-triple quadrupole tandem mass spectrometry （GC-MS/MS）. The daily intake of MPs was estimated and the carcinogenic and non-carcinogenic risks of PAEs were evaluated through a health risk assessment model. The results showed that the abundance of MPs in 21 bottled waters ranged from 48 n·L-1 to 216 n·L-1 （with the median abundance of 88 n·L-1）. The majority （72.1%） of MPs were fibrous in shape, and fragments accounted for only 27.9%. The average proportion of small-sized （10-50 µm） MPs was 33.9%, and that of large-sized MPs （>500 µm） was 4.3%. Most MPs were blue. The ∑(PAEs) in bottled water was 1.15-2.47 µg·L-1 （average 1.62 µg·L-1）. PAEs detected with high frequencies （100%） included dimethyl phthalate （DMP）, diethyl phthalate （DEP）, diisobutyl phthalate （DIBP）, di-n-butyl phthalate （DBP）, and di（2-ethylhexyl） phthalate （DEHP）, while the detection frequencies of butylbenzyl phthalate （BBP） and di-n-octyl phthalate （DNOP） were relatively low. The concentrations of DBP, DEHP, and DEP were all below the standard limits for drinking water in China. The ∑(PAEs) in the migration experiments was 0.61-2.04 µg·L-1 （average 1.33 µg·L-1）. The migration amounts of DBP and DEHP were also within the allowable range under the condition of 60℃ for 10 days. Seven PAEs were detected in both the bottles and caps, and the average content of DEHP in bottles was the highest, while DBP had the highest content in caps. The estimated intake of MPs （EDI） by drinking bottled water in different age groups of humans was 2.87 n·ï¼ˆkg·d）-1 for adults, 3.87 n·ï¼ˆkg·d）-1 for children, and 5.85 n·ï¼ˆkg·d）-1 for infants. The carcinogenic risks of DEHP in 21 bottled water samples and the migration test were less than the maximum acceptable risk level （1×10-6）, and the non-carcinogenic risk indices （HIs） of PAEs were all less than 1, indicating no non-carcinogenic risk to humans； however, the risk value of infants and children was higher than that of adults and should not be ignored.

Occurrence, migration and health risks of fluorescent whitening agents and phthalates in bottled water.

The occurrence and health risks of fluorescent whitening agents (FWAs) in bottled water were reported for the first time. FWA184 and FWA393 were the most frequently detected FWAs, with mean concentrations of 3.99-17.00 ng L-1. Phthalates (PAEs) such as dibutyl phthalate (DBP), di-iso-butyl phthalate (DiBP), and diethylhexyl phthalate (DEHP) were prevalent in bottled water, with mean levels of 40.89-716.66 ng L-1, and their concentrations in bottled water were much higher than those of FWAs. FWAs and PAEs in bottles and caps were extracted using organic solvent, and the correlation analysis showed that FWA393 and DEHP most likely originated from bottles, while bottle caps were the main sources of DBP and DiBP. The calculated risk quotients (RQs) of target substances and all age groups were considerably lower than the threshold of 0.1, indicating that consuming bottled water containing these plastic additives was unlikely to pose health risks for people of all ages. However, RQ values for underage people were several times higher than those for adults and hence cannot be neglected; therefore, special attention should be paid to understand the potential risks posed by the exposure to these plastic additives during early life stages, especially the infant stage.

Pollution characteristics, ecological and health risks of herbicides in a drinking water source and its inflowing rivers in North China.

This study measured the pollution characteristics and ecological and health risks of 19 herbicides found in drinking water sources and their inflowing rivers. The targeted herbicides were prevalent in the study area, but most concentrations were well below 10 ng L-1. Acetochlor and atrazine were the dominant herbicides, although their levels were much lower than previously reported. Total herbicide residual levels were greater in April than in December and increased from upstream to downstream, resulting in the highest pollution levels found in the reservoirs, likely due to herbicides delivered from upstream and dense agricultural planting in the surrounding areas. Only atrazine and ametryn presented moderate ecological risks, while the summed risk quotients (ΣRQs) of each sample were >0.1, indicated that the total herbicide levels represented a moderate risk in all samples. For the human health risks, the risk quotients (RQ) of all target herbicides, the total RQs of each sample, and estimated life-stage RQs were far smaller than the 0.2 threshold, indicating the absence of human health risks when the water was consumed at any stage of life. However, early life stages exhibited 3-6 times higher RQ values than adulthood and should not be overlooked. And crucially, the synergistic or antagonistic effects of mixed herbicides are not well understood, and further research is needed to understand the impact of these herbicides on the ecosystem and human health, particularly possible affects in early life stages, such as infants and children.