Co-exposure of butyl benzyl phthalate and TiO2 nanomaterials (anatase) in Metaphire guillelmi: Gut health implications by transcriptomics.

During the COVID-19 pandemic, an abundance of plastic face masks has been consumed and disposed of in the environment. In addition, substantial amounts of plastic mulch film have been used in intensive agriculture with low recovery. Butyl benzyl phthalate (BBP) and TiO2 nanomaterials (nTiO2) are widely applied in plastic products, leading to the inevitable release of BBP and nTiO2 into the soil system. However, the impact of co-exposure of BBP and nTiO2 at low concentrations on earthworms remains understudied. In the present study, transcriptomics was applied to reveal the effects of individual BBP and nTiO2 exposures at a concentration of 1 mg kg-1, along with the combined exposure of BBP and nTiO2 (1 mg kg-1 BBP + 1 mg kg-1 nTiO2 (anatase)) on Metaphire guillelmi. The result showed that BBP and nTiO2 exposures have the potential to induce neurodegeneration through glutamate accumulation, tau protein, and oxidative stress in the endoplasmic reticulum and mitochondria, as well as metabolism dysfunction. The present study contributes to our understanding of the toxic mechanisms of emerging contaminants at environmentally relevant levels and prompts consideration of the management of BBP and nTiO2 within the soil ecosystems.

Transcriptional Analysis of lncRNA and Target Genes Induced by Influenza A Virus Infection in MDCK Cells.

BACKGROUND: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. RESULTS: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. CONCLUSIONS: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

CDC20 is a potential target gene to inhibit the tumorigenesis of MDCK cells.

MDCK is currently the main cell line used for influenza vaccine production in culture. Previous studies have reported that MDCK cells possess tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, host cell DNA released after cell lysis may still pose a risk for tumorigenesis. Greater caution is needed in the production of human vaccines; therefore, the use of gene editing to establish cells incapable of forming tumors may significantly improve the safety of influenza vaccines. Knowledge regarding the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells is crucial; however, our understanding remains superficial. Through monoclonal cell screening, we previously obtained a cell line, CL23, that possesses significantly reduced cell proliferation, migration, and invasion abilities, and tumor-bearing experiments in nude mice showed the absence of tumorigenic cells. With a view to exploring tumorigenesis-related genes in MDCK cells, DIA proteomics was used to compare the differences in protein expression between wild-type (M60) and non-tumorigenic (CL23) cells. Differentially expressed proteins were verified at the mRNA level by RT-qPCR, and a number of genes involved in cell tumorigenesis were preliminarily screened. Immunoblotting further confirmed that related protein expression was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of the influenza virus; therefore, CDC20 was preliminarily determined to be an effective target gene for the inhibition of cell tumorigenicity. These results contribute to a more comprehensive understanding of the mechanism underlying cell tumorigenesis and provide a basis for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.

TGM2 inhibits the proliferation, migration and tumorigenesis of MDCK cells.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.

RSAD2 Is an Effective Target for High-Yield Vaccine Production in MDCK Cells.

Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.

Population origin, maternal effects, and hydric conditions during incubation determine embryonic and offspring survival in a desert-dwelling lizard.

While the effects of incubation environment on embryonic development and offspring traits have been extensively studied in oviparous vertebrates, studies into how genetic inheritance (population origin), maternal effects, and incubation environment interact to produce varying phenotypes, are rare. To elucidate the interactive role of those three factors during incubation in shaping offspring phenotypes through hydric conditions, we conducted a fully factorial experiment [arid and semiarid populations × maternal dry and wet treatments (MDT and MWT) × embryonic dry and wet treatments (EDT and EWT)] with a desert-dwelling lacertid lizard (Eremias argus). Female lizards in dry conditions produced larger clutch sizes but smaller eggs. The incubation period and hatching success were significantly affected by embryonic but not by maternal moisture treatments. Eggs in the EDT hatched later than those in the EWT in both arid and semiarid populations. Hatching success was lower in EDT than in EWT in the semiarid population, but not in the arid population. Hatchlings from the EDT had a slower post-hatch increase in body mass than those from the EWT. EDT would decrease the survival rates of hatchlings in the semiarid population only. In addition, structural equation models revealed that population had a stronger effect on embryonic and offspring survival than maternal and embryonic moisture. Our study demonstrates locally adaptive strategies of drought resistance at multiple life-history stages in lizard populations from diverse hydric habitats and highlights the importance of genetic factors in determining embryonic drought resistance in oviparous lizards.

Insights into Persistent Toxic Substances in Protective Cases of Mobile Phones: Occurrence, Health Risks, and Implications.

Despite the popularity of smartphones worldwide, persistent toxic substances (PTSs) in protective cases of mobile phones (PCMPs) and their health risks via direct skin contact have been ignored. This study investigated PTSs in PCMPs made in China with different materials and sales territory and their potential harm to human health. Polybrominated diphenyl ethers (PBDEs, 6.40 ng/g), new brominated flame retardants (NBFRs, 144 ng/g), organophosphate esters (OPEs, 10.1 µg/g), short-chain chlorinated paraffins (SCCPs, 3.58 µg/g), medium-chain chlorinated paraffins (MCCPs, 3.17 µg/g), and heavy metals (HMs, 72.3 µg/g) were detected. It was found that the different concentrations and compositions depend on the material, region, and use. Moreover, the raw materials used to fabricate PCMPs are of variable quality and may include recycled plastic waste. There are no standard quality specifications for PCMPs, and different materials have different properties, including specific surface area and adsorption ability. The risk assessment performed by Monte Carlo simulations indicated that the PTSs evaluated pose no health risks to the general population and may have adverse effects on individual high-exposure populations. According to the results of this work, it is suggested that more stringent global specifications for the selection of raw materials should be established, including the content and structural characteristics of PTSs, limitations on the use of additives in the production process, and the handling after use.

[Effects of Vehicle Emissions on Heavy Metals and Polycyclic Aromatic Hydrocarbons Pollution in Road Dust in Xinxiang].

In this study, samples were taken of the surface dust of main roads in Xinxiang City, and the concentrations of five heavy metals (Cd, Pb, Cr, Cu and Zn) and fifteen polycyclic aromatic hydrocarbons (PAHs) were determined by inductively coupled plasma mass spectrometry (ICP-MS) and gas chromatography-mass spectrometry (GC-MS), respectively. Meanwhile, the effects of vehicle emissions on the pollution characteristics were investigated. The results showed that the concentrations of heavy metals and PAHs ranged from 2.58 to 1560 mg·kg-1 and ND to 1.30 mg·kg-1, respectively. Overall, the concentrations of heavy metals and PAHs increased with a decrease in dust particle size. In terms of composition, the heavy metals were dominated by Zn while the high-molecular-weight PAHs were mainly homologous. In spatial distribution, the concentrations of heavy metals and PAHs were different. The total concentrations of heavy metals in road dust near Renmin Road, Xiaodian Industrial Park, and Cement Plant were the highest, while the high concentrations of PAHs appeared in the dust of Renmin Road, Upper Expressway, and 107 National Highway. Pearson correlation analysis showed that there was no positive correlation between the five heavy metals and fifteen PAHs. Then cluster analysis and factor analysis indicated that the PAHs were greatly affected by vehicle emissions, while the heavy metals were basically unaffected.