Highly Efficient Catalytic Ozonization at Ultralow Temperatures of Multicomponent VOCs over the Pt/CeO₂ Catalysts

Industrial flue gas has a great impact on the atmosphere environment and human health, and its emission temperatures are usually below 180 °C, which needs a new technology that can catalyze the removal of the multicomponent VOCs over high-performance catalysts in the presence of ozone. In this work, we prepared the Pt/CeO2 catalysts with different morphologies of Pt particles and investigated their catalytic performance for the ozonization of mixed VOCs (i.e., toluene and chlorobenzene (CB)). Among all of the as-prepared samples, Pt NRs/CeO2 with nanorod-like Pt particles showed excellent catalytic performance for the ozonization of toluene and CB. The T50% (the temperature at VOC conversion = 50%) values for toluene and CB ozonization were 40 and 48 °C at a space velocity of 40,000 mL g–1 h–1, respectively. The results of characterization revealed that the reactive oxygen species involved in the VOC ozonization were mainly the O2– and O22– species, surface oxygen vacancies of CeO2 were the active sites for the conversion of ozone to the reactive oxygen species, and the O2– species was the mainly active oxygen species in the low-temperature VOC oxidation. Furthermore, partial reactive oxygen species reacted with the Ptn+ species to generate more amount of the Pt0 species, and the metallic platinum species was the main active site for the adsorption and activation of toluene and CB. The chemisorbed VOCs at the Pt0 sites reacted with the reactive oxygen species at the interface of Pt and CeO2, resulting in the excellent low-temperature catalytic activity. Compared with the reaction without ozone participation, we find that the participation of ozone can not only decrease the reaction temperature but also reduce the production of toxic byproducts. We are sure that the Pt/CeO2 catalyst is promising in practical application for elimination of the VOCs from industrial flue gas

The emergence of new antigen branches of H9N2 avian influenza virus in China due to antigenic drift on hemagglutinin through antibody escape at immunodominant sites

Vaccination is a crucial prevention and control measure against H9N2 avian influenza viruses (AIVs) that threaten poultry production and public health. However, H9N2 AIVs in China undergo continuous antigenic drift of hemagglutinin (HA) under antibody pressure, leading to the emergence of immune escape variants. In this study, we investigated the molecular basis of the current widespread antigenic drift of H9N2 AIVs. Specifically, the most prevalent h9.4.2.5-lineage in China was divided into two antigenic branches based on monoclonal antibody (mAb) hemagglutination inhibition (HI) profiling analysis, and 12 antibody escape residues were identified as molecular markers of these two branches. The 12 escape residues were mapped to antigenic sites A, B, and E (H3 was used as the reference). Among these, eight residues primarily increased 3`SLN preference and contributed to antigenicity drift, and four of the eight residues at sites A and B were positively selected. Moreover, the analysis of H9N2 strains over time and space has revealed the emergence of a new antigenic branch in China since 2015, which has replaced the previous branch. However, the old antigenic branch recirculated to several regions after 2018. Collectively, this study provides a theoretical basis for understanding the molecular mechanisms of antigenic drift and for developing vaccine candidates that contest with the current antigenicity of H9N2 AIVs.</p

LMP promotes NDV-induced apoptosis by inducing mitochondrial dysfunction via CTSB and CTSD.

(A&B) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h or mock-transfected. The MMP level was detected by flow cytometry (A) or fluorescence microscope (B) using JC-1 probe staining following infection with Herts/33 at 0.01 MOI for 12h or mock-infected. Scale bars, 100 μm. (C&D) Quantitation of JC-1 red to green fluorescence ratio detected by flow cytometry (C) and fluorescence microscope (D) by ImageJ software are shown, respectively. (E-G) Indicated proteins were detected by Western blotting in HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints (E). The relative intensity of Bcl-2 (F) and tBid (G) were quantified by ImageJ software. GAPDH was used as a normalized control. (H-J) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h or mock-transfected. Subsequently, the indicated proteins were detected by Western blotting following infection with Herts/33 at 0.01 MOI or mock-infection for 12h (H). The relative intensity of Bcl-2 (I) and tBid (J) in NDV-infected cells were quantified by ImageJ software. GAPDH was utilized as a normalized control. (K-O) HeLa cells were transfected with si-CTSB or si-CTSD, or si-NC for 48h or mock-transfected. The expression levels of Bax and Cyt C in the cytoplasmic and mitochondrial fractions were determined by Western blotting after infection with Herts/33 at 0.01 MOI or mock-infection for 12h (K). The relative intensity ratio of Bax/GAPDH (L), Cyc/GAPDH (M), Bax/COX IV (N), and Cyc/COX IV (O) were quantified by ImageJ software, respectively. GAPDH was used as a cytoplasmic fraction control and COX IV was used as a mitochondrial fraction control. All error bars represent SDs for triplicate analyses of three independent experiments. All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.</p

The sialidase activity of the HN protein mediates the deglycosylation and degradation of LAMP1 and LAMP2.

(A) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with plasmid expressing HN protein and treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) at the indicated time points post-transfection. (B&C) HeLa cells were transfected with plasmids expressing HN protein or mock-transfected. Then the cells were treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) or DMSO or untreated for 48h. The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies. Scale bars, 20 μm (B). Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (C). (G) Sialidase activity was measured in HeLa cells transfected with the indicated plasmids for 48h and is presented as relative change to the cells transfected with plasmids expressing wild-type HN. The red dotted line indicates the difference threshold. (F) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with the indicated plasmids or mock-transfected for 48h. (D&E) The interaction between HN and LAMP1 (D) or LAMP2 (E) in HeLa cells was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids for 48h. (H) The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with the indicated plasmids or mock-transfected for 48h. Scale bars, 20 μm. (I) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are calculated by ImageJ software. (J) The apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with indicated plasmids or mock-transfection for 48h. Error bars are SDs for a triplicate analysis of three independent experiments (G&J), or SDs for 15 cells (C&H). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.</p

NDV infection induces deglycosylation and degradation of LAMP1 and LAMP2.

(A&B) HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints. The mRNA levels (A) and fluorescence images (B) of LAMP1 and LAMP2 were measured by RT-qPCR or observed by confocal microscopy, respectively. The mRNA levels of LAMP1 and LAMP2 were normalized to GAPDH using the 2−ΔΔCt method. Scale bars, 20 μm. (C-E) HeLa cells infected with Herts/33 at 0.01 MOI for indicated time points or infected with Herts/33 at indicated MOIs for 24h. The protein levels of LAMP1 and LAMP2 (C) or LAMP3, LIMPII, and LAPTM5 (E) were assessed by Western blotting. The deglycosylated LAMP1 and LAMP2 in PNGase F-treated cell lysates were analyzed by Western blotting (E). (F-H) HeLa cells were infected with Herts/33 at 0.01 MOI for 12h or mock-infected. The remaining amounts of LAMP1 and LAMP2 after treatment with CHX (20 μg/mL) for indicated time points were measured using Western blotting (F). The remaining amount of LAMP1 (G) and LAMP2 (H) was calculated as the fold change of the protein present at 0h. (I-K) NDV-infected (0.01 MOI, 6h) HeLa cells were co-treated with CHX (20 μg/mL) and NH4CL (50 mM), or Ca-074 (10 μM), or Pep A (10 μM), or 3-MA (5mM), or MG132 (5 μM) or mock-treated for 12h. LAMP1 and LAMP2 protein levels in cell lysates were analyzed by Western blotting (I). The remaining amounts of LAMP1 (J) and LAMP2 (K) were calculated as the fold change of the protein to mock-treated cells at 0h. All quantitative data represent means ± SD (n = 3). Significance was assessed using Two-way ANOVA with Dunnett’s multiple comparisons test (A), or unpaired two-tailed Student’s t-test (G&H), or One-way ANOVA with Dunnett’s multiple comparisons test (J&K).</p

HN protein induces apoptosis in HeLa cells.

HeLa cells were transfected with a plasmid expressing HN protein (1.6 μg), or empty vector plasmid (1.6 μg), or mock-transfected. The apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry after transfection for 48h. Quantitation of apoptosis rate is calculated as means ± SD (n = 3) and shown in the right panel. Significance was assessed using One-way ANOVA with Dunnett’s multiple comparisons test. (TIF)</p

Sequence for siRNA.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.</div

LMP facilitates NDV replication in HeLa and DF-1 cells.

(A&B) HeLa (A) and DF-1 (B) cells were pretreated with LLoMe (500 nM), Ca-074 (5 μM), Pep A (5 μM), or mock-treated for 3h, respectively. Subsequently, the cells were infected with Herts/33 at 0.01 MOI along with the corresponding chemicals in the cell culture medium. Culture supernatants were collected at the indicated time points, and viral titers were determined by the 50% tissue culture infectious dose (TCID50) assay. (C&D) HeLa (C) and DF-1 (D) cells were pretreated and infected as described above. Protein levels of NP and HN were assessed by Western blotting after 24h of NDV infection. The relative intensity ratio of the indicated proteins, normalized to GAPDH, was quantified using ImageJ software and is presented on the left side of the panel. All error bars are SDs for a triplicate analysis of three independent experiments. Significance was assessed using Two-way ANOVA with Dunnett’s multiple comparisons test (A&B) or one-way ANOVA with Dunnett’s multiple comparison test (C&D). (TIF)</p

CTSB and CTSD exacerbate NDV-induced LMP by inducing the generation of ROS.

(A) Intracellular ROS levels were measured by flow cytometry using DCFH-DA staining in HeLa cells infected with Herts/33 at 0.01 MOI for different timepoints or mock-infected. (B) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h, or mock-transfected, and intracellular ROS levels were measured by flow cytometry using DCFH-DA staining following infection with Herts/33 at 0.01 MOI or mock-infection for 12h. (C) HeLa cells were pretreated with LLoMe (500 nM), DMSO, or mock-untreated for 3h, respectively. Subsequently, the intracellular ROS levels were measured by flow cytometry using DCFH-DA staining cells following infection with Herts/33 at 0.01 MOI or mock-infection for 24h. (D) HeLa cells were treated with NAC (2 mM) for 24h after absorption with Herts/33 at 0.01 MOI for 1h. LMP levels were evaluated by confocal microscopy using indicated antibodies. Scale bars, 20 μm. Manders’ Colocalization Coefficients of galectin 3 with LAMP1 were quantified by ImageJ software and shown on the right. Error bars represent SDs for triplicate analyses of three independent experiments (A-C) or SDs for 15 cells (D). All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.</p

LMP promotes NDV-induced apoptosis through the involvement of CTSB and CTSD.

(A) Schematic diagram of the experimental design for (B-G). HeLa cells or DF-1 cells were infected with Herts/33 at 0.01 MOI for 24h. Then, the cells were treated with Ca-074 (20 μM), Pep A (20 μM), DMSO, or mock-treated for 6h. (B&C&E&F) The apoptosis level of HeLa cells (B) or DF-1 cells (E) was measured by AnnexinV-FITC/PI staining using flow cytometry, and quantitation of the apoptosis rate of HeLa cells (C) and DF-1 cells (F) are shown, respectively. (D&G) The NP protein in HeLa cells (D) or DF-1 cells (G) was detected by Western blotting. (H) Schematic diagram of the experimental design for (I-M). HeLa cells were transfected with si-CTSB, si-CTSD, si-NC, or mock-transfected for 48h. Then, the cells were infected with Herts/33 at 0.01 MOI or mock-infected for 12h. (I-M) The indicated proteins were detected by Western blotting (I). The relative intensity of cleaved caspase 3 (J), cleaved caspase 7 (K), cleaved caspase 9 (L), and cleaved PARP1 (M) in NDV-infected cells were quantified by ImageJ software. GAPDH was used as a normalized control. All error bars represent SDs for triplicate analyses of three independent experiments. All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.</p