Emissions and Secondary Formation of Air Pollutants from Modern Heavy-Duty Trucks in Real-World TrafficChemical Characteristics Using On-Line Mass Spectrometry

Complying with stricter emissions standards, a new generation of heavy-duty trucks (HDTs) has gradually increased its market share and now accounts for a large percentage of on-road mileage. The potential to improve air quality depends on an actual reduction in both emissions and subsequent formation of secondary pollutants. In this study, the emissions in real-world traffic from Euro VI-compliant HDTs were compared to those from older classes, represented by Euro V, using high-resolution time-of-flight chemical ionization mass spectrometry. Gas-phase primary emissions of several hundred species were observed for 70 HDTs. Furthermore, the particle phase and secondary pollutant formation (gas and particle phase) were evaluated for a number of HDTs. The reduction in primary emission factors (EFs) was evident (∼90%) and in line with a reduction of 28–97% for the typical regulated pollutants. Secondary production of most gas- and particle-phase compounds, for example, nitric acid, organic acids, and carbonyls, after photochemical aging in an oxidation flow reactor exceeded the primary emissions (EFAged/EFFresh ratio ≥2). Byproducts from urea-selective catalytic reduction systems had both primary and secondary sources. A non-negative matrix factorization analysis highlighted the issue of vehicle maintenance as a remaining concern. However, the adoption of Euro VI has a significant positive effect on emissions in real-world traffic and should be considered in, for example, urban air quality assessments

Secondary Organic Aerosol Formation from Urban Roadside Air in Hong Kong

Motor vehicle emissions are an important but poorly constrained source of secondary organic aerosol (SOA). Here, we investigated in situ SOA formation from urban roadside air in Hong Kong during winter time using an oxidation flow reactor (OFR), with equivalent atmospheric oxidation ranging from several hours to several days. The campaign-average mass enhancement of OA, nitrate, sulfate, and ammonium upon OFR aging was 7.0, 7.2, 0.8, and 2.6 μg m–3, respectively. To investigate the sources of SOA formation potential, we performed multilinear regression analysis between measured peak SOA concentrations from OFR and the concentrations of toluene that represent motor vehicle emissions and cooking OA from positive matrix factorization (PMF) analysis of ambient OA. Traffic-related SOA precursors contributed 92.3%, 92.4%, and 83.1% to the total SOA formation potential during morning rush hours, noon and early afternoon, and evening meal time, respectively. The SOA production factor (PF) was approximately 5.2 times of primary OA (POA) emission factor (EF) and the secondary particulate matter (PM) PF was approximately 2.6 times of primary particles EF. This study highlights the potential benefit of reducing secondary PM production from motor vehicle emissions in mitigating PM pollutions

Conserved function of p200 family proteins in the inhibition of type I IFNs responses.

(A) Schematic diagram of the domain structure of p200 family proteins. (B) Co-immunoprecipitation (IP, with anti-Flag) and immunoblot (IB, with anti-HA) analysis of HEK293T cells that were transfected with plasmids encoding Flag-IRF7 and HA-(IFI204, IFI205, IFI209, MNDA, IFI16, MNDAL, AIM2, IFI202 and IFI16β) for 24 hrs. Cell lysate was analyzed by immunoblot with anti-Flag and anti-HA antibodies. (C) Dual luciferase assays for analyzing the promoter activity of IFNβ or IFNα4 in Irf3−/−Irf7−/−MEFs co-transfected with IFNβ or IFNα4 promoter luciferase reporter plasmids and the indicated plasmids, respectively. The Renilla expression plasmid (pRL-TK, 10 ng) was co-transfected as an internal control (upper graphs). qRT-PCR analysis of Ifnb or Ifna4 in Irf3−/−Irf7−/− MEFs transfected with the indicated plasmids (lower graphs). 24 hrs post-transfection, cells were treated with SeV for 12 hrs. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are representative of three independent experiments (mean ± SD in C).</p

IFI204 promotes the nuclear retention of IRF7.

(A) Immunoblot analysis of phosphorylation of IRF7 with rabbit mAb against phospho-IRF7-Ser437/438 (CST D6M2I) and β-actin in NIH3T3 cells infected by lentivirus-mediated shIFI204 or shNC as negative control. Cells were uninfected (UI) or infected with SeV or transfected with poly(I:C) or ssPolyU as indicated for 12 hrs. (B) Nucleo-cytoplasmic separation analysis of IRF7 in NIH3T3 cells infected by lentivirus-mediated shIFI204 or shNC as negative control. Cells were uninfected (UI) or infected with SeV or transfected with poly(I:C) or ssPolyU as indicated for 12 or 16 hrs. Nuclear lysates were tested by western blotting using anti-IRF7 and anti-lamin b1 antibodies. Levels of IRF7 was quantified by densitometry and normalized to lamin b1 protein levels. (C) Intracellular localization of endogenous IRF7 assessed by immunostaining followed by laser scanning confocal microscopy (left panel). NIH3T3 cells infected by lentivirus-mediated shIFI204 (NIH3T3-shIFI204) or shNC (NIH3T3-WT) were infected with SeV for 12 hrs. Then cells were washed with PBS and harvested for immunofluorescence analysis. Rabbit anti-IRF7 antibody was used as primary antibody and FITC-conjugated goat anti- rabbit IgG was used to detect IRF7 as shown in green fluorescence. DAPI (4, 6-diamidino-2-phenylindole) is used for nuclei staining as shown in blue fluorescence. The white arrow indicates the different protein levels of IRF7 in perinuclear region. Relative level of IRF7 in the nucleus was quantified by densitometry (right panel). *P < 0.05, **P < 0.01 and ***P < 0.001. Data are representative of two independent experiments (mean ± SD in A-C).</p

P200 family protein IFI204 is highly expressed in MHV-infected BMDCs.

(A) Schematic of the experiment. Bone marrow cells were isolated from mouse tibia and femur and cultured for 7 to 9 days in medium containing mouse GM-CSF. BMDCs were infected by MHV (MOI = 1) and collected at 18 hpi. The cells were collected and examined for transcriptome and proteome. The graph (right panel) shows the average value of three independent experiments of transcriptome. (B) Proteome, transcriptome and qRT-PCR analyses identified higher expression of Ifi204 in MHV-infected BMDCs at 18 hpi. (C and D) qRT-PCR analysis of Ifi204 and MHV mRNA7 in MHV-infected BMDCs (C) and Ifnar-/- BMDCs (D) at different time points as indicated. **P < 0.01 and ***P < 0.001. Data are representative of three independent experiments (mean ± SD in B).</p

IFI204 interacts with IRF7.

(A and B) Schematic diagram to show the regions of IFI204 interacting with IRF7 (upper panels in A and B). Co-immunoprecipitation (IP, with anti-Flag) and immunoblot (IB, with anti-HA) analysis of HEK293T cells that were transfected with plasmids encoding Flag-IRF7 and HA-IFI204 or its mutants. Cell lysate was analyzed by immunoblot with anti-Flag and anti-HA (lower panels in A and B). (C) GST pull-down assay. Purified GST-IFI204 or GST protein was incubated with MBP-IRF7 or MBP as indicated and pulled down with glutathione resin beads. Each sample was detected by Western blotting with the indicated antibodies. GST and MBP proteins were used as a negative control, respectively. (D) Intracellular localization of IFI204 and IRF7 assessed by immunostaining followed by laser scanning confocal microscopy. Irf3−/−Irf7−/−MEFs cells were co-transfected with pFlag-IRF7 and pHA-IFI204 stimulated with SeV for 0 h (mock) and 6 hrs (SeV) at 24 hrs post-transfection. Then cells were washed with PBS and harvested for immunofluorescence analysis. Mouse anti-HA antibody was used as primary antibody and CY3-conjugated goat anti-mouse IgG was used to detect IFI204 as shown in red fluorescence. Rabbit anti-flag antibody was used as primary antibody and FITC-conjugated goat anti-rabbit IgG was used to detect IRF7 as shown in green fluorescence. DAPI (4, 6-diamidino-2-phenylindole) is used for nuclei staining as shown in blue fluorescence. Data are representative of three independent experiments.</p

Proposed molecular mechanism for the function of p200 family proteins (IFI204) to inhibit IRF7-mediated activation of type I IFNs responses.

Schematic diagram demonstrates the mechanism of p200 family proteins to suppress type I IFNs responses. Late after RNA virus infection, the highly expressed p200 family proteins, such as IFI204, interact with IRF7 in the nucleus and such interactions lead to the negative regulation of type I IFNs responses.</p

IFI204 inhibits IRF7-mediated activation of type I IFNs responses.

(A) qRT-PCR analysis of Ifnb and Ifna4 in Irf3−/−Irf7−/−MEFs transfected with IFI204, IRF3, IRF7 or IRF7-Δ247–467 mutant (IRF7Δ) as indicated. 24 hrs post-transfection, cells stimulated with HSV-1 (MOI = 1) or SeV for 12 hrs as indicated. The unstimulated (US) treatment was used as a control. (B) ELISA analysis of the expression of IFNα in Irf3−/−Irf7−/−MEFs transfected with IFI204, IRF7 or IRF7-Δ247–467 mutant (IRF7Δ) as indicated. 24 hrs post-transfection, cells were stimulated with HSV-1 (MOI = 1) or SeV for 12 hrs as indicated. The unstimulated (US) treatment was used as a control. (C) Dual luciferase assays for analyzing the promoter activity of IFNβ (left panel) and IFNα4 (right panel) in Irf3−/−Irf7−/−MEFs co-transfected with 200 ng IFNβ or IFNα4 promoter luciferase reporter plasmids and 250 ng IFI204, IRF3, IRF7 or IRF7-Δ247–467 mutant (IRF7Δ) as indicated. The Renilla expression plasmid (pRL-TK, 10 ng) was co-transfected as an internal control. 24 hrs post-transfection, cells were stimulated with SeV for 12 hrs as indicated. The unstimulated (US) treatment was used as a control. (D) Dual luciferase assays for analyzing the promoter activity of IFNα4 in HEK293T cells co-transfected with 200 ng IFNα4 promoter luciferase reporter plasmid, 250 ng IFI204 and IRF7 as indicated. The Renilla expression plasmid (pRL-TK, 10 ng) was co-transfected as an internal control. 24 hrs post-transfection, cells were stimulated with SeV for 12 hrs as indicated. The unstimulated (US) treatment was used as a control. Not significant (ns), **P < 0.01 and ***P < 0.001. Data are representative of three independent experiments (mean ± SD in A-D).</p

IFI204 inhibits IRF7 binding with its promoter.

(A) CHIP analysis of the specific binding of IRF7 and its promoter DNA. IFI204 or IFI204 mutant was co-transfected with IRF7 into Irf3−/−Irf7−/−MEFs, which were unstimulated (US) or stimulated by SeV. Gel electrophoresis of the PCR products using Ifnb promoter primers and GAPDH primers (left panel). Levels of Ifnb promoter DNA were quantified by densitometry (right panel). (B and C) EMSA analysis of IRF7 binding with its promoter region. GST-IFI204, MBP-IRF7, GST and MBP were mixed with CY5-labeled PRD I or NC sequence probes as indicated. The NC sequence, GST and MBP are negative controls. Levels of shifted DNA and free DNA were quantified by densitometry (lower panel in C). **P < 0.01 and ***P < 0.001. Data are representative of two independent experiments.</p

IFI204 inhibits the production of type I IFNs in RNA virus-infected cells.

(A) qRT-PCR analysis of Ifnb, Ifna4 and Ifna6 in NIH3T3 cells with stable knockdown of IFI204 by lentivirus-mediated shIFI204. The cells were unstimulated (US) or stimulated with SeV, poly(I:C) (10 μg/ml) or ssPolyU (10 μg/ml) as indicated for 8 hrs. (B) qRT-PCR analysis of Ifnb, Ifna4 and Ifna6 in BMDCs with knockdown of IFI204 by lentivirus-mediated shIFI204. The cells were unstimulated (US) or stimulated with MHV (MOI = 1) or SeV as indicated for 8 hrs. (C and D) ELISA analysis of the production of IFNα in IFI204-knockdown NIH3T3 cells (C) and BMDCs (D). (E) qRT-PCR analysis of Ifnb and Ifna4 in IFI16-/- and IFI16+/+ A549 cells after stimulation with SeV, poly(I:C) (10 μg/ml) or ssPolyU (10 μg/ml) for 8 hrs. US, unstimulated control. **P < 0.01 and ***P < 0.001. Data are representative of three independent experiments (mean ± SD in A-E).</p