Composition, characteristics, and treatment technologies of condensable particulate matter present in flue gas emitted by coking plants in China.

To study the total particulate matter (TPM) in flue gas emitted by coking plants, a sampling system that could be used to collect filterable particulate matter (FPM) and condensable particulate matter (CPM) was designed and developed based on Method 202 recommended by the U.S. Environmental Protection Agency in 2017 and HJ 836-2017 issued by China. Using this system, FPM and CPM in flue gas emitted by four coking furnaces named A, B, C, and D were tested in China. Further, 9 water-soluble ions, 20 elements, and organic matter present in the CPM were simultaneously examined to determine their formation mechanisms. Statistical data suggested that the FPM emission level in the coking flue gas was low and the average mass concentration was less than 10 mg/m3. However, with high CPM and TPM emission levels, the TPM mass concentrations of A, B, C, and D were 130 ± 11.1, 84.4 ± 6.36, 35.1 ± 17.0, and 63.8 ± 13.0 mg/m3, respectively. The main component of TPM was CPM, and the average mass concentration of CPM accounted for 98%, 95%, 68%, and 95% of TPM in furnaces A, B, C, and D, respectively. Water-soluble ions were the important components of CPM, and the total concentration of water-soluble ions accounted for 70%, 87%, 42%, and 66% of CPM in furnaces A, B, C, and D, respectively. Toxic and harmful heavy metals, such as Mn, Cr, Ni, Cu, Zn, As, Cd, and Pb, were detected in CPM. The formation mechanism of CPM was analyzed in combination with flue-gas treatment. It was shown that the treatment process "activated carbon- flue-gas countercurrent-integrated purification technology + ammonia spraying" used in furnaces A and B was less effective in removing CPM, water-soluble ions, metals, and compounds than that of "selective catalytic reduction denitrification + limestone-gypsum wet desulfurization (spraying NaOH solution)" in furnaces C and D. Hence, different flue-gas treatment technologies and operation levels played vital roles in the formation, transformation, and removal of CPM from flue-gas. Organic components in CPM discharged from furnace A were determined via gas chromatography-mass spectrometry, and the top 15 organic components in CPM were obtained using the area normalization method. N-alkanes accounted for the highest proportion, followed by esters and phenols, and most of them were toxic and harmful to humans and ecosystems. Therefore, advanced CPM treatment technologies should be developed to reduce atmospheric PM2.5 and its precursors to improve ambient air quality in China.

Development of a Time-Resolved Fluorescence Resonance Energy Transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Development of a Time-Resolved Fluorescence Resonance Energy Transfer ultra-high throughput screening assay for targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as acceptor. We have optimized the assay into 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Frequency-potency analysis of IgG+ memory B cells delineates neutralizing antibody responses at single-cell resolution.

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.