Listeria monocytogenes isolates from enoki mushrooms (23), smoked ducks (7), and processed ground meat products (30) were examined with respect to hemolysis, virulence genes, growth patterns, and heat resistance. The isolates that showed the highest pathogenicity and the lowest pathogenicity were analyzed to obtain the whole-genome sequence, and the sequences were further analyzed to identify genetic variations in virulence, low-temperature growth-related, and heat resistance-related factors. All isolates had β-hemolysis and virulence genes . At low temperatures, isolates with high growth  and low growth  were selected. Among them, L. monocytogenes SMFM 201804 SD 5-3 showed the highest resistance at 60°C and 70°C. The strains SMFM 201804 SD 5-3 (high-risk) and SMFM 2019-FV43 (low-risk) harbored 45 virulence genes; 41 single nucleotide variants (SNVs) were identified between these two isolates. A comparison of 26 genes related to low-temperature growth revealed 18 SNVs between these two isolates; a comparison of the 21 genes related to heat resistance revealed 16 SNVs. These results indicate that the differences in the pathogenicity of L. monocytogenes SMFM 201804 SD 5-3 and L. monocytogenes SMFM 2019-FV43 are associated with the SNVs identified in virulence genes, low-temperature growth-related genes, and heat resistance-related genes.In this study, genetic variations and characteristics of  NGS techniques are used in various fields, including verification of the functionality of probiotics and investigation of the causes of foodborne outbreaks. They have also been used to establish a database of genomic information for foodborne pathogenic bacteria and to analyze the safety of microorganisms . Whole-gutbreaks . In Irelisolates . WGS proisolates . The obj2.2.1.L. monocytogenes isolates from enoki mushrooms and other isolates (7 isolates from smoked ducks and 30 isolates from processed ground meat products) previously studied by The L. monocytogenes.A total of 127 enoki mushrooms were collected from supermarkets in Korea between August 2019 and March 2020. The samples were transported in a cooler to our laboratory. Briefly, 25 g of each sample was placed in sample bags  containing 225 mL of Listeria Enrichment Broth (Becton Dickinson and Company). The sample bags were hand-shaken for 1 min and incubated at 30°C for 48 h to enrich iap gene for the confirmatory test of L. monocytogenes [2O. The suspensions were heated at 99°C for 15 min, left at room temperature (25°C) for 3 min, and centrifuged at 5,989 × g at 4°C for 3 min; the supernatants were used as DNA templates. Real-time PCR amplification was performed using a Rotor-Gene SYBR® Green PCR Kit . Briefly, 1 μL DNA template was mixed with 2.5 μL of forward primer, 2.5 μL of reverse primer, 12.5 μL of SYBR green master mix, and 6.5 μL of dH2O. For iap amplification, an initial denaturation step at 95°C for 90 s and 35 cycles of denaturation at 95°C for 5 s, annealing at 56°C for 10 s, and extension at 72°C for 5 s were conducted. Finally, a 5-min extension was performed at 72°C. Amplified PCR products were loaded onto a 1.5% agarose gel and visualized under UV light.One-milliliter aliquots of the enriched cultures were spread-plated on Chrom agar , and the plates were incubated at 37°C for 24 h. The isolated colonies were subjected to real-time PCR analysis to investigate the presence of the ytogenes  with a 22.2.L. monocytogenes were inoculated in 10 mL tryptic soy broth with 0.6% yeast extract  and incubated at 30°C overnight. Then, 100-μL aliquots of the cultures were transferred to fresh 10 mL TSBYE and incubated at 30°C for another 24 h. The sub-cultures were streaked onto Columbia agar with 5% sheep blood  and incubated at 30°C for 48 h. Hemolytic properties  were determined by observing the zones formed around the bacterial colonies. β-hemolysis (complete hemolysis) was confirmed if a clear zone around the bacteria was observed.Isolated colonies of 2.3.L. monocytogenes isolates were suspended in 50 μL of 0.25% sodium dodecyl sulfate–0.05 N NaOH solution. One hundred microliters of sterile dH2O was added to the suspension, and the mixtures were incubated at 99°C for 15 min. Aliquots (2 μL) of the mixtures were mixed with the components of the Phire Hot Start II DNA Polymerase Kit , Taq DNA polymerase mix , 1X reaction buffer (1.5 mM MgCl2), 200 μM deoxynucleoside triphosphates (dNTPs), and 0.5 μM of each of the virulence gene primers. Five virulence genes  were detected with PCR using the primers listed in Isolated colonies of 2.4.L. monocytogenes isolates were cultured and then sub-cultured in TSBYE at 30°C overnight. The sub-cultures were centrifuged at 1,912 × g and 4°C for 15 min. Cell pellets were washed twice with phosphate-buffered saline (PBS), and the suspensions were diluted with PBS and inoculated at 5 Log CFU/mL. The aliquots (0.2 mL) of the inocula of each L. monocytogenes isolates were inoculated into tubes containing 20 mL of TSBYE and incubated at 4°C for 288 h. During incubation, the cultures of L. monocytogenes were diluted with buffered peptone water , and 0.1-mL aliquots of the diluents were plated on tryptic soy agar with 0.6% yeast extract . The plates were incubated at 30°C for 48 h, and the colonies on the plates were manually counted. The six for each strain were repeated for each time point.As described in the section “2.2. Analysis of hemolytic property,” 2.5.Aliquots (0.1 mL) of the inocula were placed on a 10-g beef rump. The inoculated samples were vacuum-packed and stored at 4°C for 20 days. The samples were analyzed every 5 days. The samples were aseptically removed from the vacuum bags and placed in filter bags (3 M™) containing 20 mL BPW. The samples were then pummeled for 1 min in a pummeler . Aliquots (1 mL) of the homogenates were diluted with BPW, and 0.1 mL of the diluents was spread-plated on Palcam agar (Becton Dickinson and Company). The plates were incubated at 30°C for 48 h, and the colonies on the plates were manually counted. The six for each strain were repeated for each time point.2.6.D-values  as follows:Aliquots (0.1 mL) of the inoculum were inoculated into tubes containing 9.9 mL of TSBYE; the tubes were preheated at 60°C and 70°C in a water bath. After 0, 3, 5, 8, and 10 min at 60°C and 0, 0.17, 3, 5, 8, and 10 min at 70°C, one-milliliter aliquots of the samples were retrieved and diluted with BPW, and 0.1-mL aliquots of the diluents were spread-plated on TSAYE, respectively. The plates were incubated at 30°C for 48 h, and the colonies on the plates were manually counted. The cell counts were then used to calculate T is temperature, t is time, Log N0 is the initial number of bacteria, and Log Nt is the number of bacteria remaining at time. The six for each strain were repeated for each time point.Where 2.7.2.7.1.g for 30 s. The cell pellets were used for extraction of DNA with the DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer’s protocol. Briefly, 5 μg of each extracted DNA sample was used to construct a library using the SMRTbell™ Template Prep Kit 1.0 (PN 100-259-100)  following the manufacturer’s instructions. Fragments smaller than 20 kb of the SMRTbell template were removed using the BluePippin size selection system  to construct large-insert libraries. The constructed library was validated using the 2100 Bioanalyzer  for quality control.After sub-culturing as described in the section “2.2. Analysis of hemolytic property” 3-mL aliquots of the sub-cultures were precipitated at 5,264  × 2.7.2.de novo assembly , and gene annotation and prediction were performed to analyze their genetic properties.The SMRTbell libraries were annealed to sequencing primers, and DNA polymerase was bound to the complex using the DNA/Polymerase Binding Kit P6 (Pacific Biosciences). This polymerase-SMRTbell-adaptor complex was loaded into SMRT cells (Pacific Biosciences) and sequenced using the PacBio RS II sequencing platform (Pacific Biosciences) which produce continuous long read (CLR). Long contigs were generated via 2.7.3.L. monocytogenes isolates were analyzed for 45 virulence factors, 26 low-temperature growth-related factors, and 21 heat resistance-related factors using CLC Genomics Workbench ver. 12.0 (Qiagen) , and genetic variations were assessed by comparing the sequences of each gene.Genetic characteristics of two high-risk and low-risk (Qiagen) . The seq2.8.® version 9.4 . Significant differences among strains were determined using analysis of covariance and a general linear model at α = 0.05.Statistical analysis was performed using SAS3.3.1.L. monocytogenes isolates from 127 enoki mushrooms (L. monocytogenes isolates (7 isolates from smoked ducks and 30 isolates from processed ground meat products) previously isolated .In this study, 23 ushrooms , and 37 isolated  were obt3.2.Listeria spp. which causes β-hemolysis is mostly pathogenic, and most of the isolated L. monocytogenes cause β-hemolysis  were detected in all L. monocytogenes isolates  and/or inlB and their interaction with the cellular receptors E-cadherin and/or Met, respectively (plcA and plcB encode phosphatidylinositol phospholipase C (PI-PLC) and phosphatidyl-choline phospholipase (PC-PLC), respectively. Both phospholipases are related to effective escape from the phagocytic vacuole into the cytoplasm , and L. monocytogenes SMFM 201803 SD 4-2 was in high growth group at five time points . L. monocytogenes SMFM 201804 SD 5-3 was in the high growth group at four points . L. monocytogenes SMFM 2019-FV43 was in the low growth group at all time points . L. monocytogenes SMFM 2020-BT30 was in low growth group at four time points , and L. monocytogenes SMFM 2019-FV42 was in low growth group at three time points . Three  with high growth and three  with low growth at 4°C were selected  had better survival under vacuum conditions than those with low growth  (L. monocytogenes isolates in low temperature survive long in vacuum conditions.Isolates with high and low growth (three each) at 4°C were further analyzed to compare their growth under vacuum conditions at 4°C. ty rates . Isolate20-BT30) . These r3.6.D60 and D70 values of the L. monocytogenes isolates, which describe heat resistance at 60°C and 70°C, ranged 2.37–3.55 min and 1.83–2.20 min, respectively  had higher D60 value than the others followed by L. monocytogenes SMFM 2019-FV42 (3.17 min), and the D70 value (2.20 min) of L. monocytogenes SMFM 201804 SD 5-3 was also higher than that of the other isolates  and L. monocytogenes SMFM 2019-FV43 (low cold- and heat-resistance) for growth at low temperature and survival in heat resistance. In the case of L. monocytogenes SMFM 201803 SD 1-1, the growth of the isolate was significantly higher than L. monocytogenes SMFM 2020-BT30, L. monocytogenes SMFM 2019-FV42, and L. monocytogenes SMFM 2019-FV43 at 4°C , in which the frequency of SNVs was high, were focused  are variations reflecting differences between a single base . The genisolates . The SNV focused . lntA innization . plcB enytoplasm . Dortet ng cells .L. monocytogenes SMFM 201804 SD 5-3. In particular, pathogenic genes  of the strain could improve attachment and invasion of host cells and affect the host cell immune response.Therefore, these variations of virulence genes may affect the high risk of 3.10.L. monocytogenes SMFM 2019-FV43 and L. monocytogenes SMFM 201804 SD 5-3 and also found genetic variations in these genes. In all, 26 genes were identified in the two isolates. Among these 26 genes, 18 SNVs were identified , in which the frequency of SNVs was high, were focused  and betL encode proteins involved in the betaine uptake system, and L. monocytogenes accumulates betaine when grown at low temperature, which functions as a cryoprotectant and osmoprotectant   in the former.We identified genes related to growth at 4°C for entified . The SNV focused . Reporteotectant . Single eratures . ltrC isg., 4°C) ; flhA anytogenes . In thisor 288 h . Thus, i3.11.L. monocytogenes SMFM 2019-FV43 and L. monocytogenes SMFM 201804 SD 5-3. We identified 21 genes related to heat resistance. For these 21 genes, 16 SNVs were identified  in L. monocytogenes under stress conditions and in infected hosts. Stress-induced clpC, clpP, and clpE proteins are crucial for virulence (L. monocytogenes SMFM 201804 SD 5-3.We analyzed heat resistance-related genes and genetic variations in entified . The SNVirulence . Regulatirulence . Stress 4.Listeria monocytogenes isolates have different risks, with different survival responses and pathogenicity under stressful conditions. These differences are caused by SNVs in virulence genes, low-temperature-related genes, and heat resistance-related genes. Our results support the position that L. monocytogenes SMFM 201804 SD 5-3 is a high-risk isolate whereas L. monocytogenes SMFM 2019-FV43 is a low-risk one.The datasets presented in this study can be found in the NCBI database. The Bioproject IDs are PRJNA958457 and PRJNA958458.JR, YC, and YY made significant contributions to the manuscript and agree to its publication. YY and YC conceived and designed the study. JR performed laboratory experiments. All authors analyzed the data, draft the manuscript, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC ( Adhatoda vasica; Malabar Nut), could attenuate airway epithelial cell (AEC) damage and ectopic MUC5AC expression in an in vitro model of WSPM-induced AEC/lung injury. Furthermore, it was investigated whether specific chemicals in Vasaka could replicate the effects of the tea. Despite the purported benefits of Vasaka in treating respiratory conditions carbazole (FICZ) [Regarding potential therapeutic opportunities based on Vasaka and use of Vasaka itself, several protective and mucosuppressing compounds were identified in the tea that could potentially be delivered systemically or directly to the lungs on an as-needed basis. These include selected flavonoids  and 9,10-EpOME. The finding that some oxylipins were TRPA1 antagonists was surprising as others have reported that they activate rodent TRPA1 , and we  in mice . While n in mice ,72 may pe (FICZ) . AccordiVasicine, vasicinone, isoorientin, isovitexin, vitexin, apigenin, 12,13-DiHOME, 9,10-DiHOME, 13(S)-HODE, 9(S)-HODE, 13-oxoODE, 9-oxoODE, 12,13-EpOME and 9,10-EpOME, 9-HpODE, and 7-ethoxyresorufin were purchased from Cayman Chemical . AITC, ionomycin calcium salt, HC-030039, and A967079 were from Sigma-Aldrich . DMSO was from Fisher Scientific .Preparation of pine WSPM was described in . Brieflyg for 2 min, followed by filtration of the supernatant using a 0.22 μm syringe filter. The Vasaka tea was used fresh by diluting into cell culture medium or stored frozen as a concentrate at −80 °C for up to 1 month. Blank medium processed in parallel was used as a negative control.Vasaka tea was prepared by incubating a suspension of organic Vasaka powder  in cell culture medium for 30 min at 37 °C. The tube was vortexed every 5 min, and after 30 min the suspension was centrifuged at 20,000× 2 atmosphere and sub-cultured every 2–3 days when cells reached ~80% confluence using trypsin. BEAS-2B cells were grown in LHC-9 medium (LHC-8 supplemented with 33 nM retinoic acid and 2.75 μM epinephrine), while HBEC3-KT cells were cultured in airway epithelial cell basal medium supplemented with a bronchial epithelial cell growth kit, 30 µg/mL geneticin, and 250 ng/mL puromycin.BEAS-2B (used only in a metabolomics study) and HBEC3-KT cells were purchased from ATCC . Cells were maintained in a humidified cell culture incubator at 37 °C with a 95% air:5% COc. cDNA (2 μg) was synthesized using the Applied Biosystems High-Capacity cDNA Synthesis Kit with RNase inhibitor. Transcript abundance was quantified using TaqMan gene expression master mix  on a Life Technologies QuantStudio 6 Flex instrument. The following TaqMan probe-based assays were used: human MUC5AC (Hs_01365616_m1); human HMOX1 (Hs01110250_m1); human DDIT3 (Hs_00358796_g1); human TRPV3 (Hs00376854_m1). The PCR programs used were according to supplier recommendations. Expression values were normalized to human β2-microglobulin , and to the average of the control samples.Cells were plated and treated at ~90% confluence. After treatment, total RNA was isolated using the Invitrogen PureLink RNA Mini Kit and quantified by UV absorbance on a Nanodrop One2 and assayed at 80–90%. Prior to assay, cells were loaded with Fluo-4 diluted 1:1 in calcium assay buffer  at ~23 °C for 1 h in the dark. After 1 h, the Fluo-4 was removed, and the cells were washed with calcium assay buffer containing 1 mM probenecid and 0.75 mM trypan red . Cells were pretreated for 30 min or 2 h with various agents by diluting in the wash buffer and in the desired treatment solutions . Assays were performed using an EVOS FL auto microscope with an on-stage environmental chamber maintained at 37 °C with a 95% air/5% CO2 atmosphere. Agonist treatments (AITC or WSPM) were added to cells at 3× the desired final concentration in calcium assay buffer. Images were captured every 6 s for 72 s. Changes in fluorescence were quantified using a custom MATLAB program as previously described [Calcium flux was measured using the Fluo-4 Direct assay kit and imaging on an EVOS FL Auto microscope at 10× magnification using a green fluorescent protein (GFP) filter as described ,46,74. CmRNA Sample Preparation kit. RNAseq was performed on a NovaSeq6000 S4 instrument with a 150 × 150 paired-end format and 100 million read-pair depth. Results were processed with University of Utah Bioinformatics core support. Optical duplicates were removed, adaptors were trimmed, and STAR alignments were generated. Differential gene expression was determined using hciR and DESeq2 (1.30.1) and a 5% false-discovery rate. Pathway analysis was performed using fgsea (1.18.0) using the Hallmarks Molecular Signatures Database. The data presented in this publication were deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE232172.RNA sequencing was performed at the High-Throughput Genomics Core Facility at the Huntsman Cancer Institute, University of Utah. Total RNA was extracted using the RNeasy Mini kit  with on-column DNase digestion. RNA quality was assessed by Agilent TapeStation, and library construction was performed using the Illumina TruSeq Stranded 2). Treatment medium was aspirated at 0, 4, and 24 h; the cells were rinsed 1× with 5 mL of cold phosphate-buffered saline, and immediately frozen at −80 °C. To each cell flask , 450 mL of cold 90% methanol containing the internal standard D4-succinic acid (Sigma-Aldrich) was added. The samples were vortexed, incubated at −20 °C for 1 h, and centrifuged at 20,000× g for 10 min at 4 °C. Supernatant (400 mL) was then transferred and mixed with D27-myristic acid (Sigma-Aldrich). Pooled quality control samples were prepared by removing a fraction of collected supernatant from each sample while process blanks were prepared using only extraction solvent, processed in parallel with the samples. All samples were then dried under vacuum prior to analysis. Samples were analyzed by gas chromatography/mass spectrometry (GC/MS) using an Agilent 7200 GC-QTOF fit with an Agilent 7693A autosampler. Dried samples were suspended in 40 µL of a 40 mg/mL methoxylamine hydrochloride (MOX)  in dry pyridine and incubated for 1 h at 37 °C. This solution (25 μL) was added to auto sampler vials to which 60 µL of N-methyl-N-trimethylsilyltrifluoracetamide  was added via the autosampler and incubated for 30 min at 37 °C. After incubation, samples were vortexed, and 1 µL of the prepared sample was injected into the GC inlet in the split mode with the inlet temperature held at 250 °C. A 5:1 split ratio was used for analysis, and any metabolite that saturated the instrument response at the 5:1 split was re-analyzed at a 50:1 split ratio. The gas chromatograph had an initial temperature of 60 °C for 1 min followed by a 10 °C/min ramp to 325 °C and a hold time of 10 min. A 30 m Agilent Zorbax DB-5MS with 10 m Duraguard capillary column was employed for chromatographic separation. Helium was used as the carrier gas at a rate of 1 mL/min. Data were quantified relative to the internal standard and relative analyte/metabolite peak areas. Processed data were then analyzed for statistical differences and metabolic pathway perturbations using Metaboanalyst 5.0 [Confluent monolayers of BEAS-2B cells grown in T-25 flasks were treated for 0, 4 or 24 h with medium containing 0.2% DMSO or WSPM  and converted to movies using an Image Express PICO . Image files (.jpg or .png) were analyzed using Cellpose 2.0 ,77 with 18 column  at 50 °C with gradient elution. The mobile phases were (A) 0.1% formic acid in H2O and (B) methanol and a flow rate of 300 mL/minute. The percentage of B varied as follows: 2% B at 0 min, 2%→95% (0 to 7.5 min), hold at 95% (7.5 to 8.5 min), 95%→2% (8.5 to 8.6 min), and hold at 2% from 8.6 to 10 min. A convex gradient (setting = 6 during the 0–7.5 min ramp) was used. The mass spectrometer was operated in positive electrospray ionization, selective reaction-monitoring mode. The MS/MS parameters were optimized by infusion of vasicine into the MS/MS system and the following precursor→product transitions were used: m/z 189.1→m/z 171.1 for vasicine, m/z 203.2→m/z 185.1 for vasicinone, m/z 449.1→m/z 431.1 for isoorientin, m/z 433.2→m/z 415.1 for isovitexin, and m/z 271.1→m/z 271.1 + 243.1 for apigenin. Data acquisition and processing were performed using Xcalibur software, and calibration curves were prepared in water using analyte peak area versus concentration.Vasicine, vasicinone, isoorientin, isovitexin, and apigenin were quantified using a Thermo Vanquish Flex UPLC System interfaced with an LTQ Velos Pro Linear Ion Trap Mass Spectrometer . Chromatographic separation of the analytes was achieved using a BEH C18 column  at 50 °C with gradient elution. The mobile phases were (A) 0.1% formic acid in H2O and (B) methanol and a flow rate of 100 mL/min. The percentage of B varied as follows: 2% B at 0 min, 2%→100% (0 to 50 min), hold at 100% (50 to 55 min), 100%→2% (55.0 to 55.1 min), and hold at 2% (55.1 to 60 min). The mass spectrometer was programmed to assay both positive and negative ions m/z 100–750 (in separate acquisitions) using the “double play” data-dependent MS/MS mode. MS/MS scans were triggered by analytes having a signal intensity set at ~5× baseline using a collision energy of 30% or 45% (separate acquisitions). The top four peaks of each scan were analyzed, and dynamic exclusion was active. The parameters were as follows: repeat count = 3; repeat duration = 5 s; exclusion list size = 25; and exclusion duration = 5 s. Data were analyzed using GNPS. The .raw files were converted to mzML files using the GNPS quickstart feature. The files were then uploaded into GNPS and processed using the Library Search feature. Default criteria were used with the exception that the minimum matched peaks = 4 and top hits per spectrum = 5.The identification of additional components of Vasaka tea was achieved using positive and negative electrospray LC/MS/MS using a Thermo Vanquish Flex UPLC System interfaced with an LTQ Velos Pro Linear Ion Trap Mass Spectrometer, coupled with dataset interrogation/spectral matching using the Global Natural Products Social (GNPS) Molecular Networking Knowledge Base . Chromat18 column  at 35 °C with gradient elution. The mobile phases were (A) 0.1% acetic acid in H2O and (B) 9:1 acetonitrile/isopropanol, and a flow rate of 250 mL/minute was used. The percentage of B varied as follows: 52.5% B at 0 min, 52.5%→57.5% (0 to 6 min), 62.5% at 6.1 min, 62.5%→77% (6.1 to 12.5 min), 77%→95% (12.5 to 16 min), 95% hold (16 to 19 min), 95%→5.25% (19 to 19.1 min), and 52.5% hold until 22 min. Negative electrospray ionization selective reaction monitoring was used. The MS/MS parameters were optimized by infusion of lipid standard  solutions into the MS/MS system. The precursor→product transitions used were as follows: m/z 313.2→m/z 194.0 + 183.1 for 12(13)-DiHOME, m/z 313.2→m/z 201.1 + 171.1 for 9(10)-DiHOME, m/z 295.3→m/z 195.1 + 171.1 for 13(S) and 9(S)-HODE, as well as 12(13)- and 9(10)-EpOME, and m/z 293.2→m/z 185.1 + 113.1 for 13- and 9-oxoODE. The precursor-to-product ion transitions used for the internal standards were as follows: m/z 317.2→m/z 185.1 for 9(10)-EpOME-d4, m/z 317.2→m/z 203.1 for 12(13)-EpOME-d4, m/z 299.2→m/z 172.0 for 9(10)-DiHOME-d4, and m/z 299.2→m/z 198.0 for 12(13)-DiHOME-d4. Data acquisition and processing were performed using Xcalibur software with calibration curves prepared in water using the ratio of analyte to internal standard peak.Quantification of 9(10)- and 12(13)-EpOME; 9(10)- and 12(13)-DiHOME, 9(S)- and 13(S)-HODE, and 9- and 13-oxoODE was achieved using a Thermo Vanquish Flex UPLC System interfaced with an LTQ Velos Pro Linear Ion Trap Mass Spectrometer. Chromatographic separation was achieved using a BEH Ct-test was used. One- or two-way ANOVA with Tukey’s or a Bonferroni post-test was used for multiple comparisons. A p-value <0.05 was considered significant.Graphing and statistical analyses were performed using GraphPad Prism 9.5.0 software. Values are represented as the mean ± SD unless stated otherwise. For comparisons between two groups, the unpaired MUC5AC expression in vitro. These effects were attributable to an attenuation of TRPA1-induced ER stress and, to a lesser extent, oxidative stress by selected flavones  and linoleic-derived oxylipins  present in the tea. It is possible that these mechanisms may underlie the purported beneficial effects of Vasaka and Vasaka tea in treating respiratory malaise, and that a standardized Vasaka tea/supplement or pure apigenin and/or 9,10-EpOME may provide therapeutic benefit.A tea prepared from a Vasaka supplement was found to inhibit the TRPA1 receptor and attenuate WSPM-induced AEC damage and ectopic


To improve the performance of the developed biosensor, the surface of the GR/DGNs/(PD/GOx)3 electrode was modified with polypyrrole (Ppy) for 5 h. A glucose biosensor based on a GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was characterized using a wide linear dynamic range of up to 39.0 mmol L−1 of glucose, sensitivity of 3.03 µA mM−1 cm−2, limit of detection of 0.683 mmol L−1, and repeatability of 9.03% for a 29.4 mmol L−1 glucose concentration. The Ppy-based glucose biosensor was characterized by a good storage stability (τ1/2 = 9.0 days). Additionally, the performance of the developed biosensor in blood serum was investigated.Reagentless electrochemical glucose biosensors were developed and investigated. A graphite rod (GR) electrode modified with electrochemically synthesized dendritic gold nanostructures (DGNs) and redox mediators (Med) such as ferrocenecarboxylic acid (FCA), 1,10-phenathroline-5,6-dione (PD), N,N,N′,N′-tetramethylbenzidine (TMB) or tetrathiafulvalene (TTF) in combination with glucose oxidase (GOx)  were developed and electrochemically investigated. A biosensor based on threefold-layer-by-layer-deposited PD and GOx (GR/DGNs/(PD/GOx) Interest in accurate, inexpensive, sensitive, selective, miniature, and rapid electrochemical biosensors ,2,3,4,5 S) are considered to be one of the types of nanomaterials with great promise in electronics and biomedical applications [4− ions are reduced to Au0; second, atomic gold clusters are formed with the increasing Au0 concentration; and third, these clusters act as seeds for DGN formation and growth [4− ion concentration, (ii) the viscosity of the solution, (iii) the overpotential [−1 cm−2 to glucose in the presence of a redox mediator [−1 cm−2 [The performance of glucose biosensors, as well as their longer operational stability and sensitivity, depends on many factors, including proper glucose oxidase (GOx) immobilization. For this purpose, the adsorption of GOx on the working electrode surface is ineffective in most cases; thus, additional crosslinking of GOx, covalent binding to a pre-modified surface or encapsulation in polymers is required ,5,9. Addications ,17,18,19d growth ,20,21. Totential , and  [2PD(red)), and then H2PD(red) is reoxidized on the working electrode surface [+ and TMB2+ ) [+ and TTF2 + (+0.36 and +0.71 V vs. Pt/Hg/Hg2Cl2(sat. KCl)) [In enzymatic biosensors, β-D-glucose is oxidized by GOx to D-glucono-1,5-lactone, whereas oxygen is reduced to hydrogen peroxide ,22,23. I/GA/GOx) . Another/GA/GOx) . In elec surface ,32. Nano surface . TMB or t. KCl))  or TTF+ t. KCl)) .2O2, which is formed during the enzymatic GOx reaction, by generating radical cations of pyrrole [Conducting polymers, e.g., polypyrrole (Ppy), have attracted great interest in clinical and environmental practices, electrocatalysis, and biomolecule immobilization due to their efficient transfer of electric charge and flexibility ,37. Elec pyrrole ,42. The  pyrrole ,36,39. TThe aim of the present study was to select a suitable redox mediator for the development of reagentless glucose biosensors based on DGNs, to investigate the impact of the polypyrrole layer on the performance of the developed biosensors, and to test an analytical system for the determination of glucose in the serum.Aspergillus niger, 208 units mg−1 protein)—were purchased from Carl Roth GmbH + Co.KG  and Fluka , respectively. The solution of sodium acetate (SA) buffer (0.05 mol L−1 sodium acetate trihydrate (CH3COONa·3H2O) with 0.1 mol L−1 potassium chloride (KCl) was prepared by mixing CH3COONa·3H2O from Reanal  and KCl from Lachema . Alfa alumina powder  was purchased from Electron Microscopy Sciences . Sodium and potassium hydroxides and sulfuric acid (H2SO4) were purchased from Merck KGaA  and Reanal ; potassium nitrate (KNO3) and pyrrole were obtained from Acros Organics ; ferrocenecarboxylic acid was obtained from Alfa Aesar GmbH&Co KG ; and hexaammineruthenium (III) chloride (Ru(NH3)6Cl3) was obtained from Fisher Scientific . Tetrachloroauric acid trihydrate (HAuCl4·3H2O), the graphite rod , hydrochloric acid, 1,10-phenathroline-5,6-dione, N,N,N′,N′-tetramethylbenzidine, and tetrathiafulvalene were obtained from Sigma-Aldrich ; 25% glutaraldehyde solution was obtained from Fluka Chemie GmbH ; and L-ascorbic acid (AA) and uric acid (UA) was obtained from AppliChem GmbH . Redox mediators were dissolved in 96% ethanol received from Vilniaus degtinė . The pyrrole was filtered before the investigations through a 5 cm column filled with Al2O3 powder.Carbohydrates-D-(+)-glucose, D(+)-saccharose, D(+)-xylose, D(+)-galactose, D(+)-mannose, D(-)-fructose, and an enzyme—glucose oxidase  was covered with long, thin, and branched DGNs, according to a previously presented methodology [−1 HAuCl4 with 0.1 mol L−1 KNO3 using a computerized potentiostat/galvanostat Autolab/PGSTAT 302N  with GPES 4.9 software at a constant potential of −0.4 V vs. Ag/AgCl(3 mol L−1KCl) for 400 s [−1 FCA, PD, and TMB or 72 mmol L−1 TTF) was added onto the GR/DGN electrode, and the solvent was evaporated at +20 ± 2 °C. After that, the GOx from 3 µL of 25 mg mL−1 solution was immobilized on the GR/DGNs/Med electrode, and the solvent was evaporated at +20 ± 2 °C. Then, the modified GR electrode was stored in a closed vessel in a 25% solution of GA for 15 min at +20 ± 2 °C  or constant-potential amperometry (CPA). Measurements were applied using working electrodes, GR/DGNs/FCA/GOx, GR/DGNs/PD/GOx, GR/DGNs/(PD/GOx)3, GR/DGNs/TMB/GOx, or GR/DGNs/TTF/GOx; a 2 cm2 platinum spiral as an auxiliary electrode; and a Ag/AgCl(3 mol L−1KCl) Metrhom  as a reference electrode. Potential scans were conducted from −0.6 to +0.9 V vs. Ag/AgCl(3 mol L−1KCl) for the GR/DGNs/FCA/GOx, GR/DGNs/PD/GOx, GR/DGNs/(PD/GOx)3, GR/DGNs/TMB/GOx, GR/DGNs/TTF/GOx, and GR electrodes and from −0.6 to +0.6 V for the GR, GR/GOx, GR/DGNs, GR/DGNs/GOx, GR/DGNs/PD, GR/DGNs/PD/GOx, and GR/DGNs/(PD/GOx)3 electrodes, with a 2.5 mV step potential, and a 0.05 V s−1 scan rate was used for the CV measurements. The CPA mode and an applied potential of +0.30 V vs. Ag/AgCl(3 mol L−1KCl) were used to register the current responses using the GR/DGNs/GOx electrode; +0.40 V vs. Ag/AgCl(3 mol L−1KCl) using the GR/DGNs/PD/GOx, GR/DGNs/(PD/GOx)3, or GR/DGNs/TTF/GOx electrodes; +0.70 V vs. Ag/AgCl(3 mol L−1KCl) using the GR/DGNs/FCA/GOx electrode; and +0.75 V vs. Ag/AgCl(3 mol L−1KCl) for the GR/DGNs/TMB/GOx electrode.Current responses were registered using a computerized potentiostat/galvanostat Autolab/PGSTAT 302N with GPES 4.9 software. Electrochemical investigations were performed in unstirred or stirred (1200 rpm) 0.05 mol L−1 H2SO4 solution in the range of 0.0 to +1.4 V. The scan rate was 0.025 V s−1. The EASA was estimated using the following equation [A—the integrated peak of gold oxide reduction, v—the potential scan rate (V s−1), and 386 µC cm−1—the charge density per unit area associated with the electrochemical reduction of a monolayer of chemisorbed oxygen on polycrystalline gold.To determine the electroactive surface area (EASA) of the electrodeposited DGNs, cyclic voltammograms were recorded in an aqueous 0.5 mol Lequation :(1)EASA=−1). The cyclic voltammograms were registered in a solution consisting of 0.1 mol L−1 KCl and 1 mmol L−1 Ru(NH3)6Cl3. The EASA was calculated for the GR/DGNs/TTF/GOx, GR/DGNs/(PD/GOx)3, GR/DGNs/TTF/GOx/Ppy(3.5 h), and GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes using the Randles–Sevcik equation:pi—maximum peak current (A), n—the number of electrons appearing in the half-reaction for the redox pair, D—diffusion coefficient (cm2 s−1), C—concentration of electroactive species (mol·cm−3), and υ—scan rate (V s−1).To determine the EASA of the modified graphite rod electrodes, the potential was swept in the range of −0.70 to 0.0 V with varying scan rates , and the apparent Michaelis constant (KM(app)). ΔImax and KM(app) were calculated as the a and b parameters of the hyperbolic function y = ax/(b + x) and were used for the approximation of the received results. The value of the LOD was estimated statistically with the software SigmaPlot 12.5 and defined as the lowest amount of glucose which provided a current response greater than the background current response value plus 3 σ.All electrochemical measurements were repeated at least three times and evaluated as the mean value. The statistic software SigmaPlot (version 12.5) was used to estimate the intercept, slope, and correlation coefficient of the calibration curve, the limit of detection (LOD), the difference in maximal current response registered during the enzymatic reaction  proceed .(3.5 h), GR/DGNs/(PD/GOx)3, and GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes in a 0.05 mol L−1 SA buffer solution of pH 6.0 at +4 °C for up to 25 days.The stability of the developed glucose biosensors was evaluated after the storage of the unmodified and polypyrrole-layer-modified GR/DGNs/TTF/GOx, GR/DGNs/TTF/GOx/Ppyg) using an IEC CL31R Multispeed centrifuge  according to the previously described method [3/Ppy(5 h) electrode. The developed glucose biosensor was tested in serum diluted 10 times with 10.0 mmol L−1 of glucose before and after the addition of 1.0 mmol L−1 of carbohydrate . This study involved an evaluation of the influence of electroactive species on the current response. Experiments were performed in serum diluted 10 times with either 10.0 mmol L−1 of glucose alone or 10.0 mmol L−1 of glucose with 0.01 or 0.1 mmol L−1 of AA or with 0.01 or 0.05 mmol L−1 of UA.The serum was diluted 10 times and centrifuged  of the graphite rod electrode.The nanostructurization of the electrode surface is one of the methods for improving the analytical parameters of the developed biosensors. In this work, electrodeposited gold nanostructures were used for this purpose. DGNs were synthesized according to a previously published study ,19. DGNs3, GR/DGNs/TMB/GOx, and GR/DGNs/TTF/GOx was studied in 0.05 mol L−1 SA buffer of pH 6.0 with 0.1 mol L−1 KCl via CV according to the methodology described in The data regarding the mechanism of the redox process and the electron transfer can be obtained in the form of a cyclic voltammogram and peak shift . The ele(3 mol L−1KCl). However, the cyclic voltammograms obtained using the GR/DGNs/PD/GOx, GR/DGNs/TMB/GOx, and GR/DGNs/TTF/GOx electrodes were characterized by clearly visible anodic and cathodic peaks ((3 mol L−1KCl) for the GR/DGNs/PD/GOx and GR/DGNs/(PD/GOx)3 electrodes and at + 0.54 V vs. Ag/AgCl(3 mol L−1KCl) for the GR/DGNs/TMB/GOx electrode. The shape of the cyclic voltammograms and the position of the anodic peak for the GR/DGNs/FCA/GOx and GR/DGNs/PD/GOx electrodes are very similar to those obtained using the GC/NPG/Cys/GA/GOx electrode in the presence of FCA (+0.316 V) [(3 mol L−1KCl) were obtained in the case of the cyclic voltammograms recorded using the GR/DGNs/TTF/GOx electrode. The presence of two peaks can be explained by the reversible oxidation/reduction process between the neutral TTF, radical cation (TTF+), and dication (TTF2+) states [(3 mol L−1KCl) [+ is considered an effective redox mediator for electron transfer [It is clear that electron transfer from the GOx redox center to electrodes without mediators does not take place when using the GR/GOx and GR/DGNs/GOx electrodes B. As canic peaks A. Anodic0.316 V)  and the 0.316 V)  electrod) states . SimilarL−1KCl) . The obt(3 mol L−1KCl) was selected for further studies, while for GR/DGNs/FCA/GOx and GR/DGNs/TMB/GOx, it was selected as +0.70 V and +0.75 V vs. Ag/AgCl(3 mol L−1KCl), respectively.To ensure efficient electron transfer on the GR/DGNs/PD/GOx or GR/DGNs/TTF/GOx electrodes, the potential value of +0.40 V vs. Ag/AgCl3, GR/DGNs/TMB/GOx, and GR/DGNs/TTF/GOx, were prepared. The calibration plots using 38 mmol L−1 of FCA, PD, or TMB or 72 mmol L−1 of TTF are shown in The nature of the redox mediator has a significant influence on the sensitivity of electrochemical biosensors. To evaluate the impacts of the FCA, PD, TMB, and TTF on the current responses, five types of working electrodes, including GR/DGNs/FCA/GOx, GR/DGNs/PD/GOx, GR/DGNs/(PD/GOx)Imax was calculated as 2.25 ± 0.92, 25.9 ± 2.8, 34.1 ± 9.6, and 69.1 ± 7.7 µA, respectively. The highest value of ΔImax was achieved for the glucose biosensor based on the GR/DGNs/TTF/GOx electrode −107 ± 7 µA. Meanwhile, in the case of the GR/DGNs/GOx-electrode-based glucose biosensor without a Med, no current response was recorded, which was expected, since direct electron transfer is difficult in the case of GOx, because FAD, as a cofactor, is located deep in the molecule globule, which is electrically well-isolated [The difference in maximal current response depends on the Med used. In the case of the FCA, TMB, PD, and threefold-deposited PD redox mediators, the Δisolated .Imax for the biosensor based on the GR/DGNs/(PD/GOx)3 electrode was 2.03 times higher than that obtained using the GR/DGNs/PD/GOx electrode. DGN fabrication leads to an increase in the electroactive surface area, and thus, an improved signal is recorded. This can be explained by the one-dimensional hollow tubular form of PD, which promotes electron transfer [3 electrodes were selected, as the highest current responses were registered for these electrodes.The value of Δtransfer . For fur3 electrodes, the enzymatic formation of the Ppy layer was performed. Firstly, the Ppy layer was enzymatically synthesized on the surfaces of the GR/DGNs/TTF/GOx or GR/DGNs/(PD/GOx)3 electrodes according to the protocol described in Imax and KM(app) were evaluated.To improve the performance of the biosensors based on the GR/DGNs/TTF/GOx and GR/DGNs/(PD/GOx)Imax values for the glucose biosensors based on the GR/DGNs/TTF/GOx or GR/DGNs/(PD/GOx)3 electrodes after the Ppy layer’s formation depended on the Ppy layer formation time. As can be seen from Imax was observed using both of the electrodes tested. In the case of the electrode prepared using TTF, when the Ppy was synthesized for 1.8, 3.5, 8, or 24 h of enzymatic polymerization  by 4.48 (ΔImax = 24.1 ± 5.8 μA), 5.68 (ΔImax = 19.0 ± 3.4 μA), 6.32 (ΔImax = 17.1 ± 3.7 μA), and 31.1 (ΔImax = 3.47 ± 1.32μA) times, respectively. In the case of the GR/DGNs/(PD/GOx)3-electrode-based biosensor modified with the Ppy layer during 2, 5, 17, 21, or 46 h of polymerization, the ΔImax decreased from 68.0 ± 4.3 µA 3 electrode) by 2.17 (ΔImax = 31.3 ± 3.3), 2.63 (ΔImax = 25.9 ± 2.4), 3.51 (ΔImax = 19.4 ± 4.5), 5.31 (ΔImax = 12.8 ± 3.4), and 6.67 (ΔImax = 10.2 ± 4.0 µA) times, respectively  value is considered to be a significant indicator of the linear dynamic range (LDR) width of the glucose biosensor. The value of KM(app), which was determined for the GR/DGNs/TTF/GOx electrode without Ppy and after 1.8, 3.5, and 8 h polymerization, increased from 5.97 to 6.86, 20.2, and 30.8 mmol L−1. The biosensors based on the unmodified GR/DGNs/(PD/GOx)3 electrodes and those modified with a Ppy layer for 2, 5, 17, and 21 h also showed increases in KM(app) from 55.5 to 61.0, 91.3, 132, and 200 mmol L−1. Although the Ppy layer on the surface of the immobilized GR electrodes reduced the current response and the sensitivity of the developed biosensors, the high value of KM(app) demonstrates the superiority of the polymerized electrodes over the unpolymerized ones in the practical determination of glucose in real samples, where a wide LDR is considered one of the main advantages.A high x- or y-axis and could be used for glucose determination. The LDRs of the GR/DGNs/TTF/GOx and GR/DGNs/(PD/GOx)3 electrodes were extended by increasing the duration of Ppy synthesis and depended on the kind of redox mediator used. In the case of the GR/DGNs/TTF/GOx electrodes  or GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes were chosen for further comparison and can be recommended as more suitable for glucose biosensor construction and the determination of glucose. The LDRs for the unmodified and selected electrodes were characterized by a correlation coefficient above 0.9900.According to the hyperbolic dependences shown in ectrodes C modifie−1 of glucose) [−1) when using a reagentless biosensor based on the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode. Thus, the LDR of this electrode was at least twice as wide as that of the other electrodes  (Au-Cys/TTF/GOx-AOx) (up to 1.0 mmol Lglucose) . Modificectrodes .(3.5 h), GR/DGNs/(PD/GOx)3 and GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes was 67.6, 11.1, 13.4, and 3.03 µA mM−1 cm−2, respectively ((3.5 h) or GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes for analyte detection in real samples.The sensitivity of the biosensors based on the GR/DGNs/TTF/GOx, GR/DGNs/TTF/GOx/Ppyectively . Althoug(3.5 h), GR/DGNs/(PD/GOx)3 and GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes in the presence of 29.4 mmol L−1 glucose were characterized by good repeatability; the relative standard deviation (RSD) was evaluated as 9.65, 8.53, 9.10, and 9.03%, respectively. After the addition of glucose into the electrochemical cell, 95% of the registered current response was reached within 8 s, and it was 1.38 times faster than that achieved using the GC/OOPpy(300 s)-GNPs/GOx electrode (11 s) [(6nm)-GOx- or Ppy/GNPs(AuCl4−)-GOx nanocomposites were used (20 s) [The biosensors developed based on GR/DGNs/TTF/GOx, GR/DGNs/TTF/GOx/Ppye (11 s)  and 2.5 d (20 s) .(3.5 h) electrodes were 9.50 and 8.76 times lower than that obtained using GR/DGNs/(PD/GOx)3 or GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes (−1) [−1) [3 electrode, a wide LDR was observed using PD in the construction of the biosensors.The type of redox mediator used and the polymerization duration affected the LOD of the developed reagentless biosensors. The LODs for the glucose biosensors based on GR/DGNs/TTF/GOx or GR/DGNs/TTF/GOx/Ppyectrodes . The LODdes (−1)  and on t−1) [−1) , respect3)6Cl3 was used as a redox probe, because its oxidation occurs in a region where the studied electrodes do not yield an additional signal (3)6Cl3 diffusion coefficient equal to 9.1 × 10−6 cm2 s−1 was used [(3.5 h), GR/DGNs/(PD/GOx)3, and GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes was calculated as 0.504, 0.648, 0.736, and 0.901 cm2, respectively, according to the results presents in The electroactive surface area of the electrodes was calculated according to the methodology described in l signal . For thewas used . The EAS(3.5 h), GR/DGNs/(PD/GOx)3, and GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes were stored at +4 °C in SA buffer solution at pH 6.0 for up to 22 and 25 days, respectively. The variation in the current responses over time using the glucose biosensors based on electrodes modified with TTF or PD is shown in The stability of biosensors is an essential parameter that defines the duration of their use . Thus, t3 (line 3) electrodes significantly decreased during the first two days, and only 12.9 or 49.0% of their initial value was retained. However, the modification of the developed electrodes with the Ppy layer could increase the stability of the biosensors. The current responses obtained using the GR/DGNs/TTF/GOx/Ppy(3.5 h) (3/Ppy(5 h) ((3.5 h) retained 44.7% of the initial current response after the first two days, whereas the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was much more stable, as it retained 92.5% of the initial current response. These results suggest that the Ppy-modified electrodes are preferable to the unmodified ones.As seen in y(3.5 h) A, line 2Ppy(5 h) C, line 4(3.5 h) or GR/DGNs/(PD/GOx)3/Ppy(5 h) electrodes retained 50% of their initial current response (τ1/2) after 2.0 and 9.0 days, respectively. It is obvious that the glucose biosensor based on the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode is 4.5 times more stable than that based on the GR/DGNs/TTF/GOx/Ppy(3.5 h) electrode. After τ1/2 = 9.0 days, the reagentless glucose biosensor based on the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was 2.73 times more stable than that obtained in our previous research using a GR/Ppy/GNPs(6nm)-GOx/GOx (τ1/2 = 3.3 days) electrode [The reagentless glucose biosensors based on the GR/DGNs/TTF/GOx/Ppylectrode .3/Ppy(5 h) electrode for the reagentless glucose biosensor’s construction and further glucose determination in blood serum.The noticeably, the poor TTF stability noted in a previous paper  and estaSaccharose, xylose, galactose, mannose, and fructose are considered as species interfering in glucose biosensing. The ascorbic acid and uric acid belong to electroactive species that can affect the current response of glucose biosensors in the blood serum ,50,51,523/Ppy(5 h) electrode to interfering and electroactive species was studied in 10-times-diluted serum according to the protocol described in 3/Ppy(5 h) electrode was evaluated, and it is summarized in 3/Ppy(5 h) electrode was almost the same after the addition of similar carbohydrates, which confirms its selectivity to glucose.The selectivity of the reagentless glucose biosensor based on the GR/DGNs/(PD/GOx)−1 of AA [−1 of UA [−1 glucose with 0.01 or 0.1 mmol L−1 of AA increased the current response to glucose obtained the using GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode by 1.80 and 5.39%, compared with the results obtained without AA (3/Ppy(5 h)-electrode-based reagentless glucose biosensor developed in this study was 1.79 and 1.14 times more resistant to 0.01 and 0.1 mmol L−1 of AA than the GR/Ppy/GNPs(AuCl4−)-GOx/GOx electrode (the interferences of 3.23 and 6.16% for 0.01 or 0.1 mmol L−1 AA were registered) [−1 glucose containing 0.01 and 0.05 mmol L−1 of UA increased the current response of the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode by 4.69 and 10.2%, respectively, compared with the results registered after adding 10.0 mmol L−1 of glucose without UA ((AuCl4−)-GOx/GOx electrode changed by 2.19 and 13.4%, respectively, after the addition of 10.0 mmol L−1 of glucose containing 0.01 or 0.05 mmol L−1 of UA [3/Ppy(5 h) electrode is characterized by good glucose selectivity and can be used for blood serum glucose analysis.The next step was to study the effect of ascorbic acid and uric acid on the current response registered in the presence of glucose. It is established that the normal physiological concentration of these electroactive species in human blood serum does not normally exceed 0.141 mmol L−1 of AA  and 0.1 −1 of UA ,54. Thesthout AA B. The GRistered) . The addthout UA B. The cu−1 of UA . In gene−1 of UA . The rea−1 [−1 [3/Ppy(5 h) electrode for glucose detection in serum was tested using the addition method. The results obtained from three measurements were expressed as average values and are presented in 3/Ppy(5 h) electrode, according to its recovery ratio (97.5–98.0%) and relative error (4.76–6.50%), is no worse than the commercial methods and sensors. As reported by Conzales’s group, the recovery ratio and relative error depend on the country, agency, method of detection, and amount of glucose [−1 (4.17 mmol L−1) of glucose, the detection criterion is 98 ± 15%; in Europe, for ≥100 mg dL−1 (5.55 mmol L−1) of glucose, it is −95 ± 15%; and for people with type 1 diabetes, it is −99% [The stated normal glucose concentration in human blood is usually below 5.6 mmol L−1 ,14,39,54 glucose . For exag dL−1 5. mmol L−13/Ppy(5 h) electrode are as follows: (i) a low LOD (0.683 mmol L−1), wide LDR (up to 39.0 mmol L−1 of glucose), and good repeatability (the RSD was 9.03%); (ii) a good storage stability (50% of the current response was retained after 9.0 days); (iii) the fast determination of glucose (approximately 8 s); and (iv) high resistance to electroactive species and suitability for glucose detection in blood serum (97.5–98.0%). The electrochemical biosensor, after some improvements and mandatory clinical validation, could be used in clinical practice for the quantitative determination of glucose.It should be noted that the advantages of the developed reagentless glucose biosensor based on the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was characterized by a wider LDR and higher stability than that based on the GR/DGNs/TTF/GOx/Ppy(3.5 h) electrode. The limitation of the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was its 3.03 µA mM−1 cm−2 sensitivity in comparison to GR/DGNs/(PD/GOx)3 electrode (13.4 µA mM−1 cm−2). However, characterized by a wide LDR (up to 39.0 mmol L−1) and good stability, the biosensor based on the GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was adapted for practical application and the determination of glucose in blood serum in the presence of interfering species. This principle of biosensor development can be adapted to other types of reagentless biosensors suitable for the analysis of real samples. The developed and improved reagentless glucose biosensor, due to its wide LDR, could be applied in various research areas, e.g., for biomedical purposes, for the control of beverages, for biofuel cell fabrication, and in bioelectronics devices.In this study, we developed an enzymatic reagentless glucose biosensor based on a GR electrode modified with electrochemically synthesized DGNs in combination with redox mediators such as TTF and PD, which had the simplicity to operate at a low cost with a quick response, high current response, and good sensitivity. After the formation of the Ppy layer, the reagentless biosensor based on the GR/DGNs/(PD/GOx)


Impairment of the type I interferon (IFN–I) signaling pathway is associated with increased severity of COVID-19 disease. Here we have undertaken a systematic review and meta = analysis on the association between the severity of COVID-19 and IFN-1 autoantibodies .Four databases, including Medline [PubMed], Embase, Web of Science, and Scopus, were systematically searched to find papers on the role of aIFN-1 and its subtype AAbs in the severity of COVID-19 infection. Data on the prevalence of aIFN-1, aIFN-α, aIFN-ω, and aIFN-β were pooled using random- or fixed-effects models. Subgroup analysis was performed based on disease severity. Odds ratios (OR) for COVID-19 disease outcome, including length of hospital stay, ICU admission and death, were calculated in relation to positive or negative plasma IFN-1 AAbs.A total of 33 studies with 13023 patients were included. The overall prevalence of circulating aIFN-1, aIFN-α, and aIFN-ω AAbs was 17.8 % , 7.2 % , and 4.4 % , respectively, and the overall prevalence of neutralizing aIFN-1, aIFN-α, aIFN-ω, and aIFN-β AAbs was 7.1 % , 7.5 % , 8.0 %  and 1.2 % , respectively. Circulating aIFN-α , neutralizing aIFN-α , and neutralizing aIFN-ω  were significantly more frequent in critical/severe patients than in moderate/mild patients . Anti–IFN–1 was more common in male subjects  and two COVID-19 outcomes including ICU admission  and death  occurred more frequently in patients with positive anti–IFN–1.Conclusion: aIFN-1 and its subtypes AAbs are associated with severe and critical COVID-19 disease and may be a predictive marker for a poor prognosis, particularly in men. •Autoantibodies against IFN-1 and its subtypes increase in COVID-19 patients.•The presence of autoantibodies against IFN-1 and its subtypes are associated with more severe COVID -19 disease.•Autoantibodies against IFN-1 and its subtypes may predict worse COVID-19 disease outcomes.•Autoantibodies against IFN-1 were more prevalent among male patients. The ini2.2All included papers met the following criteria : subject2.3Four reviewers  participated in selecting studies based on the articles' title, abstract and full text. In cases where agreement could not be reached, a fifth reviewer (A.S.) reviewed the eligibility to determine final inclusion. Two reviewers (M.A. and N.N.) independently reviewed included articles for quality. Included studies were assessed using the Joanna Briggs Institute (JBI) assessment tools, which provide an assessment tool for most study types, including observational studies . Disagre2.4Study characteristics were extracted from the included articles, including first author's last name, study conduct date, title, study design, study location (country), patient demographics, COVID-19 confirmatory method, IFN autoantibody assay, and history of systemic autoimmune rheumatic disease. The number of individuals in whom circulating or neutralizing aIFN-1/α/ω/β was detected was also recorded. In addition, characteristics of COVID-19 patients in studies comparing seropositive patients with seronegative patients to IFN-1 or its subtypes, including duration of admission, demographics, intensive care unit (ICU) admission, and mortality rate, were extracted. Data were stored in a Microsoft Excel spreadsheet . Data was collected on the plasma AABs levels in COVID-19 and non-COVID-19 patients or within COVID-19 patients according to disease severity.2.5The weighted pooled prevalence and 95 % confidence intervals (CIs) of aIFN-1, aIFN-α, aIFN-ω, and aIFN-β were calculated. The magnitudes of the estimated effects of AAbs on COVID-19 outcomes and the prevalence of AAbs in male patients were expressed as odds ratios (OR), with corresponding 95 % CIs in brackets. The prevalence of AAbs in critical/severe or moderate/mild groups was compared using OR and 95 % CIs. Sensitivity analysis was done after removing a study conducted on children . Median 3After removing 1809 duplicate papers, a total of 2343 papers were identified . Finally3.1A total of 13023 patients (from 33 studies) diagnosed with COVID-19 and had at least one of the AAbs targeting IFN-1, IFN-α, IFN-ω, or IFN-β were included in this analysis. Seropositivity of patients with different stages of disease against IFN-1 and its subtypes is detailed in Outcomes of COVID-19 in positive versus negative aIFN-1/α patients.Fourteen studies compared patients with positive and negative AAbs to IFN-1 or IFN-α. 3.2Meta-analysis showed that circulating aIFN-α was significantly more prevalent in critical/severe patients than in moderate/mild patients  . Also, t3.3Soltani-Zangbar et al. reported higher aIFN-α2 levels in severe than mild patients , though 3.4Publication bias was assessed by visual inspection of the asymmetry of the funnel plot. The figures show no evidence of publication bias; however, for some subgroup analyses, there were insufficient studies to present proper funnel plots. Funnel plots are provided for all forest plots.4This meta-analysis of 33 studies analyzed data from 13023 COVID-19 patients with respect to aIFN-1 AAbs. We estimated the pooled rate of seropositivity of circulating aIFN-1, aIFN-α, and aIFN-ω AAbs among COVID-19 patients to be approximately 17.8 % , 7.2 % , and 4.4 % , and the rates of neutralizing aIFN-1, aIFN-α, aIFN-ω, and aIFN-β AAbs were 7.1 % , 7.5 % , 8.0 % , and 1.2 % , respectively. In contrast, the aIFN-α and aIFN-ω subtypes are present in about 0.3 % of the normal population . The disRegarding the biomarker potential of aIFN AAbs, Beck et al.  suggesteIFN-1 are critical for controlling SARS-CoV-2 infection, which may explain the increased susceptibility of older adults with a reduced ability to secrete IFN-1 . The BodOur study had some limitations. Firstly, limited analysis of IFN-ω or aIFN-β levels could be performed as few studies reported them, increasing the risk of a false negative result. Secondly, another limitation is the low sensitivity of the assays used in some studies to measure aIFN-1 and its subtypes, which may have led to underreporting. Assessment of aIFN-1 in the post-COVID-19 phase may provide insights into the role of aIFN-1 AAbs in patients with long-COVID-19 and post-COVID-19 syndrome, and future studies should also investigate aIFN-γ AAbs .5In conclusion, aIFN-1 and its subtypes AAbs are associated with severe and critical COVID-19 disease and may be a predictive marker for a poor prognosis, particularly in men.Conceptualization: AA, AS. Investigation: AA, AH, MA, MMM, ZS, TJ, AS. Writing - Original Draft: AA, AH, MA, NNN. Writing - Review & Editing: TJ, AS.No external fund was received for conducting this study.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


To the Editor: Atopic dermatitis (AD) is a chronic, pruritic inflammatory skin disease affecting over 7% of US adults, with 40% having moderate-severe disease.2.5), has been associated with AD development among healthy individuals.2.5 levels within EPA standards,2.5 pollution and AD severity among AD patients, we performed a cross-sectional study of AD patients between October 19, 2017 and October 19, 2018, using the Clinformatics Data Mart Database, a de-identified national insurance claims dataset. Patients at least 12 years old with at least 6 months of continuous enrollment, an AD ICD-10 code, and either an asthma or allergic rhinitis code were included .2.5 levels were collected from EPA stations nearest to each patient’s zip code. Categorical and continuous variables were compared using Chi-squared and Student’s t-tests, respectively. Adjusting for age, sex, insurance, and urbanization, multivariate logistic regression calculated odds ratios for associations between yearlong average PM2.5 levels and moderate-severe AD. P-values < .05 were considered statistically significant.To investigate the association between yearlong PM2.5 ranged from 1.56 to 21.82 μg/m3. Mean yearlong average PM2.5 (8.35 vs 8.39 μg/m3) was comparable between moderate-severe and mild AD patients. No statistically significant associations existed between yearlong PM2.5  concentrations and AD severity  age of 46 (22) years and 64% female . Moderatseverity .Table ID2.5 concentrations declined by 37% to 8.47 μg/m3, well below the EPA’s standard of 12 μg/m3.2.5 concentration of 8.38 μg/m3 and the 95th percentile experiencing an annual average of 10.89 μg/m3. PM2.5 has only been associated with AD development or increased health care use for AD when average long-term PM2.5 levels exceeded 24.78 μg/m3 or when short-term PM2.5 increased above 80 μg/m3, respectively.,2.5 was not associated with AD severity. While a chronic air pollution threshold that exacerbates AD may exist, it does not appear to fall within ranges typically experienced in the US at this time.With increasing frequency of wildfires in the setting of climate change, there is concern over the impact of worsening pollution on conditions like AD. Balanced against this; however, are the sustained gains in air quality. Over the past 20 years, annual US average PMA.S.C. has served as a clinical investigator and/or consultant for: Regeneron Pharmaceuticals, Sanofi, AbbVie, Dermira, Eli Lilly, Pfizer. All other authors have no conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject of this manuscript, to disclose.


The mean BMI was 23.9 kg/m2 in men and 21.8 kg/m2 in women (p < 0.0001). Diabetes mellitus was found in 1282 subjects (18.7%) among men and 816 subjects (9.4%) among women (p < 0.0001). The mean Suita score was 42.0 in men and 29.6 in women (p < 0.0001). In multivariate analysis, WC (p < 0.0001), F-FF ratio (p < 0.0001), and water mass index (p < 0.0001) were independent factors linked to the Suita score for both genders. In conclusion, body composition can be associated with the Suita score in Japanese adults receiving health check-ups.The purpose of this study was to clarify the relationship between the Suita score (a prediction model for the development of cardiovascular disease) and body composition in Japanese health check-up subjects (6873 men and 8685 women). The Suita score includes 8 items . Factors associated with the Suita score within body composition-related parameters (body mass index (BMI), waist circumference (WC), fat mass index, fat-free mass index, fat mass to fat-free mass ratio (F-FF ratio), and water mass index) as assessed by bioelectrical impedance analysis were examined. The mean age of subjects was 54.8 years in men and 52.8 years in women ( Because the risk of developing myocardial infarction is extremely low among Japanese people compared to people in the West, the Framingham Risk Score (FRS), a score used in the West to predict the development of cardiovascular disease (CVD) over a 10-year period, is considered inaccurate for Japanese people ,3,4, wasBody composition refers to fat, muscle, bone, and water ,7. Body 2). Fat-free mass index (FF index) was defined as fat-free mass divided by the square of height (kg/m2). F index to FF index ratio (F-FF ratio) was defined as the F index divided by the FF index. Water mass index (W index) was defined as water mass divided by the square of height (kg/m2).Between February 2022 and May 2023, a total of 15,558 consecutive Japanese subjects with data for both the Suita score and body composition–related parameters as assessed by bioelectrical impedance analysis (BIA) were found in our medical records. Since the Suita score is for those 35 years of age and older, those under 35 were excluded [2) was calculated for men as 194 × (serum creatinine (mg/dL))−1.094 × (age (years)) −0.287, and for women as 194 × (serum creatinine (mg/dL))−1.094 × (age (years)) −0.287 × 0.739 [The Suita score is a risk score that predicts the development of CVD in urban residents and includes CKD and other risk factors. The Suita score can more accurately predict the onset of CVDs and is believed to be useful in preventing CVDs . The Sui × 0.739 .After each item is scored, the total score is defined as the Suita score . The preFirst, we investigated the relationship between the Suita score and body composition–related parameters , waist circumference (WC), F index, FF index, F-FF ratio, and W index) in both genders. Next, body composition–related parameters associated with the Suita score were examined using multivariate analysis. We obtained ethical approval for the study from the ethics committee at OMPU Hospital  and the protocol strictly observed all regulations of the Declaration of Helsinki. Subject consent with regard to the current study was waived due to the retrospective analysis of the study. t-test or Mann–Whitney U-test was applied, as appropriate. In multiple-group comparisons (continuous variables), the Kruskal–Wallis test or ANOVA was employed, as applicable. In the group comparisons , the chi-squared test was applied. In the multivariate analyses, multivariate regression analysis with multiple predictive variables using the least squares method was applied to select candidate variables. Unless otherwise noted, data were shown as numbers or mean values (±standard deviation (SD)). Considering the total number of cases exceeded 10,000, the significance level was set at p = 0.01. JMP 17.0.0 software  was employed to perform statistical analyses.In two-group comparisons (continuous variables), the Student’s n = 6873 for men and n = 8685 for women) are shown in p < 0.0001). The mean (±SD) BMI was 23.9 ± 3.6 kg/m2 in men and 21.8 ± 3.7 kg/m2 in women (p < 0.0001). Diabetes mellitus was found in 1282 subjects (18.7%) among men and 816 subjects (9.4%) among women (p < 0.0001). Habitual smoking was found in 1564 subjects (22.8%) among men and 551 subjects (6.3%) among women (p < 0.0001). The mean (±SD) Suita score was 42.0 ± 11.5 in men and 29.6 ± 11.5 in women  F index was 5.46 ± 2.35 kg/m2, 3945 subjects (57.4%) with 20 ≤ BMI < 25 kg/m2, and 2240 subjects (32.6%) with BMI ≥ 25 kg/m2. The mean (±SD) Suita score was 36.8 ± 11.9 in subjects with BMI < 20 kg/m2, 41.3 ± 11.6 in subjects with 20 ≤ BMI < 25 kg/m2, and 44.7 ± 10.3 in subjects with BMI ≥ 25 kg/m2. The overall difference of the Suita score among the three groups reached significance  with BMI < 20 kg/m2, 4239 subjects (48.8%) with 20 ≤ BMI < 25 kg/m2, and 1417 subjects (16.3%) with BMI ≥ 25 kg/m2. The mean (±SD) Suita score was 26.8 ± 10.7 in subjects with BMI < 20 kg/m2, 30.0 ± 11.5 in subjects with 20 ≤ BMI < 25 kg/m2, and 34.5 ± 11.0 in subjects with BMI ≥ 25 kg/m2. The overall difference of the Suita score among the three groups reached significance  with BMI < 20 kg/mp < 0.0001, In Japanese criteria for metabolic syndrome, WC ≥ 85 cm is the cutoff point for men . Among mp < 0.0001, In Japanese criteria for metabolic syndrome, WC ≥ 90 cm is the cutoff point for women . Among w2 in the low-risk group, 5.67 ± 2.46 kg/m2 in the intermediate-risk group, and 5.95 ± 2.31 kg/m2 in the high-risk group. The overall difference of the F index among the three groups reached significance  F index was 4.70 ± 1.98 kg/m2 in the low-risk group, 7.42 ± 3.33 kg/m2 in the intermediate-risk group, and 8.19 ± 3.18 kg/m2 in the high-risk group. The overall difference of the F index among the three groups reached significance  F index was 6.26 ± 2.62 kg/mp < 0.0001), F-FF ratio (p < 0.0001), and W index (p < 0.0001) were significant factors (set significance level: p < 0.01) associated with the Suita score (p < 0.0001), F-FF ratio (p < 0.0001), and W index (p < 0.0001) were significant factors (set significance level: p < 0.01) associated with the Suita score  found that WC . r = 0.73, p < 0.0001) and women , not shown in the results section) [In the summary of our results, body composition–related factors were closely linked to the Suita score in both men and women. Especially in women, there were significant differences in all comparisons, as shown in section) . The sigp < 0.01). WC is closely related to the accumulation of visceral fat, and visceral fat excess is a risk factor for the development of coronary artery disease [In this study, WC, F-FF ratio, and W index were extracted as independent factors associated with the Suita score for both men and women in multivariate analysis (set significance level,  disease . Adipose disease . Zhou et disease , which i disease ,29,30. I disease . BMI was disease .We should acknowledge several limitations to the current research. First, this study was a cross-sectional single-site observational research using retrospective analysis, and was limited to Japanese adults aged 35 years or older. As mentioned earlier, the Suita score is for people 35 years of age and older. Second, details regarding smoking habits and oral medications were based on individuals’ reports, potentially leading to bias. Thus, the results should be interpreted with sufficient caution and care. Nevertheless, our data indicate that WC, F-FF ratio, and W index were closely linked to the Suita score. The current large number of cases appears to be one strong point in drawing a definite conclusion.In conclusion, body composition can be associated with the Suita score, which has been well validated for CVD incidence, in Japanese adults receiving health check-ups. Daily life management with an awareness of body composition may reduce the incidence of CVD.


Short-term (lag0 and lag0–7 day) and long-term  PM2.5 concentrations at residences were extracted from the air pollutant database known as Tracking Air Pollution (TAP) in China. Multivariate logistic regression models were used to estimate associations for short- and long-term PM2.5 exposure concentrations and long-term exposure models were additionally adjusted for short-term deviations.Participants  were recruited from ten regions in China from 2018 to 2021 for the Predictive Value of Inflammatory Biomarkers and Forced Expiratory Volume in 1 s  was associated with higher odds of allergic nasal  and eye symptoms , worsening dyspnea caused by allergens , and ≥ 2 allergic symptoms , which was similar in the lag0–7 day concentrations. A 10 µg/m3 increase in the 1-year average PM2.5 concentration was associated with an increase of 23% for allergic nasal symptoms, 22% for eye symptoms, 20% for worsening dyspnea caused by allergens, and 21% for ≥ 2 allergic symptoms, similar to the 3- and 5-year average PM2.5 concentrations. These associations between long-term PM2.5 concentration and allergic symptoms were generally unchanged after adjustment for short-term deviations.A 10 µg/m2.5 was associated with an increased risk of allergic nasal and eye symptoms, worsening dyspnea caused by allergens, and ≥ 2 allergic symptoms.Short- and long-term exposure to ambient PMClinical trial ID: NCT03532893 (29 Mar 2018).The online version contains supplementary material available at 10.1186/s12931-023-02433-2. The studied population covered ten regions: Beijing, Jilin, Inner Mongolia, Shandong, Fujian, Hebei, Shanxi, Tianjin, Shaanxi, and Henan. Details of the study have been previously described . After eThe study was approved by the ethics review committees of the Peking University First Hospital (2018 Number 31). Written informed consent was acquired from all participants.Data were collected at local branch health centers. Participants underwent medical examinations, including anthropometric measurements, spirometry, blood tests, and urinary tests. A series of standard self-administered questionnaires, including demographic, risk factors, lifestyle questionnaires, St George’s Respiratory Questionnaire (SGRQ), and the allergic symptoms questionnaire  were adm2.5 concentrations, extracted from the air pollutant database known as Tracking Air Pollution in China (TAP) [2.5 concentrations in China with complete coverage at a spatial resolution of 10 km have been available since 2000. Details of the PM2.5 exposure assessment model have been described in our previous publications [2 for different years was 0.83 [2.5 exposure concentrations were estimated by assigning predicted PM2.5 concentrations to each participant’s residential address. We defined short-term exposure as the daily average of a single 0 day (lag0 day) and the moving average of 0-to-7 day concentrations (lag0–7 day) before the day before the allergic symptoms questionnaire was administered. Long-term PM2.5 exposure was estimated by calculating the 1-, 3- and 5-year averages of the PM2.5 concentrations for the calendar year before administering the allergic symptoms questionnaire. We also obtained the daily average of the ambient temperature on the day the allergic symptoms questionnaire was administered (lag0 day) at a 9 km × 9 km spatial resolution from the atmospheric reanalysis V5 (ERA5) Land dataset.Ambient PMna (TAP) , were esications . This mowas 0.83 . Residen2 test for categorical variables. Multivariable logistic regression models were used to estimate associations between short- (lag0 and lag0–7 day) and long-term  PM2.5 concentrations at residences and self-reported allergic nasal and eye symptoms, worsening dyspnea caused by allergens and ≥ 2 allergic symptoms. Effect estimates are presented as odds ratios (ORs) and their 95% confidence intervals (CIs) for each 10 µg/m3 increase in the PM2.5 concentration. We gradually added potential confounders to observe their effects, and a total of four models were developed: Model 1, no adjustments; Model 2, adjusted for age, sex, education level , BMI , geographic region , ambient temperature , the season in which the allergic symptoms questionnaire was administered ; Model 3, further adjusted for cumulative smoking exposure , passive smoking, biomass exposure, household cooking, and occupational exposure; and Model 4 (only for long-term exposure models), further adjusted for short-term PM2.5 concentration deviations. In Model 4, we examined the influence of additional adjustments for short-term variations in long-term concentrations when estimating associations between allergic symptoms and long-term PM2.5 exposure, including the 1-, 3- and 5-year average concentrations. Short-term PM2.5 concentration deviations were defined for each participant as the absolute difference between the PM2.5 concentration on the day the allergic symptoms questionnaire was administered and the 1-, 3- and 5-year average concentrations. For example, the short-term PM2.5 concentration deviation for the model of the 1-year average PM2.5 concentration was calculated as follows: Short-term PM2.5 concentration deviation = PM2.5 concentration on the day the allergic symptoms questionnaire was administered—1-year average PM2.5 concentration. The associations between ambient PM2.5 exposure and wheezing were not analyzed in this study because the prevalence of wheezing was low, and the regression model could not be run after adjusting for a range of covariates.We compared baseline characteristics between the < 2 allergic symptoms group and the ≥ 2 allergic symptoms group using the independent sample test-test for continuous variables and the χAll statistical analyses were performed using R version 4.1.2. All two-tailed tests were regarded as statistically significant at the 0.05 level.The distribution of 10,142 participants  by general characteristics and risk factors is summarized in Table 2.5 concentration was 35.19 (27.32) µg/m3 (range 1–288 µg/m3) on the day the allergic symptoms questionnaire was administered (lag0 day) and 34.73 (20.93) µg/m3 (range 2.00–197.12 µg/m3) for the moving average of 0-to-7 day (lag0–7 day). For long-term exposure, the 1-, 3- and 5-year annual average PM2.5 concentrations were 50.56 (15.27) µg/m3, 54.08 (16.92) µg/m3, and 59.79 (17.88) µg/m3, respectively  to 2020 (44.92 µg/m3) among the ten regions . PM2.5 concentrations exhibited seasonal variation, with the highest concentration in winter , followed by autumn (56.26 µg/m3), spring (55.33 µg/m3), and summer (44.99 µg/m3) .The average residential PM2.5 exposure  for allergic nasal symptoms, 1.08  for allergic eye symptoms, 1.06  for worsening dyspnea caused by allergens, and 1.07  for ≥ 2 allergic symptoms. The effect estimates were similar to those obtained using the PM2.5 data of the lag0–7 day concentrations.The results from the three models  all showed that allergic symptoms increased with higher short-term PMre Table . In adju2.5 concentrations  and allergic symptoms  and eye symptoms , worsening dyspnea caused by allergens  and ≥ 2 allergic symptoms ; these effect estimates were similar to those after adjustment for short-term PM2.5 concentration deviations in Model 4. Specifically, the OR of allergic eye symptoms for a 10 µg/m3 increase in the 1-year average concentration was 1.22  based on Model 3, compared with that of 1.22  in Model 4 after adjustment for short-term PM2.5 concentration deviations. Compared with long-term PM2.5 concentration, the short-term PM2.5 concentration deviations had a weaker impact on allergic symptoms in model 4. Each 10 µg/m3 increase in the 1-year short-term PM2.5 concentration deviation, the risk increased by 7% for allergic nasal symptoms , 6% for allergic eye symptoms , 5% for worsening dyspnea caused by allergens , 6% for ≥ 2 allergic symptoms , these effect estimates were similar to those in 3- and 5-year average PM2.5 concentration logistic regression models  increase in PM2.5 at lag0 day was associated with allergic nasal  and eye  symptoms among 2317 schoolchildren [2.5 exposure might alter tear film stability, causing the film to break up and thin by disrupting the tear lipid layer, thus causing eye irritation and discomfort [2.5 concentration was associated with the number of outpatient visits for allergic conjunctivitis during the nonpollen season [3 increase in the annual PM2.5 concentration was associated with a 20% increase in the prevalence of allergic rhinitis among preschool children in six Chinese cities [2.5 exposure levels were correlated with nasal lavage fluid eosinophils and exudation mediators in children with asthma in Paris [2.5 can increase inflammatory cytokines, induce pathological damage to the nasal mucosa and conjunctival epithelium, and worsen nasal and eye symptoms [Air pollution contributes to the onset and aggravation of allergic symptoms or diseases , 35, 37.scomfort . Accordie cities . Short-tin Paris . PM2.5 csymptoms , 40. Impsymptoms .2.5 is a potential aggravating factor for allergic respiratory symptoms or diseases [2.5 exposure and worsening dyspnea caused by allergens. A combined analysis of cross-sectional data from Lifelines and UK Biobank cohorts, each 5 µg/m3 increase in the PM2.5 concentration was associated with an increase of 16% for wheezing and 61% for shortness of breath [2.5 exposure, either alone or in combination with allergic sensitization, can induce oxidative stress, signal transduction interference, enzyme inhibition, epigenetic dysregulation, airway hyperresponsiveness and remodeling [Exposure to high levels of ambient PMdiseases , 42. Whediseases , 44. Thediseases , comparediseases , our stumodeling . Allergimodeling  and smokmodeling .2.5 exposure was more strongly associated with allergic symptoms than short-term PM2.5 exposure. The findings in our study could be attributable in part to differences between long- and short-term ambient PM2.5 exposure levels. Ambient PM2.5 exhibited the highest concentration in winter  and the lowest concentration in summer . Among the participants, 6325 (62%) were enrolled in the study in the summer, and only 373 (3.7%) were enrolled in the winter. The short-term (lag0 and lag0–7 day concentrations) ambient PM2.5 exposure levels were lower than the long-term  in this study. Meanwhile, the questionnaire collected nonperiod-specified rather than recent allergic symptoms, which may be another important reason for the stronger associations for long-term than short-term PM2.5 exposure.Our study found long-term PM2.5 concentration deviations might not significantly affect on long-term PM2.5 exposure models. Consistent with our results, a study of the LuftiBus cohort adjusting for short-term variations in nitrogen dioxide (NO2) and PM2.5 concentrations had little effect on the estimated associations between air pollution exposure and lung function parameters in long-term exposure models [Short-term ambient PMe models . Other se models , 52. The2.5 exposure, we also found that allergic symptoms were positively associated with older age, higher education level, passive smoking, household cooking, occupational exposure and family history of asthma. Household cooking and tobacco smoke are major sources of indoor air pollution. Chinese cooking emits more PM2.5 than Western cooking [Allergic diseases, which involve complex interactions of genetic, ethnic, environmental, and socioeconomic status or lifestyle risk factors, are primarily attributed to environmental factors such as indoor and outdoor air pollution, tobacco smoke exposure, and exposure to other pollutants , 44, 53. cooking . For ris cooking . In this cooking . Family 2.5 exposure and allergic symptoms. Further intervention and prospective studies are needed to verify the causality of the association in this study. Second, allergic symptoms were assessed by self-report questionnaires, making the study prone to recall bias. Third, as an issue commonly reflected in other studies, PM2.5 exposure concentrations were only estimated at the residence due to a need for more information about work addresses or time-activity patterns. This might result in misclassification. Limited by data availability, information about other ambient pollutants was not available, and we could not distinguish between associations due to PM2.5 specifically or other correlated pollutants.Several limitations of this study should be addressed. First, given the study design, it is challenging to provide causal inferences about the associations between PM2.5 exposure might have adverse effects on allergic symptoms among the middle-aged and elderly population in China, apart from other individual risk factors, including older age, higher education level, passive smoking, household cooking, occupational exposure and family history of asthma. Our findings contribute substantially to the evidence of adverse effects of ambient PM2.5 exposure on allergic symptoms in middle-aged and elderly populations. This study further supports for the urgent need to control air pollution to protect middle-aged and elderly adults.In conclusion, the findings from the PIFCOPD study showed that short- and long-term ambient PMAdditional file 1: Figure S1. Trends in annual average PM2.5 concentrations among ten regions from 2013 to 2020 year. Figure S2. Season average PM2.5 concentrations of 1-, 3- and 5-year. Table S1. Estimated risks of each independent variable in the lag0 day PM2.5 concentration logistic regression model 3. Table S2. Estimated risks of each independent variable in the lag0-7 day PM2.5 concentration logistic regression model 3. Table S3. Estimated risks of each independent variable in the 1-year average PM2.5 concentration logistic regression model 4. Table S4. Estimated risks of each independent variable in the 3-year average PM2.5 concentration logistic regression model 4. Table S5. Estimated risks of each independent variable in the 5-year average PM2.5 concentration logistic regression model 4.