Emergent evidence emphasizes the significance of cell metabolism as a sensitive target of PM exposure. However, the current understanding of the relationship between PM composition, conventional toxicity measures, and the rewiring of intracellular metabolic processes remains limited. In this work, PM10 sampled at a residential area  and a traffic-impacted location  of a Portuguese city was comprehensively characterized in terms of polycyclic aromatic hydrocarbons and plasticizers. Epithelial lung cells (A549) were then exposed for 72 h to PM10 organic extracts and different biological outcomes were assessed. UB and RS PM10 extracts dose-dependently decreased cell viability, induced reactive oxygen species (ROS), decreased mitochondrial membrane potential, caused cell cycle arrest at the G0/G1 phase, and modulated the intracellular metabolic profile. Interestingly, the RS sample, richer in particularly toxic PAHs and plasticizers, had a greater metabolic impact than the UB extract. Changes comprised significant increases in glutathione, reflecting activation of antioxidant defences to counterbalance ROS production, together with increases in lactate, NAD+, and ATP, which suggest stimulation of glycolytic energy production, possibly to compensate for reduced mitochondrial activity. Furthermore, a number of other metabolic variations hinted at changes in membrane turnover and TCA cycle dynamics, which represent novel clues on potential PM10 biological effects.Atmospheric particulate matter (PM) with diameters below 10 µm (PM Cells were seeded at a density of 1.5 × 104 cells/mL in 96-well plates and incubated for 24 h to promote cell adhesion. Then, cells were treated with eight different concentrations of PM10 organic extracts in the range of 0.125–25 µg/mL for 72 h. Control cells were treated with culture medium containing 1% DMSO (the maximum concentration present in PM10 extracts). After 72 h, 50 μL of MTT (1.0 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C. Then, the MTT solution was removed and replaced by 150 μL of DMSO, and the plate was shaken in the dark for 2 h at room temperature to dissolve the formazan crystals. The absorbance of the samples was measured using a microplate reader  at 570 nm with blank corrections. Three independent experiments were carried out, each with five technical replicates.The cytotoxicity of PM4 cells/mL in 12-well plates and incubated for 24 h. Then, cells were exposed to UB and RS PM10 extracts for 72 h at OC concentrations of 12.5 and 25 µg/mL. Control cells were incubated with 1% DMSO in culture medium. After this period, the medium was removed and the cells were washed with 500 μL of PBS (pH 7.2), followed by a 30 min incubation with 500 μL of DCFH-DA 10 μM in culture medium supplemented with 2% FBS. Finally, cells were washed, trypsinized, and resuspended in cold medium containing 2% FBS for analysis on an Attune® Acoustic Focusing Cytometer , within 45 min after cell collection. Data analysis was performed using the FlowJo software version 10.7.1 .The intracellular ROS levels were assessed using flow cytometry with the fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate , a cell-permeable compound that is cleaved intracellularly and oxidized by ROS to fluorescent 2′,7′-dichlorofluorescein (DCF). Cells were seeded at a density of 1.5 × 104 cells/mL were seeded in 6-well plates and, after cell adhesion, they were exposed for 72 h to the UB and RS PM10 extracts, as described above. At the end of the exposure time, cells were incubated with 5 µg/mL of Rho123  for 30 min. Then, cells were washed with PBS, trypsinized, and centrifuged at 700× g for 5 min at 4 °C. After that, the cell pellet was washed with 1 mL of PBS containing 1% BSA and resuspended in 500 µL of PBS containing 1% BSA and 5 µg/mL PI . Samples were then analyzed using flow cytometry with an Attune® Acoustic Focusing Cytometer  and at least 10,000 events were analyzed. The loss of MMP was visualized as indicated by the reduction in Rho123 FL.Changes in MMP were assessed using flow cytometry with the cationic fluorescent dye Rhodamine 123 (Rho123). This dye selectively accumulates in healthy mitochondria with high negative membrane potential, while a decrease in its uptake—and hence fluorescence intensity—can be associated with a decrease in the negative charge across the inner mitochondrial membrane. For this assay, 1.5 × 104 cells/mL in 12-well plates and incubated for 24 h, after which they were exposed for 72 h to the UB and RS PM10 extracts, as described above for the ROS assay. Cells were then washed with 500 μL of PBS, trypsinized, resuspended in 300 μL of culture medium, and centrifuged at 700× g for 5 min. The resulting cell pellet was then fixed with 1 mL of cold ethanol 85% and stored at −20 °C until analysis. At the time of analysis, cells were centrifuged at 112× g for 6 min at 4 °C and resuspended in 800 μL of PBS, followed by filtration through a nylon membrane (41 µm). Each sample was then incubated for 10 min with 50 μg/mL of ribonuclease A , and 50 μg/mL of propidium iodide , for additional 30 min, at room temperature in the dark. Analysis of cell cycle was performed on an Attune® Acoustic Focusing Cytometer flow cytometer . For each sample, at least 5000 events were acquired. The FlowJo software  was employed to determine the percentage of cells at Gap 0 (G0)/Gap 1 (G1), Synthesis (S), and Gap 2 (G2)/Mitosis (M) phases of the cell cycle. Two independent assays, each with two replicates, were performed for each treatment.Cells were seeded at a density of 1.5 × 104 cells/mL and incubated for 24 h to allow for cell attachment. Then, cells were exposed to UB and RS PM10 at OC concentrations of 12.5 and 25 µg/mL for 72 h. Control cells were incubated with 1% DMSO in culture medium. Four to six replicates per condition were collected within three independent assays.Cells were seeded in 100 mm diameter dishes at a density of 4 × 104) and transferred into 5 mm NMR tubes. At the end of the 72-hour exposure period, the culture medium was removed, cells were washed with cold PBS and were extracted using a dual-phase extraction, with methanol/chloroform/water (1:1:0.7) as previously described in detail . Aqueous1H observation. Standard 1H 1D spectra with water suppression (pulse program “noesypr1d”) were acquired with a 7002.8 Hz spectral width, 32 k data points, 2 s relaxation delay, and 512 scans. Spectral processing was performed in TopSpin 4.0.3  and comprised cosine multiplication (ssb 2), zero-filling to 64 k data points, manual phasing, baseline correction, and chemical shift calibration to the TSP signal at 0 ppm. The 2D 1H-1H total correlation (TOCSY) spectra and J-resolved spectra were also acquired for selected samples to aid metabolite assignment, based on matching spectral information to reference spectra available in Chenomx  and Bruker BBIOREFCODE , Rheinstetten, Germany. Spectra acquisition was carried out in a Bruker Avance III HD 500 NMR spectrometer , operating at 500.13 MHz for p-value). Metabolite variations of larger magnitude (|ES| > 0.8) were depicted in a heatmap, generated using the R software version 4.1.3 (R Core Team (2020). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/ (accessed on 30 September 2023)).For multivariate analysis, carried out in SIMCA-P 11.5 , spectral matrices were normalized to total area and scaled to unit variance. The results of Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were represented as score scatter plots and corresponding loadings plots colored by variable importance to the projection (VIP). Signal integration was performed in Amix-Viewer and the variation in selected metabolites in treated vs. control samples was assessed, along with the effect size (ES) and statistical significance (p ≤ 0.05) was used for multiple comparisons. Statistical differences are indicated as * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.Statistical analysis was performed in GraphPad Prism . One-way analysis of variance (ANOVA) with Dunnett’s test (10 extracts at concentrations that exhibited low cytotoxicity (cell viability exceeding 75%), lung A549 cells displayed increased ROS generation, decreased MMP, arrest of the cell cycle at the G0/G1 phase, and modifications in the intracellular metabolic profile. Notably, the PM10 extract derived from the RS location—which contained a higher concentration of particularly hazardous PAHs and plasticizers—induced more pronounced metabolic alterations compared to the UB extract. The observed metabolic shifts suggest that PM10 exposure triggers a strengthening of antioxidant defenses and an upregulation of energy production pathways, a response that likely reflects the protective role of metabolic adaptations in mitigating the onset of severe cytotoxic effects.Following a 72-hour exposure to PM10 extracts was not exhaustive. Although PAHs and plasticizers are well-recognized as highly toxic organic constituents of PM, the influence of other unanalyzed chemicals cannot be excluded. Secondly, while A549 cells represent a widely accepted in vitro model for human pulmonary epithelial cells, it is essential to acknowledge that the effects observed in vitro may not fully mirror the complexities of in vivo scenarios. Moreover, it is important to recognize that metabolomics offers a static snapshot of the metabolites present, which might not accurately reflect the dynamic changes that occur in vivo, and can only partly reveal the modulation of metabolic pathways. Hence, additional strategies like integrating metabolomics data with proteomics and/or transcriptomics, as well as assessing the expression of selected metabolic genes and proteins, should be explored in future studies to provide more comprehensive mechanistic insights. While informative, this study has some noteworthy limitations. Firstly, the chemical characterization of the PM


Here, we present baseline characteristics and select biomarkers of study subjects.FOOTPRINTS® has been published previously. The study population included ex-smokers with a range of COPD severities , ex-smokers with COPD and alpha-1-antitrypsin deficiency (A1ATD) and a control group of ex-smokers without airflow limitation (EwAL). At study entry, data were collected for: demographics, disease characteristics, history of comorbidities and COPD exacerbations, symptoms, lung function and volume, exercise capacity, soluble biomarkers, and quantitative and qualitative computed tomography. Baseline data are presented with descriptive statistical comparisons for soluble biomarkers in the individual GOLD and A1ATD groups versus EwAL.The methodology of FOOTPRINTSIn total, 463 subjects were enrolled. The per-protocol set comprised 456 subjects, mostly male (64.5%). The mean (standard deviation) age was 60.7 (6.9) years. At baseline, increasing pulmonary symptoms, worse lung function, increased residual volume, reduced diffusing capacity of the lung for carbon monoxide (DLco) and greater prevalence of centrilobular emphysema were observed with increasing disease severity amongst GOLD 1–3 subjects. Subjects with A1ATD (n = 19) had similar lung function parameters to GOLD 2–3 subjects, a high residual volume comparable to GOLD 3 subjects, and similar air trapping to GOLD 2 subjects. Compared with EwAL (n = 61), subjects with A1ATD had worse lung function, increased residual volume, reduced DLco, and a greater prevalence of confluent or advanced destructive emphysema. The soluble inflammatory biomarkers white blood cell count, fibrinogen, high-sensitivity C-reactive protein and plasma surfactant protein were higher in GOLD 1–3 groups than in the EwAL group. Interleukin-6 was expressed less often in EwAL subjects compared with subjects in the GOLD and A1ATD groups. Soluble receptor for advanced glycation end product was lowest in GOLD 3 subjects, indicative of more severe emphysema.®, which aims to establish correlations between biomarkers and disease progression in a representative COPD population.These findings provide context for upcoming results from FOOTPRINTSTrial registration number: NCT02719184, study start date 13/04/2016.The online version contains supplementary material available at 10.1186/s12931-023-02584-2. Chronic obstructive pulmonary disease (COPD) is a progressive disease primarily of the lungs, and is characterised by persistent respiratory symptoms and airflow limitation due to airway and alveolar abnormalities. COPD is generally attributed to exposure to tobacco and/or harmful air particles . ChangesIn the era of precision medicine, phenotyping patients with COPD based on one or more disease characteristics could improve our understanding of pathogenesis and disease progression, as patients do not generally all follow the same trajectory in their disease. As a result, this may allow for more targeted, individualised, and hence efficacious pharmacological and non-pharmacological management. Prospective longitudinal studies can help to assess whether soluble biomarkers , imaging biomarkers and clinical parameters, or combinations therein, may serve as predictors of disease progression in COPD. Several studies in COPD have either completed or are underway to help address this question, including COPDGene , ECLIPSE®  is a 3-year study aiming to assess the association between soluble biomarkers of inflammation/lung tissue destruction, and imaging and physiological biomarkers, with COPD progression in ex-smokers with mild-to-severe COPD   ≥ 80% predicted; GOLD stage 2: FEV1 ≥ 50– < 80% predicted; and GOLD stage 3: FEV1 ≥ 30– < 50% predicted), subjects with COPD and A1ATD, and an EwAL control group. All subjects in this study should have stopped smoking at least 9 months prior to inclusion ) was numerically higher than in any of the GOLD groups, but lower than in the EwAL group (573.1 m [SD 100.3 m]).At baseline, patients with the greatest disease severity (as defined by the GOLD 1–4 classification system) had the poorest lung function Table ; Fig. 2.1 was highest in GOLD 1 (2.66 L [SD 0.54 L]) and lowest in GOLD 3 subjects (1.17 L [SD 0.29 L]). Mean FEV1 was lower in GOLD groups 1–3 and subjects with A1ATD (1.85 L [SD 0.85 L]) than in EwAL (3.48 L [SD 0.77 L]). Mean percent predicted FEV1 was 87.4% [SD 9.4%] in GOLD 1 subjects and decreased with increasing disease severity to 39.6% [SD 7.6%] in GOLD 3 subjects (P < 0.0001). The mean percent predicted FEV1 was 50.4% (SD 20.3%) in subjects with A1ATD and 102.1% (SD 12.2%) in the EwAL group. Mean FVC also decreased with increasing disease severity , and, overall, was highest in subjects with A1ATD (4.54 L [SD 1.38 L]. The EwAL control group had a mean FVC of 4.42 L (SD 0.97 L). The mean FEV1/FVC ratio was 63.4% (SD 5.4%) in GOLD 1 subjects and reduced with increasing disease severity to 38.2% (SD 9.0%) in GOLD 3 subjects (P < 0.0001). Subjects with A1ATD had a mean FEV1/FVC ratio of 40.6% (SD 13.4%) and the EwAL control group FEV1/FVC ratio was 78.8% (SD 5.7%).Across GOLD groups 1–3, mean FEVTable TLC values generally increased with increasing disease severity . The highest mean TLC was observed in the A1ATD group (8.16 L [SD 1.53 L]), whereas TLC for EwAL was 6.81 L (SD 1.27 L).CO), (P < 0.0001) (Table CO was 79.8% (SD 21.3%) in GOLD 1 subjects, 63.4% (SD 18.1%) in GOLD 2 subjects, 51.5% (SD 17.5%) in GOLD 3, subjects and 51.1% (SD 12.7%) in subjects with A1ATD.Increasing disease severity and A1ATD were associated with reduced diffusing capacity of the lung for carbon monoxide (DL1) Table . The meaCO/alveolar volume ratio was highest in GOLD 1 subjects (2.25 mmol/min/kPa [SD 1.36 mmol/min/kPa]) and decreased with disease severity (GOLD 3: 1.61 mmol/min/kPa [SD 1.20 mmol/min/kPa]). Overall, mean DLCO/alveolar volume was lowest in the A1ATD group (0.83 mmol/min/kPa [SD 0.19 mmol/min/kPa] vs 1.88 mmol/min/kPa [SD 1.20 mmol/min/kPa] in EwAL). Further DLCO parameters are available in Additional file 1.The mean DLFor the majority of biomarkers presented in Fig. No association was detected between IL-6 positive expression and disease severity (P = 0.0701). IL-6 was expressed less often in the EwAL subjects compared with GOLD 1–3 and A1ATD subjects, of whom 13%–26% expressed IL-6  Fig. . EmphyseWhen assessing lung volumes by quantitative CT, a linear increase in expiratory volume and a linear decrease in PD15 and ALD were observed with increasing disease severity  Table . Mean exThe mean FRC/TLC ratio, a measure of air trapping, was 0.570 (SD 0.118) in GOLD 1 subjects versus 0.722 (SD 0.097) in GOLD 3 subjects. Ex-smoker controls had the lowest FRC/TLC ratio (0.499 [SD 0.112]). Values for GOLD 2 subjects (0.629 [SD 0.102]) and A1ATD subjects (0.653 [SD 0.121]) were between the values for GOLD 1 and GOLD 3 subjects.® study aims to identify biomarkers that are associated with COPD progression, in particular emphysema progression, over a 3-year period. Results from this study are expected to help address an unmet need to better understand different COPD phenotypes and their associated biomarkers. This could allow the use of biomarkers for diagnosis, to identify high-risk patients and to guide treatment options in a more targeted, precision-based manner. COPD diagnosis and treatment requires more than assessment of lung function. Biomarkers may help to better understand the biology, prognosis and treatment options suitable for different COPD phenotypes. In a study by Lowe et al. [The longitudinal observational FOOTPRINTSe et al. , for exa® study subjects, including lung function parameters, soluble biomarkers, qualitative and quantitative CT, and symptoms, are provided here. These data allowed us to compare characteristics between subjects grouped by COPD severity (GOLD stages 1–3), subjects with A1ATD and EwAL subjects.The baseline demographics and clinical characteristics of the FOOTPRINTSCO. Compared with EwAL, A1ATD was also associated with worsening lung function, increased lung volumes and a reduction in DLCO. Of note, TLC was highest in the A1ATD group compared with the other groups; however, this could be a result of the higher proportion of male and younger subjects in the A1ATD group. Levels of soluble inflammatory biomarkers such as WBC count, fibrinogen, hs-CRP and plasma SP-D increased linearly by disease severity, with higher levels observed in the GOLD 3 group. Previous studies have demonstrated increases in the expression of these inflammatory biomarkers to be associated with the progression of COPD [As expected, at baseline, increasing COPD severity was associated with worsening lung function, increased expiratory lung volume and a reduction in DL of COPD , 17. Of Levels of sRAGE were highest in EwAL subjects and decreased linearly with increasing disease severity, with lowest values observed in the GOLD 3 group, indicative of more severe emphysema. This is in keeping with previous studies, which have suggested lower blood sRAGE to be associated with more severe airflow obstruction and emphysema . SimilarWhen assessing lung density by quantitative CT, we noted a clear and expected decrease in PD15 with increasing COPD severity, as has been shown in previous studies . There wAs expected, as disease severity increased, symptoms tended to worsen. Values for subjects in the A1ATD group were generally between those in the GOLD 2 and 3 groups. This may not be surprising, given that the majority of the A1ATD subjects met the criteria for COPD GOLD 3  or GOLD 2 . Our study also found that mMRC dyspnoea score (< 2 and ≥ 2) and SGRQ total score (< 25 and ≥ 25) were better discriminators for the COPD GOLD 1–3 groups than CAT score (< 10 and ≥ 10), demonstrating a more consistent numerical trend across the groups.® study, there are a number of longitudinal studies that are ongoing or completed in patients with COPD, including COPDGene [® has a number of strengths. The inclusion of the A1ATD subpopulation is unique to the study and provides information on an important but rarely studied subpopulation of patients; these patients present with earlier onset and faster progression of emphysema [® was designed and began, no other study had presented an association between biomarkers of extracellular matrix degradation  and emphysema. Since then, this area has garnered the interest of several other studies [In addition to the FOOTPRINTSCOPDGene , ECLIPSECOPDGene , SPIROMICOPDGene , 5, COSYCOPDGene , CanCOLDCOPDGene  and the COPDGene . Comparamphysema . Further studies , 23–25.  studies . The Fle studies  and mort studies , 28.Tabl® study is investigating biomarkers associated with emphysema over a 3-year period to increase the understanding of COPD patient phenotypes, pathogenesis and disease progression. The data reported here document the baseline disease characteristics and biomarkers of subjects in our study, highlighting the substantial burden of COPD and A1ATD. In addition to the baseline biomarker data presented, future analyses will be conducted on sputum biomarkers. Importantly, these findings provide important context to precede the upcoming results from the 3-year study, which aim to establish key correlations between disease progression and biomarkers over time in a representative COPD population.The FOOTPRINTSAdditional file 1: Table S1. Disease history (for categories with ≥5% in any group). Table S2. Soluble biomarkers in whole blood and qualitative CT scan data. Table S3. DLCO parameters.


Oxidation represents a major pathway for the chemical degradation of pharmaceutical formulations. Few specific details are available on the mechanisms that trigger oxidation reactions in these formulations, specifically with respect to the formation of free radicals. Hence, these mechanisms must be formulated based on information on impurities and stress factors resulting from manufacturing, transportation and storage. In more detail, this article focusses on autoxidation, metal-catalyzed oxidation, photo-degradation and radicals generated from cavitation as a result of mechanical stress. Emphasis is placed on probable rather than theoretically possible pathways. The advent of biotechnology has enabled the production of recombinant proteins for therapeutic applications. A recent review of the globally highest-selling drugs in 2019 showed that out of ten drug products, seven were proteins ,4,5,6,7.4)- or ozone-induced oxidative stress [4-induced oxidative stress in rats [Oxidation represents a major pathway for the chemical degradation of proteins, which can be carried out by a range of reactive oxygen and nitrogen species, including free radicals ,9. The fe stress ,14. In c in rats .Many of the protein oxidation products that have been characterized in vivo can also form as a result of oxidative processes in therapeutic protein formulations in vitro . In addiThe exact nature and sources of oxidants in protein formulations are generally less well defined. For comparison, the biological mechanisms of oxidant production frequently rely on relatively well-characterized enzymes such as xanthine oxidase , nitric The pharmaceutical formulations of therapeutic proteins display a range of compositions for liquid, frozen and lyophilized forms. Relevant to the potential formation mechanisms of radicals is the fact that these formulations can generally contain several classes of compounds in addition to the protein, such as buffers, surfactants, amino acids, cryoprotectants, chelators and additional tonicifiers ,29. BesiThe following sections will discuss specific pathways of free radical generation with respect to the potential role of impurities and stress factors.− and O2•− represent an electron donor and superoxide, respectively, and k1 and k−1 are the rate constants for forward and reverse electron transfer.Miller et al. define true autoxidation “as the spontaneous oxidation in air of a substance not requiring catalysts” . Hence, 1 vs. log K1 (where K1 represents the equilibrium constant for redox equilibrium 1) yields a curve that can be fitted to the Marcus equation, where D− represents a series of phenolates, indophenolates and other electron donors [o2 ≈ 0.75 V for redox equilibrium 2 [A plot of log kn donors . This reibrium 2 ,33, woulThis would limit autoxidation processes to proteins that contain free cysteine residues. Monoclonal antibodies do not contain free cysteine residues, except for small quantities of incompletely folded proteins, implying that autoxidation should be a negligible problem for pharmaceutical formulations containing monoclonal antibodies.1  [A second target for potential autoxidation would be surfactants , especiae bonds) ,40, conte bonds) . Such faMorita and Tokita reported that fatty acid:oxygen copolymers are stronger initiators of lipid peroxidation in model experiments compared to simple hydroperoxides . HoweverIII does not change its oxidation state but promotes the disproportionation reaction of two complexed H2O2 molecules). A representative quantitative analysis of the elemental impurities listed in class 1 and 2A in several formulation components predicts that their levels in therapeutic protein drug products will be significantly below the PDE [2O2 for Cr(III), Fe(II), V(III) and Cu(II), indicating Fenton or Fenton-like reactivities of these metals [2O2 [2O2), potassium persulfate (K2S2O8) and potassium peroxomonosulfate (KHSO5), with nine metals, including Fe(II), Fe(III), Co(II), Ru(III), Ag(I), Ce(III), V(III), Mn(II) and Ni(II) [•) upon reaction with H2O2 [4•−) only upon reaction with K2S2O8 and/or KHSO5, oxidants that are likely not present in pharmaceutical formulations. In contrast, Stadtman et al. advocate for the formation of “caged” HO• radicals during the reaction of Mn(II) with H2O2, available for the oxidation of substrates [• radicals during reactions of V(III) and Mn(II) with H2O2 may depend on the experimental conditions; in a first approximation, the redox reactions of V(III) and Mn(II) with H2O2 would be rather comparable to the reactions of the Fe(II)/Fe(III) redox couple with H2O2. Hence, a more detailed description of the reaction mechanisms of Fe(II) and Fe(III) would serve as a model for analogous reactions of V(III) and Mn(II), and also of Cr(III) and Cu(II). Therefore, the following will entirely focus on processes of Fe-dependent radical generation through the Fenton reaction that are relevant to pharmaceutical formulations.Fenton and Fenton-like reactions represent important pathways for free radical generation. Peroxides can be introduced into formulations through excipients , primari the PDE . These e the PDE  and wille metals . Howeverals [2O2 . Anipsitd Ni(II) . Of thesith H2O2 . The othbstrates ,53,54. Tbstrates ,56, for bstrates . The forIn pharmaceutical formulations of therapeutic proteins, iron impurities can come from multiple sources, including the cell culture medium, manufacturing equipment, containers, proteins and excipients. Iron levels as high as 1–9 μM have been reported for some protein formulations ,58.III, coordinated with iron-binding ligands, L [III may be present in a variety of mixed ligand complexes, i.e., that complexes of FeIII show some heterogeneity. FeIII reacts with H2O2 according to equilibrium 3 [In general, it can be assumed that iron impurities in pharmaceutical formulations will be present as ferric iron, Fegands, L . These librium 3 ,61.LxFe3 = 69 M−1s−1 and k−3 = 0.11 s−1 have been reported for equilibrium 3 in acidic aqueous solution with pH 2.0 (where L = H2O) [III/FeII (0.77 V vs. NHE) [2/HO2− (0.79 V), the reduction of FeIII by HO2− is feasible [a = 4.8 for equilibrium 5 [2•−) can subsequently reduce an additional equivalent of LxFeIII (reaction 6) [Rate constants of kL = H2O) . Based ovs. NHE)  and HO2/feasible , so equiibrium 5 . The supction 6) ,65.LxFeIII/FeII is very sensitive to pH [xFeIII to LxFeII by HO2− must be carefully discussed with respect to these parameters. Specifically, for Lx = EDTA, the reduction potential of FeIII/FeII decreases to 0.12 V [III by HO2− may not be a major pathway of FeII formation [x = DTPA, the reduction potential decreases even further to 0.03 V [2O2 with (EDTA)FeIII yields an oxidant that converts the dipeptide Met-Met (Met = methionine) to products that are also generated via the exposure of Met-Met to a Fenton system, (EDTA)FeII/H2O2 [•) or higher-valent iron-oxo species such as FeIV=O [255 on the monoclonal antibody. In these formulations, it is likely that HO• and/or LxFeIV=O are generated via the reaction of LxFeIII with hydrogen peroxide  and RO• and/or LxFeIV=O via the reaction of LxFeII with organic hydroperoxide impurities (reactions 10 and 11). Here, LxFeIV=O (Eo′pH 7.0 ≈ 1.00 V) is the less powerful and more selective oxidant compared to HO• (Eo′pH 7.0 = 2.18 V) [However, it has been pointed out that, specifically, the reduction potential for the couple Feve to pH  and the ve to pH ,63, so to 0.12 V , suggesto 0.03 V ). HoweveeII/H2O2 , suggests FeIV=O . In thiss FeIV=O  are impo 2.18 V) .LxFeII xFeIII with H2O2 may become important, as its product, LxFeIII(−O2H), reacts significantly more efficiently with LxFeII  compared to H2O2 (k ≈ 50 M−1s−1) [In this regard, the initial reaction (reaction 3) of L M−1s−1) .LxFeIII• and/or LxFeIV=O, will have the opportunity to react with formulation constituents such as protein and excipients, generating a plethora of oxidation products and secondary oxidizing species including peroxyl radicals, alkoxyl radicals and peroxides. The initial oxidation reactions of HO• and/or LxFeIV=O may occur preferentially with the ligands L, coordinating either FeII or FeIII [• and/or LxFeIV=O with the organic constituents of the formulation. An alternative oxidant, the carbonate radical anion (•CO3−), may be generated if the formulation contains low amounts of bicarbonate (introduced through atmospheric CO2), which can generate LxFeII(CO3) [• and/or Lx(CO3)FeIV=O, may oxidize the Fe-bound carbonate to •CO3− [Ultimately, the resulting oxidizing species, HOor FeIII . PeroxyleII(CO3) . In suchto •CO3− ,70, whicxFeIII and LxFeII. Walling et al. [III/H2O2, providing evidence that oxidation reactions prevail in the presence of EDTA. Likewise, Graf et al. showed that EDTA did not prevent the oxidation of dimethylsulfoxide (DMSO)  induced by LxFeIII and hypoxanthine/xanthine oxidase [II and (EDTA)FeIII, where crystal structures demonstrate a distortion from octahedral geometry, resulting in the availability of a seventh binding site for a reaction to take place [III(H2O) with pKa ≈ 7.6 [2O2 [II, also by molecular oxygen [II efficiently reacts with H2O2, with k > 3 × 103 M−1s−1 [An important question is that of whether metal chelators can prevent the formation of oxidizing species during the reaction of peroxides with Lg et al.  demonstr oxidase . Howeverke place ,74. In ake place ,74, alsoKa ≈ 7.6 ,76. The 7.6 [2O2 ,71,77 anr oxygen . In fact3 M−1s−1 ,80.1O2 [Depending on the manufacturing environment and clinical use, protein formulations can be exposed to UVA and/or visible light , and an 1O2 ).• radicals [Tryptophan residues can form cation-π complexes ,105,106,radicals . Hence, III-dependent oxidation reactions in pharmaceutical formulations , reducing FeIII to FeII, and oxidizing the carboxylate ligand, which subsequently decarboxylates reactions (13) and (14) [In view of the discussion of Feions see  above, tand (14) ,112,113.• will add oxygen to yield a peroxyl radical, ROO•, unless R• is •CO2− (see below), while FeII reduces O2 to O2•− [2O2 [III, basal levels of FeIII in 10 mM citrate buffer, pH 6.0, were sufficient to promote the photo-oxidation of Met-enkephalin during near-UV photo-irradiation with a light dose of 25.2 Whm−2 [III levels [•CO2− during the photo-irradiation of citrate-FeIII with either near-UV or visible light, detected via spin-trapping with DMPO [•CO2− radical is a powerful reductant (Eo(CO2/•CO2−) ≈ 1.93 ± 0.22 V vs. NHE [III to FeII [2 to O2•− [•) and thiolate (RS−) [The resulting carbon-centered radical R to O2•−  and H2O22•− [2O2 . With re.2 Whm−2 , i.e., c.2 Whm−2 . In thesI levels . An impoith DMPO ,117. The vs. NHE ) that re to FeII , O2 to O to O2•− ,120 and te (RS−) ,122,123.•CO2− from citrate involves LMCT from the (deprotonated) citrate hydroxyl group rather than the citrate carboxyl groups, generating an intermediary alkoxyl radical (RO•), which undergoes α-β cleavage of the central carboxylate group 2FeIII [Mechanistic studies suggest that the formation of e)2FeIII , which sIII and His buffer. In this case, photo-induced LMCT from a deprotonated Thr residue, Thr259, led to an intermediary Thr side chain alkoxyl radical  in the presence of Fe• radicals and even O atoms [Protein formulations are exposed to various types of mechanical stress during manufacturing and transportation. Under certain circumstances, high shear stresses ,126, mix O atoms . Hence, Physicians’ Desk Reference, PDR, see [In order to ensure sterility, multidose formulations contain antimicrobial preservatives (APs) such as, e.g., phenol, m-cresol, benzyl alcohol, thimerosal or chlorobutanol ,136 . Some o• (reactions 19 and 20), where In• represents an initiating radical [•H-OH (reaction 21) [The exposure of benzyl alcohol to air leads to the slow formation of benzaldehyde, Ph-CHO . Benzald radical . Howevertion 21) . The reaTherefore, formulations containing benzyl alcohol bear a potential risk for the formation of oxygen-centered radicals  and peroxides (peroxybenzoic acid).xFeIII to LxFeII, generating a semiquinone radical (2 to O2•− (equilibrium 24), generating a quinone derivative. The latter can comproportionate with a catechol to regenerate semiquinone derivatives (equilibrium 25) [2•− will generate H2O2, which will regenerate LxFeIII through a reaction with LxFeII, generating HO• radicals  . The discals see .® or Humalog®, contain Zn(II), generally in the form of ZnO , which releases Zn2+ [2+ ions also support the formation of a native insulin hexamer [® and Humalog® also contain m-cresol and phenol, which are susceptible to hydroxylation and, subsequently, redox cycling  during the Fenton oxidation of phenols, which has been rationalized by a more persistent semiquinone radical as a result of Zn2+ complexation, generating more HO• radicals [2+ may increase the susceptibility of a formulation to Fenton oxidation.Specifically, insulin formulations, e.g., Humulin Rses Zn2+ . ZnO conses Zn2+ , but the hexamer . Both Huling see . It was radicals . Hence, 2 to O2•−, while the remaining positive hole, h+, in the valence band can oxidize adsorbed H2O/HO− to the HO• radical [3, an activity comparable to that of brookite (TiO2), but an activity lower than that of anatase (TiO2). However, ZnO was more active than anatase in the photocatalytic degradation of humic acid in aqueous solution with pH 7.88 [An alternative mechanism by which ZnO, specifically, may promote oxidation reactions is photo-degradation. ZnO is a semiconductor with a band gap of 3.2–3.7 eV , which w radical ,146. ZnO radical , showing pH 7.88 .®, Elaprase®, Vimizim®, Naglazyme®, Mepsevii®, VPRIVTM or NexviazymeTM). However, inspection of the active sites of the some of the relevant enzymes shows the presence of Cys residues, e.g., in N-acetylgalactosamine-6-sulfatase [α-formylglycine (FGly) and, therefore, are not amenable to Cys oxidation.A review of IV formulations for enzyme replacement therapy ,150 reveulfatase  and idurulfatase . These CII to a pharmaceutical formulation may generate information about the kinetics of iron-dependent oxidation degradation reactions under storage conditions, as iron impurities will likely be present as FeIII. However, the addition of FeII may lead to mechanistic information that can be used to predict certain degradation pathways in cases whereby FeIII is converted to FeII, for example, through reaction with H2O2 or hydroperoxides. A limitation of mechanistic investigations of radical-induced oxidation reactions in pharmaceutical formulations will always be that the precise quantity and nature of the radicals specifically generated under storage conditions are usually unknown. Even the monounsaturated oleic acid, the main component of polysorbate 80 fatty acid esters [Based on information on impurities and stress factors that affect pharmaceutical formulations, a series of mechanisms are formulated that could be responsible for free radical formation in pharmaceutical formulations. The focus of this article is on highly probable reactions; additional pathways may be possible in isolated cases when pharmaceutical formulations contain high levels of specific impurities that are not generally present. With respect to the design of stress tests for pharmaceutical formulations, highly probable reactions should be kept in mind. For example, it may be questionable whether the addition of Fed esters ,36, can d esters . It is u


Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07_2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tm-shift assay markers developed for SNP S07_2970381 and two linked SNPs, S07_2970276 and S07_2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNP-haplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance.Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agro-inoculated with CTV-Logan (CA/Logan) strain to locate the  Phaseolus vulgaris L.) and affects many other crops, including sugar beet (Beta vulgaris L.), pepper (Capsicum annuum L.), and tomato (Solanum lycopersicum L.) (Curtovirus within the family Geminiviridae and is transmitted in a persistent manner by the beet leafhopper (Circulifer tenellus B.). Among the 11 strains of BCTV described in the literature, Colorado (CO) and Worland (Wor) have been two of the more commonly isolated strains from plants and beet leafhoppers in recent studies in the western US. In contrast, the California/Logan (CA/Logan) strain has recently only been found in Idaho (Sinapis arvensis L.), Russian thistle , and kochia (Kochia scoparia L. Schrad.) (Beet curly top virus (BCTV) can cause severe yield loss in susceptible cultivars of snap and dry beans  . BCTV isin Idaho  and has in Idaho . EndemicSchrad.) .Bct allele identified by Bct locus, derived from the landrace ‘Jatu Rong’ (G122), was associated with a minor-effect quantitative trait locus (QTL) conferring resistance against CTV in the G122/’Taylor Horticulture’ dry bean RIL population  (BCTV [synonymous with curly top virus (CTV)] causes symptoms in common bean, including leaf curling, chlorosis, and severe plant stunting . Early spulation . The sams (BDMV) .Bct allele, is difficult because field-based screening is unreliable due to the cyclical nature of epidemics. Additionally, there is a lack of uniform disease pressure during an epidemic because of spotty feeding by the leafhopper, as common bean is a non-preferred food source  or false negatives . In addition, it was demonstrated that ‘SAS8.1550’ had reduced diagnostic value in snap bean genotypes derived primarily from the Middle American gene pool  enhance the efficiency and reliability of MAS for Bct in snap and dry bean germplasm.Our research objectives aimed to i) acquire a comprehensive understanding of the genetic basis of the http://arsftfbean.uprm.edu/beancap/). The SBDP was used in a diversity analysis of snap bean (The Snap Bean Association Panel (SnAP) is comprised of 378 historic-to-modern cultivars that span both fresh market and processing types, including large sieve, small sieve, wax, bush blue lake, Kentucky, Romano, pole, and others. The SnAP includes public and private industry-released snap bean cultivars predominantly from the United States, some landrace cultivars, and a few breeding lines. The SnAP has recently been used for Genome-Wide Association Studies (GWAS) of herbicide tolerance  and resinap bean  and for nap bean . In addihttps://npgsweb.ars-grin.gov/gringlobal/search). An additional 289 SnAP accessions were phenotyped for CTV disease reaction using an infectious CA/Logan clone of CTV and an agro-inoculation screening procedure in a controlled growth room environment at Harris Moran Seed Company in Sun Prairie, Wisconsin. The CTV CA/Logan strain was cloned into the binary vector pBIN19 in Agrobacterium tumefaciens strain GV3111 (Ti plasmid pTiB6S3SE) obtained originally from Dr. Drake Stenger. This unpublished infectious clone had increased infectivity for agro-inoculation of common bean compared to earlier-developed clones  database, cultivar registration articles, and the Germplasm Resources Information Network , and each pot contained two plants. The plants were covered and placed in a dark growth chamber for 24 h after inoculation. After 24 h, they were maintained in a growth chamber with a 14-/10-h light cycle and a temperature range of 18°C–27°C. The seedlings were transplanted into 1-gallon pots (two plants/pot) 2–6 days post-inoculation. The accessions were tested in subsets of approximately 30 accessions, in 4-day intervals, in a growth room. Each subset included two pots with two plants, each of the susceptible ‘Caprice’ and resistant ‘Hystyle’ cultivars as controls. Six accessions did not germinate, including ‘Angers’, ‘Processor’, ‘RH13’, ‘Serin’, ‘Golden Rod’, and ‘Sundial’. Disease reactions were recorded 17–24 days post-inoculation (dpi) as resistant (no visible symptoms), susceptible , or intermediate resistant (with fewer and less severe symptoms) .2O, 1 µl of total nucleic acid extract, and 0.1 µl GoTaq DNA polymerase . Thermal cycler conditions consisted of 95°C for 2 min, followed by 40 cycles of 95°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and a final extension step of 72°C for 5 min. Amplicons were visualized on a 1.5% agarose gel with ethidium bromide staining.The DBP accessions were sown during the summer of 2022 in two replications at the Washington State University (WSU) Research Station—Roza Unit (46°17′43.6ʺN 119°43′48.0ʺW) in Prosser, WA, USA, where CTV is endemic. The experimental design was a randomized complete block, and each plot consisted of four rows of 3-m row length, with a spacing of 0.56 m between rows. To determine the CTV strain(s) causing infection in the trial, leaf tissue was collected from 1 asymptomatic plant and 16 symptomatic plants during the early pod-fill R3 growth stage. The tissue was extracted using a modified Dellaporta protocol  and analDisease reaction was rated 60 days after planting on a scale from 1 to 9, where 1 = no symptoms and 0% of plants infected, 5 = moderate disease symptoms and up to 30% of plants infected, and 9 = severe disease symptoms with ≥80% of the plants dead or dying . Trait dhttps://phytozome-next.jgi.doe.gov/info/Pvulgaris_v2_1) using Bowtie2 v2.4.1  and diluted to 10 ng/μl with nuclease-free water. Multiplexed genotyping-by-sequencing (GBS) libraries were generated through digestion with ApeKI following the protocol and with barcodes detailed in 2 v2.4.1 . The ali2 v2.4.1 .Phaseolus vulgaris v2.1 as reference genome  implemented in the package fastPHASE  pipeline  with G19e genome . MaximumastPHASE . AnnotatFor the DBP accessions, the leaf tissue was collected from individual plants grown in the USDA-ARS greenhouses at Prosser, WA. Genomic DNA was isolated from 20 mg of the leaf tissue using a Qiagen DNeasy 96 Plant Kit  and sent to the US Department of Agriculture Agricultural Research Service (USDA-ARS), Soybean Genetics and Improvement Laboratory, Beltsville, MD, for the 11,292 SNPs BARC Illumina chip assay . The SNP2 threshold and the –indep-pairwise function in Plink v2.0 (https://www.cog-genomics.org/plink/2.0/), with a sliding window of 50 kb shifting by five bases. In addition, two dry bean lines, ‘BAT 93’ (representing the Middle American gene pool) and ‘Jalo EPP 558’ (representing the Andean gene pool), were added to assess the structure and diversity in SnAP. The sequence data for ‘BAT 93’ and ‘Jalo EPP 558’ was obtained by GBS, as described above.The SNP data sets used for population structure analysis in SnAP and DBP were adjusted by linkage disequilibrium (LD) pruning using a 0.5 rThe pruned set of SNPs with <20% missing values and a MAF > 0.01 were retained. Population structure was estimated using a Bayesian Markov chain Monte Carlo model (MCMC) implemented in STRUCTURE 2.3 software . Ten runGWAS analysis was conducted by mixed linear model (MLM) using the genome association and prediction integrated tool (GAPIT) . An idenBct region were identified using the gene models from G19833 v2.1 common bean reference genome. Selected candidate genes were amplified and sequenced in a subset of 14 snap and 14 dry bean genotypes from Andean and Middle American gene pools.Candidate genes annotated within the http://bioinfo.ut.ee/primer3-0.4.0/). The PCR volume was 25 µl, containing 1× Taq buffer, 1.8 mM of MgCl2, 0.4 mM of dNTPs mix , 0.25 µM of each primer (forward and reverse), 1 U of Taq polymerase , and 25 ng of genomic DNA. The amplification conditions were 2 min at 95°C, followed by 38 cycles at 94°C for 20 s, 57°C for 30 s, and 72°C for 90 s, and a final extension at 72°C for 5 min, performed in a PCR Eppendorf Mastercycler . PCR products were visualized by gel electrophoresis on 2% (w/v) agarose. Sanger sequencing from PCR products obtained was performed using the services of Eurofins MWG Operon . Sequencing data were analyzed using Geneious 9.1.8 , 0.15 μM of each primer , 1× EvaGreen® dye, and 0.1 μl of Taq polymerase (Promega) under the following thermal profile: 94°C for 2 min, then 38 cycles of denaturation at 92°C for 20 s, annealing for 20 s (the temperature was specific to each primer trio), and extension at 72°C for 20 s, and final extension at 72°C for 5 min. Melting point analysis for allele determination of the PCR products was performed with a fluorescence-detecting thermocycler . EvaGreen Fluorescent detection was performed for 1 min at 95°C, and the melting curve step ramped from 65°C to 95°C in increments of 1°C every 20 s.Tm-shift SNP markers were amplified by PCR on an Eppendorf Mastercycler . The PCR volume was 20 μl containing 20 ng of genomic DNA, 1× Taq buffer, 1.5 mM of MgCltrimmed-means one-way ANOVA using the ggbetweenstats function in the R-Package ggstatsplot. Yuen’s trimmed means test was implemented using Robust p-value fitting method using the ggbetweenstats function in the R-Package ggstatsplot ; resistant or intermediate resistance (18 accessions); or resistant, intermediate resistant, or susceptible responses (15 accessions). The disease reactions for 83 SnAP accessions, compiled from various public information sources, were categorized as 42 resistant, 32 susceptible, and 9 intermediate resistant to CTV. For upstream analysis, the qualitative reactions of each accession were transformed into a quantitative score, with resistant being coded as 1, susceptible as 0, and intermediate resistant as 2  under natural field infection. Based on the BLUP values for CTV reaction, 43 accessions were resistant (disease score between 1 to 4), 33 were tolerant (between 4.1 to 6.9), and 14 were susceptible (between 7 to 9) to the Wor strain 60 days after planting , using a pruned set of 1,833 SNPs, a likelihood of K=2 was estimated, indicating the presence of two sub-populations belonging to the Andean and Middle American gene pools, as confirmed by gene-pool checks ‘Jalo EEP 558’ and ‘BAT 93’ , using an LD-pruned set of 974 SNP markers, and K=2 was estimated .p = 1.7E−36), which was associated with resistance and intermediate resistance to CTV (Bct gene that was identified by GT) that conditions resistance to CTV found in the dry bean landrace G122 named ‘Jatu Rong’ (PI 163120) from India  and 2.79–3.60 Mb (p = 1.89.17) on Pv07, respectively (p =1.12E−45), as expected for a single major allele  from Bct region with a high p-value (p =1.84E−07)  for the Bct region based on the Andean G19833 common bean v2.1 gene models  and Phvul.L001619, which functions as a mediator of RNA polymerase II transcription subunit, showed homology with gene models from other plant species involved in the replication of geminiviruses  sequencing of the three candidate genes revealed 29 variants, 28 SNPs, and 1 InDel among a subset of 28 snap and dry bean genotypes from diverse backgrounds  protein of the sequenced ORF for gene Phvul.007G036300 did not uncover any disruptions in the EXO5 domain. Nevertheless, the missense SNP variant located in Pv07: 2,970,381 bases involves a transition of Adenine (A) to Thymine (T) base, resulting in radical replacement of a polar, uncharged amino acid, glutamine (Q), with a non-polar, hydrophobic amino acid, leucine (L), at position 279 in the EXO5 domain  (The p.Q279L) Table 2.Bct candidate gene Phvul.007G036300 in the 28 sequenced genotypes showed one cluster for susceptibility and two distinct clusters for resistance/intermediate resistance to CTV , which exhibited polymorphism between resistant and susceptible genotypes to CTV. Additionally, two Tm-shift assays were developed to distinguish introgression haplotypes of the Bct region from Andean and Middle American gene pools. The first assay, named S07_2970276, targeted the missense SNP variant Pv07: 2,970,276 bases within Phvul.007G036300 gene and differentiated genotypes from Andean and Middle American gene pools. The second assay, named S07_2966197, focuses on another missense SNP variant, Pv07: 2,966,197 bases, found in the Phvul.007G36200 gene model by A Tm-shift assay named S07_2970381 was specifically designed to detect the missense SNP Pv07: 2,970,381 in the The S07_2970381 marker exhibited a 100% correlation with the CTV phenotype observed in SnAP. For the 378 SnAP accessions, 238 (63.0%) showed resistance or intermediate resistance to the CTV CA/Logan strain and possessed the resistance allele of the S07_2970381 marker, and 100 (26.5%) were susceptible and possessed the susceptible allele of the S07_2970381 marker, while 33 (8.7%) were segregating for CTV reaction, and 7 (1.8%) had missing data (For the DBP (88 accessions) infected with the Wor strain, a correlation of 99% was observed between the S07_2970381 SNP marker and the disease score. There were 42 accessions with resistant disease scores ≤ 4.0, which possessed the resistant allele of the S07_2970381 SNP marker, and 13 with susceptible disease scores from 7.0 to 9.0, which possessed the susceptible allele  with the S07_2970381 and S07_2970276 SNP markers in the SnAP and DBP. In fact, ‘SAS8.1550’ and S07_2970276 markers were 100% identical in both bean panels.A comparison of genotypes for the S07_2970381 SNP marker and the ‘SAS8Bct region. Four haplotypes were identified: Haplo1 with AGT sequence for the S07_2966197 (A/G), S07_2970276 (G/C), and S07_2970381 (T/A) SNPs, respectively; Haplo2 with AGA sequence; and Haplo3 divided into two sub-haplotypes, Haplo3-1 with ACA sequence and Haplo3-2 with GCA sequence, based on polymorphism for S07_2966197 in Middle American accessions.The SnAP and DBP were assayed for the S07_2970381, S07_2970276, and S07_2966197 markers to identify haplotypes for the In the SnAP Table 4,pHolm-adjusted p-values, showed that the susceptible Haplo1 was significantly different from the three resistant haplotypes (p < 0.0001), while the only significant difference among resistant haplotypes mean scores was between Haplo3-1, which was more resistant than Haplo3-2 (p = 0.05)  in candidate gene Phvul.007G036300 (Exonuclease V) was identified as the most likely causal mutation conditioning Bct resistance. Moreover, GWAS of the DBP identified the S07_2970381 SNP variant as the only significant SNP for BCTV reaction in the field.The Arabidopsis homolog AT5G60370, which encodes an Exonuclease V-Like (EXOVL) protein, exhibited 43% sequence identity  with the Phvul.007G036300 Bct candidate gene. EXOVL has been reported to play critical roles in biological processes underlying morphological traits in A. thaliana . This protein is capable of degrading single-stranded DNA (ssDNA) from the 5′-end and plays a critical role in mitochondrial maintenance  from the SnAP, DBP, and additional survey of 57 dry bean genotypes clearly co-segregated with alleles of the S07_2970381 SNP marker. SVS-0863 dry bean with a tolerant disease score in the field but lacking the resistant marker allele is the only minor exception. The ‘Beluga’ dry bean with similar field tolerance but lacking the marker allele for resistance was later found to be susceptible to CTV via agro-inoculation.The Tm-shift assay marker developed for SNP S07_2970381 will be useful for tracking CTV resistance conferred by the Bct EXO5 candidate gene is nearly ubiquitous in the Middle American gene pool as 98% (140/143) Middle American snap (including admixtures) and dry bean genotypes exhibited CTV resistance (including intermediate resistance and tolerance) and possessed the resistance allele for the EXO5 candidate gene mutation (S07_2970381 SNP). Conversely, only 65% (214/327) of Andean (including admixtures) snap and dry bean genotypes tested were resistant to CTV and possessed the resistance allele for the marker. In addition, all 113 Andean snap (including admixtures) and dry bean genotypes susceptible to CTV lacked the resistance allele for the Bct EXO5 candidate gene marker. Finally, only three Middle American snap bean accessions were susceptible to CTV, and they lacked the resistance allele for the marker.The .1550’, and S07_2970276 and ‘SAS8.1550’ markers, together with distinct haplotypes for combinations of the S07_2966197, S07_2970276, and S07_2970381 SNP markers in the SnAP and DBP, suggest different origins of the Bct candidate allele. The susceptible Andean beans all possess the same haplotype (Haplo1) for the three SNP markers S07_2966197, S07_2970276, and S07_2970381 in the Bct region. Haplo2 represents a small set of Andean genotypes with resistance to CTV. Among these genotypes, there are a few Andean dry bean landrace cultivars, including IJR  and G122 from India, Canadian Wonder (ADP_010) and Kijivu (ADP_033) from East Africa, Pompadour B from the Caribbean, and California LRK, with Haplo2, which suggests that this haplotype and associated Bct resistance is of Andean origin. This ‘Haplo2’ Bct resistance source was unlikely to have been purposely used in breeding for CTV resistance. All the dry bean genotypes in the DBP with Haplo2 represent mostly breeding lines that had never been screened for CTV reaction prior to this study, and their pedigrees reveal the use of Canadian Wonder, IJR, and Kijivu landrace cultivars as parents. Moreover, only 5 of 177 CTV-resistant snap bean accessions possessed Haplo2.Resistance to CTV in snap bean was purposely initiated from crosses with Middle American dry bean genotypes like ‘Burtner’ navy bean in 1936 and small red (~ ‘Common Red Mexican’) beans during the 1950s  and dry bean (45%) genotypes in this study. Many of the dry bean genotypes with Haplo3-1 come from breeding programs that were actively selected for CTV resistance.The differences in percentage correlation between S07_2970381 and ‘SAS8he 1950s . Haplo3-Bct resistance. The mean disease score (3.5) for the Haplo3-1 dry bean genotypes indicates a significantly (p = 0.05) higher level of resistance than the mean score (4.4) for Haplo3-2 genotypes. Resistance levels for snap beans with Haplo2, Haplo3-1, or Haplo3-2 were undiscernible because only qualitative data existed for CTV reaction.Haplo3-2, on the other hand, is represented by older great northern bean cultivars  and more modern race Durango cultivars  with upright architecture obtained from race Mesoamerica introgressions. Few, if any, of the dry bean genotypes with Haplo3-2 were developed by breeding programs with active or passive selection for Bct EXO5 candidate gene should also include SNP markers S07_2966197 and S07_2970276 to monitor the different haplotypes and corresponding associations that they may have with quantitative levels of resistance observed by phenotyping. Meanwhile, based on information from this study, our recommendation is to use Haplo3-1 for MAS of CTV resistance.In light of these findings, breeding programs using S07_2970381 for MAS for CTV resistance conferred by the The original contributions presented in the study are included in the article/AS-G, JH, and PM conceived and designed the experiments. AS-G and JH implemented statistic and bioinformatic analyses. DG, AT, and CS performed and conducted phenotyping strategies. QS performed SNP chip assay genotyping. KG conducted virus detection in the field. All authors contributed to the article and approved the submitted version.


Background: Recent research has unveiled the association between microbiota and the onset and progression of breast cancer (BC). This study investigates the microbiota in breast tissue, the gut, and the oral cavity in relation to different pathological types of breast diseases, aiming to unveil the microbiota-BC relationship and provide new perspectives for BC diagnosis and treatment.Methods: The study encompassed a total of 98 breast cancer patients, with 52 diagnosed with Luminal A BC, 17 with Luminal B BC, 18 with HER2 BC, and 11 with TNBC. In addition, there were 46 patients with non-malignant breast diseases. The V3-V5 region of the 16S rRNA gene of breast tissue, feces, and the oral cavity was sequenced. Based on Amplicon Sequence Variants (ASV) representative sequences and abundance information, a series of statistical analyses were conducted including community diversity analysis, community composition analysis, species difference analysis, correlation analysis, and functional prediction analysis.Results: Notable divergences in α-diversity and β-diversity were discerned in breast tissue between BC patients and non-malignant breast disease patients. The linear discriminant analysis effect size (LEfSe) and random forest examinations pinpoint Pasteurellaceae as a significant predictor in BC cohorts. Further exploration revealed significant microbial distribution divergences across distinct pathological types of BC, with notable variations in the relative abundance of microbial species such as Streptococcus, Serratia, and Pseudomonas, underscoring the diverse microbial diversity across BC subtypes and sample origins.Conclusion: This venture sheds light on the complex microbiota milieu across varying body sites and pathological types of BC, emphasizing microbiota-BC connectivity. This articulation of a multisite microbiota-BC interrelation significantly advances a holistic grasp of BC pathogenesis. Breast cancer (BC) reigns as the most prevalent cancer among women globally, with estimations in 2023 projecting nearly 300,000 women will be diagnosed and around 43,170 women will succumb to this ailment , accentuThe human microbiome, a refined and dynamic aggregate of microbial entities, exhibits its ubiquity across a plethora of anatomical niches, spanning from the epidermis to the oral and vaginal cavities. Emerging scientific paradigms robustly accentuate the profound impact of host microbiota in modulating the tumor microenvironmental ambit. Dysbiosis within this microbial aggregate can precipitate chronic inflammatory states, reconfigure immune responses, and potentially undermine genome fidelity, thereby instigating DNA aberrations and dysregulating metabolic pathways. Such perturbations furnish a fertile substrate for BC pathogenesis and advancement .Fusobacterium nucleatum, an oral anaerobe with established oncogenic tendencies, in breast cancer, further accentuates the complexity of these interactions  promptly upon participants’ arousal (between 7 and 8 a.m.), with 5–10 mL being collected. Fecal samples were self-collected pre-operatively using stool sampling kits. Fresh-frozen breast tissues were acquired in adherence to standard biorepository protocols from individuals undergoing surgical intervention for breast cancer. A specimen container, housing 5 mL of sterile saline or water, was unveiled in the surgical suite during breast surgery to mitigate potential microbial contamination from the environment. Concurrently with tissue specimens, these environmental controls were processed. All specimens were preserved at −80°C within a 4-h window post-receipt, pending conveyance to the laboratory for processing and analysis.Salivary specimens were obtained utilizing the SalivettesGenomic DNA was extracted from the specimens employing the CTAB/SDS methodology. The concentration and purity of DNA were ascertained by electrophoresis on a 1% agarose gel. DNA was diluted to a concentration of 1 ng/μL using sterile water, the specific dilution factor being contingent on the initial concentration.® High-Fidelity PCR Master Mix, 0.2 µM of both forward and reverse primers, and approximately 10 ng of template DNA. The thermal cycling regimen entailed an inaugural denaturation step at 95°C for 4 min, succeeded by 30 cycles, each encompassing denaturation at 95°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, culminating with a 72°C hold for 4 min. Ultrapure water was utilized in the DNA extraction procedure to preclude false-positive PCR outcomes. The 16S rRNA genes from varied loci, namely, 16S V4, 16S V3, 16S V3-V4, and 16S V4-V5, were amplified employing designated primers  inclusive of barcodes. The PCR reactions comprised 15 µL of Phusion® High-Fidelity PCR Master Mix, 0.2 µM of both forward and reverse primers, and approximately 10 ng of template DNA. The thermal cycling regimen entailed an inaugural denaturation step at 95°C for 4 min, succeeded by 30 cycles, each encompassing denaturation at 95°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, culminating with a 72°C hold for 4 min.Ultrapure water was utilized in the DNA extraction procedure to preclude false-positive PCR outcomes. The 16S rRNA genes from varied loci, namely, 16S V4, 16S V3, 16S V3-V4, and 16S V4-V5, were amplified employing designated primers  inclusive of barcodes. The PCR reactions comprised 15 µL of PhusionAn equal volume of 1× loading buffer was amalgamated with the PCR outputs, followed by electrophoresis on a 2% agarose gel for detection. PCR products were pooled in equimolar proportions, thereafter undergoing purification via the Qiagen Gel Extraction Kit.® DNA PCR-Free Sample Preparation Kit, in congruence with the manufacturer’s stipulations. Index codes were integrated into the libraries. Library quality was evaluated using the Qubit@ 2.0 Fluorometer and the Agilent Bioanalyzer 2100 apparatus. The library was sequenced using Illumina NovaSeq technology, yielding 250 base pair paired-end reads. Initially, quality control and filtering, based on sequencing quality, are performed on the paired-end reads. Concurrently, the overlapping relationships between reads are utilized for assembly, yielding optimized data post-assembly. Subsequently, the DADA2 sequencing denoising method is utilized to obtain Amplicon Sequence Variant (ASV) representative sequences and abundance information. Utilizing the ASV representative sequences and abundance information, a series of statistical or visual analyses are conducted, including taxonomic, community diversity, community composition, species difference, correlation, and functional prediction analyses.Sequencing libraries were generated employing the TruSeqin situ hybridization findings. The calculation of Body Mass Index (BMI) was performed as the ratio of body weight (in kilograms) to the square of height (in meters). The surgical interventions in breast tissue were distinguished as either lumpectomy or mastectomy, contingent upon the extent of surgery.Baseline clinical and pathological data were documented, encompassing patients’ age, pre-existing basal diabetes, hypertension and diabetes history, body mass index (BMI), along with pathological and surgical details. TNM staging was evaluated according to the guidelines set forth in the 8th edition of the American Joint Committee on Cancer . Based op-value of <0.05 denoting statistical significance.Bioinformatics and statistical analysis were performed on the derived sequence data. The DADA2 method , containThe study encompassed a total of 98 breast cancer patients, with 52 diagnosed with Luminal A BC, 17 with Luminal B BC, 18 with HER2 BC, and 11 with TNBC. In addition, there were 46 patients with non-malignant breast diseases. The baseline characteristics of the cohort are in Initially, comparative scrutiny of microbial compositions across three sample types from individuals with BC and those with non-malignant breast ailments was performed.p = 0.019, p = 0.019), Shannon (p = 0.015), and Simpson (p = 0.021). The indicators of α-diversity of microbial communities in breast tissue, fecal and saliva samples are in p = 0.032) and Weighted UniFrac (p = 0.019), also demonstrated significant disparities between breast cancer patients and non-malignant cases. In feces and saliva samples, no significant differences were observed in the breast tissue, feces, or saliva samples between breast cancer patients and patients with non-malignant breast diseases. The indicators of β-diversity of microbial communities in breast tissue, fecal and saliva samples are in Employing α-diversity and β-diversity as pivotal descriptors, we gained a more nuanced understanding of the microbiota’s overall assembly and dispersion in relation to BC susceptibility. Since only the α-diversity and β-diversity of breast tissue samples showed significant differences, we conducted further analysis on the breast tissue samples using LEfSe and Random Forest, along with functional prediction analysis.p < 0.05). The results indicated that Pasteurellaceae, Prevotellaceae, Carnobacteriaceae, Lactobacil, Porphyromonadaceae, and Actinomycetaceae exhibited significant abundance differences in the breast cancer group. A subsequent random forest examination of the top 10 genera within the BC cohorts pinpointed Pasteurellaceae as having a prime predictive acumen with an area under the curve (AUC) of 68.64%, followed by Prevotellaceae (68.04%), Akkermansia (64.68%), Rhodobacteraceae (64.58%), Succiniclasticum (64.56%) and Fusobacterium (61.98%) , we estimated the effect size of abundance differences for each species  . The PIC(61.98%) .Further exploration ensued on microbiological variances across three paired samples from BC-afflicted individuals showcasing diverse pathological types, including Luminal A, Luminal B, HER2, and TNBC. The Shannon diversity index of breast tissues, fecal and saliva samples, which considers both species richness and evenness, indicate a high level of diversity within four subtypes of breast cancer . To visuMicrobial species composition and relative abundance were investigated across various pathological types within breast tissue, fecal, and saliva samples. In breast tissue samples , microbiStreptococcus and Lactobacillus abundance were noted across all pathological types, with HER2 and Triple negative types exhibiting higher levels of Pseudomonas. In fecal samples . This research has the potential to shape innovative predictive and preventive strategies, as well as personalized treatment protocols that may include microbial modulation. The dynamic relationship between microbial communities and the immune system, alongside the capacity of certain microbes to either induce genetic instability or offer therapeutic advantages, presents promising avenues for investigation. Embracing a precision medicine framework could transform BC management by integrating microbial, genomic, and environmental data to customize prevention and treatment for each patient’s unique profile.The study highlights the intricate connection between microbial structures, their functional roles, and the diversity observed among different subtypes of breast cancer. Further investigation is required to fully comprehend the possible contributions of these microbial organisms to the dynamics of breast cancer.


Clonal hematopoiesis of indeterminate potential (CHIP) increases the risk of cerebrovascular events, while its association with cerebral white matter hyperintensity (WMH) is undemonstrated. We evaluated the effect of CHIP and its major driving mutations on cerebral WMH severity.From an institutional cohort of a routine health check‐up program with a DNA repository database, subjects who were ≥50 years of age, with one or more cardiovascular risk factors but no central nervous system disorder, and performed brain MRI were included. Along with the presence of CHIP and its major driving mutations, clinical and laboratory data were obtained. WMH volume was measured in total, periventricular, and subcortical regions.DNMT3A mutation (48.8%), followed by TET2 (11.9%) and ASXL1 (8.1%) mutations. Linear regression analysis adjusting for age, sex, and conventional cerebrovascular risk factors suggested that CHIP with DNMT3A mutation was associated with the lower log‐transformed total WMH volume, unlike other CHIP mutations. When classified according to variant allele fraction (VAF) value of DNMT3A mutation, higher VAF classes were associated with the lower log‐transformed total WMH and the lower log‐transformed periventricular WMH volume, but not with the log‐transformed subcortical WMH volumes.Among the total 964 subjects, 160 subjects were classified as CHIP positive group. CHIP was most frequently associated with DNMT3A mutation is quantitatively associated with a lower volume of cerebral WMH, especially in the periventricular region. CHIP with DNMT3A mutation might have a protective role in the endothelial pathomechanism of WMH.Clonal hematopoiesis with  DNMT3A mutation is associated with lower white matter hyperintensity volume. In healthy aged population, clonal hematopoiesis of indeterminate potential (CHIP) with DNMT3A mutation, the most common driving mutation of CHIP, is associated with lower total WMH volume and WMH volume in the periventricular region. This finding is inconsistent with the previous studies which reported that CHIP increased the risk of major cardiovascular events.Clonal hematopoiesis with  Among the initially included 1154 subjects, the final study population was defined according to the following criteria: (1) ≥50 years of age, (2) without a confirmed diagnosis of a central nervous system (CNS) disorder such as dementia, stroke, tumor, or other structural or neurodegenerative disease, (3) without a major cardiac or renal co‐morbidity affecting the WMH status such as open heart surgery, heart valve disease, congestive heart failure, or end‐stage chronic kidney disease.Clinical profiles including age, sex, and presence of hypertension, diabetes mellitus, hyperlipidemia, chronic kidney disease, current smoking, ischemic heart disease, and regular use of antithrombotic agents were obtained. Laboratory data were also obtained at the time of DNA sampling, which blood parameters such as hemoglobin (g/dL), hematocrit (%), platelet count and white blood cell count (/μL), glomerular filtration rate (mL/min), hemoglobin A1c , total and low‐density lipoprotein (LDL) levels (mg/dL), C reactive protein (mg/dL), and systolic and diastolic blood pressures (mmHg).2.2APC, ASXL1, ASXL2, ATM, BCL11B, BCOR, BCORL1, BIRC3, BRAF, BRCC3, CARD11, CASP8, CBL, CD58, CD79B, CHEK2, CNOT3, CREBBP, CUX1, DDX3X, DNMT3A, EP300, ETV6, EZH2, FAM46C, FBXW7, FLT3, FOXP1, GNAS, GNB1, GPS2, HIST1H1C, IDH2, IKZF1, IKZF2, JAK1, JAK2, JAK3, JARID2, KDM6A, KIT, KLHL6, KMT2D, KRAS, LUC7L2, MAP3K1, MPL, MYD88, NF1, NFE2L2, NOTCH1, NOTCH2, NRAS, PDS5B, PDSS2, PHF6, PHIP, PIK3CA, PIK3R1, PPM1D, PRDM1, PRPF40B, PTEN, PTPN11, RAD21, RIT1, RPS15, SETD2, SETDB1, SF1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG1, STAG2, STAT3, SUZ12, TBL1XR1, TET1, TET2, TNFAIP3, TNFRSF14, TP53, U2AF1, VHL, WT1, ZRSR2) frequently involved in CHIP. Sequencing reads were trimmed by the quality and aligned to the human genome (hg19) using BWA‐MEM (v0.7.10). Reads were re‐aligned and the base quality score was recalibrated with GATK (v2.3.9). Single nucleotide variants (SNVs) were called using SNver, LoFreq, and GATK. Insertions and deletions were called using an in‐house InDel caller.After obtaining informed consent for blood storage, blood samples were collected from subjects during a routine health check‐up program. Genomic DNA was separated from peripheral blood and stored at −80°C until sequencing. Targeted sequencing was performed using Agilent SureSelectXT HS custom panel and Illumina NovaSeq 6000 with 2 × 150 bp paired‐end reads and 800× coverage. The custom panel comprises a total exon of 89 genes  any truncating mutations including nonsense, splice site mutation, or frameshift indel; (2) any variants previously reported as somatic at least 10 times in solid cancer or at least 1 time with hematopoietic or lymphoid cancer in COSMIC database v83.2.4Magnetic resonance image evaluations were performed using 1.5‐T or 3.0‐T units with protocols that included T1‐weighted, T2‐weighted, T2 fluid‐attenuated inversion recovery (FLAIR), gradient echo/susceptibility‐weighted imaging (GRE/SWI), intracranial time‐of‐flight (TOF) MRA, and neck MRA. Parameters for T2‐weighted and FLAIR images were as follows: slice number = 24–30, slice thickness/gap = 4.0–5.0/0.0–2.0 mm, repetition time/echo time = 6000–10,002/92–168.5 milliseconds, field‐of‐view = 285–220 × 285–220 mm, and matrix = 220–512 × 192–400  reviewed the MRI to exclude a preexisting ischemic or hemorrhagic stroke lesion, or a significant stenosis in the cerebral arteries. The volumetric analysis of WMH was performed by a neurologist specialist (K‐I. P.), and confirmed by a neurologist (W‐J.L.), according to previously described protocols.2.5r was used to evaluate correlations, a two‐tailed t test or Mann–Whitney U test to compare mean values, and a chi‐square to compare frequencies. Age, sex, conventional vascular risk factors, CHIP or CHIP‐driving mutations of interest, and variables with p values <0.10 in univariate analyses were included in regression analyses using enter method. In linear regression analyses, WMH volumes were log‐transformed to obtain a normal distribution  was used for all statistical analyses. Pearson's n Figure . To adju3DNMT3A mutation, 19 (11.9%) exhibited TET2 mutation, 13 (8.1%) exhibited ASXL1 mutation, and 61 (19.4%) were associated with other mutations , cerebrovascular risk factor profiles, laboratory profiles, and WMH severity profiles were comparable between the two groups , 160 (16.6%) subjects were classified as CHIP positive (CHIP group) and the remaining 804 (83.4%) as CHIP negative (non‐CHIP group). In the CHIP group, 78 (48.8%) subjects exhibited s Figure . 24 , hypertension , and current smoking  were associated with higher log‐transformed total WMH volume (Table p = 0.008) (Table R2 0.304 vs. 0.294).In the following linear regression analysis, age  Table , model 2DNMT3A mutation was significantly associated with the lower log‐transformed total WMH volume , after adjusting age, sex, and conventional cerebrovascular risk factors  , ASXL1 mutation , or CHIP with non‐DNMT3A mutation  were not associated with the log‐transformed total WMH volume  Table . CHIP wime Table .DNMT3A mutation on WMH volume . Following regression analyses, higher VAF classes of CHIP with DNMT3A mutation were associated with the lower log‐transformed total WMH volume  and the lower log‐transformed periventricular WMH volume , but not with the log‐transformed subcortical WMH volumes  ,DNMT3A mutation might have distinct clinical effect from those of other major CHIP‐driving mutations. In a study that included subjects with coronary heart disease and controls, each major CHIP‐driving mutations, DNMT3A, TET2, ASXL1, and JAK2, was associated with an increased risk of coronary heart disease, but the hazard ratio was the lowest for the DNMT3A mutation.DNMT3A mutation with diabetic polyneuropathy in patients with type 2 diabetes, whereas TET2 mutation showed a positive association.DNMT3A and TET2 in DNA methylation and hematopoietic stem cell differentiation might explain their opposite association with diabetic polyneuropathy. While TET2 is involved in DNA demethylation and myeloid stem cell differentiation, DNMT3A mediates de novo methylation of cytosine bases and inhibits hematopoietic stem cell differentiation.Considering that the proportions of CHIP among the total study population and of DNMT3A might explain the protective effect of CHIP with DNMT3A mutation. In WMH pathogenesis, aging‐related decrement of cerebral small vessel compliance has a fundamental role.Although not demonstrated in this study, distinct biological features of DNMT3A with enhanced self‐renewal and cell survival might attenuate this shear stress‐dependent pathologic processes by leukocyte‐endothelium interaction.It is recently suggested that DNMT3A mediates these shear stress‐related processes. DNMT3A expression in vascular endothelium is enhanced by fluctuating shear stress, increases methylation in the promoter of Kruppel‐like factor 4 (KLF4), and inhibits transcription of KLF4.DNMT3A mutation was associated with lower WMH volume in the periventricular region. As the periventricular region is supplied by the compliant perforating arterioles directly ramifying from the large cerebral arteries, WMH in the periventricular region is particularly susceptible to the aging‐related reduction in the small vessel compliance.DNMT3A mutation on WMH might have been more evident in the periventricular region.In this study, CHIP with DNMT3A mutation and its temporal change on the long‐term development or progression of WMH were not examined. Second, due to the small number of subjects with extensive clonal expansion , the impact of the extent of clonal expansion on the progression of WMH was not addressed in this study.DNMT3A mutation on the pathogenesis of WMH was not directly demonstrated by experimental studies. Fourth, due to the low burden of WMH in the study population, the results of this study should not be applied to the relationship between CHIP and major cerebrovascular risk. The lack of animal models that adequately represent the pathomechanism of aging‐related WMH progression might be an obstacle to overcome. Future prospective studies with follow‐up evaluations on the change in the extent of clonal expansion and WMH severity, along with experimental studies with targeted gene editing of the myeloid cells, are warranted to clarify the effect of CHIP with DNMT3A mutation on the pathomechanism of cerebral WMH.This study has several limitations to be addressed. First, as a cross‐sectional study, the impact of CHIP with In conclusion, clonal hematopoiesis with DNMT3A mutation is quantitatively associated with a lower volume of cerebral WMH, especially in the periventricular region. CHIP with DNMT3A mutation might have a protective role in the endothelial pathomechanism of WMH.H.S. and Y.K. are shareholders in Genome Opinion, Inc.Appendix S1.Click here for additional data file.


ΔH after Iodixanol treatment. The plasma-free erythrocytes revealed a pronounced shift in the maxT and a decrease in the ΔH of hemoglobin and transferrin. The total ΔH of Iodixanol treatment showed the highest decline, while Iomeprol and Iobitridol had fewer adverse effects. Similarly, the non-anticoagulated samples revealed a decrease both in the maxT and the ΔH of albumin and immunoglobulin-specific transitions. The total ΔH showed that Iodixanol had more influence on the serum. The serum-free erythrocyte samples resulted in a significant drop in the maxT of erythrocyte and transferrin (~5–6 °C). The ΔH of deconvolved hemoglobin and transferrin decreased considerably; however, the ΔH of albumin increased. Surprisingly, compared to Iomeprol and Iobitridol treatments, the total ΔH of Iodixanol was less pronounced in the non-anticoagulated erythrocyte samples. In sum, each non-ionic contrast medium affected the thermal stability of anticoagulated and non-anticoagulated erythrocyte proteins. Interestingly, Iodixanol treatment caused more significant effects. These findings suggest that conformational changes in blood components can occur, which can potentially lead to the increased prevalence of cardiovascular dysfunctions and blood clotting.To study the effect of non-ionic contrast media on anticoagulated and non-anticoagulated human whole blood samples, calorimetric measurements were performed. The anticoagulated plasma showed the greatest fall in the total  Contrast media are routinely used for contrast enhancement of human biological structures in medical imaging, such as Computed Tomography (CT), X-ray and Magnetic Resonance Imaging (MRI) ,2,3. SinIodine-based contrast media are water-soluble and can be divided into two groups: ionic and non-ionic compounds. Ionic contrast media have more adverse effects due to the high osmolality. These severe adverse effects include thyroid dysfunctions, hypersensitivity reactions and contrast agent-induced nephropathy (CIN). At the molecular level, these conditions can be related to chemotoxicity , osmotoxicity (inhibiting the transportation of water molecules through membranes) and ion toxicity of the contrast media (ionic compounds might be able to change cellular functions) . AnticoaNowadays, non-ionic contrast media are used more frequently because they lead to fewer adverse effects due to their decreased osmolality, and their application is safer compared to the ionic ones ,11. In s® 400 mg I/mL) is a non-ionic, monomeric, water-soluble X-ray contrast agent. It consists of three covalently bound iodine atoms and has low osmolality, chemotoxicity and viscosity. The osmolality and viscosity of Iomeprol is lower compared to other non-ionic contrast media -2, 4, 6-triiodo-1, 3-benzenedicarboxamide) as an active agent. Xenetix® 350 mg I/mL solution consists of 383.90 g Iobitridol -5-(2-(hydroxymethyl)hydracrylamido)-2,4,6-triiodo-N,N′-dimethylisophthalamide) (corresponds to 175.0 g iodine) as active material. Visipaque® 320 mg I/mL stock solution consists of 326.0 g Iodixanol -2,4,6-triiodo-phenyl]amino]-2-hydroxy-propyl]amino]-N,N′-bis-2,4,6-triiodo-benzene-1,3-dicarboxamide)  as an active material. Contrast media were applied 10 min before each measurement by gently mixing them with the blood samples in a concentration of 40 mM, which is twice as high as the concentration typically given to patients (20 mM). The aim of our study was to compare the effects of the applied contrast media on blood plasma, serum, and erythrocyte components.Iomeron−1 between 20–100 °C. During the sample preparation (appropriate mixture of blood sample and contrast agent), 850 µL of sample or normal saline (reference) was pipetted in conventional Hastelloy batch vessels (maxV = ~1 mL) for the measurements. The sample and reference vessels were balanced with a precision of ±0.05 mg; therefore, correction was not needed with the heat capacity of the vessels. The second denaturation procedure was used for the baseline correction of the data. At the beginning of measurements, the Hastelloy batch vessel containing the given sample is at atmospheric pressure. During measurements, the pressure can increase inside the vessel, leading to approximately 2 bars (202.6 kPa) at the end of the heating cycle. At lower temperatures, denaturation of blood components can occur, suggesting that the increased pressure has no significant effect on the thermal transition of proteins. DSC is a comprehensive method to measure the thermodynamic properties of biological and clinical samples ,38,39,40®2020 software. To quantify the thermodynamic properties of our samples, we determined the melting temperatures (mT). The mT value of the denaturation indicates the peak temperature of the calorimetric thermal curve, where 50% of the sample is unfolded. The value of the mT is a key indicator of the thermodynamic stability of proteins. The greater the mT value, the more stable the structure of the protein is [mT value is used when the protein is highly pure, and the denaturation mechanism is supposed to be a single-step transition. In our measurements, the fractionated blood samples contained multiple protein components. Therefore, maxT was used as an indicator of the thermodynamic stability of the complex system. The normalized enthalpy change (ΔH) was calculated using the area under the deconvolved thermal curves.Anticoagulated and non-anticoagulated blood plasma, serum and cellular elements of human whole blood consist of several proteins ,42,43; totein is . Tm valuIn order to compare the effects of Iomeprol, Iodibitridol and Iodixanol on the thermodynamic properties of anticoagulated and non-anticoagulated human whole blood samples , DSC measurements were performed. max1(c)T) was 62.9 °C, which is in accordance with previous findings [maxT of albumin (maxT of the deconvolved immunoglobulin-specific protein curves appeared at max2(c) =T 67.3 °C, max3(c) =T 71.2 °C and max4(c) =T 75.4 °C, respectively; while the treatment with 40 mM Iomeprol, Iobitridol and Iodixanol produced a negligible change in these maxT values  = 0.04 J·g−1, 3(cΔH) = 0.02 J·g−1 and 4(cΔH) = 0.02 J·g−1, respectively. Both the 40 mM Iomeprol and the Iodixanol treatments resulted in a considerable decrease in the enthalpy of each deconvolved immunoglobulin curve; however, no significant change was found during the treatment using 40 mM Iobitridol (2(vΔH) = 0.03 J·g−1, 3(vΔH) = 0.01 J·g−1, 4(vΔH) = 0.005 J·g−1) ΔH = 0.14 J·g−1 (~23% decrease), whereas the control value of totalΔH was 0.18 J·g−1  . In line18 J·g−1 , indicatmax1(c)T = 70.5 °C, which is similar to previous findings (72 °C) and may be due to the different scanning speed and setting parameters [maxT of the deconvolved control minor transitions were max2(c)T = 76.7 °C and max2(c)T = 81.7 °C, respectively. The maxT of the major hemoglobin peak decreased the most with the 40 mM Iodixanol treatment (max1(v)T = 68.7°C), while the effect of Iomeprol and Iobitridol was less pronounced (maxT of the deconvolved minor curves was 76.7 °C (max2(c)T) and 81.7 °C (max3(c)T). The addition of 40 mM Iomeprol and Iobitridol slightly decreased maxT, while the use of 40 mM Iodixanol caused the most significant thermal shift (max2(v)T = 74.9 °C and max3(v)T = 78.5 °C)  , Table 41(c)ΔH = 0.862 J·g−1. It did not change considerably in the presence of 40 mM Iomeprol and Iobitridol. The greatest decrease could be observed after the treatment with 40 mM Iodixanol (1(v)ΔH = 0.747 J·g−1), which resulted in a 14% fall in the ΔH (2(c)ΔH = 0.06 J·g−1 and 3(c)ΔH = 0.23 J·g−1, respectively. Interestingly, each treatment led to a significant drop in the ΔH of both minor transitions of anticoagulated erythrocyte samples (2(i)ΔH, 2(x)ΔHand2(v)ΔH), compared to the control (2(c)ΔH) value. Furthermore, the enthalpy change of the third minor peak showed a drastic fall after the treatment with Iomeprol (88%), Iobitridol (80%) and Iodixanol (84%) ΔH = 0.82 J·g−1), compared to the Iomeprol (22%) and Iobitridol treatments (13%)  . The totts (13%) . This inThese results suggest that less energy is required to unfold the major hemoglobin peak in the case of Iodixanol treatment, resulting in more adverse effects compared to Iomeprol and Iobitridol. The enthalpy changes of the minor deconvolved curves showed a more drastic decrease, suggesting that Iomeprol, Iobitridol and Iodixanol all had a similarly strong adverse effect on the thermodynamic properties of anticoagulated erythrocyte components.In sum, Iomeprol, Iobitridol and Iodixanol may primarily cause conformational changes in the minor transitional components, which can be attributed to the unknown transition and transferrin (peaks 2 and 3). These data suggest a stronger affinity of the contrast media to these proteins. Moreover, the major hemoglobin peak 1 was more influenced by Iodixanol. In summary, the total change in enthalpy decreased the most in the case of Iodixanol, indicating the most adverse effect on anticoagulated erythrocyte proteins.max1(c)T = 63.4 °C, which decreased with 1.6 °C in the presence of Iomeprol and Iodixanol, whereas the Iobitridol treatment resulted in a lower decrease (1.1 °C) in the maxT value  ,49 was Tax value , Table 7max2(c)T = 67.6 °C, max3(c)T = 71.1 °C and max4(c)T = 75.1 °C, respectively (maxT for peak 3 (1.1 °C) and peak 4 (1.6 °C), compared to Iomeprol and Iobitridol  following the Iodixanol treatment . A considerable increase of the ΔH was observed in the presence of Iomeprol (28%), Iobitridol (46%) and Iodixanol (32%) , decreasing the ol (32%) .total =ΔH 0.19 J·g−1), suggesting that Iodixanol had the strongest adverse effect on the thermodynamic properties of non-anticoagulated serum samples T = 69.6 °C, max2(c)T = 81.6 °C and max3(c)T = 85.6 °C (maxT found in previous findings (~72 °C) [−1 compared to 1 K·min−1). The presence of 40 mM Iomeprol, Iobitridol and Iodixanol caused no major changes in the maxT of hemoglobin. In addition, each contrast medium produced a drastic thermal shift (~5–6 °C decrease) in the deconvolved minor transitions (peaks 2 and 3), which can be attributed to the presence of helical erythrocyte and transferrin  , which insferrin .Interestingly, the calculated enthalpy of deconvolved non-anticoagulated erythrocyte transition curves increased by 30%, 33% and 22% in the presence of 40 mM Iomeprol, Iobitridol and Iodixanol, respectively . In contIn line with this, the total enthalpy change was slightly less pronounced in the presence of Iodixanol (27%) compared to the Iomeprol and Iobitridol treatments (33% and 31%), respectively . These dmaxT and ΔH resulted in a drastic fall in the helical erythrocyte and transferrin thermal transitions with each contrast medium, but it was less pronounced in the Iodixanol-treated samples, suggesting that Iomeprol and Iobitridol had more significant adverse effects. Iodixanol could not influence the helical erythrocyte and transferrin as strongly as Iomeprol and Iobitridol.In sum, the applied contrast media had no major effect on the melting temperature of hemoglobin. However, a significant increase was observed in the enthalpy of Iomeprol and Iobitridol-treated samples, whereas the Iodixanol treatment revealed a less adverse effect. The change in the Contrast media are widely used in routine medical imaging, such as CT, X-ray and MRI ,2,3. ThemaxT) and the change in the enthalpy (ΔH) were determined in order to characterize the detailed thermodynamic effects of the contrast media on human whole blood components.To study the effect of three non-ionic, iodine-containing contrast media, DSC measurements were carried out on anticoagulated and non-anticoagulated human whole blood samples using 40 mM of Iomeron, Xenetix or Visipaque. The melting temperature  and erythrocyte protein samples . However, Iomeprol and Iobitridol also caused considerable harmful effects  than on the plasma samples, compared to Iomeprol and Iobitridol . Moreove effects . The thermal denaturation analysis of non-anticoagulated blood serum samples indicated that 40 mM Iodixanol caused a slightly greater thermal shift in the deconvolved serum transition curves of HSA and immunoglobulin-specific proteins than Iomeprol and Iobitridol , Table 7Overall, the total enthalpy change indicates that Iodixanol had the strongest adverse effects on the thermodynamic properties of non-anticoagulated serum components .maxT (~5–6 °C decrease) in the presence of each contrast medium  . Howevert medium . The entIn contrast to the results found in the serum samples, the treatments with each contrast agent considerably increased the enthalpy change of non-anticoagulated hemoglobin transition (peak 1)  . These dTransferrin is essential in maintaining the coagulation balance . Based oOur results suggest that out of the three contrast media we used, Iodixanol can produce the most significant impact. However, Iomeprol and Iobitridol can also considerably influence the thermodynamic stability of anticoagulated and non-anticoagulated erythrocyte proteins . These findings indicate that the applied contrast media may modify protein functions and, therefore, lead to cardiovascular dysfunctions or thrombus formation.This work clearly points out some weaknesses of current contrast materials and emphasizes the need for developing a new generation of contrast media in order to avoid harmful effects on human blood components.