Understanding the composition of circulating immune cells with aging and the underlying biologic mechanisms driving aging may provide molecular targets to slow the aging process and reduce age-related disease. Utilizing cryopreserved cells from 996 Framingham Heart Study (FHS) Offspring Cohort participants aged 40 and older , we report on 116 immune cell phenotypes including monocytes, T-, B-, and NK cells and their subtypes, across age groups, sex, cytomegalovirus (CMV) exposure groups, smoking and other cardiovascular risk factors. The major cellular differences with CMV exposure were higher Granzyme B+ cells, effector cells, and effector-memory re-expressing CD45RA (TEMRA) cells for both CD4+ and CD8+. Older age was associated with lower CD3+ T cells, lower naïve cells and naïve/memory ratios for CD4+ and CD8+. We identified many immune cell differences by sex, with males showing lower naïve cells and higher effector and effector memory cells. Current smokers showed lower pro-inflammatory CD8 cells, higher CD8 regulatory type cells and altered B cell subsets. No significant associations were seen with BMI and other cardiovascular risk factors. Our cross-sectional observations of immune cell phenotypes provide a reference to further the understanding of the complexity of immune cells in blood, an easily accessible tissue. Chronic low-grade inflammation during aging  and immunosenescence are biologic processes associated with vulnerability to age-related diseases including neurodegenerative disorders, cardiovascular disease, diabetes, and cancer . Age-relThe Health and Retirement Study (HRS), a nationally representative sample of older adults from the United States, found that the numbers of naïve CD4+ and CD8+ T cells were strongly and inversely related to age in both CMV positive and CMV negative adults and were lower in men . In contUnderstanding the differences in circulating immune cells with age and the underlying biologic mechanisms driving immune cell aging may provide molecular targets to slow the aging process  and in tImmune cell phenotypes profiled from peripheral blood mononuclear cells (PBMCs) samples are shown in Sample inclusion and exclusion criteria are shown in CD8 Tn cell percentages were lower in older age groups compared to younger, while CD8 Teff and Tem cell percentages were higher in older age groups compared to younger, and CD8 Tcm cells did not show a consistent age trend within sex and CMV strata . PercentOn average, we observed a higher CD4/CD8 ratio and higher CD8 Tn/Tm ratio in the CMV negative/equivocal group compared to the CMV positive groups within each sex stratum, and higher levels in females compared to males within each CMV group. The CD4/CD8 ratio was higher in the older age groups for the CMV negative/equivocal participant group but was similar across age groups for the CMV positive groups . Both CDAmong the CD4 cell subtypes, the CMV positive and above LOD groups had lower age and sex adjusted CD4 Tn levels than the CMV negative or equivocal groups, while adjusted Teff, Tem, TEMRA, T helper type 1 (Th 1) and Granzyme B producing cell subtype levels were higher in the CMV positive and above LOD groups than in the negative and equivocal groups. We observed similar patterns among the corresponding CD8 cell subtypes, the one exception being that CD8 Tcm cells also differed by CMV status.B cell levels were similar in negative and equivocal CMV groups, and lower in the CMV positive groups. Finally, all three ARIP measures tested were significantly different across the CMV groups, with slightly higher ratios in the CMV equivocal group compared to the negative group, and lower ratios in the CMV positive and above LOD participant groups compared to the negative group.The age and sex adjusted CMV associations for all 119 immune cell phenotypes for individuals with CMV measurement are presented in As shown in CD3+ T cell levels were lower for each increasing age group. CD4 Tn cell levels were the only CD4 cell subtype with significant differences by age: levels were lower in the older groups compared to the younger. The two youngest age groups differed by only 0.01 SD unit, and the two oldest age group means differed by only 0.02 SD units. CD8 Tn, Teff, and Granyzme B producing CD8 cells all differed by age group at FDR<0.05. CD8 Tn cell levels were lower in each increasing age group, while the other two cell types had higher levels in older age groups.The adjusted mean CD4 Tn/Tm ratio was similar in the two younger age groups, and lower in the three older age groups. The CD8 Tn/Tm ratio had larger differences across age groups than the CD4 Tn/Tm ratio, and the ratio was lower in each subsequent increasing age group. Together, these data are consistent with an age-related shift from naïve cells to a more senescent/exhausted phenotype, with the shift occurring in CD8 cells more evident than CD4 cells.The multivariable adjusted associations with age groups for all 119 immune cell phenotypes are presented in Sex associations for all 119 total immune cell phenotypes and ARIP measures are presented in APOE genotype, obesity status, type 2 diabetes, prevalent CVD or AF at exam 7, hypertension treatment, lipid treatment, SBP, TC, and HDL levels, or diagnosis of chronic blood cancer prior to exam 7 . Estimated effect sizes for each group, p–values, and FDRs are reported in Adjusting for Model 2 covariates, we did not observe significant associations of the 116 immune cell phenotypes and the 3 ARIP measures with education, Among nearly 1000 Framingham Offspring cohort middle-aged and older adult men and women we characterized the circulating innate and adaptive immune system by profiling 116 immune cell phenotypes including subtypes of CD4 and CD8 T cells, B cells, NK cells, and monocytes, and calculated three ARIP measures based on T cell subtypes. We confirmed and extended known associations with CMV, age, and sex in models adjusting for important covariates. Finally, we identified associations of specific immune cell phenotypes with smoking status but not with other cardiovascular disease risk factors and cardiovascular disease. Thus, these factors, including hypertension, cardiovascular disease, atrial fibrillation, and diabetes, as well as body mass index, lipid treatment, systolic blood pressure, total cholesterol, and high-density cholesterol levels, are unlikely to confound the associations between immune cell levels and age, sex, CMV status, and smoking status that we observed in this study, or associations with outcomes of interest that will be explored in future studies. Our cross-sectional observations of immune cell phenotypes in a large community sample provide a reference for the scientific community to further our understanding of the complexity of the immune cell subsets and diversity of the immune system in the blood, a tissue that is easily accessible in human population studies.As expected nearly half the participants in our study sample were CMV positive with women making up about two-thirds of the CMV positive group above LOD. Viral infection, particularly from CMV, is known to associate with alterations to the immune system , 12, 19,Our findings largely confirm other studies that identified increases in memory and TEMRA T cell subsets with CMV positive status , 21, 22.We expand upon a recent report from the Health and Retirement Study of T cells, CD4 and CD8 cells and their naïve, effector, effector memory, and central memory subtypes  to incluConsistent with previous reports , 16, 19,There are recognized differences in the immune system in men and women with women at greater risk for autoimmune disease. Differences increase at older ages (after age 65 years), with men observed to have higher levels of innate activity (inflammaging) and lower adaptive immunity compared to women , althougFinally, we also observed lower levels of B cells in men compared to women consistent with other reports , 33 and Although B cells were not significantly different between current smokers and non-smokers, many of the B cell subsets were. Specifically, the memory B cells (CD19+CD27+CD43-) were higher in smokers, while the percent of the memory B cells  that express IgD+ and/or IgM+ were lower in smokers. Previous studies have shown similar results with memory B cells for current smokers and demonstrated that former smokers had memory B cells similar to that of never smokers. They additionally saw a similar class-switch in the memory B cells in current smokers, measured by a loss of IgM . The swiEqually important in this study is the lack of association of these immune cells with cardiovascular risk factors. While there were some weak associations with prevalent cardiovascular disease, diabetes, and hypertension, none remained significant using an FDR<0.05. These weak associations have been previously reported in studies with fewer immunophenotypes , 14, 20.APOE genotyping permitting association analyses that may provide a reference for future studies of immune cells and various clinical conditions. The participants in this study continue under surveillance for clinical outcomes and have up to 20 years of follow up data that can be used in future studies to examine the relationship between immune cell phenotypes and incident cardiovascular, cognitive, and other health outcomes. The study also has limitations. The FHS Offspring participants are predominantly white and European ancestry, and therefore do not reflect the diversity of the United States population. The immune cell profiling was conducted at one time point limiting our ability to examine longitudinal relations of changes in immune cell proportions with age. Circulating immune cells are not necessarily representative of tissue-resident T cells. Immune cell data was presented as a proportion rather than an absolute count, due to a lack of a complete blood cell count with differential, and this may explain differences in data compared with other reports. CMV was assayed at one point in time but was measured in mid to later adulthood when most exposures would have occurred [This study has several strengths. Properly stored PBMC samples for immune cell profiling came from participants in a richly characterized community-based cohort who were dementia-free. While fresh whole blood is the optimum media for immune cell profiling, data has shown that cryopreserved cells show excellent correlation to whole blood profiles . Study poccurred . Our stuWe profiled 116 immune cell phenotypes including subtypes of CD4 and CD8 T cells, B cells, NK cells, and monocytes in the community-based Framingham Offspring cohort. Our observations confirm and extend known associations of immune cell subtypes with CMV and age that show a shift from a naïve phenotype towards an exhausted phenotype. We report sex differences, with males exhibiting a more exhausted, cytotoxic landscape than females. We identified associations between CD8 exhausted cells and B cell subsets, but not overall B cells, with smoking status. Importantly, we did not identify significant immune cell associations with other risk factors, such as body mass index, prevalent cardiovascular disease, hypertension or diabetes. While further studies in larger, more diverse sample and more than one time point with immunophenotypic data are needed, this work will provide a valuable resource for future studies of the association of immune cell phenotypes and incident age-related disease.The FHS is a community-based prospective cohort study. Starting in 1948, the FHS recruited 5209 participants residing in Framingham, Massachusetts who were primarily white American adults of European ancestry as the Original cohort , followeFor the current study, we identified 1332 Offspring participants who attended exam 7 (1998 to 2001) and had 2 or more vials of PBMCs stored (to ensure the resource was not exhausted) that can be used to profile immune cells. Among this group of Offspring participants, we identified a study sample of 1000 individuals aged 40 years and older who were dementia-free at exam 7. Immune cell phenotyping protocols followed previously published protocols used in other large population studies , 42. BriFor surface labeling panels , samples are incubated with a fixable live/dead stain for 15 minutes. After centrifugation to remove the live/dead stain, cells were incubated with the indicated antibodies or appropriate isotype for 15 minutes in the dark at concentrations recommended by manufacturers. Cells were pelleted, washed with media, resuspended in 1% paraformaldehyde, and stored in the dark at 4C until run on the flow cytometer.For intracellular staining, samples are stimulated with phorbal myristic acetate/Ionomycin in the presence of Brefeldin A for 3 hours at 37C as previously described , 42. CelFlow cytometry was performed on a MACSQuant 16. Single color compensation beads were used to set machine compensation. Isotype controls and FMO controls were used to aid in gate setting. FCS Express 6.0 was used to analyze data. A Vericell PBMC control sample (Biolegend) was analyzed with each run to assess run variability, with coefficients of variation (CVs) ranging from 2.5% for CD3+, 12.3% for CD4+, 17.5% for CD8+ and 7.8% for CD14+ monocytes.All data are presented as a percent of their main parent population as listed in We excluded 13 immune cell phenotypes with very low variability , leaving 116 cell phenotypes. The number of participants with data and summary statistics for each immune cell phenotype profiled are presented in We calculated three ARIP measures using immune cell phenotypes profiled in our study: the CD4/CD8 ratio, and two measures of the ratio between naïve cells and memory and effector cells for both CD4 T cells and CD8 T cells. For the ratio between naïve T cells and memory T cells, Tn/Tm, we used the formula Tn/Tm = Tn / (Teff + Tem + Tcm) separately for the CD4 and CD8 Tn cells, Teff cells, Tem cells, and Tcm cells . The numCMV is a common pathogen that influences T cell subsets in individuals infected with CMV. Therefore, we assayed CMV IgG by ELISA using the Creative Diagnostics CMV IgG kit  using an existing plasma sample from Offspring exam 7 from 943 of the participants with immune cell phenotyping. We obtained quantitative values in U/ml; the CV for the CMV assay was 5.9%.Our analyses excluded 4 participants with missing data for most of the immune cell subtypes, resulting from failure of these samples for multiple assays. The 116 immune cell phenotypes had sample sizes ranging from n=922 to 996. The number of participants with data and summary statistics for each immune cell phenotype profiled are presented in We computed unadjusted mean percentages of immune cell phenotypes for For all association analyses, immune cell phenotypes were rank-based inverse normal transformed and treated as the outcome variable. To test for association between CMV status and immune cell phenotypes, we used individuals with CMV status available and adjusted for age and sex (Model 1) using a linear mixed effect model, accounting for correlations among related individuals using a random effect with the kinship matrix.APOE genotypes, and other CVD risk factors including BMI, smoking, presence of diabetes, prevalent CVD, prevalent AF, SBP, on hypertension treatment at exam 7, TC, HDL, on lipid treatment at exam 7, and prevalent cancer prior to exam 7. All covariates except APOE ε4 carrier status and education level were directly measured at the FHS Offspring exam 7. Education level was ascertained at FHS Offspring exams 2 and 8, and during off-exam-cycle neuropsychological testing. We used highest education level reported and categorized it into four levels: attended high school, high school graduate, attended college, and college graduate. APOE genotype groups were defined as ε3ε3, ε2 carriers, and ε4 carriers with the ε3ε3 group treated as the reference level. BMI was grouped into three levels: normal (BMI < 25), overweight (25 ≤ BMI < 30), and obese (BMI ≥ 30). The presence of diabetes was determined if any of the following were satisfied: fasting blood glucose level of 126 mg/dL or higher, random blood glucose level of 198 mg/dL or higher, or use of antidiabetic medications. Prevalent CVD was defined at exam 7 based on current or previous diagnosis of coronary heart disease , transient ischemic attack, intermittent claudication, and congestive heart failure determined by adjudication of a panel of senior investigators.To test for associations of immune cells with age, sex, smoking status as current smokers vs. non-smokers (never or former), and other covariates, we used a multivariable model (“Model 2”) that included CMV, age, sex, education level, APOE genotypes, all immune cell phenotypes, and available exam 7 variables were used in the imputation. For each immune cell phenotype outcome and each imputed data set, we fit a linear mixed effect model adjusting for Model 2 covariates accounting for correlations among related individuals using a random effect with the kinship matrix. Effect estimates and likelihood ratio p-values were pooled across data sets.We used multiple imputation by chained equations  to creatTo account for multiple testing, false discovery rates  (FDRs) ap–values from the fitted models were carried out using the mice package [We used a threshold of FDR < 0.05 to declare significance. All analyses were conducted in R software version 4.0.2 . The mul package . Linear  package .Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9Supplementary Table 10Supplementary Table 11Supplementary Table 12Supplementary Table 13Supplementary Table 14Supplementary Table 15Supplementary Table 16Supplementary Table 17Supplementary Table 18


This shift has resulted in a reversal of the diurnal warming trend, expanding the diurnal temperature range over recent decades. The intensified Tmax warming is attributed to a widespread reduction in cloud cover, which has led to increased solar irradiance at the surface. Our findings underscore the urgent need for enhanced scrutiny of recent temperature trends and their implications for the wider earth system.In the latter half of the twentieth century, a significant climate phenomenon “diurnal asymmetric warming” emerged, wherein global land surface temperatures increased more rapidly during the night than during the day. However, recent episodes of global brightening and regional droughts and heatwaves have brought notable alterations to this asymmetric warming trend. Here, we re-evaluate sub-diurnal temperature patterns, revealing a substantial increase in the warming rates of daily maximum temperatures (T The authors find a significant increase in daily maximum temperature warming rates, while daily minimum temperatures remain stable over land in recent decades. This may be due to reduced cloud cover, leading to increased incoming solar radiation. In addition to daily average temperatures, the diurnal temperature range (DTR), defined as the difference between the daily maximum temperature (Tmax) and daily minimum temperature (Tmin), provides useful information about the climate4. Changes in DTR have received considerable attention because it is closely linked with crop yields7, plant growth11, animal wellbeing13 and human health16. Existing studies found that the surface warming since the 1950s has been associated with larger increases in Tmin than in Tmax, i.e., decreases in DTR, which is commonly known as night warming or asymmetric warming20.The surface air temperature (SAT) is a commonly used measure of land surface climate change due to its ability to represent terrestrial energy exchange with reasonable accuracy23, solar radiation25, aerosols26, precipitation28, planetary boundary layer height29, land use change31 and deforestation33. For example, an increase in total cloud cover reduces DTR due to a decrease in daytime surface insolation and an increase in night-time downwards longwave radiation34. Increases in precipitation and soil moisture can reduce Tmax and therefore DTR through increased evaporative cooling35. However, considering known changes in these processes, the higher warming rate of Tmin than that of Tmax is seemingly inconsistent with two recent phenomena. One contradiction is the increase in surface solar radiation that has occurred since the late 1980s, which is referred to as brightening after dimming38. Solar radiation affects Tmax more than Tmin26, thus, brightening should contribute to further warming of Tmax. The second contradiction is the increased occurrence of drought events and heatwaves, especially in spring and summer41. This phenomenon indicates that the cooling effect of soil moisture may have been weakened, which should lead to a faster increase in Tmax. Motivated by these different changes, we re-evaluated the warming rates of Tmax and Tmin over the period of 1961–2020 and investigated the causes behind the associated changes in DTR.Changes in DTR are complex; the changes are subject to many factors, including cloud covermax and Tmin using two station observation-based datasets from Berkeley Earth Surface Temperatures (BEST)42 and the Climatic Research Unit Time-Series version 4.07 (CRU TS)43. Both datasets were gridded and gap-filled over the land masses, and BEST was used to detect global surface temperature changes in the Intergovernmental Panel on Climate Change (IPCC) sixth assessment report44. As shown in Fig. max calculated using a 30-year moving window increased faster than that of Tmin during 1961–2020. The warming rate in global average Tmax reached the warming rate in Tmin in recent decades, with an earlier surpassing moment in BEST than in CRU TS. In the last 30-year window (1991–2020), both datasets exhibit a slight (CRU TS dataset) or substantially more pronounced (BEST dataset) rise in the global average of Tmax compared to that of Tmin. Spatially, a stronger warming rate of Tmax was found in almost 25% of the land area in the earlier time window (1961–1990); the area expanded rapidly, and in the recent time window (1991–2020) the area of stronger warming rate of Tmax was found in at least half of the area  compared to the correlation between CRU TS’s DTR data and the GSOD station dataset . The findings revealed a significantly stronger correlation between BEST’s DTR data and the GSOD station dataset  was found during 1961–1990. For the period 1991–2020, the globally averaged DTR increased at a rate of 0.06 °C decade−1 (p < 0.05)  in DTR were detected in spring, summer and winter, which were mainly caused by a significant increase in Tmax  employed as independent variables monthly total cloud cover from the fifth-generation ECMWF reanalysis (ERA5)56 dataset, aerosol optical depth from Modern-Era Retrospective analysis for Research and Applications, version 2 (MERRA-2)57, and soil moisture from the Global Land Evaporation Amsterdam Model (GLEAM)58 dataset, while DTR was calculated from the BEST dataset as the dependent variable spanning 1981 to 2020. Ridge regression is a linear regression method with regularization that can effectively address the issue of multi-collinearity. By introducing a penalty term in the cost function that discourages overly large parameter values, it improves upon the ordinary least squares regression model in scenarios where there exist strong correlations among independent variables. An evaluation of the ridge regression (Methods) shows that the ridge regression model can capture the majority of the explained variance in DTR . Random Forest is a machine learning technique based on decision trees and can capture non-linear relationships. The results from the Random Forest regression consistently corroborate our prior findings, indicating that total cloud cover is indeed the dominant driving force behind DTR fluctuations in terrestrial regions, encompassing a substantial proportion (79.1%) of the land area  negative correlation was found between the annual average global surface total cloud cover and DTR. The mean bootstrapped partial correlation coefficient ) between these two variables was higher than that of the mean correlation between DTR and soil moisture , as well as the correlation between DTR and aerosol optical depth .To further elucidate the influence of different environment variables on DTR, we analyzed the changes of DTR, total cloud cover, aerosol optical depth and soil moisture over the global land area during recent decades  and the recent decades (1991–2020) Fig. . Specifi25. The above results indicate that the recent global decrease in cloud cover62 has increased incoming solar radiation at the surface, resulting in a greater increase in Tmax than in Tmin, ultimately expanding DTR.To further elucidate the relationship between incident solar radiation and DTR, we conducted a partial correlation analysis between DTR and incident shortwave radiation extracted from ERA5 (1961–2020), CERES (2001–2020), and MERRA-2 1981–2020), while adjusting for the influence of soil moisture . Given the complexity of LULCC’s effects, our study specifically focuses on exploring one potential impact: changes in albedo. However, we found that there was no significant positive or negative correlation between changes in albedo and DTR across the majority of land areas  and the diverse range of sources (8 sources) integrated within the BEST temperature dataset could potentially explain its higher correlation with the observed temperatures from the stations in the GSOD dataset, as compared to the CRU TS temperature dataset. In addition to disparities in station selection, variations in gridding and interpolation methodologies69, as well as the implementation of quality assurance procedures73, may also contribute to the differences in detected regional DTR trends between these two datasets. With that said, irrespective of the dataset employed, it is evident that more than half of the global land surface has displayed a discernible increasing trend in DTR over the past three decades. This finding signals a fundamental shift in the pattern of asymmetric warming.The reversing asymmetric warming is supported by regional increasing DTR trends in Europe74, atmospheric water vapor75, vegetation76, LULCC78 and regional atmospheric circulation79. This omission may introduce further uncertainty. Increased precipitation, for example, is closely linked to decreased DTR, which can be attributed to the strong association between precipitation, cloud cover, atmospheric water vapor, radiation, and soil moisture74. Future studies are needed to disentangle the effects of these highly correlated variables on DTR. Prior research conducted in specific regions, such as China and India78, has demonstrated that LULCC exerts a notable influence on DTR. In light of the intricate nature of LULCC’s effects, our study specifically focuses on exploring the potential impact of changes in albedo. However, we found that there is a relatively weak correlation between DTR and albedo across the majority of land areas. This could be attributed to the fact that LULCC encompasses other processes, such as vegetation dynamics81. Further studies are needed to gain a comprehensive understanding of the entire scenario.The results of the ridge regression analysis in this study suggest that statistical models incorporating total cloud cover, aerosol optical depth, and soil moisture as independent variables can effectively capture the variations in DTR across most land areas. The model fit is relatively low in regions such as South America and Africa 43 and Berkeley Earth Surface Temperatures (BEST) datasets42. The CRU TS temperature dataset is derived from a blend of data sources including weather station records, ship logs, and more recent satellite observations. This dataset undergoes meticulous calibration to account for biases and variations in measurement methods, making it an extensively used resource in climate research for documenting long-term temperature trends and variations. It provides spatiotemporal resolutions of 0.5° × 0.5° on a monthly basis, covering the period from 1901 to 2022.The TThe BEST dataset encompasses a larger sample of approximately 39,000 records. It employs advanced statistical techniques to quantify and adjust measurement biases, ensuring a highly accurate representation of global temperature trends. This dataset offers spatiotemporal resolutions of 1° × 1° on a monthly basis, spanning from 1850 to the present.max and Tmin records were acquired from the Global Surface Summary of the Day (GSOD) database, downloaded in September 2021 from https://data.nodc.noaa.gov/cgi-bin/iso?id=gov.noaa.ncdc:C00516. The GSOD dataset originates from the Integrated Surface Hourly (ISH) database developed by the US National Climatic Data Center. This dataset contains meteorological variables, including temperature, precipitation, and wind speed, from over 9000 weather stations. The original observations undergo rigorous quality control procedures to ensure their accuracy84. Specifically, we selected daily maximum and minimum temperature data with relatively complete records from all stations covering the period 1991 to 2020. We employed stringent selection criteria to exclude incomplete data series, only including stations with missing values not exceeding one year of the analysis period (1991 to 2020) and with complete records for all 12 months. Furthermore, we required each monthly value to be derived from at least 15 days of data. Consequently, our trend analysis encompassed a total of 2557 stations.In situ T56 on a 0.25° × 0.25° regular latitude–longitude grid (the native resolution of ERA5 is about 31 km). In addition, monthly total cloud cover was obtained from the MODIS MCD06COSP data at a spatial resolution of 1° × 1° after 2003. ERA5 total cloud cover was used for the main analyzes.Monthly data on total cloud cover were obtained from the fifth-generation ECMWF reanalysis (ERA5) dataset85 dataset at a spatial resolution of 1° × 1° after March 2000, and the Modern-Era Retrospective analysis for Research and Applications, version 2 (MERRA-2)57 dataset with a spatial resolution of 0.625° × 0.5° after 1980.The monthly incident shortwave radiation data in all-sky conditions were obtained from the ERA5 dataset on a 0.25° grid, the Cloud and the Earth’s Radiant Energy System energy balanced and filled edition 4.1 (CERES–EBAF)58, which is a global dataset spanning 40 years from 1981 to 2020 and based on satellite and reanalysis data. The cloud cover, radiation, aerosol optical depth, and soil moisture data sets were aggregated to a common 0.5° grid.The monthly average aerosol optical depth and surface albedo at a spatial resolution of 0.625°×0.5° was obtained from the MERRA-2 dataset beginning in 1980. The monthly surface soil moisture at a spatial resolution of 0.25° was obtained from the Global Land Evaporation Amsterdam Model (GLEAM) version 3.5 datasett-test.We assessed the accuracy of the temperature gridded data using GSOD station temperature dataset. The evaluation period spanned from 1978 to 2020, during which the GSOD data records were relatively complete. Following a similar methodology used in analyzing station-based DTR trends, we selected a total of 2,058 stations with missing observations comprising less than 5% of the evaluation period for temperature-gridded data assessment. In the evaluation process, we extracted the time series of DTR from the gridded BEST and CRU TS datasets corresponding to each station’s location. Daily data were averaged to obtain yearly data and missing values at these stations were filled using linear interpolation. We then computed Pearson correlation coefficients between the annual station observation series and the observed DTR sequence at each station, serving as an indicator of accuracy. The significance of the correlation coefficient differences between the DTR station data and the two sets of DTR gridded data was tested by the one-tailed Student’s To analyze DTR variations by season, we defined the seasons as follows: March, April, May for spring (autumn), June, July, August for summer (winter), September, October, November for autumn (spring), and December, January, February for winter (summer) in the Northern Hemisphere (Southern Hemisphere). Correspondingly, the annual average value of a variable in one year is defined as the average of 12 months from December in the preceding year to November of that year.^ represents the estimated regression coefficients, yi is the dependent variable, β0 is the intercept term, and βi signifies the regression coefficient for the independent variable xi. The tuning parameter λ governs the extent of shrinkage applied to the coefficients. When λ is set to zero, the regularization term exerts no effect, and ridge regression reduces to ordinary least squares regression. However, as λ increases, the penalty term gains influence. The larger λ becomes, the more pronounced is the shrinkage applied to the coefficients. Consequently, ridge regression effectively mitigates the impact of multi-collinearity.Ridge regression is a method for estimating coefficients in multiple linear regression models in scenarios characterized by high correlations among the independent variables. In ridge regression, a regularization term is introduced to the standard least squares objective function, aiding in stabilizing the estimated coefficients. This regularization term is controlled by a tuning parameter denoted as λ. The ridge regression objective function can be expressed as follows:Prior to performing the ridge regression analysis in this study, all-time series were detrended by subtracting the linear trend and transformed into z-scores by subtracting the monthly climatology means dividing by the monthly climatological standard deviations from 1981 to 2020. For validation, the z-scores were randomly divided into an 80% calibration dataset and a 20% validation dataset. The dataset in the training set was used to train the ridge regression model, while the dataset in the validation set was employed to assess the performance of the ridge regression model. Afterwards, all the datasets were merged to determine the ridge regression coefficients.i² denotes the coefficient of determination between the ith independent variable and all other independent variables. A higher VIF value indicates stronger multi-collinearity within the regression model’s independent variables. Here, a VIF value less than 3 suggests an acceptable level of multi-collinearity86.The ridge regression was performed on the variables in each grid cell, and the tuning parameter λ was determined for each grid cell based on the Variance Inflation Factor (VIF) of the independent variables within each individual grid cell. VIF serves as a measure of multi-collinearity among the independent variables in the regression model. The following formula was used to calculate the VIF:Throughout the regression analysis process, the initial value of λ was set to 0 and incremented by a step size of 0.01. As λ increased, the degree of multi-collinearity decreased, subsequently resulting in a decline in the VIF value. The incrementation of λ ceased when the VIF value dropped below 3, with this λ value being determined as the tuning parameter at this grid point. The significance of the ridge regression analysis was assessed utilizing an F-test at a significance level of 0.05. The accuracy of the ridge regression model was evaluated by employing the coefficient of determination on the validation dataset.The color of the cyan-magenta-yellow (CMY) composite was determined by the relative contributions from the magnitudes of the ridge regression coefficients (R.c), which can be expressed as follows:x1, R.cx2, R.cx3 represent the R.c of DTR to variables x1, x2, and x3, respectively. We denoted the relative contributions of variables x1, x2, and x3 to DTR as C, M, and Y, respectively. These contributions then served as the brightness values for the cyan, magenta, and yellow channels, respectively, culminating in the generation of a CMY image.Here, R.c59. The input to the Random Forest was standardized in the same way as for the ridge regression analysis, and the data was also randomly partitioned into an 80% calibration dataset and a 20% validation dataset for the validation. During the modeling process at individual grid cells, the Random Forest algorithm leveraged the provided sequences of independent variables and dependent variables in the calibration dataset to train 100 decision trees. Each decision tree independently predicted DTR values based on the given predictor variables. Moreover, we employed out-of-bag (OOB) prediction error estimation, an inherent capability of the Random Forest algorithm. Additionally, we assessed the importance of predictors by enabling the OOB Predictor Importance feature, providing insights into the relative contributions of total cloud cover, aerosol optical depth, and soil moisture in predicting DTR. Similar to the ridge regression coefficients, these Predictor Importance features were subsequently utilized in generating a CMY image.We also utilized the Random Forest algorithm for regression analysismax or Tmin at individual grid points, while controlling for the effect of soil moisture, we conducted a partial correlation analysis. Partial correlation analysis is a statistical technique used to assess the relationship between two variables while controlling for the influence of one or more additional variables. Here, both the variables involved in the correlation analysis and the variable being controlled for were specific to each individual grid cell. The significance of the partial correlations was evaluated at a threshold of p < 0.05.To determine the magnitude of the relationship between annual solar radiation and DTR, T87. Specifically, we generated 1000 bootstrap samples through random samples with replacements from the original data to create samples of equal size to the original dataset. For each bootstrap sample, we computed the partial correlation coefficient between DTR and an environmental factor while adjusting for the potential confounding effect of other variables. Subsequently, we calculated the mean correlation coefficient across all bootstrap samples and determined the 95% confidence interval using the 2.5th to 97.5th percentile of the bootstrap distribution.The uncertainties of the partial correlations between global annual DTR and total cloud cover, soil moisture, or aerosol optical depth were assessed using the bootstrap methodSupplementary InformationPeer Review File


Previous studies have examined the optimal mode of breech delivery extensively, but there is a scarcity of publications focusing on the birth injuries of neonates born in breech presentation. This study aimed to examine birth injury in breech deliveries.In this retrospective register-based nationwide cohort study, data on birth injuries in vaginal breech deliveries with singleton live births were compared to cesarean section with breech presentation and cephalic vaginal delivery between 2004 and 2017 in Finland. The data were retrieved from the National Medical Birth Register. Primary outcome variables were severe and mild birth injury. Incidences of birth injuries in different gestational ages and birthweights were calculated in different modes of delivery. Crude odds ratios of risk factors for severe birth injury were analyzed.n = 4344), there were 0.8% of neonates with severe birth injury and 1.5% of neonates with mild birth injury compared to 0.06% and 0.2% in breech cesarean section  and 0.3% and 1.9% in cephalic vaginal delivery . Brachial plexus palsy was the most common type of injury in vaginal breech delivery. Increasing gestational age and birthweight had a stronger effect on the risk for injury among cephalic vaginal deliveries than among vaginal breech deliveries.In vaginal breech delivery (Birth injuries were rare in vaginal breech deliveries. The incidence of severe birth injury was two times higher in vaginal breech delivery compared to cephalic vaginal delivery. Brachial plexus palsy was the most common type of injury in vaginal breech delivery.The online version contains supplementary material available at 10.1007/s00404-022-06772-1. Approximately two to three percent of neonates diagnosed with birth injury , 2 are aThe risk for birth injury in breech presentation is considered comparable to that of cephalic vaginal delivery  , 8, as t+0 weeks of gestation with breech presentation was higher after an attempt of VD than after planned CS in two large population-based studies [The incidence of morbidity and mortality of neonates after 37 studies , 10 and  studies . Similar studies , 12. Nev studies , 14 and  studies –17.A considerable amount of literature has been published on morbidity and mortality rates, whereas only a few studies have focused on birth injuries among neonates born in breech presentation. Since the incidence of birth injuries has been described to be relatively low, a large nationwide register was chosen as a study cohort. This study aims to examine the type and rate of birth injuries in vaginal breech deliveries (breech VD) compared to CS with breech presentation (breech CS) and cephalic vaginal deliveries  in Finland, where breech VD in selected women is still a common practice. We also aim to describe the incidence of birth injuries in different gestational weeks and explore the risk factors involved, especially those associated with severe birth injury in different types of delivery.This nationwide population-based cohort study was conducted using data from the Finnish Medical Birth Register (MBR) and the Care Register for Health Care. Both registers are maintained by the Finnish Institute for Health and Welfare. All Finnish hospitals are required to report clinical data to these national registries. The MBR includes data on pregnancies, deliveries, and information on the health of neonates. The data are completed by information obtained from the Central Population Register and the Cause-of-Death Register. The Care Register for Health Care contains information on patient diagnoses and operations performed during the hospital stay. The coverage and accuracy of these registers have been shown to be excellent , 19.The study period was from 2004 to 2017, and it focused on singleton breech deliveries that resulted in a live birth. Breech VD and breech CS were studied separately. Planned and unplanned CS were analyzed together (breech CS) since birth injuries were infrequent after CS. Neonates with cephalic presentation born by spontaneous vaginal delivery or vacuum-assisted delivery formed a cephalic VD group, which was used for comparison. Forceps deliveries were excluded as they were rare , and the presentation of the neonate could not be reliably defined in all cases. Figure +0 weeks of gestation: Breech VD is an option if the mother is motivated to vaginal delivery, the estimated fetal weight is < 4000 g, and the fetus is in a frank, complete, or incomplete breech position with the head in a flexed position during the delivery. Often, adequate measurements of the maternal pelvis are confirmed by magnetic resonance pelvimetry. CS is preferred if intrauterine growth restriction is suspected, or the fetus is otherwise at high risk for distress during delivery. According to national guidelines for preterm deliveries, CS may lower the risk for morbidity in primiparas before 32 gestational weeks [In Finland, five universities with medical faculties offer the education of medical doctors and trainees in gynecology and obstetrics. The management of breech delivery is included in the curriculum of gynecology and obstetrics. There are no national guidelines for term breech pregnancies. However, according to an inquiry addressed to the tertiary level obstetrics centers in Finland, there are common well-established clinical practices for managing breech pregnancies and deliveries after 37al weeks . HoweverThe two primary outcome variables were severe and mild birth injury. Birth injuries detected during the early neonatal period (0 to 6 days) were coded with the Finnish implementation of the 10th Revision of International Statistical Classification of Diseases and Related Health Problems (ICD-10) codes and retrieved from the MBR. In addition, hospital visits linked to any birth injury diagnosis recorded in the Care Register for Health Care during the first year after birth were included to increase the coverage. Severe birth injury was defined according to Muraca et al.  and incl2) were included after 2006, as values from several hospitals were missing for the years 2004 and 2005. Birthweight above + 2 standard deviations (SD) or below − 2 SDs were defined as large for gestational age and small for gestational age standardized for parity, sex, and gestational age in a Finnish population [Tables pulation . The uset-test and Mann–Whitney U-test were used for comparisons of continuous variables.The incidences of severe and mild birth injury were calculated and stratified by gestational age. Variables were described as frequencies and proportions for categorical variables, and as means and standard deviations or medians and interquartile ranges for continuous variables. Welch two sample The risk factors for severe birth injury were calculated. The results are presented as odds ratios and risk differences with 95% confidence intervals (CI). Poisson regression model was used to assess the incidences of birthweight and gestational age using the number of cases per gestational weeks/birthweight as an offset term. The model was used separately for mild and severe birth injuries and in different modes of delivery. Regression analysis was limited to birthweight < 4000 g, as the clinical practice in Finland mainly recommend breech VD when the estimated fetal weight is < 4000 g. Statistical analysis was performed using R Statistical Software version 4.0.3.In total, 650,528 neonates were included. Of these, 4344 neonates (0.7%) had breech VD, 16,979 neonates (2.6%) had breech CS, and 629,182 neonates (96.7%) had cephalic VD, either spontaneous (90%) or vacuum-assisted (10%)  and 40+2 (interquartile range 39+2–41+1), respectively (Table The birthweight of neonates with severe birth injury was 3320 g (SD 483) in the breech VD group and 4071 g (SD 518) in the cephalic VD group. Gestational ages were similar in both groups, 40+0 and 27+6 41% (51/124), 28+0 and 31+6 29% (55/187), 32+0 and 36+6 30% (500/1654), 37+0 and 40+6 19% , and 41+0 and 42+6 30% (402/1344) of fetuses with breech presentation had VD. There were no severe birth injuries, and three neonates (at 31 weeks of gestation) in the breech VD group had a mild birth injury between 24+0 and 31+6 weeks of gestation. In the cephalic VD group, there was a similar finding of single injuries. After 32 weeks of gestation, the incidence of injury remained stable with some sporadic fluctuation up to 42 weeks of gestation among the breech VD group  or the incidence of severe birth injury and gestational weeks . In contrast, the incidence of birth injury showed an increasing trend with higher gestational age in the cephalic VD group , Fig. Between gestational weeks 24Furthermore, no association was found in Poisson regression between birthweight 500 g to 4000 g) and incidence of mild birth injury  or between birthweight and severe birth injury  in breech VD. In the cephalic VD group, the incidence of mild birth injury  and the incidence of severe birth injury  showed an increasing trend along with higher birthweight. In the breech CS group, however, the incidence of mild birth injury seemed to show a decreasing trend with increasing birthweight  Fig. . There w000 g andIn this population-based study, the incidence of severe birth injury was higher in the vaginal breech delivery group than in the cesarean section with breech presentation group and the cephalic vaginal delivery group. However, the incidence remained low in all groups. A brachial plexus palsy was the most frequent injury in vaginal breech delivery. Perhaps because of the more rigorous selection of women for vaginal delivery, high birthweight did not seem to be as important a risk factor for birth injury in vaginal breech delivery as in cephalic vaginal delivery.The total incidence of birth injury in breech VD was similar to that previously reported , 9, 11. Risk factors for severe birth injury, mostly representing the risk factors for BPP, found in cephalic VD were comparable to the risk factors reported for BPP in previous studies that mainly concerned neonates in cephalic presentation  . In the The incidence of birth injury was low in all gestational ages in neonates with breech presentation, and no evidence was found of an association between gestational age and birth injury. It has been suggested that CS reduces perinatal morbidity and mortality in preterm breech neonates , 12, 27,Furthermore, the significance of high birthweight remains unclear. The guidelines recommend preferring CS when the estimated birthweight is  > 3800 to 4000 g –30, but This study provides valuable information on the risks associated with breech deliveries. The strength of this study was the nationwide study population and the long study period that enabled us to study rare incidents such as birth injuries. In Finland, reporting to the registers is mandatory, the medical treatment of pregnancies is homogenous even without national guidelines for breech pregnancies, and the rate of breech VD has remained stable during the twenty-first century . Thus, rOur study confirmed that risk for birth injury is low in breech VD and breech CS. Nevertheless, the risk for severe birth injury, specifically BPP, was higher among breech VD than breech CS or cephalic VD. Birth injuries in neonates with breech presentation were sporadic, and no clinically relevant risk factors were found. These findings suggest that careful selection of women is required to ensure safe vaginal breech delivery.Supplementary file1 (PDF 14 KB)Below is the link to the electronic supplementary material.


Atomic force microscopy (AFM) has been used to studythe mechanicalproperties of cells, in particular, malignant cells. Softening ofvarious cancer cells compared to their nonmalignant counterparts hasbeen reported for various cell types. However, in most AFM studies,the pericellular layer was ignored. As was shown, it could substantiallychange the measured cell rigidity and miss important information onthe physical properties of the pericellular layer. Here we take intoaccount the pericellular layer by using the brush model to do theAFM indentation study of bladder epithelial bladder nonmalignant (HCV29)and cancerous (TCCSUP) cells. It allows us to measure not only thequasistatic Young’s modulus of the cell body but also the physicalproperties of the pericellular layer (the equilibrium length and graftingdensity). We found that the inner pericellular brush was longer forcancer cells, but its grafting density was similar to that found fornonmalignant cells. The outer brush was much shorter and less densefor cancer cells. Furthermore, we demonstrate a method to convertthe obtained physical properties of the pericellular layer into biochemicallanguage better known to the cell biology community. It is done byusing heparinase I and neuraminidase enzymatic treatments that removespecific molecular parts of the pericellular layer. The presentedhere approach can also be used to decipher the molecular compositionof not only pericellular but also other molecular layers. Atomic force microscopy (AFM3) is a versatile technique capable of recording distinct signalsrelated to mechanical5 or rheological7 oradhesive8 properties of biological samples.Such broad AFM functionality is a strong driving force for introducingAFM to clinical practice as a fundamentally new type of nonlabelingbiomarker for the diagnostics and/or prognosis of cancer and probablyother diseases.Various techniques used to identify themechanical properties ofsingle cells or even tissues have shown that cell mechanics correlateswell with cancer progression.10 Cellinteractions with ECM components, such as proteins or proteoglycans,regulate cellular functions, including maintaining cell shape, adhesion,migration, proliferation, polarity, differentiation, and apoptosis.11 In pathological conditions, increased synthesis of specific ECMcomponents can contribute to cancer growth and progression.13 The cellular response depends on the nature of the adhesion receptors,i.e., integrins and cadherins. The change in microenvironment stiffnesscan initiate alterations in their expression. Integrins are linkedto the actin molecules of the cytoskeleton. Thus, changes in theirexpression contribute to cell deformability. Findings show that mostcancer cells are characterized by larger deformability, which is believedto be one of the causes of altered cell adhesive and migratory properties.15 The altered adhesive properties of cancer cells have been a targetfor numerous research attempts to identify molecules for inhibitingcancer invasion due to their accessibility from the cell exterior. The cell surface contains numerous adhesivemolecules, such as integrins16 or cadherins.17 These molecules, together with proteoglycansattached to the pericellular membrane18 like syndecans,19 form a layer calledglycocalyx.20It has been demonstrated that cancer cells adaptand change inresponse to external stimuli or mechanical properties of the extracellularmatrix (ECM), changing to more invasive phenotypes.21 The AFM indentation data were processed through the brush model,22 the approach that allows the separation of the mechanical propertiesof the cell body and its pericellular layer. It has been shown thatAFM can detect both the presence of glycocalyx and corrugation ofthe pericellular membrane (like microridges and microvilli23). Despite the relative complexity of the model,it was shown that it is rather robust; i.e., it allows for ratherprecise separation of the mechanical deformation of the cell bodyfrom the physical properties of the pericellular brush layer.25 For example, it was shown that a possible error in the definitionof the quasistatic Young’s modulus of the cell body does notexceed 4% due to the model and experimental uncertainties. To amplify,the brush model allows us to derive the quasistatic Young’smodulus of the cell body accompanied by information on the biophysicalproperties of the pericellular brush layer in a robust self-consistentway.The alteration of glycocalyxon malignant cells was studied withthe AFM technique for cervical cancer.26 Although this cancer is effectively treatedif captured early, a 50–80% recurrence rate requires continuousmonitoring of patients for recurrence and/or progression to a moreadvanced stage . The monitoring includes invasiveoptical bladder examination (cystoscopy) and possible tumor resectionfor pathology examination. The requirement for frequent cystoscopymakes BC the most expensive cancer per patient to diagnose, monitor,and treat. Numerous global authorities recognize it as a major healthissue incurring a significant burden on healthcare systems.27The present study focuses on human bladder epithelial cancercells.Bladder cancer (BC) is one of the most common cancers worldwide. Itis characterized by high incidence and mortality rates.28 andthe greater adaptability of these cells to altered microenvironments.30 Despite that, it was possible to distinguish cancer cells from nonmalignantor benign cells based on their mechanical properties.31 Changes in the mechanical properties of epithelial cells can berelated to actin cytoskeleton organization. The alterations in actinorganization might be related to the adhesive properties of the bladdercells. Immunohistochemical analysis has already shown that normalhuman urothelium expresses integrins built of such subunits as α3,αV, β1, and β4.32 In parallelstudies, a strong correlation has been found between the metastaticactivity of cells and glycocalyx composition in cancer cells, includingsialylation, fucosylation, O-glycan truncation, and N- and O-linkedglycan branching.20 The biological functionsof glycocalyx-associated molecules lie in the ability to interactwith various ligands modulating the interaction of cells with ECM;thus, they can be involved in such processes as epithelial–mesenchymaltransition (EMT) and carcinogenesis.33The bladder is characterized by a largedegree of mechanical flexibilitylinked with its physiological role35 The expression of HS can be altered after the transformation fromnoninvasive to invasive states.36 The abnormallyhigh presence of SA residues is also considered a distinctive featureassociated with malignancy and the invasiveness of cancers.37 Both proteoglycans and glycans, present on thecell surface, could be used as cancer identification and treatmenttargets. Due to their complexity and heterogeneity, methods for analyzingtheir properties in the cell surface context are strongly needed.Atomic force microscopy allows obtaining of high lateral resolutionof mechanical properties of the pericellular layer. However, it isfrequently difficult to understand the biological significance ofthis information. Therefore, it is instrumental to find a way to translatethe obtained mechanical information into biomolecular data, whichis the ground for biological and medical understanding of the significanceof the pericellular layer. Here we suggest the use of enzymatic treatmentto remove particular molecular parts of the pericellular coat andto correlate it with the AFM measured mechanical properties of thepericellular layer. Specifically, we aim to investigate sialic acid(SA) and heparan sulfate (HS) residues of the glycocalyx proteoglycans.HS is one of the proteoglycan family encompassing polysaccharide-basedchains bound to such proteins as glypicans or syndecans.5The presence of HS chains and SA residues is evaluated here usingAFM by cleaving the residues with specific enzymes. We use heparinaseI (hep) and neuraminidase (neu) enzymes to selectively remove HS andSA residues from the cell surface. By analyzing the indentation forcecurves with the help of the brush model, one can obtain the parameterof the pericellular brush layer, which includes glycocalyx. Here wefound that cancer and nonmalignant HCV29 cells possess two brush layers(inner and outer). The inner brush in nonmalignant HCV29 cells wasshorter than that of transitional cell carcinoma TCCSUP cells butof the same effective molecular density. The accompanying outer cellbrush was larger and denser than in the case of cancer cells. Furthermore,we found that changes in the cellular brush depend on the type ofenzyme applied. A substantial change in the pericellular brush layerobserved after enzymatic treatment indicates the presence of eitherHS chains or SA residues. The mechanical properties of the cell bodyor the inner part of the pericellular brush layer were not changedafter hep treatment, neither in HCV29 nor in TCCSUP bladder cells.In the case of neu treatment, the stiffening was observed only afterneu treatment in TCCSUP cells and not in nonmalignant HCV29 cells.Notably, a substantial difference in the rigidity of the cell bodyagrees with the previous observations showing that nonmalignant bladdercancer cells were considerably more rigid.38 Proteoglycans are frequently dysregulated during cancer progression,39 and therefore, they possess the potential tobe a target for inhibiting cancer invasiveness, already demonstratedfor heparan sulfates.40 Altered sialylation,a hallmark of cancer progression, is critical for glycans as the additionof sialic acids at the terminal end of glycans changes a charge bearingby these molecules charge41). As the sialylation of moleculesresponsible for cell adhesion can increase the metastatic potentialof many cancers,42 their desialylationmay inhibit the dissemination of cancer cells.43 Consequently, the structure and function of various glycans,glycoproteins, or glycolipids are altered.44 Thus, it is plausible to expect that cancer cells show specificalterations in their glycocalyx.The glycocalyx is a dense networkof glycans bound to glycoproteins,glycolipids, and proteoglycans, playing a crucial role in the interactionsof cancer cells with the microenvironment.45Besides comparing nonmalignantand malignant (cancerous) bladderepithelial cells, our study includes a quantitative description ofglycocalyx properties before and after the enzymatic cleavage of proteoglycansand terminal sialic acid residues by heparinase I and neuraminidase.Such a treatment cleaves the glycocalyx containing those residues,thereby changing the mechanical properties of the pericellular brushlayer. AFM is a suitable method for examining the physical propertiesof cell glycocalyx.47 Thus, we started the experiments to characterizethe actin content and filament organization in nonmalignant HCV29and transitional cell carcinoma TCCSUP cells. The actin filamentswere stained with fluorescent Alexa Fluor 488 dye conjugated withphalloidin. Phalloidin binds specifically to F-actin, a polymerizedform of actin, at the binding site between F-actin subunits.48 Therefore, this dye is widely applied to visualizefilamentous forms of actin. The cell nucleus was labeled with theHoechst 33342 dye. Confocal images showed that both cell lines aremorphologically different . Heparinase I removes heparan sulfate chains constitutingthe proteoglycans.34 In such a case, largerfragments of heparan sulfate chains are cleaved  compared to neuraminidase cleavage.Knowing from our previous studiesthecell 1 C,D.Itthecell 2A. The e49 The results showed a larger deformability of cancerous cells inrelation to the control, nonmalignant cells. However, in those studies,the AFM measurements and data analysis were conducted without consideringthe presence of the pericellular brush layer surrounding cells. As was shown,25 the mechanicalcontribution of the pericellular layer is significant; ignoring thepericellular brush layer results in the dependence of Young’smodulus of the cell on the indentation depth. Thus, it does not allowus to compare the mechanical properties of cells directly and to measurethe absolute values of the Young’s modulus. The brush modelused in the present work allows for deriving the quasistatic Young’smodulus of the cell body in a robust self-consistent way, in whichthe modulus does not depend (or just weakly depends) on the indentationdepth. Thus, the present observation of a significant softness ofthe malignant compared to the nonmalignant cells is a rather nontrivialand important result.The mechanical properties of bladder cancer cellshave alreadybeen measured.n =33), 4.39 ± 1.72 (n = 25), 5.87 ± 3.23kPa (n = 30) for nontreated, hep- and neu-treatedcells, respectively. In the case of cancerous TCCSUP cells, the quasistaticYoung’s modulus remained at the same level as nontreated cellsafter removing heparan sulfate fragments; however, removing sialicacid residues induced some stiffening of cells. The quasistatic Young’smoduli were 1.23 ± 0.70 kPa (n = 35), 1.05 ±0.48 kPa (n = 35), and 1.69 ± 0.77 kPa (n = 30), respectively.Supporting Information, Figure S7). The effect was cell- and enzyme-dependent  remained unaltered upon hep treatment ,but it decreased when neu was applied to the cells .In the case of cancer TCCSUP cells, the nontreated cell (control)height was 3.09 ± 1.09 μm (n = 35 cells).It increased for cells treated with hep to 4.81 ± 0.99 μm andremainedsimilar to control cells after neu treatment . Changes in cellheight indicate remodeling of the cell cytoskeleton, which might contributeto cell mechanics. However, the lack of direct correlation with thecell mechanical properties indicates that, most probably, such remodelinginvolves short actin filaments, and stress fibers remained responsiblefor the mechanical properties of cells .The height of HCV29 cells . Negatively charged sialicacid residues provide charge repulsion and prevent unwanted cellularinteractions.54 Thus, removing these residueschanges the properties of the glycocalyx, probably leading to itspartial collapse, as has already been seen for endothelial cells.55 Such collapse can form a stiffer shell aroundthe cell. The results presented in Neu treatment  did not affect themechanical properties of nonmalignant HCV29 cells, although the cellheight decreased. Glycans containing terminal sialic acid residuesare attached to the cell adhesion molecules, such as integrins orcadherins. Thus, their desialylation may affect direct or indirectattachment to the actin cytoskeleton. Lack of rigidity changes inHCV29 cells can be linked to a smaller amount of sialic acids in thesecells than in bladder cancer cells.56 (21 it was shown that the pericellularlayer of cancer cells could be characterized by a brush with two characteristicsizes (0.45 and 2.6 μm long). Using guinea pig fibroblasts,57 it was demonstrated that the pericellular layermight consist of a shorter and relatively rigid inner part, whichcomprises corrugations of the pericellular membrane, and an outerpart comprised mainly of oligosaccharides and glycoproteins . Here the brush model was appliedto obtain not only the mechanical properties of the cell body butalso the physical characteristics of the pericellular brush layer,its thickness (length), and effective surface density . We considered the properties of the inner and outer brush layersseparately.The pericellular brush is composed of two main parts. Oneis theglycans that can be considered as a layer of polymeric chains graftedto the cell membrane, while the second part is the membrane protrusionsthat can be considered as random asperities. Deformation of both partscan be described with the help of exponential force dependence. Therefore,we used the Alexander DeGenne model  for polymerbrush to approximate the pericellular brush56 6 and 7.  density 4. It sho–2. The outer brush length (n = 33 cells) than for TCCSUP , showing only weak statistical significance(p < 0.05). The corresponding brush density  for control and 170 ± 90 nm (n = 25) forhep and 290 ± 200 nm (n = 30) for neu. The correspondingbrush density also showed no changes, 140 ± 75 μm–2 (n = 33) for control, 120 ± 38 μm–2 (n = 25) for hep, and 110 ±76 (n = 30) for neu. For TCCSUP cells, no significantchanges in brush length were also observed for control and enzymatic-treatedcells, i.e., 550 ± 290 , 620± 360 , and 580 ± 340 nm. The corresponding inner brush densitydid not change too, 130 ± 64 μm–2 , 120 ± 46 μm–2 , and 130 ± 62 μm–2 . According to ref  brush shows thatthis brushis longer for nonmalignant HCV29 than for cancerous TCCSUP cells6.n = 33cells) to 17 ± 24 μm (n = 25), with densitydecreasing from 1.60 ± 2.0 μm–2 (n = 33) to 0.60 ± 0.70 μm–2 (n = 25). The action of neuraminidase showed nosignificant effect on length or density in HCV29 cells (47 ±40 μm and 2.3 ± 2.1 μm–2 (n = 30). For TCCSUP cells treated with either heparinaseor neuraminidase, no significant changes in the outer brush lengthwere detected. The only significant change is an increase in the outerbrush density, i.e., 0.59 ± 1.3 μm–2  to 1.8 ± 2.4 μm–2 .The enzyme treatment brought similar results forHCV29 cells; i.e.,after applying heparinase I, the outer brush became shorter by morethan 2 times  and large errors, we confirm distinct adhesive propertiesof cell surfaces by applying principal component analysis (PCA) tothe obtained data. It clearly separated the work of adhesion for cancercells, denoting nontreated, 0.12 ± 0.12 fJ, and enzyme-treated,0.21 ± 0.23 and 0.15 ± 0.16 fJ for hep and neu treatedTCCSUP cells. Interestingly, cells treated with hep I had differentadhesive properties than cells treated with neu. For nonmalignantHCV29 cells, there is a clear separation between nontreated (0.087± 0.120 fJ) and enzyme-treated cells .The separation between hep- and neu-treated cells is very small, indicatingsimilar adhesive properties of cells before and after the enzyme treatment.Heparan sulfate and sialic acid residue cleavage change the adhesiveproperties of both cell types by altering the structure, charge, andconformation of the glycocalyx. Hemispherical silicon nitride AFMprobes adhere differently to nontreated cells, revealing that nonmalignantHCV29 cells are less adhesive than cancerous TCCSUP cells. The enzymatictreatment decreased and increased the adhesiveness of nonmalignantand cancer cells, respectively. Since no specific interactions areexpected, the adhesive properties are defined by the area of contactbetween the AFM probe and sample surface. The differences in the contactarea could be explained by the roughness of the pericellular membrane,which is essentially the inner brush. Furthermore, a denser innerbrush should definitely contribute toward higher hydrodynamic dragforce, resisting the AFM probe from pulling out of the contact. Comparingthe amount of the inner brush shown in The results show a lower work of adhesion for nonmalignantHCV29than for cancerous TCCSUP cells 8B. BecauBased onthe obtained results, we present the differences in theglycocalyx structure in nonmalignant and cancerous cells in a graphicalway 9. This f56 the pericellular brush layer canbe substantiallydifferent for normal and cancer cells. The bladder epithelial cellsstudied here revealed a similar pericellular brush structure, whichis characterized by inner and outer layers (inner and outer brushes).Nonmalignant HCV29 cells are characterized by the shorter inner andlonger outer brushes of various densities (the inner is denser thanthe outer brush) compared to the cancer TCCSUP cells. Further enzymatictreatment of the pericellular layer shows that only the outer brushresponds to the treatment. It implies that the inner brush is mainlythe corrugation of the pericellular membrane, which agrees with previouslystudied guinea pig cells.23As already reported for human cervical epithelialcells,60 that can potentially be used to identify bladder cancers.61 Syndecans are not the only HS-bearing molecules. Others includeperlecan or glypicans.62 This and the effectof hep on HCV29 suggest that nonmalignant cells possess a higher levelof HS chains that are not attached to syndecans. Simultaneously, theincreased outer brush density after neu treatment in cancerous cellsindicates a higher level of sialic acid residues, reported in theliterature.44Distinctcell mechanics and adhesion accompany changes in thepericellular brush properties. Nonmalignant cells are stiffer thancancer cells and reveal a smaller nonspecific adhesion. The enzymatictreatment affects the adhesive properties of cells. Adhesion becomeseven smaller for nonmalignant cells but larger for cancerous cells.In parallel, changes in the pericellular brush properties are observedin an enzyme-dependent manner (probably related to the location ofthe cleavage sites). The most abundant core proteins possessing HSare four members of the syndecan family57 Thus, it was interesting to verify if these changes stay when usingthe self-consistent brush model, which allows obtaining of the quasistaticYoung’s modulus with an error of just a few percent.25 Here we observed that bladder epithelial cancer cells are stillsofter than nonmalignant ones, which qualitatively agrees with thepreviously reported observations.Here we studied the physical propertiesof bladder human epithelialcells with the help of the AFM indentation technique. The use of thebrush model allowed us to study the mechanical properties of the cellbody as well as the pericellular layer. Although it was previouslyreported that malignant cells were softer than nonmalignant, ignoringthe pericellular layer, it substantially changes the derived valuesof the quasistatic Young’s modulus.The most nontrivial resultsof this work are in the study of thephysical properties of the pericellular layer. We found a substantialdifference in the structure and properties of the pericellular layerof both cell types, which demonstrated double-size brush behavior.The inner pericellular brush was longer for cancer cells, but itsgrafting density was similar to that found for nonmalignant cells.The outer brush was much shorter and less dense for cancer cells.To understand the biochemical nature of the detected pericellularbush layer, we performed enzymatic treatment of cells with heparinaseI and neuraminidase. This treatment showed significant effects onlyon the outer brush of the pericellular layer of both nonmalignantand cancerous cells. Neu affected mainly the cancerous cells by increasingsubstantially the effective grafting density of the outer brush. Itimplies that the number of sialic acid residues is substantially higherin the cancerous cells, which is in agreement with the previouslyreported data. Moreover, the increase of grafting density presumablyindicates supramolecular restructuring of long polysaccharide moleculesin a stiffer molecular construct. A substantial decrease of the outerbrush was observed for both nonmalignant and cancerous cells aftertreatment with hep. It tells us that a major part of glycocalyx proteoglycansof the bladder epithelial cells contains heparan sulfate chains. Theseresults show that subsequential treatment of cells with various enzymesapplied to AFM experiments allows the detection of the molecular componentsand physical structure of the molecules of the pericellular layer.It is an important bridge between physics and molecular biology, byallowing translation of the measured physical properties of the pericellularlayer into the language of biomolecules.2 and relative humidity above 98%. Cells were passaged when theirconfluence reached 80–90%. HCV29 and TCCSUP cells were detachedfrom the surface using 0.05% and 0.25% trypsin–EDTA solution(Sigma) for 4 min, respectively. After a few passages, cells wereseeded on a Petri dish (passages 6–8) for AFM measurementsand on glass coverslips for fluorescence imaging.Two human bladder cancer cell lines wereused: nonmalignant cells of the ureter  and transitional cellcarcinoma . HCV29 cells were culturedin RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum. TCCSUP cells were cultured in Eagle’s minimumessential medium  supplemented with 10% FBS.These different media represent the physiological conditions specificto each cell line. Cells were grown in culture flasks (Sarstedt) inan incubator (Nuaire) at 37 °C in 95% air and 5% CO2, and 0.01% BSA) and neuraminidase ) were preparedat the concentration of 200 U/mL and 25 U/mL, respectively. The cellswere initially cultured in the corresponding culture medium containing10% FBS, then rinsed with 1% FBS and kept in it. Enzymes (hep or neu)were added to the cells for 1 h at the final concentration of 1 U/mLand 0.25 U/mL for hep and neu, respectively. After the treatment,cells were washed with PBS  buffer, and the correspondingculture medium containing 1% FBS was added to a Petri dish.The stock solutions of heparinase I  was applied todetect G- and F-actin. Bands were visualized using horseradish peroxidase-coupledsecondary anti-rabbit antibody . The ratiobetween G/F actin was calculated using ImageJ software based on thedensitometry approach.The G-actin/F-actin invivo assay kit  was applied to quantifythe ratio between G-actin and F-actin. Cells were homogenized in 300μL of LAS2 buffer containing detergents, disrupting the cellmembrane. As a result, only G-actin was solubilized, while F-actinwas not. Next, the centrifuging of cell lysate at 100.000Cultured on the surface of thechambered coverslips placed in a Petri dish , the cells were fixed with 3.7% paraformaldehyde in PBS for20 min at room temperature (RT). Next, they were washed with PBS (3times for 2 min), treated with 0.2% cold Triton X-100 in PBS for 4min at 4 °C, and washed again with PBS. Next, the cells wereincubated for 30 min at RT with phalloidin conjugated with Alexa Fluor488 (1:200 in PBS). Next, the cells were washed in PBS and incubatedwith Hoechst 33342 dye  for 15 min at RT. After rinsing with PBS, cells were keptin PBS for confocal imaging. Images of actin filaments were acquiredusing a confocal microscope (Leica TCS SP8 WLL) equipped with new-generationHyD detectors. Fluorescent dyes were excited at 405 nm (Hoechst 33342via UV CW laser) and 499 nm (Alexa Fluor 488 via WLL white laser).Images were acquired using an oil immersion 63× objective lens(HC PL APO CS2 NA 1.40).63 to calibrate the sensitivityof the photodetector. The resonance frequency of the used cantileversranges from 6 to 12 kHz, corresponding to a range of spring constantsfrom 12 to 50 mN/m. At the free end of each cantilever, a hemisphericalprobe, i.e., a cylinder ended with a half spheroid with a radius of5 μm, was mounted.MLCT-SPH-DC silicon nitride cantilevers were chosen. Each cantilever was precalibratedby the manufacturer using a laser Doppler vibrometer. The fact thateach cantilever was independently calibrated allows us to know thereal cantilever spring constant used in the experiments and applythe SNAP protocol57The AFM-based indentation measurementswere conducted on the top of the cell, i.e., over the whole cell.Each cell was mapped with a grid of 15 pixels ×15 pixels seton each map (scan size from 50 μm × 50 μm to 70 μm× 70 μm). At each point, an individual force curve wasrecorded . The relative sample position was linearlychanged at a speed of 6 μm/s. The maximum load force was setto 7–8 nN. The cell geometry was saved with collection ofthe force curves. The latter allowed one to calculate the cell radius.p < 0.05.Statistical significance wasdetermined based on Kruskal–Wallis ANOVA nonparametric testusing the confidence level starting with 24 This approach is called the brush model. It was demonstrated thatthis model unambiguously separated the elastic properties of the cellbody from the force signature of the pericellular brush layer, allowingself-consistent calculations of the quasistatic (elastic) Young’smodulus and, in addition, the properties of the pericellular brush.64 Later the model was further elaborated. Themost comprehensive analysis of the model was done recently to evaluateits robustness, i.e., a weak dependence of the model results in possibleuncertainties in the model itself and ambiguities in the experimentaldata65Ifone wants to use the concept of elastic modulus, it has to be consistentwith the definition of such a modulus; i.e., the material should behomogeneous and isotropic. Although a cell can hardly be treated asa homogeneous material, it can be a good approximation for relativelysmall deformations. As was shown previously, the approximation ofhomogeneity works well if and only if (1) one takes into account thepresence of an essentially nonlinear pericellular brush layer and(2) one uses a relatively dull AFM probe.Thebrush model is described in detail in the above references. Here webriefly describe the major steps used to process the AFM force curvesthrough this model. Z0 is theposition of the undeformed cell body, h is the distancebetween the AFM probe and the surface of the cell body, and i is the deformation of the cell body. The latter can becalculated using the hertz model:E is the quasistaticYoung’s modulus, k is the spring constantof the AFM cantilever, and Rprobe and Rcell are the radii of the AFM probe and cell,respectively. The Poisson ratio of a cell is chosen to be 0.5. Assumingthat the PB layer is softer than the cell, at the maximum indentationthe brush layer is almost squeezed (h = 0), and themodulus of the cell body is calculated by fitting the indentationcurve within the squeezed brush region.The following equation describes the distance betweenthe cellbody and the spherical indenter:67 The followingcorrection is used for the hertz model:Z represents the relativevertical scanner position of the cantilever, d isthe cantilever deflection, Z0 is the undeformedposition of the cell body, h is the separation betweenthe cell body and AFM probe, E is the elastic modulusof the cell body, k is the cantilever spring constant,andht is the cell height. The effective radius, R*, is defined as Rprobe and Rcell are the radii of curvature of the AFM probeand cell, respectively.The glass slide on whichthe cells are sitting is a rigid substance,which can overestimate the quasistatic Young’s modulus. Theeffect was described in the literature.69 for an entropicpolymer brush:kB is theBoltzmann constant, T is the temperature, N is the surface density of the brush constituents (graftingdensity or effective molecular density), and L isthe equilibrium thickness of the brush layer. Note that this formulais valid provided 0.1< h/L <0.8. In the case that the indentation curve demonstrated a doubleslope behavior in logarithmic scale, the longer and softer brush andthe shorter and more rigid brush can be described by a simple sumof two brush forces:64N1, L1, and N2, L2 are the parameters of the larger and smallerbrush, respectively.A clear exponential dependence, a characteristicfeature of theforce due to the brush layer, agrees with a molecular brush describedby the Alexander–de Gennes model71 Therefore, the observed pull-off force is presumably a combinationof a viscous hydrodynamic interaction between the AFM probe and cellsurface. Electrostatic forces could also quickly form a bond, butthey are not expected to be prevalent because of the negatively chargedsilicon nitride surface and mainly negatively charged molecules onthe cell surface. The work of adhesion was calculated as the areaincluded within the adhesive part of the force curve.The retraction part of the force curveswas analyzed to determine the work of adhesion, i.e., the work neededto detach a bare hemispherical probe from the cell surface. Developingeven nonspecific adhesion between nonfunctionalized silicon nitridehemispherical probe and cell surface typically requires a much longertime than the duration of the contact during the described measurements.


Background and objective: In patients with asthma, fractional exhaled nitric oxide (FeNO) is a biomarker for eosinophilic airway inflammation. However, the correlation with the blood eosinophil count, immunoglobulin E (IgE), and spirometric values is not well established. We aimed to investigate the correlation between FeNO, blood eosinophils, IgE, and spirometric values in asthmatic patients.Methods: Data were extracted from the electronic medical records of adult asthmatic patients, and included the FeNO, blood eosinophil count, IgE, and spirometric values. The correlation between the markers was investigated using receiver operating characteristics (ROC) and the area under the curve (AUC).3, and 42% had a blood eosinophil count > 300 cells/mm3. There was a significant positive correlation between FeNO and the blood eosinophil count  as well as with the IgE . There was significant negative correlation between FeNO and FEV1% .Results: A total of 135 adult patients  were analyzed. The mean FeNO was 36 ppb and almost half (48%) had a FeNO > 25 ppb. The mean blood eosinophil count was 377 cells/mmConclusion: The blood eosinophil count, IgE, and spirometric values were correlated with the severity of the eosinophilic airway inflammation (high FeNO). Asthmatic patients with a higher FeNO level had a higher eosinophil count, higher IgE levels, and lower FEV1 values. Asthma is a heterogeneous disease characterized by chronic airway inflammation . It is dBecause asthma is a heterogeneous and complex disease, an assessment based on symptoms and spirometry alone does not accurately represent the underlying airway inflammation, the patient characteristics or profile (phenotype), or the underlying mechanism of airway inflammation (endotype) . We aimeThe FeNO measurement is an easy and non-invasive test. A FeNO concentration greater than 25 parts per billion (ppb) in adults is a marker of eosinophilic airway inflammation and predicts the likelihood of corticosteroid responsiveness ,4. An elSeveral studies attempted to establish a correlation between the blood eosinophil count and FeNO, but with contrary findings. A study reported no relationship between FeNO and the blood eosinophil count in uncontrolled asthma ; howeverAs most of the studies investigating the correlation between the biomarkers in asthma reported conflicting results with variable conclusions, we aimed to investigate the correlation between FeNO, the blood eosinophil count, serum IgE level, and spirometric values in patients with asthma.This was a retrospective analytical study, conducted at the Pulmonary clinic at King Abdulaziz Medical City in Riyadh, Saudi Arabia. Both genders, 15 years and older, with a diagnosis of asthma, and FeNO, spirometry tests, and the blood eosinophil count results available in the electronic medical records system, from June 2019 to June 2022, were included in the study. The sampling technique was non-probability purposive sampling. Smokers and patients with chronic pulmonary disease or significant co-morbidity which can affect the value of FeNO  were excluded.3) and low blood eosinophil count (< 300 cell/mm3). Asthma control was assessed using the Asthma Control Test (ACT > 20 was considered as controlled asthma and < 20 uncontrolled). The patients were subdivided into impaired or normal lung function values . Bronchodilator reversibility was defined as >200 mL and > 12% post-bronchodilator. The study protocol was approved by the Institutional Review Board of King Abdullah International Medical Research Center.The data collected consisted of the baseline characteristics, including age, gender, body mass index (BMI), and duration of asthma diagnosis as well as an asthma control test (ACT). The other variables were the FeNO, eosinophil count, IgE, forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and the ratio of FEV1/FVC as well as bronchodilator reversibility. In this study, the level of FeNO was categorized into two groups: high FeNO (FeNO ≥ 25 ppb) and low FeNO (FeNO < 25 ppb). The level of the blood eosinophil count was categorized as a high blood eosinophil count  and the area under the curve (AUC). A p-value ≤ 0.05 was considered statistically significant.3 and the mean percentage was 4.6%. Less than half (42%) had an absolute eosinophil count > 300 cells/mm3. The mean IgE level was 311 KU/L, and the majority (68%) had an IgE > 100 KU/L. The mean FEV1%, FVC%, and FEV1/FVC ratio were 85%, 91%, and 78% respectively. Of the group with reduced spirometry values (ratio less than 75%), the mean FEV1%, FVC%, and FEV1/FVC ratio were 56%, 63%, and 64% respectively. The mean bronchodilator reversibility value was 192 mL, and 48% had a bronchodilator reversibility response (> 200 mL).A total of 135 patients with asthma met the inclusion criteria. The baseline characteristics of the patients are displayed in Table There was no significant difference between the high and low FeNO groups in terms of age, gender, BMI, ACT and the duration of the disease. There were significant differences between the two groups and the eosinophil count and IgE. The patients in the high FeNO group also had a higher eosinophil count and IgE in comparison to the patients in a low FeNO group . Regarding the spirometric values, there was a significant difference between the two groups as the patients in the high FeNO group had a lower FEV1 (mean 78%), FEV1/FVC ratio (mean 74%) and bronchodilator reversibility compared to the patients in the low FeNO group  , and between the FeNO levels and the IgE levels  , and a weakly positive significant correlation between the FeNO level and bronchodilator reversibility  . In addition, a positive correlation was found between FeNO and serum IgE . However, a significant but negative correlation existed between FeNO and FEV1 , and a significant correlation between FeNO and bronchodilator reversibility . There was no significant correlation between FeNO and asthma control (p = 0.12). Patients in the high FeNO group had a higher eosinophil count, higher IgE levels, and lower spirometric values.As inflammation is the main pathognomonic feature of asthma, measurement of its biomarkers is important to establish the diagnosis of asthma, assess the severity of the disease and monitor the response to therapy. FENO is a non-invasive, inexpensive, and easy-to-perform test used with the blood eosinophil count as a biomarker for eosinophilic airway inflammation, and the level correlates well with type 2 asthma and the severity of inflammation. The current findings confirm the correlation between FeNO and the blood eosinophil count as an indication of the severity of eosinophilic airway inflammation, also supported in the literature. FeNO was significantly correlated with the blood eosinophil count in uncontrolled childhood asthma 8 as well as in uncontrolled adult asthmatic patients . A studyThe current findings demonstrated a significant and positive correlation between FeNO and the serum IgE level. Patients in the higher FeNO group had higher IgE levels. Strunk et al.  reportedHowever, we found a significant negative correlation between FeNO and FEV1% predicted. The spirometry measurements are lower with increasing inflammation (increasing FeNO level). A study indicating no correlation between FENO and the spirometric values considered them as different physiological parameters for diagnosing asthma . FeNO anInterestingly, the current findings found no significant correlation between FeNO and asthma control . A patient may have a high FeNO (airway inflammation) but controlled asthma (ACT>20) and vice versa. This can be explained by the definition of severe eosinophilic asthma, which is not the same definition as uncontrolled asthma  reportedThe current study had several advantages. These included a large sample size, exclusion of several confounding factors such as smoking and chronic lung diseases other than asthma, using optimal cut-off points of 25 ppb for FeNO as recommended by the American Thoracic Society, and finally, an optimal cut-off point of 300 cell/mm3 for the eosinophil count, as recommended by GINA .A limitation of the study is the retrospective analysis of data from a single pulmonary function laboratory. In addition, some of the variables have been measured on separate occasions which were minimized by establishing a three-month data collection period between all the tests. Another limitation is not investigating the correlation between the FeNO and the risk of exacerbation. Choosing an analytic retrospective medical record study did not allow for studying such a correlation. Another limitation is that the sputum eosinophil count was not measured, but such a measurement is not routinely available in most centers and laboratories as it is time-consuming and needs special preparation.In conclusion, the FeNO, blood eosinophil count, IgE, and spirometric values correlated well with the severity of the eosinophilic airway inflammation in adult asthmatic patients. Additional prospective multi-center studies with multiple measurements of the biomarkers with a larger sample size are required to explore and verify the current results and ultimately support appropriate monitoring of asthma management.


Background: Several closure devices are routinely used for percutaneous arterial access, while a relatively low number is available for the management of large bore venous accesses. The Woggle technique is a modification of the purse-string suture which was introduced several years ago in patients undergoing hemodialysis. Methods: A population of 45 patients who underwent transvenous femoral structural heart interventions was retrospectively evaluated. The Woggle technique consists of a purge string suture with a collar to maintain the tension as stable over time and a suture lock to tighten the suture. Results: Sheaths magnitude ranged from 8 French (F) to 14 F. A rapid post-procedural hemostasis was achieved in the whole population, and in 95% of cases, definite hemostasis was obtained after the first single release; the mean time of release was 302 ± 83 min. Although no relevant bleedings were reported, a significant reduction in hemoglobin levels was found in the whole population. This decrement was statistically significant only in the group with sheaths higher than 12 F. A single mild local hematoma was recorded in the group in which smaller sheaths were used. Seventy-two percent of patients were pre-treated with a dual antiplatelet therapy. Conclusions: The Woggle technique has shown to be a simple, effective, and safe approach for the management of large bore venous in percutaneous structural heart interventions. Vascular access sites for percutaneous structural heart interventions can be either venous or arterial. Several closure devices are routinely used for arterial access , while aThus, the aim of the present study was to evaluate either the safety of the Woggle technique or the feasibility of the management of venous access sites used for percutaneous structural heart interventions which are mostly represented by left atrial appendage occlusion (LAAO) and patent foramen ovale (PFO) closure.2). Hypercholesterolemia was defined as a previous history of hypercholesterolemia, chronic treatment with any lipid-lowering drug  [We performed a retrospective cohort study of patients who underwent percutaneous structural heart interventions, LAAO and PFO closure, needing venous access sites with a large bore. Data were collected from May 2020 and May 2023 at San Carlo Borromeo Hospital and Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. From May 2020, we routinely used the Woggle technique, and almost all patients treated with trans venous percutaneous structural heart intervention were included. A large bore was defined as when the procedure needed a sheath over 8 French (F). All demographic and clinical data were prospectively collected in a dedicated database. The study was conducted according to good clinical practice, institutional guidelines, national legal requirements, European standards, and the revised Declaration of Helsinki. All patients provided informed consent for the use of personal data. Hypertension was defined as a previous diagnosis of arterial hypertension under optimal treatment or measurement of systolic blood pressure at rest (BP) >140 mmHg and/or a diastolic BP >90 mmHg. The diagnosis of diabetes was based on a previous history of diabetes treatment, fasting glycemia >126 mg/dL or glycosylated hemoglobin >6.5%. Body mass index was calculated as weight/height (Kg/mstatins) . Local hThe Woggle technique is a modification of the purse-string suture . The purt-test was used to compare differences between means. The statistical significance of p-values was set at 0.05 and the confidence intervals (CI) at 95%.Statistical analysis was performed with the IBM SPSS Statistic Release v28. Continuous variables were tested for normal distribution with the Shapiro–Wilk test and expressed as mean ± standard deviation if normally distributed, or median and interquartile ranges if not. Categorical variables were expressed as absolute and as percentage frequencies, and differences were tested using the chi-squared test. A Mann–Whitney test was used for non-normal quantitative variables, while Student’s 2. Five patients (11%) were on hemodialysis, and only one had a history of previous hemorrhage. In all patients, procedures were performed through venous femoral access with sheath magnitudes ranging from 8 to 14 F, and 20 patients (44%) had a size larger than 12 F. Unfractionated heparin was infused during all procedures, with a mean cumulative dose of 92 ± 22 UI/kg; protamin to reverse heparin was never administered at end of the procedures. At the time of intervention, 40 patients (89%) were on therapy with aspirin and 26 (58%) were on dual antiplatelet therapy (DAPT) with clopidogrel. The mean procedural time was 65 ± 35 min with a success rate of 100%. The Woggle technique was successful in the achievement of immediate post-procedural hemostasis for the whole population and, in 95% of cases (43/45 patients), definite hemostasis was achieved after the first single release that was performed at a mean time of 302 ± 83 min after the procedure. The time of first release was decided by the first operator based on the heparin’s total half-life and the current antiplatelet therapy. In the two patients without definite hemostasis, the first release was sufficient to re-stretch the suture and lock it again with the suture lock. A second attempt was performed after roughly 30 min. Technical achievement for hemostasis, defined as no supplementary requirement for further manual compression, manipulation, or protracted examination, was accomplished in 100% of patients. There were no differences in creatinine before and after the procedure, while a significant difference in hemoglobin value was found (12.8 ± 2.2 vs. 12.4 ± 2.1 gr/dL, p = 0.01, A total of 45 patients were recruited and all were treated with the Woggle technique for large-bore venous closure. According to the total of the procedures, 33% were LAAO and 66% were PFO closures. LAAO was performed in patients who were affected by chronic atrial fibrillation and with a high risk of bleeding, whereas PFO closures were performed in patients affected by ischaemic stroke ,12. The The main findings of the present study are the safety and efficacy of the Woggle technique to achieve hemostasis after percutaneous venous large bore procedures for LAAO and PFO closure. Indeed, to the best of our knowledge, few studies have evaluated the Woggle technique to achieve hemostasis mostly in the hemodialysis arterio-venous fistula ,5. The rThe Woggle technique is a simple, effective, and safe method for the management of large-bore venous percutaneous structural heart interventions.