Added sugars intake deciles ranged from <3.8 to >23.3% of calories among adults 19+ y, with a median intake of 11.0% of calories. Significant associations (p ≤ 0.01) between added sugars intake deciles and percentage of the population below the Estimated Average Requirement (EAR) were found for magnesium, vitamin C, vitamin D, and vitamin E; only the association with magnesium remained significant after dropping the two highest and lowest deciles of intake, suggesting a threshold effect. Intakes below approximately 18% of calories from added sugars were generally not associated with micronutrient inadequacy. However, even at the lower deciles of added sugars, large percentages of the population were below the EAR for these four micronutrients, suggesting that adequate intakes are difficult to achieve regardless of added sugars intake.There is inconsistent evidence regarding the impact of added sugars consumption on micronutrient dilution of the diet. We examined the associations between added sugars intake deciles and nutrient adequacy for 17 micronutrients in U.S. adults 19+ ( Dietary guidelines in North America and worldwide call for restrictions on the intake of added sugars. The 2015–2020 Dietary Guidelines for Americans (DGA) recommend reducing added sugars intake to <10% of calories . SimilarConcerns about high added sugars intake, and hence the call for dietary restrictions, stem from the scientific literature on added sugars and health. The majority of the evidence is from studies of sugar-sweetened beverages, which have been shown to be associated with weight gain , type 2 Little is known about the potential for micronutrient dilution at higher added sugar intakes in the diet of the U.S. adult population. One study based on the National Health and Nutrition Examination Survey (NHANES) 2003–2006 has shown an inverse relationship with added sugars intake for some micronutrients ; howeverWe therefore examined associations between added sugars intake and micronutrient adequacy in U.S. adults, in whom there are different considerations than for children in terms of food intake and sources of added sugars and micronutrients. We analyzed these associations for 17 nutrients with an Estimated Average Requirement (EAR) across deciles of added sugars intake using NHANES 2009–2014.What We Eat in America via two 24-h dietary recalls, the first collected in person in the health examination and the second conducted via telephone 3–10 days after the first. Both 24-h recalls were collected using the Automated Multiple-Pass Method [NHANES is a nationally-representative, cross-sectional survey conducted by the National Center for Health Statistics, part of the U.S. Centers for Disease Control and Prevention. An overview of NHANES, including the purpose, study population, sampling strategy, survey procedures, and response rates is provided elsewhere ,22. Bries Method . For ourAdded sugars intake was determined using the USDA Food Patterns Equivalent Database (FPED) for each NHANES release . The FPE12, B6, C, D, and E. The National Cancer Institute (NCI) Method was used to estimate usual intake (UI) and distribution of intake of nutrients for each age group [Intakes of 17 micronutrients with an EAR were obtained from NHANES dietary intake files ; these wge group . Given mge group , the EARge group . Micronup ≤ 0.01 .Linear regression analyses were conducted within the three age groups  to determine whether relationships existed between decile of added sugars intake and micronutrient adequacy. Dummy-coded deciles of intake (1–10) were used as the independent variable. Regression coefficients were generated to examine the magnitude of significant relationships and showed the change in percentage of the population with inadequate intakes (below the EAR) as added sugars intake increased across deciles of intake. Sensitivity analysis was performed to examine the influence of the extremes of intakes of added sugars to the main findings, first by restricting the analysis to deciles two through nine, and second by restricting the analysis to deciles three through eight. Additionally, to assess whether the relationship of added sugars intake with prevalence of micronutrient inadequacy was curvilinear, regression analyses were conducted using the linear and quadratic term of dummy variable for decile number; if the quadratic term was significant, then a curvilinear relationship was present. Statistical significance was set at n = 61) as determined by USDA National Center for Health Statistics staff, those pregnant or lactating (n = 240), those with zero calories (n = 16), and those with only a single dietary recall (n = 1867), the final sample was 13,949 , with 7424  participants 19–50 y and 6525  participants 51+ y. The average age (standard error) of those 19+ y was 47.1 (0.4) y, and the average age of those 19–50 and 51+ y was 34.6 (0.3) and 63.6 (0.2) y, respectively.After excluding those with unreliable dietary records  across deciles of intake ranged from <3.8 to >23.3 in adults aged 19+ y . Median Among adults aged 19+ y, there was a significant association between added sugars intake and micronutrient inadequacy for four of the 17 micronutrients we examined: magnesium (β ± SE: 4.0 ± 1.0 percentage units per decile), vitamin C (2.4 ± 0.7 percentage units per decile), vitamin D (0.7 ± 0.09 percentage units per decile), and vitamin E (2.0 ± 0.3 percentage units per decile) . For exaWhen linear regression analyses were repeated with the lowest and highest deciles dropped (1 and 10), the positive associations between added sugars intake and percentage of the population below the EAR persisted for both magnesium and vitamin E in adults 19+ y . In thos12, B6, and C) in adults 19+ y, indicating that as added sugars increased, the degree of micronutrient inadequacy increased at a faster rate  and lower among older adults (10.1%), which is a demographic trend also seen in previous surveys in the United States , and in Our estimate of median added sugars intake of 11.0% of calories indicates that half of U.S. adults were either below or very close to the DGA recommendation of <10% of calories from added sugars ,16,18,19The types of foods and beverages contributing to added sugars consumption at different intake levels may partly explain the associations we found. A recent study has shown that among adults consuming the highest amount of added sugars (decile 10), sweetened beverages, coffee, and tea together accounted for 62% of their added sugars intake . The lacFurther elucidation of the impact of added sugars intake on micronutrient adequacy can be gleaned from our truncated analyses (running regressions after dropping extremes in added sugars intake). When we repeated linear regressions across added sugars deciles excluding the two lowest and highest deciles, associations for three of the micronutrients  were apparent among adults 19+ y; and an association with vitamin E was apparent among younger adults (19–50 y). Together with our evaluation of curvilinear relationships of added sugars intake and micronutrient adequacy, these results suggest the impact of added sugars on micronutrient adequacy may only be an issue at the highest levels of added sugars intake . This interpretation is consistent with other research showing a threshold effect, whereby micronutrient inadequacies were found to increase significantly only above a certain level of added sugars intake  ,19. A grMagnesium, and vitamins C, D, and E are among the micronutrients that have been characterized as shortfall nutrients in the U.S. diet . SeveralTo achieve nutrient adequacy and help prevent chronic disease, the DGA emphasize “healthy eating patterns” . StudiesOur study has some strengths and limitations. Using NHANES data from three cycles provided a large amount of data collected under commonly accepted procedures. Regression analyses using NHANES weights and design criteria allowed us to estimate the magnitude of change in the percentage of the population below the EAR as added sugars intake increased. Furthermore, we used deciles of added sugars intake versus arbitrarily selected set points, allowing us to examine points along the continuum of added sugars intake where micronutrient intakes are sufficient and/or begin to be a concern. In order to minimize misclassification errors, we used the two-day average intake of added sugars rather than one-day intakes or modeling intake  to define the added sugars deciles. We also ran regression analyses with added sugars intake entered as either a decile variable  or as an actual intake variable , and consistencies in the results of these regressions confirmed that misclassification was unlikely to impact our results dramatically.As for limitations, even using a two-day average of added sugars intake rather than a measure of long-term habitual intake may lead to biased estimates of added sugars intake. The error in the added sugars intake variable due to within-person variation would also cause an attenuation of any affect, meaning we would be less likely to see associations between added sugars intake and micronutrient adequacy. When we conducted the same analyses with UI to define added sugars intake, the results were similar , suggesting our analyses using a two-day average of added sugars intake did not limit our detection of relationships. Other limitations include dietary intake data that are self-reported, and while the best procedures were used to assess micronutrient intake, self-reported dietary data rely on memory and the ability of subjects to accurately report the foods and amounts of foods consumed. It is also unknown if there is an under-reporting bias regarding foods with added sugars; if such under- reporting exists, it would impact our added sugars intake estimates.In conclusion, using NHANES 2009–2014, we found that the median intake of added sugars among U.S. adults was 11.0% of calories, suggesting that half of the population was either below or very close to the DGA limit of <10% of calories from added sugars [


Ice storms are a type of extreme winter weather event common to north temperate and boreal forests worldwide. Recent climate modelling studies suggest that these storms may become more frequent and severe under a changing climate. Compared to other types of storm events, relatively little is known about the direct and indirect impacts of these storms on forests, as naturally occurring ice storms are inherently difficult to study. Here we describe a novel experimental approach used to create a suite of ice storms in a mature hardwood forest in New Hampshire, USA. The experiment included five ice storm intensities  applied in a single year, and one ice storm intensity (12.7 mm) applied in two consecutive years. Results demonstrate the feasibility of this approach for creating experimental ice storms, quantify the increase in fine and coarse woody debris mass and nutrients transferred from the forest canopy to the soil under the different icing conditions, and show an increase in the damage to the forest canopy with increasing icing that evolves over time. In this forest, little damage occurred below 6.4 mm radial ice accretion, moderate damage occurred with up to 12.7 mm of accretion, and significant branch breakage and canopy damage occurred with 19.1 mm of ice. The icing in consecutive years demonstrated an interactive effect of ice storm frequency and severity such that some branches damaged in the first year of icing appeared to remain in the canopy and then fall to the ground in the second year of icing. These results have implications for National Weather Service ice storm warning levels, as they provide a quantitative assessment of ice-load related inputs of forest debris that will be useful to municipalities creating response plans for current and future ice storms. Thesrobursts –9. Theseng years .post facto analyses, a growing number of controlled experiments are emerging to help elucidate and model ecosystem responses to changes in the frequency, intensity and severity of extreme events [e.g., As an alternative to ts e.g., , 12. Thets e.g., , 6, 13. post facto studies, these changes can lead to a cascade of changes in forest ecosystem structure and function, including increased inputs of coarse and fine woody debris to the forest floor, increases in light below the forest canopy, reductions in photosynthetic capacity and stored plant carbohydrates, alterations of soil temperature and moisture, changes in belowground plant and microbial processes, and altered leaching and gaseous nutrient losses from the soil [Ice storms are a type of extreme winter weather event that are a major driver of forest disturbance in temperate and boreal ecosystems worldwide –18. Climthe soil –27.Despite this general understanding of the impacts of ice storms on forest ecosystems, manipulation of icing intensity and frequency followed by investigation of ecosystem effects have not been attempted to date. Given the widespread occurrence of these extreme weather events in north temperate and boreal regions, their immediate and lasting impacts, and projections for increased frequency and intensity under a changing climate, there is an urgent need to better understand the impacts of a range of ice loadings and return intervals on forest ecosystems.The objectives of this paper are to (1) provide a detailed methodological approach for creating experimental ice storms, (2) discuss the efficacy of the design , (3) describe possible artifacts of the treatments, and (4) present results on the initial impact of these experimental storms on nutrient transfers in fine and coarse woody debris and tree canopy damage assessments. This work was conducted at the Hubbard Brook Experimental Forest (HBEF) in New Hampshire, builds on a feasibility study at the same site , and comThe HBEF is a 3,160-ha valley in central New Hampshire, USA  . The cli2/ha and was comprised of eight species: sugar maple (Acer saccharum Marsh.) (34%), red maple (Acer rubrum L.) (25%), American beech (Fagus grandifolia Ehrh.) (17%), yellow birch  (16%), red spruce (Picea rubens Sarg.) (3%), bigtooth aspen (Populus grandidentata (Michx.) (2%), balsam fir  Mill.) (2%) and paper birch (Betula paprifera Marsh.) (1%)  (1%) . Canopy h.) (1%) .Ten 20×30 m plots were established in May 2015 and were blocked by location, with plots 1 through 5 in one block on the eastern side of the study site, and plots 6 through 10 on the western side . Within The entire 20×30-m plot was iced during treatments. All response measurements were made within an inner 10×20-m area, allowing for a 5-m buffer zone . The inn® centrifugal pump that fed high pressure water to a nozzle mounted next to the pump. Pump nozzles were pressurized to flow at approximately 300 liters per minute. All pumps were gasoline powered. One UTV operated along each of the two long sides of the plot, with each spraying water the full length and half the depth of the plot  was taken from Hubbard Brook by two low pressure/high volume pumps and supplied to two track-equipped utility terrain vehicles (UTV) via moveable fire hoses. Each UTV was equipped with a BB4the plot ; S1 Fig.® HMP 60 thermistors housed in gill solar radiation shields, with one located near plot 9 and one near plot 2. Wind speed was measured with a RM Young® model 05103 anemometer at a weather station approximately 5 km east of the experimental plots . Application dates were chosen to meet criteria assuring effective ice accumulation on canopy surfaces (air temperature < -4°C) with minimal drift of mist (i.e. wind speed < 4.5 m/s) as detailed by Rustad and Campbell [Air temperature was measured using two Campbell ScientificCampbell . Much of4), nitrate (NO3) and chloride (Cl) on a Metrohm® Ion Chromatograph; calcium (Ca), magnesium (Mg), potassium (K), and sodium (Na) on an Agilent® 730 ICP optical emission spectrometer; dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) on a Shimadzu® TOCV with a TNM-1 nitrogen (N) detector; and ammonium (NH4) by colorimetry on a SEAL Analytical AQ2 discrete analyzer at the Louis C. Wyman Forest Sciences Laboratory, Durham, NH. Dissolved organic N (DON) was calculated as the difference between TDN and inorganic N (NO3-N + NH4-N). Total mass of elements applied to the canopy as iced ‘precipitation’ was calculated as the ‘precipitation’ chemistry from the stream aliquot multiplied by the ‘precipitation’ volume for each plot.Volume of water applied per plot was calculated as the product of application time and water flow. To construct chemical budgets for the treatments, a 60 ml aliquot of Hubbard Brook stream water was collected prior to each icing, and analyzed for sulfate , L = length of the dowel (cm); V = volume of melted ice (mL), and ρi = density of glaze ice (g cm-3).Radial ice accretion on collectors was measured with two methods. First, ice thickness was measured in the field immediately following icing with digital calipers at multiple points along the six dowel arms. Second, after ice thickness measurements were made in the field, each of the six arms of the collectors were cut at the base with a reciprocating saw, being careful to keep the ice on the dowel intact. Ice-coated dowels were placed individually in labeled zip lock bags and transported back to HBEF Headquarters where the ice was melted at room temperature and the volume of meltwater from each dowel measured. The radial ice accretion equivalent (Req) was calculated using the equation from :Req=-D24, NO3, Cl, Ca, Mg, K, Na, NH4, DOC, TDN and DON by the same methods as for the stream water aliquots. Total throughfall flux for each element in each plot was calculated as the mean plot chemistry multiplied by the mean plot volume.Canopy throughfall volume was measured with 20 L (28.7 cm diameter) buckets placed on the ground in the center of each subplot (eight per plot). Two of these buckets were acid washed and designated for chemical analyses. After icing, buckets were retrieved, thawed, and the volume of canopy throughfall in each bucket was measured. Aliquots from the two acid-washed buckets per plot were poured off, refrigerated, and analyzed for SO® #40 mesh sieve, and analyzed for total carbon (C) and nitrogen (N) on a Thermo Flash® EA 1112 Series CN Analyzer® and for total Ca, Mg, K, and phosphorus (P) by microwave digest and analysis on an ICP [Fine woody debris  and foliar litter were collected in plastic baskets (52L×37W×27H cm) that were placed in the center of each of seven subplots for all plots. Foliar litter was collected during the pre-treatment period 5 August, 2015 to 17 January, 2016. Fine woody debris was collected between two to four weeks after icing treatments in 2016 and ten weeks after icing treatments in 2017 to allow all the near-term broken branches to reach the ground. In instances where larger branches lay on the litter baskets, twigs less than 2 cm were clipped around the perimeter of the basket and were included as part of the sample. All foliar litter and FWD samples were oven dried at 60°C to constant weight, weighed, ground through a Wiley Milln an ICP at the LoCoarse woody debris  was collected on a 2m×2m quadrat located within each of the three subplots designated for destructive measurements on each of the main plots . On NoveA qualitative tree canopy damage assessment was made pre- and post-treatment, following methods used after the 1998 ice storm and the HBEF pilot ice storm experiment . In this-2) and mass (mg m-2) of 10 analytes ; explanatory variables included treatment and a random effect for plot. The lme4 package in R was used to conduct mixed effects analyses [A series of linear mixed effects models were used to analyze throughfall ice accretion, water volumes and chemistry. Response variables included ice accretion (mm), water volume , sampling period (for CWD) and treatment × sampling period (for CWD). Plot was incorporated as a random effect. An α of 0.05 was used to compare treatments (for FWD and CWD) and sampling periods (for CWD). Cumulative link mixed models were used to assess decay class as an ordered, categorical variable . CWD masn2017 = 2, n2018 = 6) because they were not found or were not assigned a damage class. Multiple comparisons testing with an α of 0.05 were used to compare treatments and sampling periods. We conducted three rounds of analyses: 1) all species including all treatments, species was not included as a fixed effect in this model; 2) American beech, sugar maple, and yellow birch including all treatments; and 3) American beech, red maple, sugar maple, and yellow birch, including the Control, Mid, Mid×2, and High treatments as there were no red maple trees in the Low treatment. Weighted means of damage classes are presented as numbers to illustrate differences among treatments, sampling periods and species.Cumulative link mixed models were used to assess damage as an ordered, categorical variable. Fixed effects were treatment × sampling period × species; and tree was used as a random effect. Only trees within the inner 10×20 m buffer were included in the analysis. Eight trees were excluded from analyses from one year of sampling  exceeded those of natural ice storms and comprised inputs comparable in magnitude to annual precipitation inputs at the site , 45 (Plot 3) to 68 (Plot 2) percent of the water sprayed onto plots was retained by the canopy as ice . During The two methods for measuring ice accretion were in close agreement for smaller treatments (< 10 mm) with few icicles. Specifically, ice accretion measured by calipers was 111 and 99 percent of ice accretion measured by melted water volume for the Low and Mid treatments, respectively . HoweverIn general, the Low, Mid×2-yr1 and Mid×2-yr2 treatments were close to target levels , whereas the Mid and the High treatments were below targets .In February 2016, the first ice applications resulted in a general stepwise increase in the FWD from the Control to the High treatment plots . StatistThe mass of FWD nutrients generally followed the same patterns as for total mass for both years. .-2, mean C mass ranged from 225.79 to 750.79 g m-2, mean N mass ranged from 1.48 to 2.72 g m-2, mean P mass ranged from 0.07 to 0.12 g m-2, mean Ca mass ranged from 1.75 to 4.19 g m-2, mean Mg mass ranged from 0.12 to 0.36 g m-2, and mean K mass ranged from 0.20 to 0.55 g m-2. No significant pre-treatment differences were observed among the treatment plots for total mass or any of the elemental masses. Weighted mean decay class across all treatment plots was 3.5, with no significant differences among the treatments.Total mass and element content of CWD removed from the plots in November 2015 was highly variable, reflecting the localized history of inputs. Across all treatment plots, mean CWD mass ranged from 475.08 to 1564.05 g mIn July 2016, the first ice applications resulted in a general stepwise increase in CWD mass from the Control to the High treatment plots . SignifiP < 0.001). Little additional CWD fell between July and November 2017 . In August 2016, the combined mean tree canopy damage indices were significantly greater in the Mid and High treatment plots compared to the Control and Low treatment plots, with the Mid×2 at an intermediate value. In August 2017, the combined mean tree canopy damage indices were significantly greater in the Mid×2 treatment plots compared to the Control plots . In AuguThe combined mean canopy tree damage indices increased in all treatments, including the Control, over the five years of the study, but the Mid, Mid×2 and High treatment plots showed a greater increase in damage over time than the Control and Low treatment plots, which resulted in the significantly higher damage indices for the assessments completed in the summers of 2018 and 2019 .For individual species, American beech had numerically the lowest initial values for mean canopy tree damage indices (1.2 in the Control treatment to 1.6 in the Mid treatment), followed by yellow birch (1.2 in the Mid×2 treatment to 2.3 in the Mid treatment), red maple (1.3 in the Mid×2 treatment to 2.6 in the Control treatment), and then sugar maple (1.6 in the Mid treatment to 2.6 in the Control treatment) .For American beech, no significant differences were observed in mean tree canopy damage indices among treatments prior to icing (May 2015) . By AuguDue to small sample size, few of the species year × treatment comparisons were statistically significant . Of the Our methods and results demonstrate the feasibility of (i) creating a gradient of experimental ice storms and (ii) creating experimental ice storms in consecutive years. The treatment levels were chosen to reflect the amount of ice accretion that triggers National Weather Service ice storm warnings from the Mid-Atlantic to the New England and New York states , and then to extend this to a more extreme level , and to include an extreme frequency . This initial work establishes the framework to evaluate the response to and recovery from this experimental suite of ice storms for decades to come.Radial ice accretion was used as a measure of disturbance intensity. Ice accretion is difficult to characterize and quantify as its buildup varies on surfaces of different materials, diameters, shapes and inclinations . MoreoveThe most immediate ecological impact of ice storms on forests is creation of canopy gaps as twigs, branches, tree tops and eventually entire trees break and snap under the load of the accumulated ice. In a related paper on the canopy impacts of this ice experiment, Fahey et al.  showed sResults reported here complement the work of Fahey et al.  by quantCarbon inputs in FWD and CWD, in general, followed mass inputs, with step-wise increases in input commensurate with icing in 2016 and a pulse of C input in the Mid×2-yr2 treatment plots in 2017. The FWD C inputs following the single events in this ice storm experiment ranged from 18% of the average annual fine litter C inputs at HBEF in the Low treatment to 82% of the average annual inputs in the Mid×2-yr2 treatment . The CWD2, respectively)   after thctively) . The CWDctively) .This current experiment was designed after a pilot ice storm experiment conducted at HBEF in 2012 at a similar elevation and forest type to this experiment . Fine woDifferences in FWD C and CWD C inputs among the several experimental and the natural icing events at HBEF reflect the many factors that interact to influence forest response to ice storms. These include natural variations in species composition, stand age, tree and stand health, soils, and antecedent or postcedent event weather conditions, as well as experimental artifacts such as the more rapid icing (~4 hours) during the experiments versus the natural event (~3 days).In addition to C inputs, fine and coarse woody debris play an important role in recycling nutrients in northern forest ecosystems. Nitrogen, P and Ca can all be limiting in these forests . AdditioThe results from the tree canopy damage assessments demonstrated that tree response to ice-induced crown injury unfolds over a period of years. For all species combined, the indices of canopy damage increased over time, with significant difference between Mid and High treatment plots vs Control and Low treatment plots only emerging after two to three years following icing . SimilarFour deciduous species were present in all the treatments and provided limited evidence for species-specific differences in response to icing. For example, red maple and yellow birch trees showed the greatest change in tree canopy damage indices over the four years of the study. In contrast, American beech had the lowest initial tree canopy damage indices for all the species, and showed the smallest change in this index in response to the repeated ice applications. It is notable that these responses are for the overstory American beech which were relatively healthy, with little sign of beech bark disease, a major disease that impacts the health of American beech across its range . ObservaDespite these patterns over time, few significant pair-wise species differences in tree canopy damage emerged among the four deciduous species. This may be due, in part, to the small sample size for most species, or to the fact that not all species were present in all treatments, which precluded additional species in the pairwise comparisons. Notably, sample sizes for the two coniferous species present in the plots—red spruce and balsam fir—were not robust enough for pair-wise comparisons. Coniferous species are known to be more resilient to ice storms than deciduous trees . In ordeApplication of an experimental gradient approach to ice storm damage allowed for the identification of several thresholds for this forest stand. Light icing  caused little twig or branch breakage. Although this amount of icing causes slippery conditions on roads and walkways, it had little impact on FWD and CWD in this forest, as reflected in the relatively low amounts of FWD and CWD in the Low treatment plots, which were not significantly different than the Control plots Figs  and 6. TIn conducting field-scale experiments, potential methodological artifacts that may confound results must be considered. For icing experiments, an artifact can be collateral impacts on forest hydrologic and chemical budgets from the water used to generate ice treatments. Here, the water source for icing was the nearby Hubbard Brook. The chemistry of this stream, studied for over 50 years , is char3-+ NH4+ + DON) for the High treatment represented 7% of the average annual precipitation inputs for the site; total base cations input ranged from 63% (K+) to 300% (Ca2+) and 317% (Mg+) of the average annual precipitation inputs  occur in back-to-back years. Our results suggest that damaged trees, branches or whole trees that do not fall in the first year are more susceptible to further damage in an ensuing year.This experiment was the first of its kind in creating a suite of experimental ice storms of different intensities and frequencies in a forest ecosystem. Recommendations for future work include: (i) increase intensity of icing to simulate a tipping point that marks a state change characterized by long-lasting alterations of stand composition or structure; (ii) increase frequency of icing within a single year and over a greater number of consecutive years to better understand how resilient trees are to repeat ice storms and identify tipping points characterized by a state change; (iii) conduct a similar gradient of ice storms in different vegetation types and age classes to better understand what vegetation types and age classes are more or less susceptible to ice damage; (iv) conduct the experiment under different wind speeds, as greater damage is likely with higher wind velocities; and (v) increase plot size to encompass the complete canopies and root systems of a larger number of trees to minimize belowground interactions from adjacent non-iced trees. More work is also recommended to develop simple, low cost measurement protocols for ice accretion, including a refined design for the passive collector that optimizes collector surfaces, angles, and approaches to minimize icicle formation . Such collectors would allow researchers and citizen scientists to gather more standardized information on the intensity and frequency of icing events across the landscape.The most interesting results from this experiment are likely those that will evolve over time as this forest ecosystem reorganizes and recovers from this suite of experimental ice storms. This study provides an experimental framework to study the short- and long-term impacts of these experimental ice storms on the structure and function of this northern hardwood forest now , 55, 56 S1 Fig(JPG)Click here for additional data file.S1 Photo license form(PDF)Click here for additional data file.


FBN1). Cardiovascular complications are the leading causes of mortality among MFS. In the present study, a whole-exome sequencing of MFS in the Chinese population was conducted to investigate the correlation between FBNI gene mutation and MFS. Forty-four low-frequency harmful loci were identified for the FBN1 gene in HGMD database. In addition, 38 loci were identified in the same database that have not been related to MFS before. A strict filtering and screening protocol revealed two patients of the studied group have double mutations in the FBN1 gene. The two patients harboring the double mutations expressed a prominent, highly pathological phenotype in the affected family. In addition to the FBN1 gene, we also found that 27 patients had mutations in the PKD1 gene, however these patients did not have kidney disease, and 16 of the 27 patients expressed aortic related complications. Genotype-phenotype analysis showed that patients with aortic complications are older in the family, aged between 20 and 40 years.Marfan syndrome (MFS) is a dominant monogenic disease caused by mutations in fibrillin 1 ( FBN1) [FBN1 was identified as the pathogenic factor of MFS [FBN1 gene. More than 90% of patients diagnosed with MFS revealed mutations in the FBN1 gene [FBN1 mutation(s), express a range of phenotypes from mild to severe disease. Thus, single FBN1 mutation cannot fully explain the heterogeneity of clinical phenotypes in MFS patients, who can express different pathologies in the eyes, skin, musculoskeletal, cardiovascular, and pulmonary systems. Despite other complications, cardiovascular pathologies remain the major risk factor for death in MFS patients [Marfan’s syndrome (MFS) is one of the most common autosomal dominant connective tissue diseases, with an incidence of 2–3/10000, caused by mutations in the fibrillin-1 gene  [FBN1 mutation and FBN1 truncation mutations had a high risk of developing an acute aortic event or requiring surgical intervention [The differences in the clinical phenotypes of patients with MFS exist even within the same family harboring the same genetic mutations. Moreover, the onset of the disease at different ages may further lead to different clinical phenotypes (REF). So far, no strong genotype–phenotype correlation between hnology) . In 2017rvention .de novo gene sites or additional genes contributing to the phenotypic differences among MFS patients or whether there are two mutations in MFS patients.However, the previous studies and findings thus far don't account to some clinical phenotypic differences expressed in MFS patients. Therefore, we investigated whether there are The cohort consisted of 131 MFS patients, including 82 probands and their family members. Among them, there were 69 males and 62 females. The average age of the patients at the time of enrollment was 25.00 ± 14.54 (range 0.4–64), and the average age of cardiovascular involvement was 22.64 ± 14.67 (range 0–64). The diagnosis of MFS patients was performed in accordance with the 2010 revised Ghent criteria [Genomic DNA extracted from peripheral blood of each patient was fragmented to an average size of 180–280 bp and subjected to DNA library creation using established Illumina paired-end protocols. The Agilent SureSelect Human All ExonV6 Kit  was used for exome capture according to the manufacturer’s instructions. The Illumina Novaseq 6000 platform  was utilized for genomic DNA sequencing in Novogene Bioinformatics Technology Co., Ltd  to generate 150-bp paired-end reads with a minimum coverage of 10× for ∼99% of the genome (mean coverage of 100×).After targeted NGS sequencing, the mutation obtained was further verified in the proband and remaining affected family members by Sanger sequencing. PCR amplification was performed using the forward primer (caactcctgtgagctgttgc) and reverse primer (acgttgtccacagtgagtcc). The obtained sequence was compared with the FBN1 reference gene (NM_000138.4) to identify mutations.After sequencing, basecall files conversion and demultiplexing were performed with bcl2fastq software (Illumina). The resulting FASTQ data were submitted to in-house quality control software for removing low quality reads, and then were aligned to the reference human genome (hs37d5) using the Burrows-Wheeler Aligner (bwa) , and dupAnnotation was performed using ANNOVAR (2017June8) . AnnotatFirstly, Filtering of rare variants was performed as follows: (1) variants with a MAF less than 0.01 in 1000 genomic data  , esp6500in vitro or in vivo functional studies supportive of a deleterious effect on the gene or gene product”. (1) A missense variant that created or destroyed a cysteine residue. 2) A missense variant that affected conserved residues in the EGF-like domain consensus sequence (D/N) X (D/N) (E/Q) Xm (D/N) Xn (Y/F) (m and n represent variable numbers of residues). Positive result: pathogenic or likely pathogenic variant(s) in a known disease gene associated with the reported phenotype.In order to better predict the pathogenicity of the variants, this part refers to the previous research classification of our center . The Amein trans with a pathogenic/likely pathogenic variant in a gene that explains the reported phenotype.Variant(s) in a known disease gene possibly associated with the reported phenotype. This category includes novel variants, including missense variants or in-frame insertions/deletions in disease genes that overlap with the provided phenotype for the patients, and a single rare or highly suspicious novel variant of uncertain significance (VUS) known to be Variant(s) predicted to be deleterious in a gene that has not been previously implicated in MFS or for which the published data supporting an association may not yet be definitive were considered potential novel candidates. Supporting data were based on model organism data, copy number variant data, tolerance of the gene to sequence variation, data regarding tissue or developmental timing of expression, or knowledge of the gene function and pathway analysis.de novo occurrence and determining the phase of variants in recessive disorders). This category also includes recessive conditions that overlap with the phenotype provided for the fetus in which only a single pathogenic/likely pathogenic variant is identified.VUS in a known disease gene and a patient phenotype consistent with the reported disease spectrum  frequency: reserve variants with MAF less than 0.0001 in gnomAD data (gnomAD_ALL and gnomAD_EAS) and in house Novo-Zhonghua exome database from Novogene; (2) reserve variation with functional loss . At least two software in Sift, polyphe2, and mutationastar predict harmful missense variation; classic splicing site + - 3 to 10 mutations, retain two software predicted mutations affecting splicing . At the t test. A value of P<0.05 was considered significant. Statistical analysis was performed by SPSS 22.0.The quantitative data and variables are respectively presented as the mean ± standard deviation (SD) and frequencies or percentages. Quantitative variables were compared using Student’s FBN1 gene . Both patients presented with widened aorta, and the younger brother of P_136 was also an MFS patient, but the phenotype was not as obvious as in patient P_136. MFS onset in the two patients was at a relatively earlier age, P_101 disease onset at 6 years of age and P_136 at 7 years.We found eight patients  had more than one SNP or INDEL on FBN1 . The ageFBN1 , and finFBN1 gene. These genes were PKD1, PKD2, FLNA, NKX2-5 and ACVRL1. Mutations in PKD1 gene appeared at much higher frequency than others. Twenty-seven patients  have mutations in PKD1 which is located on chromosome 16 in humans. The average age of disease onset is 27.27 years in these patients. Sixteen of the patients suffered from cardiovascular complications. Additionally we found that 14 of 27 patients have missense mutations in FBN1. Although mutations in other genes  have been detected but they didn't appear to contribute to MFS according to the low pathogenicity after filtering.Surprisingly the sequencing results revealed the presence of mutations in genes other than the FBN1, MED12, TGFBR2, SMARD, FBN2, TP53, CDH1, FN1, COL4A3, and COL4A2.In order to construct a genotype–phenotype correlations map, the sequencing results along with various databases were combined to screen and sort the affected genes. Genes were sorted based on the strength of the correlation between genetic mutations and MFS . The topTTN, NEFH, PLEC, CASQ2, and SYNE1 . Whereas no significant difference was found between the age of patients showing no aortic phenotype and those with only aortic widening (P>0.05)  .FBN1 gene showed prominent MFS phenotypes compared to other family members. Moreover, the external manifestations of patients are also obvious, but not every patient has aortic complications.The patients harboring two-mutations in theFBN1 gene have been reported to cause MFS pathologies affecting peripheral tissues, skeletal anomalies and aortic complications [FBN1 loci. The findings in the present study expand the spectrum of the FBN1 site mutation information database. The novel loci were not detected in the early stage of genomic testing [Pathogenic mutations in the  testing . It is p testing . HoweverFBN1 gene that exacerbate the disease pathology in MFS patients. The mutations we report in the present study are: exon26:c.A3142G:p.I1048V and exon14:c.G1622A:p.C541Y from one patient (P_101), exon11:c.G1220A:p.G407D and exon65:c.C8080T:p.R2694X from a second patient (P_136). Previous reports indicate that all patients with two mutations in FBN1 have obvious MFS phenotypes. Nonetheless the incidence is extremely low in the population, and the clinical significanceis still unknown [FBN1 manifested external phenotypes and early disease onset, which is consistent with previous reports [FBN1 in MFS patients is still not clear, the current reports pointed out that the probability for such mutation to cause severe MFS is very high [FBN1 gene.To this extent, one of our novel findings is the detection of two mutations in the  unknown ,24. In oery high ,26. ThisPKD1 gene mutations, but the mutations are located on the patient’s chromosome 16. Previously, it has been reported that PKD1 mutation is one of the main genes that cause polycystic kidney disease in patients. The pathogenic genes are all located on chromosome 15 of patients, and the disease is a late-onset disorder [PKD1 gene mutation suffers from aortic dissection [PKD1 gene mutation and aortic related complications were found in some patients, polycystic kidney did not appear in these patients. In addition, PKD1 gene mutations in this group of patients are located on chromosome 16, so far, there is no report on whether the mutation of PKD1 gene in addition to chromosome 15 will also cause polycystic kidney disease in MFS patients. Moreover, polycystic kidney disease is a late onset disease while MFS patients are all early onset, in which the appearance of other phenotypes still need further follow-up, but we did not find any renal lesions in the existing older MFS patients. Therefore, the detection of this genetic mutation in patients with MFS, whether it alone causes aortic related complications, or whether it works together with FBN1 gene, needs further research and demonstration.In this group of MFS patients, we also found 27 patients with disorder . In recessection . AlthougFBN1 gene in the exon 24-32 region [FBN1 frameshift mutations and nonsense mutations are prone to aortic dissection, and missense mutations are prone to aortic aneurysms [Although peripheral tissues of Marfan patients are often affected, aortic complications are the main mortality risk factors. Previous studies have shown that MFS patients with severe phenotypes are prone to mutations in the 2 region . Early rneurysms . Based oFBN1 gene, and these mutations can have an impact on the prognosis and phenotype of patients. We are working to obtain a large sample size and multicenter data to analyze gene information of introns in patients with MFS in the near future in order to improve genetic information of patients with MFS. And, for other gene loci in the article, such as PKD1, etc., since there are no samples during data analysis, we cannot perform sanger verification for these genes. However, as shown by the data provided, these sites’ sequencing data are good . We believe that these site mutations are very reliable. However, we will study this part in-depth in the future.In the present study, only exons from MFS patient’s DNA were studied, but related reports showed that MFS can also be caused by mutations in the intron region of the In the present study, through the whole-exome sequencing of MFS patients, we found many new MFS mutation sites and patient double mutation sites, which further verified the pathogenicity of the FBN1 gene to MFS patients. At the same time, it is believed that FBN1 gene mutation, which the main factor leading to patients with aortic dissection or aneurysm, and double mutation sites are a crucial factor that aggravates the patient’s phenotype. Besides, we also found that the age of MFS patients with related aortic complications in the same family is generally between 20 and 40 years, which is older than other MFS patients in the same family with no or mild aortic phenotype, which can provide a basis for clinicians to have better disease prognosis and prevent the occurrence of aortic adverse events. The clinical difference of MFS patients is vast, which may be caused by modification factors, such as the gene loci found on PKD1 in this sequencing. We consider these loci may be modified factors, but they do not occur on the disease-causing chromosomes. Due to the lack of samples in this part of the sequencing process, we cannot verify this part of the data. However, for this research, we are only preliminary research, and the relationship between these factors needs further research and discussion in the future.


Cell viability, DNA fragmentation, PARP-1 and γH2AX expression were examined to evaluate genotoxic effects of the treatment. Cell cycle progression, p53 and p21 expression, apoptosis, cytoskeleton morphology and cell migration were also investigated. In addition, global DNA methylation, DNMT1 mRNA levels and nuclear/mitochondrial sirtuins were studied as epigenetic biomarkers. Rapha Myr® exhibited low antioxidant capability and exerted antiproliferative and genotoxic effects on 1321N1 cells by blocking the cell cycle, disarranging cytoskeleton structure and focal adhesions, decreasing the integrin α5 expression, renewing anoikis and modulating some important epigenetic pathways independently of the cellular p53 status. In addition, Rapha Myr® suppresses the expression of the oncogenic p53 mutant protein. These findings promote Rapha Myr® as a promising chemotherapeutic agent for integrated cancer therapy of human astrocytoma.Brain and other nervous system cancers are the 10th leading cause of death worldwide. Genome instability, cell cycle deregulation, epigenetic mechanisms, cytoarchitecture disassembly, redox homeostasis as well as apoptosis are involved in carcinogenesis. A diet rich in fruits and vegetables is inversely related with the risk of developing cancer. Several studies report that cruciferous vegetables exhibited antiproliferative effects due to the multi-pharmacological functions of their secondary metabolites such as isothiocyanate sulforaphane deriving from the enzymatic hydrolysis of glucosinolates. We treated human astrocytoma 1321N1 cells for 24 h with different concentrations (0.5, 1.25 and 2.5%  Currently, the strategies involved in the prevention, progression, remission and relapse of cancer represent the key priority aims in public health worldwide ,2.Anaplastic astrocytomas and glioblastomas (GBM) represent the most frequent and aggressive primary malignancies of the central nervous system. So far, they are incurable diseases due mostly to their highly invasive phenotype, and the poor efficiency of the available therapies further reduced by the blood brain barrier that limits the delivery of drugs into the brain. Moreover, the current chemotherapy may lead to drug resistance in GBM treatment. Therefore, the treatment of these tumors constitutes a fundamental challenge for neuro-oncology and there is an urgent need to fully understand the molecular background of these cancers and to develop new promising therapeutic strategies .The hallmarks of tumorigenesis and cancer progression are genetic instability, deregulation of epigenetic mechanisms, oxidative homeostasis, cell cycle progression, tumor microenvironment, and cell death program activation  . AnoikisIn addition, a body of literature data highlights that epigenetic aberrations such as alterations in chromatin state, DNA methylation, histone modifications and the posttranscriptional regulation of gene expression by miRNAs play a crucial role in the tumor suppressor silencing, oncogene activation, or in the cell fate transitions, thus giving rise to all of the classic hallmarks of cancer ,9,10.All these aspects of cancer biology are widely regulated by environmental and lifestyle factors including physical activity, nutrition, dietary supplements  and synthetic agents. Many natural bioactive compounds have demonstrated their capability in counteracting tumorigenesis as well as tumor progression in various cancer types, through their impact on genomic instability, tumor-promoting inflammation, dysregulated metabolism, immune system evasion and epigenetics ,11,12. TCruciferous vegetables , a rich source of glucosinolates, which are metabolized to isothiocyanate compounds (ITCs), exert many anticancer effects both in vitro and in vivo models . SulforaNotably, SFN exerts its effects through the Nrf2-mediated induction of phase II detoxification enzymes, with the suppression of oxidative stress-induced DNA damage, inhibition of proliferation, histones modulation, anti-inflammatory and pro-apoptotic activity ,17,18,19Although SFN is water-soluble and well absorbed after oral administration both in humans and animals ,26, the Sita et al. (2018) reviewed a number of studies reporting the use of adjunctive SFN therapy in GBM for better targeting resistance and synergistically complementing and improving standard treatments as it induces apoptosis, and inhibits both growth and invasion of GBM cells .cellular tumor antigen p53 (mut p53 R213Q) as a model, because the p53 gene is mutated in approximately 40% of astrocytic tumours and dissection of the molecular pathogenesis of astrocytic tumors has identified defects in the p53 pathway as one of the most common molecular alterations observed in human astrocytoma involved in both the early transforming events and progression from low-grade to high-grade neoplasms. The functional elimination of these critical cell cycle regulatory and apoptosis-inducing factors is believed to contribute to the aggressive and invasive nature of these tumors  with 15% fetal bovine serum.Human Foreskin Fibroblast cells  for 24 h. The morphological changes were observed and the images were captured under an inverted light microscope .1321N1, U87, SHSY5Y and HFF1 cells were seeded in 12-well plates at 1.5 × 10® extract was evaluated by MTT, a colorimetric method which measures the reduction in MTT, a yellow tetrazolium salt, to a purple formazan by the mitochondrial dehydrogenase enzyme of living cells /%TDNA [MspI])*100] was chosen as the parameter to assess global DNA methylation.® Reverse Transcription Kit  [® Green RT-PCR Kit) on Rotor-Gene Q 5PLEX . Validated primers were purchased from Qiagen Inc: DNMT1 (QT00034335) and RPL0 (QT00075012). The comparative threshold cycle method (ΔΔCt model) was performed for relative quantification and RPL0 was chosen as the reference gene. The results were expressed as Fold changes (2Ct−ΔΔ) relative to control (set as 1).Total RNA was isolated from 1321N1 cells using TRIzol reagent  according to the manufacturer’s instructions. The reverse transcription was carried out on 1 µg of total RNA using the QuantiTectMD, USA) . The DNM® extract (0.5 and 1.25% v/v) for 24 h by using the ab113474 kit . Nuclear sirtuins activity was evaluated by using the Universal SIRT Activity Assay Kit . The absorbance was measured with a microplate spectrophotometer reader  at λ 450 nm. The activity for each sample was expressed as OD/min/mg proteins and was calculated according to manufacturer’s protocol [Nuclear extracts were obtained from cells untreated and treated with different concentrations of Rapha Myrprotocol .p < 0.05 as the minimum level of significance. All the analyses were performed using Graph Prism version 5.All the results were obtained by three independent experiments each performed in triplicate and expressed as mean ± standard deviation (SD). Statistical differences among different treatments were assessed by one-way ANOVA. Post hoc comparison was performed according to the Bonferroni test. We applied ®, a broccoli seed extract plus active myrosinase, on human astrocytoma cells with mutant p53 R213Q. Rapha Myr® showed antiproliferative and cytotoxic effects exerted by modulating some epigenetic pathways and inducing significant alteration of cytoarchitecture morphology. This data, together with the induction of apoptosis, inhibition of α5 translocation and low adhesion on ECM, reduction in cell migration and integrin α5 expression, allowed us to suggest a contribution of the mixture in restoring anoikis sensitivity in human astrocytoma cells through epigenetic regulation and the functional reduction in the p53 oncogenic isoform responsible for the aggressive and invasive nature of this tumor. Rapha Myr®, due to the mix of sulforaphane and myrosinase, has been proven to be a particularly efficient natural supplement, which could be ascribed among the nutraceuticals. Besides the necessity of further in vivo and in vitro studies to fully elucidate the chemopreventive/chemotherapeutic properties of SFN and other isothiocyanates, we surely can point out Rapha Myr® as a valuable compound for integrated oncology strategies in the prevention and treatment of human astrocytoma.SFN has been recognized as a safe agent for its low toxicity towards normal cells; it can rapidly cross the blood brain barrier and accumulate in the CNS after administration and can overcome the chemoresistance of tumor cells . Our wor


Escherichia coli surveillance in the Netherlands. The change to reporting acute, more severe infections appears to be a good compromise between workload, redundancy, and public health relevance, provided isolates remain available for typing and sequencing.We describe the consequences of 2 major changes in notification criteria for Shiga toxin–producing  Escherichia coli (STEC) is a zoonotic pathogen that causes illnesses ranging from mild diarrhea to hemolytic uremic syndrome (HUS) and death . Information gathered includes sex, birth year, date of illness onset, symptoms, hospitalization, and death. From its inception in January 1999, STEC surveillance in the Netherlands has been enhanced by laboratory surveillance in which laboratories are requested to voluntarily send STEC isolates to RIVM for further typing.stx) in feces or bowel contents; and 2) notification for every person with HUS or with STEC O157 confirmed via a specific antibody response for STEC O157, a culture, or demonstration of Stx or stx1 or stx2 genes in feces or bowel contents. In July 2007, notification criteria shifted from notification for STEC O157 to notification of all STEC serotypes, using the same 2 criteria. Then, in July 2016, the first criterium was changed to notification of every person with diarrhea, blood in stool, vomiting or any combination, and <21 days between date of symptom onset and date of fecal sample taken in combination with STEC confirmation via a culture, demonstration of the combination of stx1 gene with the eae or escV gene, or demonstration of stx2 gene. When STEC notification began in 1999, there were 2 criteria: 1) notification for every person with diarrhea, stomachache, or both; STEC O157 confirmed via a culture; or demonstration of Shiga toxin (Stx) or Shiga toxin genes  and during 2011–2015 (70–90 cases/year). In other years, 32–64 cases were notified. The proportion of HUS cases was highest (12%–26%) during the early years of surveillance, 1999–2004; since 2005, HUS incidence varies from 2%–11% per year. The median number of HUS cases were 12.5 (range 5–23) cases during 2008–2015 and 21 (range 12–22) cases during 2017–2019. During July 2016–December 2019, median age among HUS cases was 29 years compared with median age of 18 years during 1999–2007 and 22 years during July 2007–June 2016. Hospitalization rates for STEC remained stable, fluctuating from 29% to 54% per year. stx1 + hly and stx1 + stx2 + hly were seen less often in non-O157 STEC, but profiles stx1 + eae + hly and stx2 + eae + hly became more common  were diagnosed STEC O26 infections. In addition, STEC O26 prevalence increased from 12% during July 2007–June 2016 to 27% during July 2016–December 2019 . STEC O1e common .The introduction of PCR facilitated detection of all STEC in the Netherlands. However, STEC is a heterogeneous group and some serotypes are more prone to cause severe disease than others. Expanding surveillance to all STEC caused a 20-fold increase of reported cases, some of which were from cases with mild and long-term symptoms. Because PCR is faster, cheaper, and easier than culture, it might be requested more for rapid results in cases of less severe disease. Furthermore, many laboratories implemented reverse transcription PCR multiplex assays in which a specimen is tested for several diseases in a single run, instead of testing for 1 disease at a time. A study in Norway showed that in laboratories introducing a multiplex assay as standard detection method, the number of STEC reports, especially of low-virulence STEC, increased substantially compared with laboratories without this method (>1 of 3 predefined symptoms increased the public health relevance of surveillance. The data did not show large effects of the criteria changes on STEC O157, which implies no noticeable effects on notifications of relatively severe disease within the surveillance. However, a new challenge emerged. Isolates are needed to provide information on confirmed STEC cases and circulating serotypes and are used for nationwide outbreak detection using whole-genome sequencing. In an era of increased molecular diagnostics, less culturing is performed by the regional laboratories, especially as serotype information is not relevant for treating patients. The current notification criteria in the Netherlands appear to be a good compromise between medical laboratory workload, redundancy of less public health-relevant cases, and the ability to carry out public health actions. However, we stress that national surveillance is threatened by reduced culturing and urge public health institutes and laboratories to coordinate to safeguard against loss of cultures in the future.Surveillance of STEC serotypes and virulence gene profiles remains vital and relevant. The confinement of the notification criteria to acute disease onset with


Many compounds bound to particulate matter, such as polycyclic aromatic hydrocarbons (PAH), are suspected to be genotoxic, mutagenic, and carcinogenic. In this study, PAHs in the PM1 particle fraction were measured for one year (1/1/2018–31/12/2018). The measuring station was located in the northern residential part of Zagreb, the Croatian capital, close to a street with modest traffic. Significant differences were found between PAH concentrations during cold  and warm (April–September) periods of the year. In general, the mass concentrations of PAHs characteristic for car exhausts (benzo(ghi)perylene (BghiP), indenopyrene (IP), and benzo(b)fluoranthene (BbF)) were higher during the whole year than concentrations of fluoranthene (Flu) and pyrene (Pyr), which originated mostly from domestic heating and biomass burning. Combustion of diesel and gasoline from vehicles was found to be one of the main PAH sources. The incremental lifetime cancer risk (ILCR) was estimated for three age groups of populations and the results were much lower than the acceptable risk level (1 × 10−6). However, more than ten times higher PAH concentrations in the cold part of the year, as well as associated health risk, emphasize the need for monitoring of PAHs in PM1. These data represent a valuable tool in future plans and actions to control PAH sources and to improve the quality of life of urban populations.Airborne particles are composed of inorganic species and organic compounds. PM Particulate matter (PM) is assumed to be among the most hazardous of all ambient pollutants. Particle pollution contains “inhalable coarse particles” with diameters larger than 2.5 μm and smaller than 10 μm and “fine particles” with diameters of 2.5 μm or smaller. One of the most significant organic groups bound to PM in terms of health risk are polycyclic aromatic hydrocarbons (PAH). These compounds can exist in the atmosphere in the vapor phase (PAHs with low molecular weight), whereas heavier ones (PAHs with high molecular weight) are mostly adsorbed on the particle phase . Catalyt2.5), and especially particles with aerodynamic diameter of less than 1 μm (PM1), may play an important role in affecting human health. Squizzato et al. [1 and its content have so far not been included in routine measurement programs, although they would provide more information about potential PM sources and could be used to improve PM control strategies and health protection. A recent study by Yang et al. [1 and PM2.5 levels were associated with poorer lung function in children, with stronger associations for PM1 compared to PM2.5, pointing to the importance of regulating finer PM fractions. Furthermore, a lot of previous health risk estimations were carried out taking into account larger fractions of particulate matter , which do not enter deeply into the respiratory system [Although particle composition has been determined by many authors, there is still not much research related to the study of PAHs in airborne fine particles. Fine particles, with an aerodynamic diameter of less than 2.5 μm  was determined. Potential pollution sources were assessed using PAH diagnostic ratios, Spearman’s regression, and principal component analysis (PCA). In a previous study at the same location [1 in this part of Europe.In this study, the mass concentrations of individual PAHs in the PMlocation , factor 1 particle fraction were measured continuously from January to December 2018, together with gaseous pollutants . The measuring station  was situated in the northern residential area of Zagreb, the Croatian capital (~800.000 inhabitants). It is a low-rise building area with small inhabitant density and mild traffic density. Residential (domestic) heating relies mostly on gas, but some households still use oil or wood for heating and cooking.Concentrations of 11 PAHs in PM1 particle fraction were collected on quartz filters with low-volume sequential automatic sampler (Sven Leckel) from about 55 m3 of air per day. Filters were collected 24-h a day, and the total number of samples was 363. The sampler inlet was located approximately 1.5 m above ground and 15 m away from the road. The PM1 samples were kept frozen in aluminum foil at −18 °C until PAH analysis to avoid PAH losses and sample degradation. The filters were extracted no later than two months after sampling.Samples of PM2 was determined using a HORIBA APSA 370 device according to the norm (EN 1421:2014). NO2 was measured by a HORIBA APNA 370 device (according the norm EN 14211:2012), O3 was measured using a HORIBA APOA 370 device (according the norm EN 14625:2012), and CO was measured using a HORIBA APMA 370 device (according the norm (EN 14626:2012). Measurements of SO2, CO, NO2, and O3 are part of the local air quality monitoring network funded by the City of Zagreb, City Office for Economy, Energetics and Environment Protection.Gaseous pollutants were measured using automatic devices. SOwww.meteo.hr).Meteorological data  were obtained from the nearest meteorological station (Maksimir) of the Croatan Hydrological and Meteorological Service anthracene, chrysene, benzo(k)fluoranthene, benzo(a)pyrene, and indenopyrene, but for fluoranthene, benzo(j)fluoranthene, benzo(b)fluoranthene, dibenzo(ah)anthracene, and benzo(ghi)perilene the calibration range was from 0.01 ng μL−1 to 0.16 ng μL−1. Samples were analyzed for the following PAHs: fluoranthene (Flu), pyrene (Pyr), benzo(a)anthracene (BaA), chrysene (Chry), benzo(j)fluoranthene (BjF), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), dibenzo(ah)anthracene (DahA), benzo(ghi)perylene (BghiP), and indenopyrene (IP). The limit of detection (LoD) was from 0.001 ng m−3 for BaA to 0.03 ng m−3 for BjF. The quantification limit (QL) varied from 0.002 ng m−3 for BaA to 0.1 ng m3 for BjF. The accuracy of the method was determined by analyzing the standard reference material  provided by the National Institute of Standards and Technology (NIST) and ranged from 88% for Flu to 109% for BkF. Samples of SRM 1649a were processed the same way as real samples. The details of detection and quantification limits, as well as accuracy are shown in Extraction of PAHs from filters was performed with a solvent mixture (toluene:cyclohexane 7:3) in an ultrasonic bath. Further preparation included centrifugation 10 min, 3000 rpm) and evaporation to dryness. After that, samples were redissolved in acetonitrile. Concentrations of PAHs were determined by Agilent Infinity 1260 high-performance liquid chromatography (HPLC) with a fluorescence detector. Zorbax Eclipse PAH column (100 × 4.6 mm) was used for separations of PAHs. The mobile phase was acetonitrile and water (60:40), with a flow rate of 1 mL min−1 ,30. Cali00 rpm anp < 0.05). The Shapiro-Wilk test was used to test the normality of variables. Seasonal differences between warm and cold period for each PAH concentrations, heavy and light PAHs, and PM1 particles were tested by Mann–Whitney U test.The statistical results were processed by Microsoft Excel and the Statistica 13 (Tibco Software Inc.) program. Statistical significance was set at 5% pyrene was used as an indicator of health impact of the PAH mixture to human health. Many studies have shown that BaP was present at more than 50% in total carcinogenic activity and that made it a good indicator of carcinogenic activity of the PAH mixture. The carcinogenic activity of individual PAHs was estimated on the basis of toxic equivalency factors (TEFs) from the literature. Different TEF schemes are developed by different authors, based on experiments in animals ,37,38,39eq) were calculated by multiplying the mass concentration of an individual PAH with its respective toxic equivalency factor. Total carcinogenic potency (TCP) was calculated by summing up the BaPeq of each measured PAH. The equation used to calculate the TCP is presented below (1):For calculating the risk of the PAH mixture in ambient air, the carcinogenic potencies of individual PAHs are expressed relative to the potency of BaP. BaP equivalents . In this study we tried to estimate the most probable scenario for three age groups: infant (0–1 year), children (5–19 year), and adult (20–70 year). If we assumed that people spent an average of ten hours at their job/school and eight to ten hours at home (including sleeping), we can assume that they were elsewhere for the rest of their day, due to the fact that people spend approximately 25% of their time outdoors, that is 6 h per day.1-bounded PAHs was computed following Equation (2) [The incremental lifetime cancer risk (ILCR) posed by exposure to PMtion (2) .ILCR = inh—inhalation cancer slope factor of BaP/kg day mg−1SFeq daily dose/ng day−1IEL—BaP−1EF—exposure frequency/day year−1ED—daily exposure level/μg gBW—body weight/kgAT—average time/day−6)Cf—conversion factor , inhalation rate , and daily exposure time span . The equation used to calculate the IEL is presented below (3):In this study, a SFinh of 3.14 kg day mgand Liao . The BaP1 particle fraction during the whole calendar year are shown in p < 0.001) of PM1 concentrations with high values during the cold period of the year  and lower values during the warm period (April–September). The average 24-h concentrations of PM1 varied from 0.7 to 55.3 μg m−3 with an annual average of 13.6 μg m−3. Monthly average concentrations were also calculated for the PAH sum and for individual PAHs, and the results are presented in −3 during June to 21.497 ng m−3 during December, and the annual mean mass concentration was 6.354 ng m−3. The highest mass concentration during the cold period was measured for BbF, while in the warm period, the highest mass concentrations were determined for BghiP. High BghiP mass concentrations in the warm period in comparison to other hydrocarbons were probably due to BghiP stability at high temperatures. The lowest mass concentrations for both periods (cold and warm) were determined for DahA. The range of the monthly average concentrations for DahA was from 0.010 ng m−3 (May) to 0.386 ng m−3 (December). The average monthly mass concentrations of BaP ranged from 0.038 ng m−3 (June) to 2.826 ng m−3 (December), while the annual mass concentrations were 0.765 ng m−3. The target value in the European Union set by Directive 2004/107/EC for BaP content in the PM10 fraction is only 1 ng m−3 averaged over a calendar year. The reason for higher concentration exclusively during the cold part of the year can be explained because of the long temperature inversion periods that occur in Zagreb. Such weather conditions are characterized by high pressure and impaired air mixing, which favors the pollutants accumulation. These stable atmospheric conditions combine with increased emissions from heating are the basic origins of elevated concentrations of PAHs during the cold season. In the study area, gas, oil or, wood are mostly used for domestic heating, while coal has not been in use for more than thirty years.The monthly average mass concentrations of the PM1 fractions [1 particle fraction during both the heating and non-heating seasons at different locations in Poland compared with Zagreb. Much higher winter concentrations were also found in Ostrava-Radvanice , in Kladno-Švermov and Brno (urban sites) in Czech Republic, while levels of PM1 and ΣPAHs were similar in Košetice and Čelakovice. In a coastal area in Poland , BaP levels were much higher than in this study, while in a coastal area in Greece PM1 the levels were similar as in Zagreb but particle-bound PAHs were significantly lower. Similar PAH concentrations were also found in Guadalajara Metropolitan Area in Mexico while PAH concentrations determined in the Metropolitan Area of Porto Alegre, Brazil, were much lower during winter but higher during summer [The relevant literature comprises a very limited number of papers related to PAHs in the PMractions ,28,40,41ractions , and Majractions  reported1 particle fraction could originate predominantly from car exhausts [As significant differences were found between cold and warm months, we divided the results into two groups, separately for the cold  and the warm (April–September) period of the year. Summary statistical parameters for all of the measured PAHs for these two periods are shown in exhausts ,43,44.1-bounded PAHs during warm periods can easily evaporate from particles to gas phase, so their concentrations in the gas phase increased, but in the particle phase their concentrations decreased. The reason behind this could be that for these PAHs the dominant sources were biomass burning and during the warm period the effect of these sources were minimal. These results also indicated that traffic (diesel or gasoline) could be the main pollution source of PAHs in PM1 in this area. To confirm the assumption, we performed diagnostic ratio and principal component analysis to identify possible pollution sources.According to the number of rings and molecular weight, PAHs can also be classified into two groups: heavy and light. Heavy PAH concentrations were calculated as the sum of PAHs with five or more aromatic rings, and light PAHs represent the sum of PAHs with four aromatic rings. Heavy PAHs are usually characteristic for car exhausts, while light PAHs originate mostly from domestic heating or biomass burning ,44 Figu. Figure Specific ratios of individual PAHs are characteristic for some combustion processes and many authors used diagnostic ratios of PAHs to identify potential pollution sources ,11,12,131 originated predominately from diesel or gasoline emission. The contributions of the individual PAHs to the sum of total PAH mass concentration were calculated as well and are presented in In this paper, the average IP/(IP + BghiP) ratios were 0.5 during both the cold and warm periods, suggesting that the produced PAHs stemmed from the emission of diesel vehicles. Those average values (0.5) for cold and warm period also corresponded to the 97th and 99th percentile value, respectively, i.e., during the cold period 97% of days had a ratio value lower than 0.5, while during the warm period 99% days were with a ratio of <0.5. The average BaP/(BaP + Chry) and Flu/(Flu + Pyr) ratios were 0.5 during both periods. The average ratio BaP/(BaP + Chry) in the same time represents the 60- and 67-percentile for cold and warm periods, respectively. The other ~40% of results were higher than average value, and the individual value did not exceed 0.7, indicating both diesel and gasoline combustion as potential sources. The average ratio value Flu/(Flu + Pyr) was marginal between biomass burning, emissions from gasoline or diesel vehicles, and combustion of other liquid fossil fuels, but during the warm period there were more days (15%) with a ratio value characteristic for wood burning (during the cold period 96% of results was <0.5). Because of that, it could be concluded that during the warm period mixed sources are probably present (wood combustion and emissions from gasoline or diesel vehicles). The BaP/BghiP ratio was 0.9 during the cold and 0.6 during the warm period. In the warm period, the value was characteristic for emission from diesel , but in 10 particle fractions showed similar results during heating season [From g season . These rg season  and in Pg season . ResultsHowever, Katsoyiannis et al.  presenteFurthermore, for investigating the similarities and differences between samples, PCA was applied, both for the cold and warm period. In the cold and warm period, the eigenvalues of the first two PCs were larger than 1, which indicated their significance. In the cold period, the first two principal components represented 98.08 cum.%, with the first and the second PCs contributing to the total variance of the data set with 89.09 cum.% and 8.99 cum.%, respectively. Results of the PCA during the cold period are presented on the PC1-PC2 loading a and scoAll variables had a negative effect on PC1, while all PAHs had positive values on PC2 except for Flu, Pyr, and Chry. Chry showed the lowest negative values. The highest negative effect on PC2 was for Pyr and Flu. In the warm period the first two principal components represent 97.74 cum.% whereas the first and the second PCs contributed with 95.20 cum.% and 2.54 cum.%, respectively, to the total variance of the data set. Results of the PCA during the warm period are presented on the PC1-PC2 loading a and scoAll of the variables also had a negative effect on PC1 as in the cold period, while Flu, Pyr, and Chry exhibited positive values on PC2, but the rest of PAHs showed negative values. Based on these parameters, two different factors were extracted during both periods. In the cold period, Flu and Pyr were grouped separately because their common origin was different from the rest of the PAHs. These are PAHs characteristic for biomass burning, and their sources probably could have been domestic heating ,28,48,49PCA analysis extracted two factors during the cold and warm periods: the first factor separated Flu and Pyr, and the second factor the rest of the PAHs. Because of that, wind roses were shown for two groups, the sum of Flu and Pyr concentrations (ƩFlu+Pyr) and the sum of the remaining nine PAHs (ƩRest PAHs). The dependence of PAH concentrations on wind direction is shown on Winds coming from ENE were the most frequent during the cold and warm periods, followed by winds from NNE and SE-ESE sector, while the winds from other directions were relatively rare. On the other hand, winds of high velocities came from north-western and south-western directions, as well as from NE-ENE and SE sections. In the cold months, the highest concentrations of ƩFlu+Pyr primarily came from the west  and then from the south . The difference in concentrations in the cold and warm part of the year is greater than 80%. For the ƩRest PAH wind roses look similar as for ƩFlu+Py, but in the cold months of the year the dominant direction was SSE and then W, while in the warm months the dominant source of pollution was the east . In the −3 and in the cold 2.139 ng m−3. These results were similar to the TCPs obtained previously in Zagreb [−3 during summer to 4.503 ng m−3 during winter. TCP values similar or lower than the ones in this study were reported by other authors who used the same TEF scheme [The BaP equivalent concentration and total carcinogenic potency were calculated according to Equation (1) and are shown in n Zagreb . In a stn Zagreb , TCP valF scheme ,28, whilF scheme ,51 and tF scheme .10 or PM2.5) particle fractions [Contributions of BaP to the carcinogenic potency in this study exceeded 60%. These values were similar for both measuring periods and were on average 62% and 67% for the warm and cold season, respectively. This indicates that benzo(a)pyrene had an important role in the carcinogenic activity of the PAH mixture. The same contribution was reported by Jakovljevic et al.  for the ractions ,52.To determine the daily population exposures, total carcinogenic potency was used to calculate the daily dose according to Equation (3). In this study, we tried to estimate the most probable scenario for three age groups: infant (0–1 year), children (5–19 year), and adult (20–70 year). If we assumed that people spent an average ten hours on their job/school and eight to ten hours at home (including sleeping), we can assume that they were elsewhere for the rest of their day, due to the fact that people spend approximately 25% of their time outdoors. The incremental lifetime cancer risk (ILCR) posed by exposure to PM-bounded PAHs was computed following Equation (2) .We used Equation (3) for calculations of IEL for the warm and cold period, for three age group and the results are shown in As the variables in the ILCR calculation belong to certain distributions taking the average value into account can be misleading for yielding the risk estimation. Therefore, we employed a probabilistic Monte Carlo (MC) simulation to feed the distributions to the ILCR model for infants, children, and adults in cold and warm seasons. All of the sampled variables were sampled randomly and independently. The information on variable distributions was taken from Chen and Lia  and Liu −1, 21.0 ng day−1, and 27.7 ng day−1 in the warm period for infants, children, and adults, respectively. In the cold period, the BaPeq daily dose was higher; 76.5 ng day−1, 319.2 ng day−1, and 420.2 ng day−1 for infants, children, and adults, respectively. Exposure time is a parameter that influences the inhalation risk most strongly. The incremental lifetime cancer risk (ILCR) was calculated according to Equation (2) and it was 3.35 × 10−10 for infants, 4.56 × 10−9 for children, and 1.08 × 10−8 for adults in the cold period, but in the warm period the ILCR was 15 times lower than in the cold. According to US EPA [−6) is considered to be an acceptable level risk. A lifetime risk of one in a thousand (ILCR = 10−3) was considered to be a serious health threat. The results determined in this study were much lower than the acceptable risk level of 10−6. Higher ILCR values were found by Majewski et al. [1 particle ranged from 5.49 × 10−8 (Warsaw) to 1.43 × 10−7 (Gliwice). However, these results were also below the acceptable risk level of 1 × 10−6.IEL was lower during the warm than cold period; 5.04 ng dayo US EPA , a one ip < 0.05). The correlation between PAHs was very strong and coefficients ranged from 0.52 to 0.99 in the cold and from 0.81 to 0.99 in the warm period. These very high correlation coefficients suggested their shared sources and activities. Negative correlations were established between all of the PAHs and temperature and between PAHs and rainfall in both measuring periods (cold and warm). Humidity and pressure were negatively correlated with concentrations of PAHs in the warm period. During the cold part of the year, long temperature inversion periods may occur. Such weather conditions are characterized by high pressure and impaired air mixing, which favors pollutant accumulation. These stable atmospheric conditions combined with increased emissions are the basic origins of elevated concentrations of PAHs during the cold season.Spearman’s correlation matrix  showed t2, and all PAHs, as was a positive correlation between NO2 and PAHs with high molecular weight, while a negative weak correlation but still statistically significant was established between heavy PAHs and O3. Similar correlations were found during the warm period except between PAH and SO2, where the correlation was not significant. As there is no industry at the location, SO2 was mostly emitted from vehicles [2 concentrations have been continuously decreasing over the last ten years, the reason for which is the fact that coal has not been in use for more than thirty years at this location [2 were in good agreement with other studies [2 is one of the main traffic-related air pollutants, and in reaction with PAHs, it can produce nitro PAHs in the atmosphere [The negative correlation with temperature can be explained with the ability of PAHs to easily vaporize from particle to gas phase ,54. A sivehicles . Previoulocation . Positiv studies ,57. Prevmosphere . In bothmosphere ,58. Posimosphere .1 particle fraction at an urban location in Zagreb, Croatia, showed seasonal differences with higher mass concentrations of PAH in the cold than in the warm period. The annual mass concentration for BaP was 0.765 ng m−3, which indicated that the value set by Directive 2004/107/EC for BaP of 1 ng m−3 has not been exceeded. During both measurement periods, the contributions of heavy PAHs, characteristic for vehicle emissions, were much higher than those of light PAHs. Results of diagnostic ratios and PCA showed that the main emission source of PAHs associated with PM1 in this study area was engine combustion of diesel or gasoline during the warm period but that in the cold period emission from domestic heating is, in addition to diesel, the dominant source. The total carcinogenic potency was estimated using toxic equivalency factors. The average TCP was 0.14 ng m−3 and 2.14 ng m−3 for the warm and cold period, respectively. The incremental lifetime cancer risk (ILCR) was determined for three age groups of the population: infants (0–1 year), children (5–19 year), and adults (20–70 year) and was below the maximum acceptable level (1 × 10−6), revealing that carcinogenic risk posed to the three age groups via inhalation is acceptable. However, more than ten times higher PAH values in the cold part of the year, as well as associated health risk, emphasize the need for regular monitoring of PAH levels in smaller particle fractions, such as PM1. These data are a valuable tool in future plans and actions to control PAH sources and to improve the quality of life of urban populations.Measurements of PAHs in the PM