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.


MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio  carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly , shortened less , and exhibited slower shortening rates  and relaxation rates . Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent’s iPSCs recapitulated the phenotype of the proband’s iPSC-CMs, and conversely, correction of the R443P variant in the proband’s iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the  GJA1  were observed in 10% of HLHS patients, and that cardiac transplant-free survival was reduced in HLHS subjects containing MYH6 variants in comparison with HLHS patients without MYH6 variants. Furthermore, cardiac tissue from MYH6 variant carriers exhibited significant upregulation of sarcomere genes, including ACTA1 , MYL2 (myosin light chain 2), TNNT2 (cardiac troponin T), and the MYH6 homolog MYH7, which encodes the beta-myosin heavy chain (β-MHC) isoform. Using cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) obtained from an HLHS patient carrying an MYH6-R443P variant, we discovered that sarcomere structure was dysmorphic (Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease (CHD) characterized by atresia/stenosis of the aortic and mitral valves, and severe hypoplasia of the left ventricle and aorta . HLHS afGJA1 , NKX2.5   cultures, and declines to ∼86% at D14 (MYH6 variants associated with HLHS.Two myosin heavy chain proteins (α-MHC and β-MHC) are expressed in the human heart. During development, α-MHC is expressed in both the primitive atrium and ventricle until gestational day 35, after which its expression continually decreases in the ventricle , 1991. I% at D14 . This liwea) zebrafish, which harbor myh6 mutations and exhibit defects in both cardiac chambers, including defective atrial contraction along with abnormal sarcomere organization and an underdeveloped ventricle  during cardiogenesis . Our finentricle .MYH6, begins beating even earlier, during the third week of development. Thus, the pathological changes associated with HLHS are likely present long before we are able to detect them. Given these limitations, developmental defects such as HLHS are often investigated using murine models. However, the chamber-specific MHC expression in rodents is the opposite of humans, making them an unsuitable option for our studies. Here we studied human cardiac tissue and patient-specific iPSC-CMs to identify structural characteristics of atrial and ventricular tissue of HLHS patients and contractile properties of human iPSC-CMs with an MYH6 variant. The latter permitted us to monitor early stages of cardiomyogenesis in CMs of HLHS patients in vitro. Our hypothesis is that the MYH6 variant iPSC-CMs exhibit depressed extent and velocity of shortening that can be rescued by correcting the variant in proband-iPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing. Importantly, we show the feasibility of employing iPSC-CMs to ascertain functional consequences of the MYH6-R443P variant, demonstrating that changes in the myosin isoforms confer contractile limitations.Current non-invasive imaging methods allow detection of HLHS as early as 16 weeks gestational age , long afStudy subjects have been previously described . All subMYH6-R443P were identified using ZiFiT Targeter (Version 4.2). Three guide RNAs were cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9  to introduce MYH6-R443P into the heart healthy parent’s iPSCs. CRISPR efficiency was determined using the Cel-1 surveyor assay in HEK293 cells. The variant MYH6-R443P was flanked approximately 60 bp on either side by single strand oligonucleotides (ssODNs) (All CRISPRs were designed as previously described . Briefly(ssODNs) . Silent MYH6-R443P variant in proband iPSCs, we designed CRISPR targets  into iPSCs (1 × 106 cells/transfection). iPSCs from each transfection were then seeded into one well of a geltrex-coated 24-well plate (5 × 105 cells/cm2) for recovery in mTeSR1 supplemented with 5 μM ROCK inhibitor. The following day (D1), iPSCs were cultured with mTeSR1. Two days post-transfection (D2), iPSCs from one well of 24-well plate were sub-cultured at a clonal density (∼5 × 104 cells/cm2) into all wells of a 6-well plate pre-seeded with mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells  in human ESC medium. ESC medium was comprised of Knockout DMEM  supplemented with 20% Knockout Serum Replacement , MEM-NEAA , 2 mM L-glutamine , Penicillin/Streptomycin, 0.1 mM β-mercaptoethanol , 10 ng/ml human basic fibroblast growth factor , 50 ng/ml L-ascorbic acid  and 5 μM ROCK inhibitor. Medium was changed 2 days later (D4) to ESC medium without ROCK inhibitor. Five days post-seeding (D5), medium was changed to puromycin (0.5 g/ml)-supplemented MEF-conditioned medium with bFGF and L-ascorbic acid, which was replaced every 2 days for 1 week. Following maintenance (∼D12), distinct colonies (∼1 mm diameter) were manually transferred to a single well of a 24-well plate pre-coated with geltrex in mTeSR1 medium plus ROCK inhibitor without MEFs. Following genotyping (see below), clones were expanded to a geltrex-coated well of a 12-well plate in mTeSR1 plus ROCK inhibitor and further expanded and frozen in mFreSR freezing medium . Isolated CRISPRed iPSC lines were frequently sub-cloned to ensure homogeneity of the population.Prior to transfection, iPSCs were pretreated for 3–4 h with 5 μM Rho-associated kinase (ROCK) inhibitor , dissociated with accutase  to ensure single cell suspension, and transferred to an electroporation cuvette. For each transfection (D0), 1 μg of the gene-specific CRISPRs, 80 μM of the relevant ssODN, 1 μg of each of the two AAVS1-specific cassettes, and 1 μg AAVS1 donor plasmid were added to 100 μl P4 solution  and electroporated using program CB-150 on a 4D NucleofectorMYH6-R443P gene were sequenced at the conserved region in MYH7 as well to check the CRISPR’s off-target activity.To isolate genomic DNA from iPSCs, 30 μl Quick Extract Solution  was added to each cell pellet and incubated for 15 min at 65°C, followed by 5 min at 95°C. PCR was carried out using gene-specific primers , and 2 μ2 and 4% O2. All experiments were performed on cells during passages 10–50. One day prior to inducing cardiomyocyte (CM) differentiation, the iPSCs were seeded at a cell density of >1 × 105/cm2 on a 10 μg/cm2 geltrex-coated  tissue culture dish. The cells were cultured in mTeSR1 medium  supplemented with 5 μM ROCK inhibitor. The cells were induced with 10 μM CHIR99021  and 10 ng/ml Activin-A  in insulin-free RPMI/B27 . On D3, the medium was exchanged for insulin-free RPMI/B27 with 5 μM IWP . Starting at D7, at 2-day intervals the medium was changed to RPMI/B27 with insulin . Cultures were evaluated for % of cardiac troponin T (cTnT) positive cells via flow cytometry and MHC (MF20) expression by immunostaining. The cardiomyocytes were either frozen at D15, or were sub-cultured between D10–D15 after dissociation into single cells with 0.25% trypsin; specifically, cells centrifuged at 1,000 rpm for 5 min were either frozen at a density of >2 × 106 in cryovials with 10% DMSO and 90% FBS, or were re-plated at a density of 2 × 104–5 × 104/cm2 on a geltrex-coated 12 cm2 round cover glass for single cell-based contractility studies, or at >5 × 104/ cm2 for automated contractility video analysis .Induced pluripotent stem cell (iPSC) lines were generated from dermal fibroblasts donated by HLHS probands and their parents. Fibroblasts were reprogrammed to pluripotent stem cells using Sendai reprogramming as previously described . PluripoThe iPSC-CMs and tissues were co-immunostained with anti-sarcomeric α-actinin (AF594)  to image sarcomere organization, and anti-MHC monoclonal MF20 (AF488)  to inform myocyte identity. Sarcomeres in iPSC-CMs were visualized with a Nikon inverted fluorescent microscope at 400x.2+ Tyrode’s solution (35°C) mounted on the stage of an inverted microscope (Nikon TE2000). Spontaneously contracting iPSCs were visualized at 40x and contractile events recorded using a MyoCam-S video 250 Hz camera and a CFA300 cell framing adapter . The left and right edges of iPSC-CMs were defined and contractions per minute (CPM), shortening (μm), % cell shortening, and rates of shortening and relaxation (μm/second) were measured using the IonWizard image detection program . Ten cells on a 12 cm2 round cover glass were evaluated for 10 contractions each. For a subset of D20 iPSC-CMs, the same parameters were determined for responses to 1 and 2 Hz stimulation or to 1 μM isoproterenol. As an alternative method, an automated contractility software1  , D34 (D34–D37), and D41 (D41–D43). The iPSC-CMs were transferred to a recording chamber containing 1 mM Ca States) , 2015 wa90) was defined as the time (ms) from the start of the action potential until membrane potential returned to 10% of its peak height.Action potentials of iPSC-CMs were obtained with microelectrodes (50–70 MΩ) filled with 2.7 M KCl and 10 mM HEPES and recorded at 35°C using Clampex9.2 software . For each iPSC-CM measured, the lowest membrane potential , threshold potential, peak spike potential, and action potential duration were averaged from 5 action potentials per cell. Action potential duration . Then, cover glasses were inserted into a quick-release chamber  bathed in 1 mM Ca2+ Tyrode’s solution at 35°C. Whole cell Ca2+ transients were recorded using Fura-2 AM as described previously  and ratio units/s . Ten Ca2+ transients were averaged for every iPSC-CM.The iPSC-CMs were plated on an 18 mmeviously . Briefly2+ Tyrode’s solution and centrifuged for 5 min at 16,100 × g. The pellet of iPSC-CMs was collected in 10 μl SDS sample buffer  and stored at −80°C until studied.Following contractile studies, iPSC-CMs were scraped from cover glasses into 1 mM CaMHC composition was determined by silver staining 5% SDS-PAGE gels as previously described . BrieflyDiluted samples were run on a 4–15% gradient gel for 1 h at 150 V and transferred to a 0.2 μm nitrocellulose membrane using a Mini Trans-Blot cell  at 100 V for 50 min. The gel was run in Tris-glycine running buffer and transferred with running buffer containing 20% methanol. Following transfer, membranes were blocked for 3 h in 5% non-fat dry milk in TBS-0.1% Tween pH 7.6 (TBST) and probed with primary antibodies overnight at 4°C. Primary antibodies for MHC , GAPDH , and ventricular myosin light chain-2 (MLC2v)  were diluted in 5% non-fat dry milk in TBST. Then, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies  in 5% non-fat dry milk in TBST for 1 h at room temperature. Protein expression was detected on autoradiography film  by chemiluminescence. ImageJ  was used for densiometric analysis and protein expression was normalized to GAPDH.MYH6 variant and one from an HLHS subject with the MYH6-R443P variant. The 25 non-neonatal tissues were from subjects between 227 days to 24-years old. These samples were divided into three study groups: control , HLHS subjects without an MYH6 variant, and HLHS subjects with an MYH6 variant. In each study group, when available, we included atrial and ventricular tissues. In all cases, the samples were pieces of tissue dissected and phenotyped at the time of surgery. They were snap frozen immediately after removal from the patient. Some tissues came at the time of heart implantation, and some came from the time of reparative surgery.A total of 27 samples from atrial and ventricular tissues were examined, including two 4-day old neonatal atrial tissues, one from an HLHS subject without an Cryo-sections (10 μm) of the frozen tissues were prepared. The tissues were co-immunostained with anti-sarcomeric α-actinin (AF594)  to image sarcomere organization, and anti-MHC monoclonal MF20 (AF488)  to inform myocyte identity. Sarcomeres in tissue sections and iPSC-CMs were visualized with Structured Illumination Microscopy (SIM) at 1000x magnification.t-test using Excel (Microsoft). If the data were not normally distributed, comparisons were made using the non-parametric Wilcoxon signed-rank test. Statistical significance was set at P < 0.05.Significant differences between two groups were analyzed through an unpaired two-tailed Student’s MYH6-R443P) at timepoints D20, D27, D34, and D41. Post hoc pair-wise comparisons were performed using Tukey’s test. All subsequent studies  were restricted to D20. Data were analyzed with a one-way ANOVA, except for stimulation data, which were analyzed with a two-way ANOVA. Statistics were performed using Minitab v18.1 with significance set at P < 0.05.When necessary, data were transformed to meet assumptions of normality and homogeneity of variance. For the time course study, a two-way ANOVA was performed to compare iPSC-CM contractility of wild-type (WT) vs. variant  lines, as well as lines having one allele (inserted +/VAR) or both alleles inserted (inserted VAR/VAR). The CRISPR target site has one base-pair mismatch, and the sequences of both MYH6  in the proband’s iPSCs  iPSC-CMs at time points D20, D27, D34, and D41  and VAR  and increased velocity (P < 0.0001) compared to Probandcc iPSC-CMs.In mass cell cultures, using a separate automated contractility software , we also analyzed velocity of corrected WT iPSC-CMs at D20-D22. With this analysis, we obtained similar results as single-cell studies. Corrected WT iPSC-CMs exhibit faster contraction : 26.5 for MYH6-R443P , MYH6-K849-), MYH6-E1503V)]. The MYH6-R443P variant is in the head domain whereas MYH6-K849- and MYH6-E1503V are in the tail domain. The atrial and the ventricular tissues in MYH6- K849-, while the atrial and the ventricular tissues in MYH6-E1503V. At the neonatal stage, sarcomeres are organized in both atrial tissues from HLHS patients with and without MYH6 variants    while thre 26.6) .MYH6-R443P variant on HLHS, we used CRISPR/Cas9 gene editing to generate isogenic controls. Insertion of the MYH6-R443P variant into the heart healthy parent’s iPSCs recapitulated the dysmorphic sarcomere phenotype , and decreased rates of shortening and relaxation. These defects occurred early (by D20) and likely resulted from reduced development of the SR including the SERCA pump and the increased expression of the slow β-MHC isoform in the variant iPSC-CMs  and the threshold potential were not different between cell types, this suggests that the rate of spontaneous depolarization to threshold may have been faster in the WT. However, our recordings did not show an obvious difference in the rate of depolarization to threshold between cell types  also showed sarcomere disruption (MYH6-R443P (26.5), MYH6-K849- (25.9), MYH6-E1503V (33) and DMD-I288N (26.6) had the most apparent disruptions in sarcomere structure in atrial tissues ex vivo. One limitation was lack of paired controls, however tissue samples showed convincing evidence of abnormalities  muscle is the sarcomere, a highly ordered structure containing contractile and regulatory proteins. Disruptions in sarcomere proteins have been implicated in many cardiac diseases . Here, w tissues . This resruption . Furthermalities .MYH6-R443P cell lines were created using CRISPR/Cas9 gene editing and these lines recapitulated the phenotypes of patient-specific MYH6-R443P iPSC-CMs. An additional limitation is the heterogenous population of iPSC-CMs, including both atrial and ventricular cells. There are MLC-2a, MLC-2v, as well as double-positive iPSC-CMs in our cultures upon immunostaining at D31-D35 (data not shown). As our protein expression  in a non-biased, blinded fashion. A WT line was always differentiated along with experimental lines to compare differences among cell types at each differentiation stage. Additionally, isogenic pression  and autopression  were anaposition .MYH6-R443P allele showed morphological and physiological differences from WT. iPSC-CMs could be used for clinical applications such as pharmacological drug testing, but these applications require mature CMs. A number of strategies have been employed to promote the maturity of iPSC-CMs, including: the administration of biochemical agents [adrenergic receptor agonists, Triiodothyronine  myh6-mutant zebrafish exhibits a hypoplastic ventricle  protocols by the Children’s Hospital of Wisconsin . Subjects were consented through the CHD Tissue Bank  and the Wisconsin Pediatric Cardiac Registry , IRB-approved research databases housed at CHW prior to inclusion in the study. Both biorepositories provided all cardiac tissue as well as iPSCs, from patients and family members, with associated clinical outcome variables.AT-M and RF designed and directed the project. AT-M, RF, MEM, JWL, and AG were involved in planning and supervising the work. M-SK, RF, BF, JL, MC, MA, SS, LK, LT, and MM processed the experimental data, performed the analysis, and interpreted the data. M-SK took the lead in writing manuscript with support from AT-M, RF, BF, JL, LK, LT, MM, MA, and SS. All authors discussed the results and commented on the manuscript.AT-M and MEM are cofounders of TAI Diagnostics , a biotechnology company involved in transplant diagnostics, and members of its scientific advisory board. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Succinic acid, extracted from amber, is widely used in cardiovascular therapy. The effect of succinic acid on the activity of cytochrome P450 (CYP450) enzymes was investigated in this study.in vitro. The inhibition of CYPs was fitted with competitive or non-competitive inhibition models and corresponding parameters were also obtained. The effect of succinic acid (100 μM) on the activity of eight isoforms of CYP450  was investigated compared to the specific inhibitor and blank controls in pooled human liver microsomes 50 values of 12.82, 14.53, and 19.60 μM, respectively. Succinic acid inhibited the activity of CYP3A4 in a non-competitive manner with the Ki value of 6.18 μM, and inhibited CYP2D6 and 2C9 competitively with Ki values of 7.40 and 9.48 μM, respectively. Furthermore, the inhibition of CYP3A4 was found to be time-dependent with the inactKI/K value of 6.52/0.051 min−1·μM−1. Succinic acid exerted inhibitory effect on the activity of CYP3A4, 2D6, and 2C9 with the ICin vitro inhibitory effects on the activity of CYP3A4, 2D6, and 2C9, which indicated the potential drug-drug interactions. Succinic acid should be carefully co-administrated with the drugs metabolized by CYP3A4, 2D6, and 2C9. Succinic acid showed  Cardiovascular disease is a ubiquitous heart disease that threatens human health  enzymes are responsible for the metabolism of a wide array of endogenous compounds and xenobiotics, which exist mostly in the liver , testosterone (CYP3A4), coumarin (CYP2A6), chlorzoxazone (CYP2E1), dextromethorphan (CYP2D6), diclofenac (CYP2C9), S-mephenytoin (CYP2C19) and paclitaxel (CYP2C8). The effect model was also investigated by kinetic studies.The activity of CYP1, CYP2, and CYP3 is closely related to the metabolism of exogenous compounds and could be strongly affected by the presence of environmental pollutants and dietary bioactive compounds , NADP+, phenacetin (≥98%), acetaminophen (≥98%), 4-hydroxymephenytoin (≥98%), 7-hydroxycoumarin (≥98%), 4′-hydroxydiclofenac (≥98%), sulfaphenazole (≥98%), quinidine (≥98%), tranylcypromine (≥98%), chlorzoxazone (≥98%), 6-hydroxychlorzoxazone (≥98%), paclitaxel (≥98%), 6β-hydroxytestosterone (≥98%), clomethiazole (≥98%), and furafylline (≥98%) were obtained from Sigma Chemical Co . Montelukast (≥98%) was obtained from Beijing Aleznova Pharmaceutical . Coumarin (≥98%), diclofenac (≥98%), dextromethorphan (≥98%), and ketoconazole (≥98%) were purchased from ICN Biomedicals . Pooled HLMs were purchased from BD Biosciences Discovery Labware. All other reagents and solvents were of analytical reagent grade.Succinic acid (≥98%) and testosterone (≥98%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products . The chemical structure of succinic acid is shown in O-deethylation for CYP1A2, testosterone 6β-hydroxylation for CYP3A4, coumarin 7-hydroxylation for CYP2A6, chlorzoxazone 6-hydroxylation for CYP2E1, dextromethorphan O-demethylation for CYP2D6, diclofenac 4′-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, and paclitaxel 6α-hydroxylation for CYP2C8. All incubations were performed in triplicate, and the mean values were utilized. The typical incubation systems contained 100 mM potassium phosphate buffer (pH 7.4), NADPH generating system , the appropriate concentration of HLMs, a corresponding probe substrate and hispidulin (or positive inhibitor for different probe reactions) in a final volume of 200 μL.As shown in The concentration of succinic acid was 100 μM, and the positive inhibitor concentrations were as follows: 10 μM furafylline for CYP1A2, 1 μM ketoconazole for CYP3A4, 10 μM tranylcypromine for CYP2A6, 50 μM clomethiazole for CYP2E1, 10 μM quinidine for CYP2D6, 10 μM sulfaphenazole for CYP2C9, 50 μM tranylcypromine for CYP2C19, 5 μM montelukast for CYP2C8. Probe substrates, positive inhibitors , and succinic acid were dissolved in methanol, with a final concentration of 1% (v/v), and 1% neat methanol was added to the incubations without inhibitor. The final microsomal protein concentration and incubation times for the different probe reactions are shown in 50). iK values were obtained by incubating various concentrations of different probe substrates  in the presence of 0–50 μM succinic acid.Succinic acid (100 μM) was used to initially screen for its direct inhibitory effects towards different human CYP isoforms. For the CYP isoforms whose activities were strongly inhibited, secondary studies were performed to obtain the half inhibition concentration  was pre-incubated with HLMs (1 mg/mL) in the presence of an NADPH-generating system for 30 min at 37 °C. After incubation, an aliquot (20 μL) was transferred to another incubation tube  containing an NADPH-generating system and probe substrates whose final concentrations were approximate to KI and inactK values for the inactivation of CYP3A4, the incubations were conducted using higher probe substrate concentrations  and various concentrations of succinic acid (0–50 μM) after different preincubation times (0–30 min), with a two-step incubation scheme, as described above.To determine the maxV and mK. Inhibition data from the experiments that were conducted using multiple compound concentrations were represented by Dixon plots, and inhibition constant (iK) values were calculated using non-linear regression according to the following equation:iK is the inhibition constant, S is the concentration of the substrate, and mK is the substrate concentration at half the maximum velocity (maxV) of the reaction. The mechanism of the inhibition was inspected using the Lineweaver-Burk plots and the enzyme inhibition models. The data comparison was performed using Student’s t-test and performed using IBM SPSS statistics 20 (SPSS Inc.).The enzyme kinetic parameters for the probe reaction were estimated from the best fit line, using least-squares linear regression of the inverse substrate concentration versus the inverse velocity (Lineweaver-Burk plots), and the mean values were used to calculate p < 0.05, 50 was obtained as 12.82 μM  (Romand et al. in vivo interactions and effects on the activity of P-gp and UGT.The limitation of this study is that in vitro inhibitory effect of succinic acid on the activity of CYP3A4, 2D6, and 2C9 was found in this research. The concentration of drugs and the incubation time are two important factors that influenced the activity of CYPs. These results indicated the potential interaction between succinic acid and drugs metabolized by CYP3A4, 2D6, and 2C9, but further in vivo studies are needed to verify these potential drug-drug interactions.The


Quantitative analysis of MAs in DM showed a plausible agreement between ICGA and the DCP in SS-OCTA. These findings contribute to the imaging methodology in DM.Diabetic maculopathy (DM) is a microvascular dysfunction clinically characterized by microaneurysms (MA) leading to edema and central visual deprivation. This prospective explorative study investigated 27 eyes of 17 patients with DM by fluorescein/indocyanine green angiography  and by swept source-optical coherence tomography angiography  to identify clinically relevant MAs. The SS-OCTA cubes were split into the superficial capillary plexus (SCP) and the deep capillary plexus (DCP) according to the automated segmentation. The images of all modalities were superimposed for alignment by an Early Treatment Diabetic Retinopathy Study grid overlay and compared to each other. In total, the mean number of MAs in FA was 33.4 ± 22 (standard deviation) , in ICGA 24.9 ± 16.9 , in the SCP 6.5 ± 3.7  and in the DCP 18.1 ± 10.5 . Mixed effects models between ICGA and the DCP were borderline significant ( Clinically significant macula edema (CSME) is an expression of fluid accumulation in a predefined area within the macula impairing the central vision4. The fluid derives from a breakdown of the inner blood–retina barrier, namely protrusions of the capillary walls or so called microaneurysms (MA)5. These outpouchings of capillaries are typically the earliest clinically visible signs of retinal damage and a hallmark of DM2.The exact identification of MAs is important as their location and number yields prognostic information on the severity of DM as well as on the clinical relevance for treatment guidance of focal or diffuse edema6.Diabetic maculopathy (DM) is a major cause of vision loss in developed countries9. It is highly sensitive as it is able to visualize even smallest hyperfluorescent dots. However, it represents all vascularized structures of the fundus in an en-face image with obscuration of deeper layers in case of extensive leakage even in early phases such as present in diffuse edema. Therefore, its application for clinical management might not be favorable at all stages of the disease. Indocyanine green (ICG) has a higher molecular weight (775 g/mol) and binds to different transport proteins in the blood circulation with higher capacity and a tendency to form aggregates in solvents without extravasation10. The attributes of ICG angiography (ICGA) could be preferential for the quantification of clinically relevant MAs in DM and the planning of individual treatment algorithms13. Undesirable adverse events like nausea and emesis or even rare but serious anaphylaxis are addressed to the use of intravenous dye.Fluorescein angiography (FA) has been the gold standard to examine the retinal vasculature and graduate DM15. It enables the visualization of flow signals at several distinct layers namely the superficial capillary plexus (SCP), the intermediate capillary plexus (ICP), the deep capillary plexus (DCP) or the choriocapillaris. Numerous studies investigated the potential of OCTA technology in the axial detection of macular MAs preferentially located in the DCP, where CSME almost exclusively originates20.Optical coherence tomography angiography (OCTA) is a non-invasive dye-independent imaging technique based on the principle of motion contrast: the decorrelation of an OCT signal amplitude due to red blood cell movementIn the light of the above, we evaluated the quantitative appearance of macular MAs based on their clinical manifestation according to the Early Treatment Diabetes Retinopathy Study (ETDRS) grid in dye-based angiography and compared the results with those observed in the SCP and the DCP imaged by swept source (SS)-OCTA.For this prospective explorative study 27 treatment-naive eyes of 17 diabetic patients were recruited from our tertiary eye care center  with a male preponderance (59%). The median age at the time of recruitment was 58 (range 36–76) years. Both eyes of 10 patients and 7 sole eyes (86% right eyes) were included. All patients underwent a complete ophthalmic examination including best-corrected visual acuity (BCVA) using the ETDRS charts at 4 m—counting every correctly read letter—and converted to Snellen. BCVA was 20/32 + 1 (0.92 ± 0.3 ETDRS) on average (range 0.06–1.26 ETDRS). Eyes with a funduscopic appearance of DM and diabetic retinopathy (DR) ranging from mild non-proliferative (NPDR) to proliferative (PDR) changes without the urgent need of surgical intervention were included. Mild to severe NPDR was present in 22 eyes with a CSME evident in 11 eyes, leaving 5 eyes with PDR. In addition to routine fluorescein angiography (FA), indocyanine green angiography (ICGA) and swept source-optical coherence tomography angiography (SS-OCTA) split into the superficial capillary plexus (SCP) and the deep capillary plexus (DCP) by automated segmentation were conducted. Exclusion criteria included previous treatment for DM or DR independent of cataract surgery and bad image quality due to motion artefacts in the cases of opaque media or fixation error. In total, 9 (33%) eyes have already had successful cataract surgery, while 10 (37%) eyes showed cataract formation to some degree.21. In the rare case of grading disagreement between two independent readers beyond 3 MAs within one sector, a senior clinical advisor was consulted. To investigate the interrater agreement, intraclass correlation coefficients (ICC) were calculated separately for the different device settings. Overall, a good ICC > 0.9 could be detected  between all imaging modalities for the total grid as well as for each sector were listed in Table p < 0.05). A statistically significant difference between FA and ICGA could be observed only in the inferior sector of the 6 mm diameter   on average in FA, 24.93 ± 16.87  in ICGA, 6.48 ± 3.7  in the SCP and 18.07 ± 10.47  in the DCP. A mean of 21.3 ± 14.2  MAs were visible in both dye-based angiograms, while 4.98 ± 3.1  MAs where present in both SS-OCTA layers. A detailed list of each imaging modality and ETDRS grid sector was displayed in Table 21. FA showed the highest number of MAs in total as well as in each quadrant, followed by ICGA and—closely—the DCP. The mean MA numbers in FA and ICGA were different , although only one sector showed a significant value when analyzed independently. The median was more useful in this setting than the mean due to the high numbers of MAs in only a few quadrants, also graphically illustrated by the box plot charts and their spikes  images for grading DR and correlated them with color fundus photography as used in ETDRS30. They summed up all vascular plexus and found MAs present in 91% of cases in contrast to fundus photography. As a conclusion, they suggested SS-OCTA as a screening tool even in early DR as it might be more sensitive than presumed.In our study, least MAs in SS-OCTA were found in the SCP from the inner limiting membrane (ILM) to the inner plexiforme layer (IPL), while the majority was situated in the DCP between the IPL and the outer plexiforme layer (OPL). These results conform to earlier studies on the quantitative appearance of MAs preliminary found in the deeper segmentations of SS-OCTA31. They averaged the images up to ten times but could not find a correlation with either averaging time or ICG angiograms. Their work showed a significant correlation between different morphological characteristics—particularly the bulge types—and visibility. MAs must have the capability to accumulate dye or ascertain flow to be detected by one of the technologies. Although we did not investigate morphology separately, only MAs with dye staining or flow signal in SS-OCTA were eligible for enrollment. We meticulously counted hyperfluorescent dots in dye-based angiography as well as MA subtypes in the respective plexus of SS-OCTA and found good agreement in numbers between ICGA and the DCP with a preponderance towards depth-independent ICGA.The median numbers in ICGA and the DCP of SS-OCTA resembled each other in most sectors of the ETDRS grid Fig.  while on32. A more homogenous collective would emphasize the clarity of our data. Grading was performed by two readers, while another advisor was only consulted in the rare incidence of disagreement above 3 MAs. ICC was excellent for all but the SCP as listed in Table Major limitations of this research include its low numbers. This is partially attributed to the explorative character despite its comparative nature. ICGA is associated with dye-dependent risk factors but not a first line diagnostic tool for graduating diabetic maculopathy. Therefore, it was not integrated into our clinical routine. Due to the lack in numbers we could not distinguish between cubes consisting of 320 or 512 B-scans, although a higher scan density would implement a better resolution and hence should lead to a more accurate detection rate. Also, eyes with and without CSME as well as NPDR and PDR were included as long as the macular region was not endangered by tractive detachment or obscuration by blood. Edema is expressed as early leakage in FA but could also lead to a miscount of MAs in other imaging techniquesTo conclude, ICGA showed similar outcome values compared to the DCP in SS-OCTA regarding the detection rate of clinically relevant diabetic macular MAs. We believe that our results contribute to the imaging methodology in DM.The clinical trial with the registration number EK-17-083-0517 was approved by the Viennese ethical commission and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from voluntary participants after they had been informed about the aims of the study, the procedures involved, their potential present and future benefit as well as possible risks.All examinations in this prospective explorative observational study took place between September 2018 and August 2019. Indirect slit-lamp biomicroscopy  with dilated pupils using 0.5% tropicamide  drops, multimodal imaging including spectral domain (SD)-OCT  and FA were performed as standard of care for the detection of DM but immediately extended with ICGA . In addition, the central retina was scanned with Topcon’s DRI-OCT Triton Plus  device using SS-OCTA consecutively on the same day. Heidelberg’s SPECTRALIS scanning laser ophthalmoscope provides 30° en-face images with an isotropic transversal resolution between 11 and 40 µm in high-speed settings independent of the administered dye or laser diode and is equipped with an eye-tracker. In each patient, both eyes were examined parallel with a series of consecutive pictures starting approximately at 30 s after dye injection with dilated pupils. The distribution of dye changed over time and images were recorded up to 15 min after dye injection. Early FA pictures were hence investigated in each eye for comparability due to severe leakage in later phases  provides 3 × 3 mm, 4.5 × 4.5 mm, 6 × 6 mm, and even 9 × 9 mm macular cubes with automated subdivision into four different en-face layers: the SCP, the DCP, the outer retina and the choriocapillaris. Topcon’s device does not come with an automated segmentation of the ICP and therefore this interesting analysis could not be performed. Standard 6 × 6 mm cubes with a volume of 320 B-scans or higher resolution 512 B-scan cubes were captured. While the digital axial resolution was 2.6 µm, the transverse resolution ranged from 9.4 to 18.8 µm depending on the selected cube. The integrated software was used to delineate the SCP and DCP automatically. The SCP was defined as a slab between 2 lines drawn from the ILM to the outer limits of the IPL  was calculated for main findings. All analyses were performed using R, release 3.3.3. Diagrams were illustrated by using SPSS 20 . Figures were composed utilizing Affinity Photo 1.7 .No correction for multiplicity was performed due to the exploratory character of the study. The global statistical significance level was set to 0.05 and


Cystatin C, first described in 1961 and formally named in 1984, is a non-glycosylated, basic protein encoded by the CST3 gene that is found in virtually all nucleated cells. It is a potent inhibitor of lysosomal proteinases and extracellular inhibitors of cysteine proteases that play a pleiotropic role in human vascular pathophysiology, in particular regulating cathepsins, which are overexpressed in atherosclerotic and aneurysmal lesions,The role of serum cystatin C as a measure of kidney function (filtration) is now well established. Cystatin C is generated at a mostly constant rate, is freely filtered, reabsorbed and catabolized in the proximal tubule of the nephronBeyond its use in the estimation of kidney function, cystatin C-based eGFR has been shown to be a superior predictor of cardiovascular disease (CVD) and mortality when compared to serum creatinine-based eGFRet al. assess another specific sub-population where cystatin C may have additional utility over serum creatinine and other markers, namely subjects with end stage kidney diseaseOther potential roles for cystatin C include being an earlier marker for acute kidney injury, a superior marker of kidney transplant function, CVD risk and transplant failure, and a more accurate measure of function in specific sub-populations such as liver cirrhosis and oncologyet al. assessed serum and peritoneal dialysis effluent cystatin C in 52 patients treated with peritoneal dialysis (PD) in a single dialysis center. In addition to the assessment of residual renal function (RRF) they explored the correlation of cystatin C and cardiovascular events as well as an assessment of body composition using bioimpedance. Like the general population, higher levels of cystatin C was associated with a two-fold increase in the likelihood of cardiovascular disease (odds ratio (OR) 2.65), although the 95% confidence interval was wide, reflecting significant uncertainty in the result (0.84 to 8.37). This is not surprising given the limitations of the small sample size and the cross-sectional nature of the study design and analysis. The assessment of RRF in patients treated with PD is critical given its importance in prognosis and total overall dialysis clearance. The current study reaffirms our understanding that cystatin C may be a good marker of assessing RRF. Patients who have been on PD longer have a higher cystatin C level correlating to the gradual loss of RRF seen after dialysis commencement. Weekly total creatinine clearance was significantly negatively correlated with cystatin C levels as would be expected. It is important to note that PD also removes cystatin C and hence its levels will be determined by both RRF and dialysis clearance. Also as expected, cystatin C levels positively correlated with relative lean tissue mass. Moreira et al. raises more questions than it answers due to the size and study design, given the role of cystatin C as an established tool for the assessment of kidney function in the general and CKD population as well as an additive marker of cardiovascular risk assessment, further large prospective studies in the PD population are warranted in order to delineate whether cystatin C will have a role in the management of the PD population. While the study by Moreira


This paper shows that below a critical value of dimensionality that lies between two and three, the potential between objects begins to fall as the energy levels increase. For dimensionality below two, the potential becomes constant irrespective of separation and the force between them disappears, which represents a new paradigm of asymptotic freedom. Since asymptotic freedom is at the basis of many applications such as those associated with strange metals, unconventional superconductors, and fractional quantum Hall states, the new paradigm can have novel applications. It also is of relevance to the study of anomalous mechanical effects that are important in metamaterials. In this paper, we investigate a further implication of this theory to the anomalous situation of strong interaction at large distances and much weaker interaction at short distances, which is a characteristic of asymptotic freedom4.It was recently showne and since Nature chooses optimality, this should be the dimensionality of physical space5 and the inverse square law itself may be seen as a consequence of noninteger dimensionality6. The information efficiency per dimension is d = 1, with peak at e) and the intrinsic dimensionality of data is also e8. As dimensionality and information are foundational to the understanding of physical reality, these ideas ought to be of relevance in the understanding of asymptotic freedom.From the perspective of information theory, the optimum number of dimensions is the noninteger  number mentclass2pt{minim9, and it plays a role in novel states of matter that include strange metals, unconventional superconductors11, and fractional quantum Hall states14. As a property of some gauge theories, it offers a mechanism for confinement at large distance by invoking properties of the mathematical structures used to describe these interactions.Asymptotic freedom is counterintuitive15. In materials that exhibit negative linear compressibility, there is expansion in one or more directions during the process of uniform compression18 and this anomalous mechanical behavior has found applications in the design of pressure sensors, artificial muscles and actuators21.Other counterintuitive phenomena are associated with negative differential response, and they are to be found not only in physics but also in chemistry and biological processes22, which provides a way to view seemingly inconsistent descriptions as two sides of the same reality. Although, the principle arose originally in quantum theory, it has been argued that it should apply much more broadly to diverse fields of science23, including biological phenomena24. Complementarity is the description of the same phenomenon in distinct, categorically different ways that cannot be done in the same spatial, temporal, or situational context.This counterintuitive nature is sometimes understood through the lens of the complementarity principle27, which may be seen as an example of increasing the energy scale, with prospective applications to superconductivity, ferroelectricity, information communication, sensing and detection30.The notion of dimension applies to physical reality at all conceivable scales, therefore one must also consider anomalous mechanical properties of materials that emerge from compressing three-dimensional volumes into lower dimensions31, information is physical and, therefore, information-theoretic ideas must play a key role in the understanding of the above-mentioned anomalous phenomena.Some of the inconsistencies in the interpretation of phenomena could be the consequence of different ways space is considered in the theory. As stressed by LandauerIn this paper, rather than examining the application of noninteger dimensionality to any of the above phenomena directly, we show that a novel version of asymptotic freedom is associated with noninteger dimensionality. This represents an unexpected new framework with potential applications similar to those of anomalous mechanical properties of materials. Also, since gauge transformations relate to the very nature of space, dimensionality may be of relevance to gauge theories.6. This is seen most clearly when we visualize a 2-dimensional space obtained from a 3-dimensional space which will cause all the points in the third dimension to collapse to the extant two dimensions.We can visualize a given noninteger space sitting within the container of the ceiling integer space . It may be argued that a fundamental characteristic of a noninteger space is that of a continual shrinking of the metrical relationships between objectsd, which we shall compare with its ceiling function d the smallest integer greater than it. Since dimensionality is an additive property, we can distribute the fractional part along any direction. Thus d = 1.8  would imply a dimension of 0.9 along the one-dimensional infinite line in any direction.Consider the example of a noninteger space with dimension tend to shrink in proportion to the value of dimensionality, and the tendency to do so is a function of the ratio d is the ceiling function of d, and d = 1.8, the shrinking is a function of The view of the noninteger dimension that there are gaps in the space is complementary to the view that relative measures on the line The tendency for space to contract constitutes a potential, making points associated with events that the observer witnesses to tend to come closer to each other. This potential leads to dynamics that emerge thus from the very nature of space. This potential is not assumed to have an a priori existence, independent of dimensions, and therefore it represents a view different from the current understanding.d characterizes the space, the potential between two objects with unit measures must also be inversely related to the separation so that objects that are further apart have less influence on each other than those that are near. Points on a 1-dimensional infinite space stay where they are, whereas those on a 0.8-dimensional infinite space will have a potential to come closer by the 0.8 measure. Consider two objects with unit measures with a separation of r, and let the potential be defined by Since r has surface equal to r should be modified to Sd for 3- and 2- and 1-dimensional worlds is 4πr2, 2πr, and 0, respectively, and these are the surface area of a sphere, the circumference of a circle, and the length of a point. Corrected for reduced density, the expression for d < 3 and 1 < d < 2, that is r.The sphere representing the equipotential surfaces at a distance of d, over the range 2 < d < 3, the potential is proportional to 1/r and therefore the forces between objects in such a space will be proportional to 6.The forces corresponding to these potentials will be the derivative of the expressions (3). For a fixed r as a variable) are:The potentials as functions of dimensionality alone , but would depend on external constraints for an engineered system.Some further constraints are needed to find plausible candidates for A matched distribution. If physical structures are compared to the optimal filter corresponding to natural distribution, then the use of optimum signal theory32 will require that the potentials be matched to the efficiencies associated with the d-values.d and call it the DED  f(d) with the range from 1 to the maximum of N:N that is of interest to those looking at physical dimensions is 3. For such a case e, the expected value of dimension is substantially less at 2.148 with a variance of 0.268.One can associate a pdf with the dimension N = e,If d-dimensional space acting as a matched filter, one may choose the following function:d = 2.455.To return to the entclass1pt{minimaNow we consider two ad hoc functions that satisfy the constraints of zero values at 2 and 3. The first of these is the same as that of Fig. d = 2.4495, whereas the peak for the red curve  reduces the strength of interaction. Increasing dimensionality beyond the critical dimension also reduces this strength, and this behavior has obvious applications for understanding evolution in cosmology.The continuing reduction of dimensionality is further injection of energy into the system. Figures r implies that objects in such a space will experience the same potential and, therefore, will behave as if in a bag, and experience no force that varies with the separation. There will be a dependence on d, that can be put to use in engineering applications.For situations where the ceiling dimensionality is 2, the potential function asymptotic freedom that is arrived at by squeezing the dimensionality of space. This fact represents a most unexpected property associated with the dimensionality of noninteger spaces.Since there is no dependence with respect to separation, this represents a case of 1 < d < 2, there could be dynamics as a consequence of externally applied forces that need to be analyzed separately.Although there is no noninteger space generated intrinsic dynamics for the case of dcrit somewhere around the mid-point of 2 and 3, the potential begins to fall, signifying a reduction in the interactions amongst the particles. At d = 2 and below, it becomes constant, irrespective of separation, and the force between objects disappears, which represents a case of asymptotic freedom that can be the basis of novel applications. This is a surprising property of noninteger spaces.This paper explored properties of noninteger dimensionality and examined how interaction potentials across objects vary. It was shown that as the dimensionality falls below We have shown that objects in a 2-dimensional space will not interact with each other, but as the dimensionality increases beyond 2, the interactions begin to increase in strength until the point of maximum strength. Systems could be engineered where the difference in these regimes is exploited to perform specific signal processing or anomalous mechanical function. This also opens up the question of how to control the value of the critical dimension that is likely to be an important variable in the design of metamaterials.The work in this paper provides a dimensionality-based explanation of interactions becoming weaker as energy scales increase. Whether this phenomenon based on dimensionality of space has any connection with other models of asymptotic freedom remains to be investigated.


In the heart, hyperactivation of NHE1 has been linked to the development of different pathologies. Several studies in animal models that reproduce the deleterious effects of ischemia/reperfusion injury or cardiac hypertrophy have conclusively demonstrated that NHE1 inhibition provides cardioprotection. Unfortunately, NHE1 inhibitors failed to reproduce these effects in the clinical arena. The reasons for those discrepancies are not apparent yet. However, a reasonable clue to consider would be that drugs that completely abolish the exchanger activity, including that its essential housekeeping function may not be the best therapeutic approach. Therefore, interventions tending to specifically reduce its hyperactive state without affecting its basal activity emerge as a novel potential gold standard. In this regard, a promising goal seems to be the modulation of the phosphorylation state of the cytosolic tail of the exchanger. Recent own experiments demonstrated that Sildenafil, a phosphodiesterase 5A inhibitor drug that has been widely used for the treatment of erectile dysfunction is able to decrease NHE1 phosphorylation, and hence reduce its hyperactivity. In connection, growing evidence demonstrates cardioprotective properties of Sildenafil against different cardiac pathologies, with the distinctive characteristic of directly affecting cardiac tissue without altering blood pressure. This mini-review was aimed to focus on the regulation of NHE1 activity by Sildenafil. For this purpose, experimental data reporting Sildenafil effects in different animal models of heart disease will be discussed.The cardiac Na This + extrusion through the NHE1 contributes to maintain intracellular pH (pHi) of cardiac cells in a healthy narrow range, fulfilling its crucial basal housekeeping function   . HoweverSildenafil was found to trigger a direct NHE1 inhibitory action in rat , 57 and We have also demonstrated that the inhibitory effect of Sildenafil on the NHE1 requires activation of the p38MAPK , since b2O2 production under ischemia (Another controversial subject is the possible role of acute Sildenafil on ROS. In this regard, it has been shown that this drug did not reduce ROS-induced lipid peroxidation in hearts subjected to regional ischemia/reperfusion  but reduischemia , 69. Desischemia . Longer ischemia –70.+2 channels (2+, therefore eluding the calcium/calmodulin-NFAT pro-apoptotic pathway (Different studies were aimed to explain the subcellular basis of the beneficial actions of chronic PDE5A inhibition by Sildenafil in the myocardium. Among them, it is interesting to recall the described inhibitory effect on voltage-operated L-type Cachannels , and on channels , with th pathway . The res pathway . Interes pathway .Ca channels (+2-induced mitochondrial swelling (The possibility that chronic effects of Sildenafil could also involve mitochondrial actions should be considered. Chronic Sildenafil treatment improved respiratory rate and reduced ROS production . These echannels , preventchannels , and/or channels . In connswelling . In addiswelling . Consideswelling . Figure As it was mentioned in a previous section, since the approval and commercialization of Sildenafil more than two decades ago , the phaWe have briefly reviewed the role of Sildenafil on NHE1 regulation and its possible pathophysiological relevance. Classical Sildenafil targets were presented, but the main focus of this revision was centered on the novel inhibitory action of this drug on the NHE1. We were particularly interested in stressing the potential benefits of specifically targeting NHE1 hyperactivity, avoiding its complete blockade, which suggests that Sildenafil treatment would be a better therapeutic approach than traditional NHE1 blockers in the field of cardiology.Finally, considering that Sildenafil has been safely used during more than two decades  and thatAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptococcusmutans (ATCC 25175), and to lower the pH in liquid culture media. In a second phase, these foods (n = 4) were examined in terms of their ability to inhibit the microorganisms in contact with early carious lesions in children and to reduce the pH of mixed cultures combined with the food. The results revealed that, of the foods tested, three inhibit the growth of microorganisms in carious lesions and, at the same time, lower the pH of the culture by more than 2.5 units. The food with the highest inhibitory capacity  showed a similar effect among patients (P > 0.05), which together with the fact that its sugar concentration is less than 10%, makes it an ideal candidate for clinical study.Caries rates in school-age children are still high enough to be the cause of serious concern for health systems in different countries. The biotechnology strategies studied to decrease these rates include the consumption of probiotics—available via a variety of functional foods obtainable on the market—that are able to inhibit bacteria associated with this disease. In this vein, the purpose of this study was to determine the effect of these foods on the growth of microorganisms in early carious lesions in children aged between 6 and 12. In the first phase, an agar well diffusion method was applied to selected foods, available in supermarkets, which contain probiotics that have already been shown to inhibit  A diet rich in fermentable carbohydrates is one of the reasons for this, as cariogenic microorganisms in dental biofilm can metabolize these carbohydrates very rapidly, producing the acids that demineralize teeth2. This is one of the reasons for which organizations such as the World Health Organization (WHO) recommend a daily sugar intake of less than 10% of total caloric intake, and even advise that a 5% intake would be even more beneficial to oral health3.Dental caries represent the most widespread non-communicable disease globally and, for this reason, constitute a public health problem. Despite the contribution of fluoride to reducing the rates of dental caries, the disease continues to have a high level of incidence from an early age4. The first and second permanent molars erupt between the ages of 6–12. The former are most at risk for caries and, in fact, are the most frequently affected by them, given their retentive tooth morphology and the fact that they are difficult to see because of their location5. In Colombia, the last Estudio de Salud Bucal (ENSAB IV)  reported that 54.2% of 12-year-olds already had caries in their permanent teeth, a figure that motivates further research on strategies that could complement the role of fluoride in preventing this disease6.The consequences of dental caries can affect quality of life, given the acute and chronic medical conditions that they can cause, as well as the socioeconomic problems that contribute to inequality that affects a country’s development and, in turn, a population’s access to healthcare8.One of these strategies involves the consumption of foods with components that inhibit or reduce the effects of cariogenic microorganisms. These include milk, cacao, some fruits such as grapes, and propolis among others, or functional foods defined as foods enriched with components that provide health benefits9.These cover foods supplemented with probiotics, which are microorganisms, mainly bacteria, that are safe for human consumption, and that when ingested in sufficient quantities can be beneficial for human healthLactobacillusrhamnosus GG, Lactobacillusreuteri, Lactobacilluscasei, Lactobacillusbrevis, Lactobacillusacidophilus, Bifidobacteriumlactis, Bifidobacteriumlongum) have also been studied in order to find their possible capacity to control oral cavity diseases such as caries. This is due to their known functions such as the inhibition of cariogenic microorganisms by means of the production of toxic substances and competition for nutrition and space, and by encouraging increased oral cavity immune responses17.There is a wide variety of functional foods supplemented with probiotics on the market that improve the immune system’s response and enhance the functioning of the digestive system. The majority of these probiotic bacteria  followed by Bifidobacteriumlactis (19%)  and Lactobacillusbulgaricus (44%) . The vast majority of these products were fermented milk products (83%), followed by milk powders for babies (9%)   from the initial pH of the foods  and one was a fermented milk product that is not categorized as a yoghurt (No. 9). The probiotic bacteria reported to be contained in these foods were S. mutans 25175 , it was found that foods with a sugar concentration higher than 10 g/100 g had a significantly greater capacity (P < 0.01) to inhibit 175 Fig. a. Foods 175 Fig. b. Howeve175 Fig. c, no sig175 Fig. d.Figure The majority of the children were male (58%), with an average age of 9.6 ± 1.7. All of the patients had an ICDAS of 2, and 87% had a medium risk of caries Table .Table 3CThis study found that, of the mixed cultures taken from the dental biofilm of carious lesions of 15 children with ICDAS 2, food No. 14 produced the largest inhibition diameter, which was statistically significant (P < 0.01). The inhibition zones produced by this food measured a median of 14 mm (IQR 13–14), followed by products 1 and 9, which produced a statistically similar median inhibition (P > 0.05) of 13 mm (12–13) (Table No significant differences were found between patient samples (P > 0.05) in terms of the inhibition of the mixed cultures of dental biofilm from carious lesions by functional food No. 14, which produced the largest inhibition diameter . The most commonly occurring median decrease among patients was greater than 2.80  2.43–3.52) , of which three were identified as having a greater ability for inhibition and a lower effect on reducing the pH of the culture medium. The three products included two yoghurts and a fermented milk product that did not meet the categorization for yoghurt20. Of these foods, No. 14 displayed the greatest inhibition capacity along with a lower sugar concentration.This study identified commercially available functional foods with added probiotics that have the capacity to inhibit the growth of S. mutans in the saliva of patients who have consumed such foods17. Fermented dairy products have a high potential for caries control, as, in addition to being made up of a consortium of probiotic and/or fermentative bacteria, their metabolites and other antimicrobial substances, they also contain the benefits of milk products such as calcium, phosphate, and lipids, as well as casein phosphopeptides (CPP). These, both contribute to the remineralization of teeth21, and inhibit the growth of cariogenic bacteria22.Several studies have revealed the efficacy of yoghurts and other fermented milk products in inhibiting the growth of microorganisms associated with caries. The results report a statistically significant decrease in the quantity of Lactobacillusacidophilus, B. animalis subsp. lactis BB12, and Bifidobacterium sp. Furthermore, S. thermophilus and L. bulgaricus were also present as fermentative bacteria in yoghurts. The inhibition caused by the bacteria present in these foods has been attributed to the production of bacteriocins, organic acids, and hydrogen peroxide26.The bacteria announced on the labels of the foods that were the most successful in inhibiting the growth of the mixed culture of bacteria taken from the early carious lesions in children  included B. animalis subsp. lactis BB12 and L. acidophilus which have shown significant inhibition of S. mutans in the saliva of patients in different age ranges17. The present study found that the food that contained B. animalis subsp. lactis BB12 as its only probiotic and fermentative bacteria (No. 9) presented the greatest inhibition of S. mutans 25175. However, when evaluating this food with a mixed culture of microorganisms present in the biofilm of carious lesions, its inhibitive capacity was lowered. These results concur with those found in a clinical trial involving 1-month-old newborns, who were given B. animalis subsp. lactis BB12 for almost 2 years, and where, despite the reduction in the numbers of S. mutans, there was no impact on caries experience27. This is probably associated with the vast diversity of microorganisms implicated in the formation of caries, where an inhibition of their growth is not as high as it is with S. mutans. The authors of this study also concluded that the probiotic did not permanently establish itself in the oral cavity, a result similar to that found in other studies showing that these types of probiotics are transient colonizers28.Of these bacteria, the most frequently assessed are S. thermophilus and L. bulgaricus present in yoghurts, Petti et al.29 showed the inhibitory effects of these bacteria on S. mutans 6519T and other bacteria from the viridans group such as S. oralis 25671, confirming the important inhibitory properties of these types of bacteria in yoghurt.In relation to lactic acid bacteria such as S. thermophilus, L. bulgaricus, Lacidophilus and B. lactis. This can be associated with the synergic action of bacteria present in this food, which potentiate bioactivity such as the foods’ inhibitory capacity against pathogenic microorganisms. This has been demonstrated by studies focusing on the control of other types of diseases30.Regarding the effect of the foods selected in this study to determine the inhibition of mixed cultures from caries lesions, the yogurt that generated the greatest diameter of inhibition (No. 14) was the one containing a combination of 3.The importance of studying the effect of the bacteria present in the food on lowering the pH of the culture medium, is given by the fact that considering their lactic acid properties, the release of a high concentration of acids can be counterproductive when supplying them for caries control. The first phase of this study consisted in a selection process, where products No. 3 and 13 were excluded, in spite of presenting good inhibition. These foods significantly lowered the pH of the culture medium (P < 0.05) compared to the other foods selected as candidates for the second phase, probably because they were the foods with the highest sugar concentration (14 g/100 g). This value exceeds the daily sugar concentration recommended by WHO 31. These types of commercially available foods tend to have high sugar concentrations that could contribute to the appearance of carious lesions in cases where an individual does not apply appropriate diet and hygiene practices.It is important to point out, at this stage, that of the candidates selected in the first phase , the sugar concentrations of two of the four foods (No. 1 and 9) also exceeded this recommendation, although to a lesser extent (12 g/100 g). A study conducted using the nutritional information of different types of yoghurts commercially available in England found that, although there was evidence that these provided health benefits, most of them had a median quantity of total carbohydrates of over 10 g/100 g, including those developed for childrenS. mutans 25175. Some of the foods with a sugar content of over 5 g/100 g caused greater inhibition, and therefore demonstrate the role that sugar plays in the biological activity of the bacteria present in fermented milk products32. In this respect, and as has been shown in other studies, these types of bacteria cannot be considered ideal as probiotics for use in the oral cavity given the acidification of the medium33.Thus, commercially available foods that contain sugar concentrations within the limits of low-sugar product categorization and with levels known to have additional health benefits (5 g/100 g) could be considered ideal candidates in terms of supporting oral health. However, in the present study, these types of foods that represented 52% of the evaluated products (n = 12), did not produce a significant inhibition in the growth of Despite this, it is important to highlight that including these types of bacteria in the diet can contribute to inhibiting the growth of cariogenic microorganisms and therefore complement the effects of other preventative measures for this disease. In fact, the present study identified that three of the four selected candidates  inhibited the growth of the mixed culture of microorganisms taken from early carious lesions, and No. 14 produced the highest significant inhibition, had a lower sugar concentration, and reduced the pH to a level similar to the other foodstuffs.Despite the limitations of this study, such as the lack of tests on biofilm models and a greater diversity of bacteria involved in caries, it was possible to select a functional food supplemented with probiotics that homogeneously inhibited the microorganisms present in the dental plaque of the initial caries lesions of 15 children who attended the dental clinic at three different points in time.This study therefore identified a commercially available food with added probiotics suitable for clinical trial. This candidate produced the largest diameter of inhibition for microorganisms present in the biofilm of early carious lesions in 6–12-year-old children and contains less than 10 g/100 g of sugar. Although the bacteria present in these foods are not ideal candidates for oral cavity probiotics, they can help to inhibit the growth of cariogenic microorganisms, and so complement the role of fluoride and good oral health habits. Furthermore, as this food is a milk-based product, it boosts the buffer system of the oral cavity and the remineralization of the tooth enamel.S. mutansATCC 25175 and a reduced pH lowering capacity. In the second phase, the inhibition and pH lowering capacity of the selected foods was evaluated against microbial mixed cultures from dental biofilm of children with caries. This study was approved by the subcommittee of ethics (039-2018) at Universidad Cooperativa de Colombia and all procedures were in line with the ethical standards established by the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.In vitro experimental study carried out in two phases. In the first phase consisted on the selection of a number of commercial foods supplemented with probiotics. These presented a greater inhibitory capacity against the reference strain Different functional foods supplemented with probiotics available in chain supermarkets in Villavicencio, Colombia, were collected in order to assess only those foods sold in all of Colombia’s cities. The collection criteria were as follows: the foods had to be liquid or dehydrated food for reconstitution with liquid, they had to be suitable for consumption by people of all ages, they had to specify the added probiotics or fermentative bacteria on the packaging, they had to have a sell by date of more than a week, and not be sold in the pharmacy or natural remedies section. These foods were taken to the microbiology laboratory where the information included on their labels was recorded . Prior to processing, the pH of each one was measured using a pH meter  with a microelectrode .S. mutans ATCC 25175 , using the spread plate technique, in a concentration of 1–2 × 108 UFC/mL18. Then, holes were bored in the medium, creating 9 mm diameter wells for the inoculation of 100 µL of the foodstuffs. Clorhexidina at 0.2% (positive control) and a sterile saline solution at 0.9% (negative control) were used as controls for the inhibition. The cultures were incubated at 37 °C with 5% CO2 over 15 h to determine whether inhibition had taken place. If so, the diameter of the zone of inhibition or halo was measured. To determine acid production and therefore the pH reduction generated by the bacteria present in the functional foods, 100 µL of the foods were inoculated in BHI broth (Oxoid) and the pH was measured before and after incubation at 37 °C with 5% CO2 over 15 h. Inhibition and pH lowering tests were performed in triplicate on different days using 3 different lots in Villavicencio and 2 in Medellín, Colombia. According to the results of this study, foods were selected for the next phase, taking into account those that presented greater inhibition of S. mutans and lower acid production.For the inhibition test, 20 mL of the solid Brain Heart Infusion (BHI) (Oxoid) was inoculated with the strain riesgograma19, which determines 7 risk factors: history of caries, the presence of dental biofilm, local/systemic factors of plaque retention, content of the diet, frequency of eating during the day, use of fluoride and reason for last dental appointment. Furthermore, the patients had to be free from systemic illnesses; have no orthodontic appliances fitted; should not have taken antibiotics in the last 3 months; and, at the time of sample taking, should not have brushed their teeth in the 8 h prior. For the microbiological analysis, dental plaque was taken from the lesion using a microspoon until filling an inoculation loop calibrated to 2 µL. The samples were deposited independently in Eppendorf tubes with glass pearls and 1,000 µL of saline solution, and immediately transported to the laboratory.For this phase, parents or caregivers of children aged 6–12 years authorized their children's participation in the study, by means of a signed written informed consent, while the children agreed to participate by giving their written assent. They were interviewed by a pediatric dentist calibrated in ICDAS  in order to find the child's clinical history, and subsequently examine and define whether they fell in line with the selection criteria. The criteria included having ICDAS 1 and 2 caries lesions, and moderate or high caries risk according to individual caries risk assessment using a 2, the inhibition and size of the halo was determined. For the pH reduction analysis, the pH was determined before and after incubating 100 µL of the dental biofilm sample and 100 µL of the foodstuff in 1,000 µL of BHI broth. These analyses were carried out using samples taken from a lesion from the same child on three different days and in triplicate. The foods originated from three distinct lots.For the inhibition tests, the samples of dental plaque were mixed by vortex mixer  for 1 min. Then, 100 µL of the dental plaque was inoculated in 20 mL of solid BHI (Oxoid) using the spread plate technique. The holes had a diameter of 9 mm where 100 µL of the selected foods and controls were deposited. After incubating at 37 °C for 15 h with 5% COThe SPSS version 25  software was used to conduct the analyses. Data normality was determined using the Kolmogorov–Smirnov test and variance homogeneity using the Levene test. The Kruskal–Wallis test was used to determine whether there were differences in terms of inhibition and pH reduction generated by the bacteria present in the different foods. To define the foods with the best response, the results were compared with the food with the highest inhibition level and with the food with the lowest degree of pH reduction, using the U Mann–Whitney test.To analyze the microorganism inhibition and pH reduction capacity of selected foods in mixed cultures of lesions with caries, the results pertaining to selected foods were examined using the Kruskal–Wallis test and compared using Dunn's post-hoc test.