Identification of early signs of atherosclerosis in young adults have the potential to guide early interventions to prevent later cardiovascular disease. We therefore analyzed measures of vascular structure and function and biomarkers of cardiovascular risk in a sample of young healthy adults.Pulse-wave velocity (PWV), carotid-intima media thickness (cIMT) and augmentation index (AIX) were measured in 834 healthy non-smokers (ages 18.0–25.9). Emphasis was put on discriminating between individuals having a vascular structure and function associated with a higher or lower risk, and cluster analysis algorithms were employed to assign the subjects into groups based on these vascular measurements. In addition, a vascular status score (VSS) was calculated by summarizing the results according to quintiles of the vascular measurements. The associations between VSS and cardiovascular biomarkers were examined by regression analyses.The cluster analyses did not yield sufficiently distinct clustering . VSS proved a better classificatory variable. The associations between VSS and biomarkers of cardiovascular risk were analyzed by univariable and multivariable regressions. Only body fat percentage and C-reactive protein (CRP) were independently associated with VSS.A VSS calculation, which integrates PWV, cIMT, and AIX measurements is better suited for cardiovascular risk evaluation in young adults than cluster analyses. The independent associations of VSS with body fat percentage and CRP highlight the decisive role of adiposity and systemic inflammation in early atherosclerotic progression and suggests a subordinate role of insulin and lipid metabolism in this age span. The progression from a silent presence of atherosclerosis to symptomatic disease is characterized by a gradual buildup of plaques that are initially asymptomatic , 2. AutoPhysiological measurements of vascular stiffness and vessel wall thickness serve as surrogate risk markers of future cardiovascular risk , 11. WelIn female subjects, contraceptive use was included in the model to examine its interaction with vascular structure and function. Studies examining the effect of exogenously administered estrogen on vascular measurements have yielded conflicting results –19, and The purpose of the observational Lifestyle, Biomarkers and Atherosclerosis study (the LBA study) is to identify risk factors and biomarkers related to the early stages of the atherosclerotic process, and to make use of the findings to improve the preventive management of cardiovascular disease. In this study, we employed cluster analyses, mathematical pattern recognition methods used to identify subgroups within a given data set . This waThe LBA sample consists of 834 Swedish adults between 18 and 25.9 years of age. Subjects were recruited by advertising at the Örebro University, and a local newspaper. A validated computerized questionnaire was used to assess the subjects’ lifestyle habits and served as verification that they met the inclusion criteria of being nonsmokers not suffering from chronic diseases . The subHeight was measured with a fixed stadiometer to the nearest 0.5 cm, with the subjects standing without shoes, heels together, back straight, and arms extended alongside the body. Body fat percentage was measured using a bioelectrical impedance body composition analyzer . Adjustments were made with 1 kg for clothes and the standard setting was used.Blood pressure was measured after 15 min of rest in the left arm using a digital automated device  with Dura-Cuf . The subjects came for two visits on two different days to the university examination room and their blood pressure was registered on both occasions.The cIMT was measured using a high-resolution ultrasound B-mode system,  with a 12 MHz linear array transducer, as previously described . An averStiffness measures (PWV and AIX) were registered using applanation tonometry, using SphygmoCor  as previously described –33.PWV was measured in the supine position. Carotid and femoral pulse waves were recorded with simultaneously ECG recording, and the PWV (m/s) calculated as PWV = distance between measurement locations (m) / transit time (s) for the pulse wave. A higher PWV indicates an increased vessel stiffness.For AIX, the radial artery tonometry was performed at the subject’s right wrist. The aortic pressure waveform was derived from the radial waveform by a validated transfer function. AIX is calculated from the aortic pressure waveform and adjusted to heart rate 75 beats per minute (AIx_HR75). An average of AIx_HR75 from three measurements was reported for each subject. A higher AIX indicates an increased vessel stiffness .Samples were collected after an overnight fast into sodium citrate fluoride vacutainer tubes for glucose analysis and serum and plasma vacutainer tubes for the rest of the analyses . Serum was left to clot for at least 30 min before centrifugation and subsequent analysis. CRP, Orosomucoid, Apolipoprotein A-1 (Apo A-1) and Apolipoprotein B (Apo B) were analyzed on a Siemens ADVIA 1800 Chemistry instrument with a coefficient of variation (CV) of 5% at 0.74 mg/L with the Siemens High Sensitivity CRP Assay . The Apo A-1 assay had a CV of 4% at 0.9 g/L and the Apo B assay a CV of 5% at 1.5 g/L. Orosomucoid had a CV of 4% at 0.47 g/L using the DAKO orosomucoid immunoturbidimetry assay . Total cholesterol (CHOL), Triglycerides (TG), high-density lipoprotein (HDL) and glucose were assayed colorimetrically with Vitros MicroSlide technology . Direct low-density lipoprotein (LDL) was assayed by a two-step colorimetric assay with Vitros MicroWell technology. CHOL (3% CV at 3.9 mmol/L), TG (CV of 4% at 1.3 g/L), HDL (6% CV at 1.0 mmol/L), LDL (5% CV at 2.4 mmol/L) and glucose (4% CV at 4.6 mmol/L) were analyzed on a Vitros 5.1 system . Insulin was analyzed with the Abbott Architect Insulin Assay, a sandwich immunoassay using chemiluminescence detection with a CV of 7% at 8.0 mIU/L on an Architect i2000SR unit .Statistical analyses were performed with R ver 3.4.3 . The vascular measures used in the cluster analyses were PWV, AIX and cIMT. The following cluster analysis algorithms were used: k means clustering, agglomerative nesting hierarchical cluster analysis (AGNES) and partitioning around medoids cluster analysis (PAM). Some of the algorithms allow machine identification of the ideal number of clusters in a model, while others require a trial and error approach, where each analysis is done with a predetermined number of clusters. Silhouette analyses permit the creation of silhouette plots visualizing the separation distance of the clusters, serving as an assessment of the quality of the cluster analysis models based on the number of predetermined clusters. A high silhouette index is suggestive of a good fit of the produced clusters. The optimal number of clusters was also evaluated using the Elbow method where a bend in the plot of the total within-cluster sum of squares is generally considered an indicator of the appropriate number of clusters.Median, VSSTertile and VSSQuintile. A higher VSS indicates an unfavorable vascular structure and function.In addition to the cluster analyses, an alternative model was devised in order to determine subgroups within the population. First, the vascular measures were transformed into z scores, one for each variable, calculated separately for males and females. The z scores were categorized into Vascular Status Scores (VSS) based on either median, tertiles and quintiles for the cIMT, PWV and AIX measurements, respectively. Each category was assigned a number, starting with 0, corresponding to the lowest category, adding 1 for each additional category. The range of scores was thus 0–3 for the median based score and 0–6 and 0–12 for the tertile and quintile based, respectively. For each individual, the assigned numbers of the three variables were added, forming the VSSUnivariable regression models were performed to compare the VSS to the established risk factors. Multivariable regression models were then performed with the variables found to be significantly associated with the VSSs, to examine possible confounding factors and identify risk factors independently associated with an unfavorable vascular structure and function in a young population.Baseline characteristics of the study population are shown in Table The three measures of vascular structure and function, cIMT, PWV and AIX, were entered into cluster analyses using the different cluster analysis algorithms, AGNES, PAM and k-means clustering. None of these clustering algorithms yielded distinct clusters, corresponding to groups of individuals with a vascular structure and function distinctly associated with a higher or lower risk of CVD. A substantial overlap was seen between the formed clusters Fig. . The k-mQuintile). In women, estrogen contraceptive use did was not significantly associated with either of the VSSs. A recalculation after reclassification of the individuals who did not report the name of their contraceptives gave rise to the same result (data not shown). In general, the analyses yielded similar results and p values, irrespective of the type of VSS used. The VSSQuintile was chosen for further multivariable analysis. To examine possible independent associations between the VSS and the biomarker variables, the variables with a p value lower than 0.1 in the univariable analyses . The relationship between the VSS calculations with the established markers of cardiovascular risk was analyzed by univariable regression analyses, using the VSSs as the dependent variable, in separate analyses. As seen in Table es Table  were inces Table . Among tTheoretical advances in the understanding of how risk factors for atherosclerosis start operating long before any clinical diagnoses have become apparent are increasingly recognized , 37, andQuintile, were not identified as constituting an unfavorable subgroup by any of the cluster analyses. A variable oriented approach (VSS) thus performed better in singling them out as belonging to an extreme category with respect to their vascular status.Cardiovascular risk profiling based on blood biomarkers is often improved when based on more variables , 49. SimQuintile, but not HDL, Apo B/Apo A-1 ratio, glucose, orosomucoid or estrogen contraceptive use in women. In multivariable analyses, only body fat percentage and CRP remained independently significant predictors of VSSQuintile  in this study of young healthy individuals, probably due to the close collinearity of these measures. Therefore, we devised a variable oriented approach, by scoring the measures of vascular structure and function to compose VSS as a surrogate endpoint in the context of the LBA population. The serum concentration of C-reactive protein and the body fat percentage were the only independently significant predictors of VSSAdditional file 1. Sensitivity analysis recalculation of the main Tables


The excessive mobilization of fatty acids from dairy cows’ adipose tissue increases blood non-esterified fatty acid concentrations and could have a negative effect on the fertility parameters and milk yield, as well as increase the risk of metabolic disorders and also result in early-lactation culling risk. Propylene glycol and rumen-protected starch from maize grain are commonly used as glucose precursors reducing nonesterified fatty acid levels; however, no such comparisons are available, thus it was decided to assume it as the aim of this study. Propylene glycol had a positive effect on shortening the period to first ovulation. Propylene glycol and maize grain improved the first service conception rate and decreased the number of services per conception in cows. In conclusion, both treatments with propylene glycol and maize grain had a slight effect on the metabolic profile and no effect on milking performance, yet they improved fertility parameters, which could indirectly enhance milk production economics.The aim of the study was to determine the effect of propylene glycol and maize grain content by-pass starch supplementation during the transition period and the first 56 days of lactation on blood metabolic indices, milk production and fertility parameters in dairy cows. Seventy-five Polish Holstein-Friesian dairy cows were assigned to treatment 21 days before calving. The treatments included: TG—2.5 kg triticale grain/cow per day supplemented from 14 days prepartum to day 56 postpartum, PG—2.5 kg triticale grain/cow per day supplemented from day 14 before parturition to day 56 postpartum, and 400 g propylene glycol/cow per day from 14 days prepartum to 14 days of lactation and MG—2.5 kg maize grain/cow per day supplemented from day 14 before parturition to day 56 postpartum. PG and MG had an effect resulting in the highest glucose concentration at 28 d of lactation. Cows assigned to the PG and MG groups had significantly higher cholesterol levels confronted with TG group at day 14 of lactation, while at days 28 and 56 the same difference was observed only between the MG and TG groups. PG had an effect on shortening the period to first ovulation. PG and MG improved the first service conception rate and decreased the number of services per conception in cows. In conclusion, both treatments of dairy cows with PG and MG improved their fertility parameters, while they had a slight effect on their metabolic profile and no effect on their milking performance. During the past decades intensive genetic selection of dairy cows, which main goal was to increase milk yield, has brought about a level of production in which nutrition allowances are difficult to supply . NutritiPropylene glycol (PG) has been applied in ketosis treatment since the 1950s  and is sThus, we hypothesized that propylene glycol and/or maize grain contain by–pass starch, which digestion in the small intestine to glucose exerts a better glucogenic effect compared to excessive rumen degradation of starch, while it might inhibit lipogenesis as well as improve the metabolic status, milking performance and fertility. Therefore, the aim of the study was to determine the effect of propylene glycol and triticale grain as propionic acid sources in the rumen and maize grain content by-pass starch supplementation during the transition period and the first 56 days of lactation on metabolic profile indices, milk production and fertility parameters in dairy cows.All animal procedures were performed in accordance with the guidelines of the Polish Council of Animal Care. The protocol for this study was approved by the Local Animal Care Committee no. 10 of the Poznan University of Life Sciences.Seventy-five Polish Holstein-Friesian dairy cows were assigned to treatment 21 days before the expected calving date (estimated pregnancy period—280 days). Primiparous cows (seven per each group) were divided according to calving date, while multiparous cows (18 per each group) were divided with respect to calving date, parity (2–4), prior milk production , body weight and body condition score. The treatments included: TG , PG , MG (2.5 kg maize grain/cow per day supplemented from day 14 before parturition to day 56 postpartum). The nutritional values of the feed components were calculated on the basis of the analyzed nutrient contents using the PrevAlim 3.23 software . The diets were balanced according to the French INRA system recommendations , neutraThe body condition score (BCS) was performed according to the methodology presented by Edmonson et al.  during tBlood sampling was performed (four hours after morning feeding) 3 weeks and 1 week before calving and on days 14, 28 and 56 of lactation. Samples were collected into tubes with polystyrene separating granules covered with a clot activator, rotated, frozen and stored (−20 °C) for further analyses. Serum was thawed and analyzed for the concentration of nonestrified fatty acids (NEFA), according to Duncomb’s colorimetric method . The conThe cows were milked twice a day, individual milk yields were recorded daily during 56 days of lactation. Milk samples were collected during milkings twice a day (morning and afternoon) at weekly intervals. Samples were collected into tubes with 2-bromo-2-nitropropane-1,3-diol, next refrigerated and delivered to a commercial laboratory  for analyses of fat, protein and lactose. Energy corrected milk (ECM) was calculated according to Reist et al. Reist et al. (2003) as [(0.038 × g crude fat + 0.024 × g crude protein + 0.017 × g lactose)] × kg milk/3.14 .Fertility parameters, such as days to the first ovulation, first-service conception rate, services per conception and day open, were also recorded. The cows were observed visually around the estrus period and the first ovulation was confirmed using an ultrasound scanner equipped with a 7.5 MHz convex transducer . During the study cows’ health status was monitored.®/STAT software package . One-way analysis of variance was conducted using the GLM Procedure and the Duncan test. Significance was declared at p ≤ 0.05.The obtained results were processed statistically using the SAS 9.1  SAS®/STAp > 0.05, Propylene glycol and maize grain had no effect on BCS and condition changes during the last 21 d of the dry period as well as during the first 2 months of lactation (p > 0.05) (There were no significant differences between the groups in mean dry matter intake from −21 day to 56 day of lactation ( > 0.05) .p ≤ 0.05; Propylene glycol and maize grain had an effect on the hematic glucose concentration at 28 day of lactation that resulted significantly higher compared to TG group. (p ≤ 0.05). Propylene glycol and maize grain had no effect on triglyceride concentrations during the transition period (p > 0.05); nevertheless, at 56 d lower blood triglyceride concentrations were recorded in cows assigned to PG (p ≤ 0.1) and MG (p ≤ 0.05) compared with the TG group. Both treatments, propylene glycol and maize grain, had no effect on NEFA and BUN concentrations as well as ASPAT activity in blood (p > 0.05). No significant differences were found between the groups in terms of mean milk yield and mean energy corrected milk production  had a positive effect on fertility parameters such as shortening the period to first ovulation, higher first services conception rate and lower services per conception. Shrestha et al.  argued tLowered energy uptake in early lactation have an effect on an inferior metabolic status of cows, which is connected with a longer interval to first ovulation . In the The treatments  were found to have no effect on milk yield, energy corrected milk production or milk composition. Probably similar milk performance results may show that homeostasis of dairy cows from all the groups was maintained. Replacement of 2.5 kg of triticale grain containing starch excessively degraded in the rumen with 2.5 kg maize grain with by–pass starch, which is digested in the small intestine, as well as supplementation of 400 g of propylene glycol were insufficient to influence milk performance. Similar results showing no effect on the milk yield were reported after 600 g of propylene glycol administration as an oral dose during the first week after calving  and 200 In conclusion, both treatments of dairy cows with PG and MG improved fertility parameters, while they had a slight effect on the metabolic profile and no effect on milking performance in dairy cows. Fertility parameters are strongly connected with reproductive performance, which is a key factor affecting profitability of milk production. Thus PG and MG could be reconsidered in the close–up and fresh diets.


Lead (Pb) exposure in early life affects brain development resulting in cognitive and behavioral deficits. Epidemiologic and experimental evidence of sex as an effect modifier of developmental Pb exposure is emerging. In the present study, we investigated Pb effects on behavior and mechanisms of neuroplasticity in the hippocampus and potential sex differences. To this aim, dams were exposed, from one month pre-mating to offspring weaning, to Pb via drinking water at 5 mg/kg body weight per day. In the offspring of both sexes, the longitudinal assessment of motor, emotional, and cognitive end points was performed. We also evaluated the expression and synaptic distribution of N-methyl-D-Aspartate receptor (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits at post-natal day (pnd) 23 and 70 in the hippocampus. Neonatal motor patterns and explorative behavior in offspring were affected in both sexes. Pb effects in emotional response and memory retention were observed in adult females only, preceded by increased levels of GluN2A and GluA1 subunits at the post-synapse at pnd 23. These data suggest that Pb exposure during development affects glutamatergic receptors distribution at the post-synaptic spine in females. These effects may contribute to alterations in selected behavioral domains. Early-life exposure to environmental chemicals interferes with developmental programming and induces subclinical alterations that may result in pathophysiology and behavioral deficits later in life, consistently with the Developmental Origins of Health and Disease (DOHaD) hypothesis ,2,3.Lead (Pb) is a systemic toxicant primarily affecting the central nervous system (CNS). Exposure of the general population predominantly occurs via the diet, whereas workplace exposure is of concern in specific occupational settings ,5,6. Pre01) of 12 μg blood Pb/L  as the reference point for intellectual deficits in children measured by the Full Scale IQ score and concluded that there is concern at current levels of Pb exposure for effects on neurodevelopment , sex ratio , and body weight of pups at birth .Data collected at birth did not show overt detrimental effects of gestational Pb exposure on number of delivered pups .As for sensorimotor development, all pups showed a gradual decline across days in latency to righting reflex and negative geotaxis . Latencyp < 0.05, Pb × pnd interaction F  = 2.963 p < 0.05], head rising , and wall climbing [main effect of Pb F  = 7.79 p < 0.01]. Pb pups spent less time in locomotion compared to Veh at pnd 10 in favor of head rising and wall climbing indicating a stereotyped/perseverative profile.The analysis of spontaneous movements indicated other Pb effects on selected motor patterns A, namelyWhile displaying these spontaneous movements, Pb pups emitted a number of calls comparable to Veh, showing a similar temporal profile of emission, with peak of emission of USVs at pnd 7–10 B. NumberOn pnd 13 during the homing test, Pb and Veh pups took similar time to reach the nest arm and spent similar time there compared to Veh pups, indicating comparable olfactory discrimination and preference in the two groups .p = 0.02]: Pb rats performed less entries compared to Veh rats, suggesting decrement of explorative behavior  exhibited during exploration of the novel environment (open-field test), behavior D. Notablp < 0.01, Pb × sex interaction F  = 3.842 p = 0.06] and spent less time there  exhibiting a more anxious profile  = 4.112 p = 0.05], p = 0.01], a posture related to risk assessment behavior when approaching a novel area, with Pb rats exhibiting less SAP episodes than Veh rats  at adulthood, Pb effects were found selectively in female sex. Pb female rats visited the open arms with lower frequency compared to Veh female rats  that failed to discriminate between the novel object and the one previously explored (familiar), As for long-term recognition memory , Pb effect was found only in female rats  and travelled longer distance [F  = 14.029 p < 0.01] than Veh rats , Pb rats significantly decreased their escape latencies to reach the hidden platform across days in a similar fashion to Veh rats A. As forVeh rats C,D. Analale rats E.Organization of the glutamatergic response during development, to shape excitatory synaptic connectivity, depends both on the balanced expression of NMDAR and AMPAR subunits and their proper distribution between the synaptic and extra-synaptic site . To evalNMDAR and AMPAR subunit total expression and amount at the post-synaptic site were not different between sexes, with the only exception of GluA1 subunit of AMPAR that was higher in female TIF at pnd 23 .Exposure to Pb significantly increased GluN2A A and GluThe molecular alterations observed at pnd 23 were completely restored and no other effect occurred after a 47 days withdrawal period from Pb. Thus, Pb did not affect both total and TIF expression of any of the considered subunits at pnd 70 .Finally, we focused our attention on Pb modulation of spine density, which is under the control of NMDAR subunits distribution at the synaptic spine ,39 and rIn this study, we aimed at evaluating the sex dependent vulnerability to 5 mg Pb/kg b.w. per day exposure during gestation and lactation in a rodent model, considering both behavioral outcomes and neurobiological correlates in a longitudinal perspective.We observed: (i) behavioral changes induced by Pb exposure regardless of sex that include alterations in neonatal spontaneous movements and spatial memory retention ; (ii) beThe effects of Pb on early motor patterns have not been investigated in detail in previous rodent studies. They concern both sexes, and consist mainly in increased “stereotyped” explorative responses  at the expenses of locomotion, possibly mirroring the inability to inhibit inappropriate responding and perseveration described in both rodents and humans exposed to Pb .−1 in drinking water [−1 was administered [As adults, Pb exposed rats acquire the spatial task at a rate comparable with that of Veh rats, but they show a mild deficit in spatial memory retention as revealed by the longer latency to reach the target quadrant during the probe trial. Spatial memory is a behavioral domain affected by developmental exposure to Pb: previous data collected in rats reported marked impairment in the MWM performance after early life exposure to Pb at exposure levels ranging from 150 to 2000 mg Lng water ,46,47,48nistered . The useSex differences became evident at adulthood. Two behavioral domains appear specifically sensitive to sex dependent vulnerability to Pb, namely anxiety and recognition memory. The anxiogenic effects of developmental exposure to Pb have been previously reported at doses higher than the one of the present study, without considering differences in the two sexes ,51,52,53Additionally, female rats exposed to Pb show a marked impairment in long-term memory. More specifically, they failed to show the expected preference for the novel object compared to the familiar one in the NOR task  and dispThe present findings are consistent with studies in which Pb exposure through gestation and lactation led to impairment in learning and memory and depressive-like behavior in female but not male rats ,64,65,66The formation and maturation of developing glutamatergic circuitry is the result of modification in synapses as ruled by patterns of neuronal activity and modulation in the expression and trafficking of NMDA and AMPA receptors subtypes, which contribute to the molecular basis of learning and memories ,39,68. A−1), the observation obtained in both sexes with no distinction, the different rat strain (Long–Evans versus Sprague Dawley) and the examination of animals that had been previously handled in behavioral protocols. The last point is particularly relevant considering that exposure to behavioral test may influence gene expression in the hippocampus [Notably, polymorphic variants in GRIN2A leading to altered NR2A expression are associated with neuro-behavioral anomalies in children ,75,76. Opocampus . The molpocampus . Intrigupocampus . Of notepocampus , in accoRecent overviews have included Pb among EDCs, hypothesizing a direct interference with sex hormones throughout early development to explain its sex dependent effects ,82,83,84Overall, our behavioral and molecular data indicate more pronounced effects in females than males developmentally exposed to Pb, which by interfering with biochemical trajectories linked to the maturation of the excitatory circuits early in life may set the stage for behavioral alterations later in life. Of note, in epidemiological studies where sex effects were examined, adverse outcomes from developmental Pb exposures have been described more frequently in males than in females ,36,90,91Evidence derived from experimental models addressing the issue of sex differences in brain and behavior might enhance the attention of epidemiology towards the proper consideration of sex/gender dependent vulnerability in population studies.−1 Pb (corresponding to 5 mg Pb/kg body weight per day), respectively, via drinking water 4 weeks prior to breeding throughout pregnancy and lactation until weaning (pnd 23). Female rats were mated with males  for 4 to 5 days to cover the duration of an estrous cycle.Following the adaption period, female rats  were randomly assigned to one of the two experimental groups  to receive 0 or 50 mg LDay of birth was designated as pnd 0. On pnd 1, litter were culled to equal numbers to standardize litter size, with an aim to have ten pups, sex balanced, per litter. At pnd 1 the sex of all pups in each litter was attributed on the basis of anogenital distance.ad libitum, under a 12h-12h light/dark cycle (lights on from 7:00 a.m. till 7:00 p.m.).At weaning (pnd 23) the sex of each pups was again checked to confirm correspondence with sex attribution at birth. Male and female offspring were separated and housed 4 per cage up to the end of all experiments. All the animals were kept under standard animal housing  with food and water All experimental procedures were carried out in accordance with the European and Italian legislation  and were approved by the Ethical Committee  of the Italian National Health Institute  and by the Italian Ministry of Health .2·3H2O) was purchased from Sigma Aldrich . Test solutions for animal treatment were prepared in soft tap water, slightly acidified (pH 4.6) with acetic acid, and containing no Pb acetate (Veh) or Pb acetate at a concentration 50 mg L−1 of Pb (as element). The medium ensured complete dissolution of the Pb salt and acceptance by the animals was the same as plain tap water. The Pb concentration in the test solutions was analytically checked  and stability was verified for 5 days. The solutions were prepared afresh every 3–4 days and used as drinking water for animal treatment.Lead acetate (Pb(Ac)Neonatal stage: one female and one male offspring from 6 litters for each experimental group  underwent the neurodevelopmental test battery described below, including somatic, motor and sensorial assessment on pnd 4, 7, 10, 12, and 13.For identification purposes, on pnd 4 pups were tattooed on the paw with animal tattoo ink .Juvenile/adult stage: female and male rats (from 6 Veh and 9 Pb litters) not subjected to neurodevelopmental test battery, were assessed in the following behavioral test: Open-Field , Spontaneous alternation , Elevated Plus Maze , Novel Object Recognition  and Morris Water Maze . Number of animals undergoing each behavioral test are reported in Figure legends.All apparatus were cleaned with 70% alcohol following each animal testing. All behavioral procedures were carried out between 9:00 a.m. and 3:00 p.m.On each day of testing , a single pup was placed into an empty glass container , located inside a sound-attenuating Styrofoam box, and USVs were recorded during a 3-min test as described in . Number Concomitant with the USV recording, the spontaneous movements of the pups were also videotaped. In accordance with previous studies focused on neonatal rodent behavior , the folFrequency and duration of each behavioral pattern were analyzed by using The Observer XT software .At the end of the 3-min recording session, each pup was assessed for reflex development and somatic growth (body weight and length) from pnd 4 to 12, as previously described .The righting reflex was assessed by placing the pup on its back over a flat surface: the time needed to return to the natural position  was measured using a stopwatch. The reflex was tested once in each day of assessment with a cut-off latency of 60 s.The negative geotaxis was assessed by placing the pup on a 30-cm incline plane (20°), in a head down position. The time required to reorient to a head up position was recorded using a stopwatch and the cut-off time was 60 s.To assess early discriminative performances and maternal preference behavior, on pnd 13, pups were separated from the dam and kept for 30 min in an incubator  at 28 ± 1 °C. The apparatus consisted in a grey Plexiglas T-shaped maze . Each pup was gently placed in the start arm and allowed to freely explore the maze for 3 min. The time taken by the pup to reach the goal arm (containing nest litter), number of entries and time spent in the three arms were recorded and analyzed by The Observer XT software .To assess the locomotor activity during the exploration of novel environment, rats were tested in the OF test. The OF apparatus consisted of a black Plexiglas box (80 × 80 × 60 cm). Each subject was scored individually after being placed in one corner of the apparatus and spontaneous locomotor activity of the animals were video-recorded for 10 min (as described in ). DistanTo assess the spatial working memory and explorative activity, rats were tested in the Y-maze test. The apparatus consisted of three identical arms (50 × 16 × 32 cm) diverging at 120° angle one to the other and an equilateral triangular central area. Each animal was placed in the center of the Y-maze and was free to explore the arena for 5 min. The following dependent variables were registered and analyzed by The Observer XT software : total number of arm entries and a sequential list of arms entered to assess number of alternations. An arm entry was scored when rat placed the four paws within that arm. An alternation was defined as an entry into three different arms on consecutive choices. Spontaneous alternation was calculated using the following formula:  × 100.To assess the anxiety-like behavior, rats were tested in the EPM based on the natural conflict between the exploration of new areas and avoidance of unsafe areas. The EPM comprised two open arms and two closed arms that extended from a common central platform. The apparatus was constructed from Plexiglas  and elevated to a height of 60 cm above the floor level. Rats were individually placed on the central platform facing a closed arm and allowed to freely explore the maze for 5 min (as described in ). ConvenIn order to evaluate the recognition memory of one previously explored object (familiar) compared with one novel object, rats were tested for the NOR test. NOR was performed in OF apparatus . Rats were tested during three sessions  separated by an interval of 24 h. In the familiarization session (duration 10 min), each rat was faced with two identical objects placed in two adjacent corners  and the time exploring freely each object was recorded. In the retention trial (duration 10 min), one of the two familiar objects was replaced by a novel object and the time exploring each was recorded. Exploration time (computed when the snout pointed to the object at a distance ≤1cm) was scored by The Observer XT software . Discrimination index score was calculated using the following formula: (tn − tf)/(tn + tf), where tn—the amount of time rats explored the novel object and tf—the amount of time rats explored the familiar object.To assess the spatial learning and memory rats were tested in the MWM test. MWM was performed in a circular black pool (diameter 210 cm) filled with 24 °C water rendered opaque by the addition of atoxic acrylic paint . An escape platform (diameter 10 cm) was submerged 2 cm below the water level. Animals used distal visual-spatial cues  to find the hidden escape platform located in the center of the target quadrant.Each rat underwent to two phases of MWM: a spatial learning phase (training) of four days duration followed by a single Probe trial 24 h after the last training trial.During the spatial learning phase  rats were released into the pool from one of the four starting points (one per each quadrant) and trained to find the hidden escape platform. The order of the sequence was changed pseudo-randomly between days. A trial was finished when the animal found the escape platform or when 60 s had elapsed. If a rat failed to find the platform, it was gently guided to the platform by the experimenter. Rats remained on the platform for 30 s. After each trial rats were dried and when the session finished they were returned to their colony room.Twenty four hour after training, spatial memory was assessed with a probe trial in which the escape platform was removed and rats were released from the quadrant opposite to target quadrant (which contained the escape platform during the training phase) and allowed to swim for 60 s in searching for it.Latency to reach the escape platform, time spent in each quadrant, latency and distance moved to first entry to target quadrant and path efficiency were recorded and analyzed by ANY-Maze software .Blood and brain dissection (cortex and hippocampus) were obtained from rats sacrificed at pnd 23; blood samples were collected in EDTA (0.2 mg/100 mL) from the left atrium of the heart of rats anesthetized by isoflurane. Samples were stored at −80 °C.3, Carlo Erba Reagenti, Rodano, Italy) and H2O2  were of ultrapure grade. The Pb calibration standard and internal standard (Rh) were prepared from certified standards solutions of 1000 ± 3 µg mL−1 . Blood was diluted 1:50 with 0.2% v/v HNO3 and 0.005% v/v Triton X-100 (J.T. Baker). Procedural blanks submitted to the same sample handing of blood specimens were prepared and their concentration subtracted from that of study samples. Tissues were digested by microwave irradiation with HNO3 and H2O2 by means of an Ultrawave closed-vessel system equipped with temperature and pressure control . An 8800 Triple Quad mass spectrometer from Agilent Technologies  was used for ICP-MS determinations. The standard addition method was used for quantification. Accuracy was checked by the control material Seronorm Whole Blood L1 , for blood, and the certified reference material BCR 8414 Bovine Muscle , for tissues.Sample treatment was performed in a clean room laboratory. A Milli-Q Element System  was used to obtain high purity water for sample preparation. Nitric acid  in presence of a complete set of proteases  and phosphatases inhibitors . An aliquot of the homogenate was collected and immediately frozen in dry ice: this fraction represents the homogenate sample (HOMO). The HOMO was then centrifuged at 13,000x g for 15 min. The resulting supernatant was removed and the pellet was resuspended in ice-cold buffer  and centrifuged at 100,000× g for 1 h. The supernatant was discarded and the pellet obtained was homogenized in a glass-glass potter in 20 mM ice-cold Hepes and then stored at −80 °C until processing.The homogenate and the post-synaptic Triton-insoluble fraction (TIF) were prepared from hippocampal tissue as previously described in . BrieflyProtein content of HOMO and TIF’s samples was quantified using Bio-Rad dye reagent protein assay . After measuring protein concentration, all samples were standardized at 1 μg/uL concentration and 15 μg of protein per sample were loaded in each lane of the gel. After gel electrophoresis, proteins were transferred to a nitrocellulose membrane  and blocked for 2 h at room temperature with I-block solution  before antibody incubation. Western blot analysis were performed using antibodies raised against NMDA glutamate receptor subunits GluN2A, GluN2B and GluN1, and AMPA glutamate receptor subunits GluA1, GluA2 diluted 1:1000, and actin diluted 1:2500. Secondary antibodies were diluted 1:10,000 and all dilutions were made in I-block solution. Quantification of Western blotting analysis was performed by means of computer-assisted imaging (Image Lab Software from Bio-Rad) after normalization on actin levels. All the reagents were purchased from Sigma-Aldrich . Monoclonal GluN2B and GluA2 antibodies were purchased from NeuroMab ; polyclonal GluA1 from Calbiochem ; monoclonal GluN1 antibody from Invitrogen ; monoclonal GluN2A, actin and secondary anti-mouse antibodies from Sigma Aldrich ; and secondary anti-rabbit antibody from Bio-Rad .Reproductive performances of dams exposed to Pb were presented as means +/− SD (standard deviations).Biodistribution data were reported as medians and interquartile ranges (IQR: 75th–25th centile). Comparisons between Veh and Pb animals were carried out by the Mann–Whitney Wilcoxon test (two-tailed).U-test was used.Behavioral data were analyzed by analysis of variance (ANOVA) with group and sex as between-factors and day/trial/quadrant as repeated measures followed by post-hoc Tukey’s analysis on significant interaction effects. When parametric assumptions were not fully met, the non-parametric Mann–Whitney For the protein expression data, statistical analysis between Veh and Pb groups was performed, within each sex, by using unpaired Student’s t-test.p < 0.05) was considered statistically significant.Values deviating from the mean by more than 2.5 times the standard deviation (SD) were discarded as considered outliers (cut off = 2.5 SD). A 95% level  affects selected behavioral domains and glutamatergic receptor expression and distribution in a sex-dependent fashion. Here we investigated one of several molecular mechanisms potentially underlying the behavioral abnormalities observed, providing the basis for further insights such as glutamatergic receptors functional studies. Overall, our behavioral and biochemical data indicate more pronounced effects in females than males exposed to Pb during development. We suggest that Pb may set the stage for altered behavior later in life by interfering with biochemical trajectories linked to early maturation of the excitatory circuits.


Biocomposites based on poly(lactic acid) (PLA) and biochar (BC) particles derived from spent ground coffee were prepared using two different processing routes, namely melt mixing and solvent casting. The formulated biocomposites were characterized through rheological, thermal, and mechanical analyses, aiming at evaluating the effects of the filler content and of the processing method on their final properties. The rheological characterization demonstrated the effectiveness of both exploited strategies in achieving a good level of filler dispersion within the matrix, notwithstanding the occurrence of a remarkable decrease of the PLA molar mass during the processing at high temperature. Nevertheless, significant alterations of the PLA rheological behavior were observed in the composites obtained by melt mixing. Differential scanning calorimetry (DSC) measurements indicated a remarkable influence of the processing method on the thermal behavior of biocomposites. More specifically, melt mixing caused the appearance of two melting peaks, though the structure of the materials remained almost amorphous; conversely, a significant increase of the crystalline phase content was observed for solvent cast biocomposites containing low amounts of filler that acted as nucleating agents. Finally, thermogravimetric analyses suggested a catalytic effect of BC particles on the degradation of PLA; its biocomposites showed decreased thermal stability as compared with the neat PLA matrix. The increasing demand for environmentally sustainable products has stimulated a growing research effort in the last years towards the production of new bio-sourced materials, characterized by a lower environmental impact with respect to the traditional fossil-fuel counterparts ,2,3. In Recently, biochar (BC) has attracted increasing interest as far as its utilization as filler for polymer-based composites, either thermoplastics or thermosets, is concerned ,20. BC iGenerally speaking, biochar is currently exploited as a cheap, functional material, already employed for several applications  and undoubtedly it is a way for recovering wastes at the end of life, offering them a new added value .co-terephtalate) was melt compounded with BC derived from agriculture commodities to produce composites potentially suitable for food packaging applications [Few reports dealing with the formulation of BC-containing composites based on biopolymers are present in the literature ,30. For ications . BC addiIn this work, PLA-based biocomposites containing BC particles derived from spent ground coffee were formulated through two different processing methods, namely melt mixing and solvent casting. BC was introduced within the polymer matrix at different contents, ranging from 1 wt.% to 7.5 wt.%, aiming at evaluating the effect of the processing route and of the BC loading on the ultimate properties of the resulting biocomposites. The dispersion of the embedded BC particles and the extent of the established polymer–filler interactions were assessed through morphological and rheological analyses; besides, the thermal and mechanical behavior of the biocomposites was investigated, assessing the possible influence of the processing on the PLA intrinsic characteristics. Finally, the thermal stability of biocomposites was evaluated and compared to that of neat PLA.Two different PLA matrices were used, PLA 3251D and PLA 4042D, both supplied by IngeoTM Natural Natureworks . PLA 3251D is an injection molding grade and has a melt flow index of 13 g/10 min ; conversely, PLA 4042D is suitable for film applications and shows a melt flow index of 6 g/10 min .A waste brewed coffee powder  was used as starting material for the production of biochar (BC). In order to remove the leftover chemicals in waste coffee powder after brewing, the sampled waste brewed coffee powder was washed many times with demineralized water. The sample was further centrifuged and filtered and the obtained waste brewed coffee powder was then dried in an oven at 90 °C for 10 h. The pyrolysis of the material was subsequently performed at 700 °C for 1 h in a nitrogen atmosphere (gas flow: 120 mL/min). The heating ramp rate of the tubular furnace was set at 5 °C/min; after the pyrolysis step, the material was manually ground in a mortar, achieving an average size of 10 μm. Further characteristics of the obtained BC particles are reported in .PLA and PLA-based composites containing 1, 2.5, 5, and 7.5 wt.% of BC were processed through two different methods, namely melt mixing and solvent casting. PLA 3251D was used to formulate solvent cast composites, owing to its lower average molar mass and enhanced solubility in the selected solvent as compared with PLA 4042D.In melt mixing (MM), the BC powder was compounded with the PLA4042D matrix using a Brabender Plastograph mixer and operating at 170 °C, 100 rpm for 5 min. Neat PLA was subjected to the same processing. Specimens for the various characterization were obtained by means of a compression molding step, using a laboratory press , working at 170 °C, under a pressure of 100 bar for 2 min. Prior to the processing, PLA and BC were vacuum dried overnight at 70 °C. The melt mixed materials were designed as PLA+XBC_MM, where X indicates the BC loading (wt.%).In the solvent casting (SC) process, PLA 3251D pellets were dissolved in chloroform with vigorous stirring at room temperature. BC was added after the complete dissolution of PLA and the obtained solutions were stirred for 5 h. The solutions were then cast onto a glass Petri dish and allowed to dry for about 24 h at room temperature. The resulting solvent cast films were placed in a vacuum oven at 40 °C for three days in order to remove all remaining solvent. The solvent cast systems were designed as PLA+XBC_SC (X accounts for wt.% BC loading) −1 to 102 rad/s at 170 °C, under a nitrogen atmosphere. The strain amplitude was selected for each sample in order to fall in the linear viscoelastic region. Time sweep tests were carried out at in air at 170 °C, ω = 1 rad/s, γ = 10%, and for 3600 s.Rheological measurements were performed using an ARES  strain-controlled rheometer in parallel plate geometry (plate diameter: 25 mm). Strain sweep tests were carried out at 170 °C and ω = 1 rad/s. The complex viscosity and storage and loss moduli were measured performing frequency scans from 10Microstructures were observed using a LEO-1450VP scanning electron microscope  (beam voltage: 20 kV) on the cross-sections of the investigated samples fractured in liquid nitrogen.2 gas (20 mL/min) using samples of around 8 mg in sealed aluminum pans. All the materials were subjected to the following cycle:-First heating up from 30 to 200 °C at 10 °C/min;-Cooling down from 200 to 0 °C at 10 °C/min;-Second heating up from 0 to 180 °C at 10 °C/min.Differential scanning calorimetry (DSC) analyses were carried out using a QA1000 TA Instrument apparatus . All the experiments were performed under dry Ng (glass transition temperature), Tcc , Tm (melting temperature), and Xc  in controlled conditions. Xc was calculated as the ratio between the heat of fusion of the sample , considering only the polymer fraction, and the heat of fusion of a 100% crystalline PLA .2 flow (35 mL/min). T5%, T10% , and Tmax values  were calculated; besides, the final residue at 600 °C was measured.Thermogravimetric analyses (TGA) were performed using a Pyris1TGA apparatus  . Samples (about 10 mg) were placed in alumina pans and runs were carried out in the range of 50–600 °C, with a heating rate of 10 °C/min, under N® 5966 dynamometer . The measurements were carried out at two different crosshead speeds: 1 mm/min until 0.2% of deformation is reached, and then 100 mm/min up to sample breakage. At least five specimens for each system were tested and the were results averaged.Tensile tests were performed using an InstronThe linear viscoelastic behavior of neat PLA and all formulated composites was evaluated through oscillatory measurements; in Looking at the results of the solvent cast systems, a different scenario can be observed. In particular, a progressive increase of the complex viscosity values with respect to those of the neat PLA matrix is noticed, as the filler loading increases. However, different to what was observed in the case of melt mixed composites, the presence of BC particles does not induce the appearance of a stress yield behavior, and BC-containing systems exhibit a frequency-dependence of the viscosity curves very similar to that of neat PLA. In other words, the presence of BC filler causes a mere vertical shift of the complex viscosity curve towards higher values, without actually modifying the relaxation spectrum of PLA macromolecules. Usually, this behavior is observed in polymer-based composites characterized by a low level of polymer–filler interactions, in which the embedded particles are not able to significantly modify the macromolecules dynamics . As a reFurther important information about the interactions existing in polymer-based composites can be derived from the analysis of the rheological response under large deformations; To gain a better understanding of the thermally-activated reactions leading to a decrease of the PLA molar mass and to verify the possible effect of BC on the degradation of the matrix in the melt state, the temporal evolution of complex viscosity was evaluated through time sweep measurements at 170 °C in air, for both series of composites. In Some typical SEM micrographs of the fractured surface of composites containing 7.5 wt.% of BC obtained through melt mixing and solvent casting are shown in g and Tcc values as compared with the neat matrix, indicating an enhanced mobility of PLA macromolecules owing to the molar mass decrease resulting from degradation phenomena occurring during processing. Additionally, beyond 1 wt.% of BC loading, a double melting peak appears. However, the total content of the crystalline phase in melt mixed samples remains almost unchanged as compared with neat PLA, and the materials exhibit an amorphous structure.As far as melt mixed systems are concerned, the presence of BC causes a slight decrease of Tg, Tcc, and Tm values with respect to the neat matrix are observed, suggesting an enhanced crystallization capability. In addition, a remarkable increase of the crystallinity degree can be noticed for composites containing up to 2.5 wt.% of BC; a further increase of the filler content does not favor the crystallization process, and for PLA+7.5BC_SC, only a slight increase of Xc as compared with the neat PLA was achieved. The observed behavior can be explained considering two concurrent opposite phenomena exerted by the embedded BC particles: a nucleating effect and an interference on the crystallization process of PLA macromolecules. At low BC loadings, the first effect is prominent and the composites show improved crystallization ability; conversely, for higher amounts of BC, the presence of disperse fillers hampers the crystallization process of polymer macromolecules, and a lower content of crystalline phase is formed.Looking at the results shown by solvent cast systems, lower T5%, T10%, TMax, and the final residues at 600 °C in both air and nitrogen atmospheres. In nitrogen, the degradation of the composites occurs in a single degradation step; the weight loss observed for solvent cast systems in the temperature range of 90–180 °C can be ascribed to the removal of the residual solvent in the films [5% and T10% values can be noticed as a function of the filler content, as well as a gradual shifting towards lower values of the maximum degradation temperature. A detrimental effect of BC particles derived from different sources on the thermal and thermo-oxidative stability of PLA and other bio-polyesters matrices has already been reported in the literature and was attributed to the catalytic effect of potassium, usually contained in BC, on the decomposition of the polymer matrix [he films . The prer matrix ,41. The r matrix  may taker matrix .The mechanical properties of the PLA-based systems were evaluated through tensile tests; the obtained mechanical properties, namely tensile modulus and tensile strength, are plotted in Two different processing methods, that is, melt mixing and solvent casting, were exploited for preparing PLA-based biocomposites containing BC particles obtained from pyrolysis of spent coffee grounds. The rheological results suggested that melt mixing method is more effective in dispersing BC particles owing to the high shear force that systems experienced during processing, though the high temperature, at which the processing is performed, caused the occurrence of degradation phenomena in PLA, leading to a severe reduction of the polymer molar mass. Conversely, the solvent casting method, carried out at room temperature, preserved PLA from degradation, but the weak dispersing stresses involved in this processing did not promote the establishment of strong polymer–filler interactions, owing to the bigger size of BC and its sponge-like structure. Furthermore, the selected processing route significantly affected the thermal behavior of formulated materials; in fact, melt mixed systems showed an amorphous structure, while solvent casting composites exhibited crystalline phases, owing to the nucleating effect of the embedded BC particles. Finally, irrespective of the processing strategy, an improvement of the tensile modulus was documented for all biocomposites, as compared with neat PLA.From an overall point of view, the obtained results clearly showed that the ultimate properties of the PLA/BC biocomposites can be profitably modulated by selecting the content of BC and the suitable processing method, which are able to govern the morphology of the materials, as well as their rheological and mechanical behavior.3) in the preparation of polymer biocomposites with tailored properties, useful for packaging, automotive, and electronic applications. Besides, the low production cost of this carbon filler, together with the possibility of deriving it from different waste sources, clearly represents an added value toward the circular economy approach. The combination of a waste-derived filler together with a biopolymer is thus a very effective tool for the improvement of materials sustainability.All these findings may suggest the suitability of BC for replacing some common fillers (such as carbon black, talc, and CaCO


Piezo1 gain-of-function variant common in individuals of African descent with glaucoma-related phenotypes. We analyzed whole genome sequences to identify Piezo1 variants and their frequencies among 1565 human participants. For the most common variant (e756del), we compared phenotypes between heterozygotes, homozygotes, and wildtypes. Longitudinal mixed effects models of visual field mean deviation (MD) and retinal nerve fiber layer (RNFL) thickness were used to evaluate progression. Based on trends in the models, further investigation was conducted using Piezo1 gain-of-function mice. About 30% of African descent individuals had at least one e756del allele. There were trends suggesting e756del was associated with higher IOPs, thinner RNFLs, lower optic nerve head capillary densities, and greater decreases in MD and RNFL thickness over time, but these did not reach statistical significance. Among mice, increased Piezo1 activity was not significantly associated with IOP or retinal ganglion cell density. Our study confirms that the Piezo1 e756del gain-of-function variant is a frequent polymorphism present in African descent individuals but is unrelated to examined differences in glaucoma phenotypes. Ongoing work is needed to elucidate the role of Piezo1-mediated mechanotransduction in glaucoma.Glaucoma disproportionately affects individuals of African descent. Prior studies of the PIEZO1 mechanoreceptor have suggested a possible role in glaucoma pathophysiology. Here, we investigated associations between a  Current management is focused on reducing intraocular pressure (IOP), the only proven modifiable risk factor3.Glaucoma is the leading cause of irreversible blindness worldwidePiezo1 and Piezo2) were identified in 20105. PIEZOs have been shown to have critical roles in many functions dependent on force sensation, such as touch8, proprioception9, baroreception10, red blood cell function12, and vascular development13. Several studies have suggested that Piezo1 may potentially have a role in glaucoma pathophysiology. Choi et al.14 found that Piezo1 is expressed in the mouse optic nerve head and may contribute to astrocytes’ responsiveness to traumatic or glaucomatous injury. Koser et al.15 demonstrated that RGC axon growth in Xenopus was aberrant when Piezo1 was knocked down. Similarly, Piezo1 was found to inhibit axon regeneration in Drosophila16. In human donor eyes, Piezo1 was identified in the trabecular meshwork17.Increasing knowledge of mechanoreceptors over the last decade may help shed light on pressure-sensitive biological processes. PIEZO1 and PIEZO2 are mechanosensitive ion channel proteins, whose encoding genes  and the Diagnostic Innovations in Glaucoma Study (DIGS). The characteristics and examination protocols of ADAGES and DIGS have been described previously24. In brief, these cohorts included glaucoma patients and control participants without glaucoma recruited from five study centers in the United States. Participants provided written informed consent at enrollment after explanation of the nature and possible consequences of the study. The study was approved by the UCSD Institutional Review Board and adhered to the tenets of the Declaration of Helsinki. Second, we conducted experimental studies in mice to evaluate for associations between Piezo1 genotypic variants with intraocular pressure and RGC density. All animal procedures were approved by the Institutional Animal Care and Use Committees of The Scripps Research Institute (TSRI) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.First, we investigated potential phenotypic associations with Piezo1 genotypic variants (a1998v and e756del) and determine the frequencies of these alleles in this study population. These were chosen given their identification as gain-of-function variants concentrated within populations of African descent11. Whole genome sequencing and variant calling was conducted by Human Longevity, Inc, leveraging the ‘Issac’ pipeline  using the hg38 reference genome25. Variants were extracted for the Piezo1 gene region  using bcftools, biomaRt, and tabix, and then filtered to only include high confidence calls. Any single nucleotide polymorphisms (SNPs) that cause the a1998v mutation or the e756 deletion were tabulated for wildtype, heterozygous, and homozygous, then mapped to the respective patient identifiers.We analyzed whole genome sequences from the human participants to identify Admixture genotype mapping was conducted to ensure accuracy over self-reported ancestry. We used a panel of 100,000 ancestry informative markers to determine the percentage of admixture for each individual. Each individual was then recharacterized based on the highest percentage of admixture ancestry. Subsequent analyses were limited to African descent individuals based on admixture mapping to remove race as a potential confounder of phenotypes.Because the a1998v variant was present at low frequency in the study population , we focused subsequent analyses on the e756del variant in African descent individuals. We examined whether there were any significant differences in systemic phenotypes such as age, gender, blood pressure, and body mass index (BMI). We then evaluated whether the e756del variant was associated with a range of ocular phenotypes relevant to glaucoma based on data from the most recent documented visit. These included intraocular pressure (IOP), axial length, spherical equivalent of refractive error, and central corneal thickness (CCT). Data from Spectralis optical coherence tomography (OCT) imaging  included retinal nerve fiber layer (RNFL) thickness from the optic nerve head circle scan and ganglion cell complex (GCC) thickness. Additional phenotypes included mean deviation (MD) on Humphrey Visual Field 24-2 perimetry testing  and capillary density as measured by optical coherence tomography angiography (OCTA) in both the macula and optic nerve . Genotype–phenotype associations were evaluated for all African descent individuals in aggregate and then additional analyses were performed stratified by glaucoma status .To examine whether e756del may influence the rate of glaucoma progression, longitudinal analyses were performed examining changes in MD and RNFL thickness over time. To evaluate changes in visual field MD, we required at least five years of follow-up and ten visits. To evaluate changes in mean global RNFL thickness as measured on OCT, we required at least one year of follow-up and three visits. Predictors in multivariable mixed effects models of these progression markers included baseline age, follow-up in years, and presence of the e756del variant, and zygosity . Longitudinal analyses were performed for all subjects in aggregate and also for glaucoma eyes only.Piezo1 gain-of-function mice (Piezo1GOF) were generated previously11. Mice were maintained on a C57BL/6 background. The experimental cohort was generated by mating two heterozygotes (P1GOF/+ X P1GOF/+), resulting in 11 wildtype P1+/+, 25 heterozygotes P1GOF/+, and 11 homozygotes P1GOF/GOF ranging in age from 5 to 17 months. The mice were housed in a 12 hr light/dark cycle (lights on 6am to 6 pm) in a temperature-controlled room (24 degrees Celsius) with free access to food and water.Constitutive Mouse IOP measurements were obtained using an Icare Tonolab rebound tonometer for rodents . All measurements were obtained between 10 a.m. and 4 p.m. Individual mice were briefly anesthetized immediately prior to and during IOP measurement. Anesthesia was performed with isoflurane . IOP was measured according to the manufacturer’s protocol. Three consecutive measurements were measured and averaged for the reported value. A single examiner obtained all mouse IOP measurements and was masked to genotypes.2 equidistant from the optic disk. An average of the three measurements was used for statistical comparisons. Genotypes were masked throughout this process.Mouse RGC density was measured by manual counting. Mice were deeply anesthetized with isoflurane prior to cervical dislocation. Eyes were immediately extracted and fixed in 4% paraformaldehyde (PFA) for 30 min. Retinas were then extracted from whole eyes and fixed for an additional 60 min in 4% PFA. RGCs were stained with anti-RBPMS antibody (Millipore: ABN1376) followed by a fluorescent secondary antibody for visualization. Retinas were whole-mounted and imaged at 40 × with a NikonC2 confocal microscope . RGCs were counted from a max projection through the entire ganglion cell layer (GCL). RGCs were counted in three sample areas of 150 µmt-tests for continuous variables and Fisher’s exact test for categorical variables. Genotypic associations with ocular phenotypes in humans were evaluated using linear mixed effects models with random intercepts to account for within-subject correlation. In addition to evaluating for any significant differences across all three groups , we also evaluated differences between groups in a pair-wise fashion. Multivariable mixed effects models were generated for changes in MD over time and changes in global mean RNFL thickness over time. Interaction terms between baseline age and years of follow-up and between the presence of e756del and years of follow-up were incorporated. All statistical analyses for human clinical data were conducted in R version 3.5.126. For mouse phenotypes, a standard one-way analysis of variance (ANOVA) with a Tukey post-test for multiple comparisons was used to compare IOP and RGC density measured between wildtype, heterozygous and homozygous GOF mice. Statistical analyses for mouse data were conducted using Prism software . For all analyses in humans and in mice, statistical significance was defined as p < 0.05.To evaluate associations between genotype and systemic phenotypes in humans, significance was determined based on two-sample pwr package in R27, which allows for multiple regression power calculation. Using the sample size of 683 individuals of African descent, a significance level of alpha = 0.05, f2 (effect size) of 0.02 , we had 88.2% power to detect a statistically significant difference in phenotypes across 3 genotype groups . For evaluating visual field progression longitudinally, we only had 317 eyes from 170 patients with sufficient visual field data for those odels. With this sample size, we had 99.9% power to detect a modest effect size (f2 = 0.15) and 54.3% power to detect a small effect size (f2 = 0.02).Our analyses of human data were constrained by the number of individuals who had undergone whole genome sequencing and had sufficient ophthalmic data available. Thus, we evaluated our power given a set sample size using the power.anova.test29 was used to determine the power to detect a difference in mean RGC density of 15 cells per 150uM2 of mouse retina between groups of varying genotypes. This equated to a between-group variance of 225. With three groups , a significance level designated at alpha = 0.05, and 11 mice per group, the study had a power of 91.4% to detect a statistically significant difference in RGC density.For power calculations in the mouse experiments, the R function Piezo1 genotypic variants e756del and a1998v. Of these, based on racial admixture mapping of genome sequencing data, there were 683 (43.6%) individuals of African descent, 653 (41.7%) of European descent, 36 (2.3%) of Latino descent, 3 (0.2%) of East Asian descent, 2 (0.1%) of Central or South Asian descent, and 188 (12.0%) of other racial admixtures. Table In total, there were 1565 participants who underwent whole genome sequencing and identification of the Piezo1 e756del allele. In contrast, there was only one individual heterozygous for e756del among 653 European descent individuals . The a1998v variant was infrequent among African descent individuals , and there were none among European descent individuals. All individuals with a1998v also had the e756del variant.Almost one-third of African descent individuals  had at least one p < 0.01, Supplementary Table Table There were several trends suggesting possible associations between the e756del mutation and ocular phenotypes in individuals of African descent, although these did not reach statistical significance Table . IndividTo analyze glaucoma progression based on visual fields, we required at least five years of follow-up and ten visits, resulting in 170 subjects  and 317 eyes . Being heterozygous or homozygous for e756del carried negative coefficients in the model, suggesting that there was potentially a trend of greater decreases in mean deviation over time in eyes of individuals with the mutation, although this association did not reach statistical significance Table . To analPiezo1 allele on IOP regulation and glaucoma we generated a cohort of constitutive Piezo1GOF mice11 and assessed IOP and RGC density. We assayed 47 mice in total: 11 wildtype (Piezo1+/+), 25 heterozygotes (Piezo1GOF/+), and 11 homozygotes (Piezo1GOF/GOF). Measured IOP ranged from 10 to 22 mmHg . Given the age dependence of glaucoma-related phenotypes, we also assessed the relationship between age and IOP in our cohort  Piezo1 function on RGC survival, we measured RGC density in our Piezo1GOF cohort . Additionally, no obvious differences in the impact of age on RGC density were observed . Our goal was to explore whether interesting trends arose that would warrant further investigation with mouse experimental studies, where there would be less environmental and genetic variability. And indeed, our analyses of human data revealed that e756del homozygotes tended to have higher IOPs in healthy subjects and those with glaucoma  and thinner RNFLs (despite younger age). Mixed effects models of changes in RNFL thickness over time in both the cohort as a whole and among glaucoma subjects only suggested trends of e756del homozygosity being associated with more rapid decreases in RNFL thickness over time compared with wild-type , so these analyses were also relatively underpowered.There were some limitations associated with the clinical data from humans. First, there is a known effect of aging on changes in visual field and RNFL over time, among both glaucomatous and healthy eyesPiezo1 activity on IOP or ganglion cell numbers. However, there were several limitations to these experiments that are worth noting. Due to the small size of the eyes, in part, IOP measurements in mice are less accurate and reproducible than in humans. Additionally, our mice were anesthetized during IOP measurement, which is known to impact IOP40. Further, our measurement of ganglion cell numbers was done by sampling areas around the retina without regard for nasal/temporal/dorsal/lateral location of the sample. This method would miss any regional impact of the Piezo1GOF allele in the ganglion cell layer. With a more complete analysis of retinal ganglion cell density (throughout the entire retina), there may be an impact of the Piezo1GOF allele. Third, while our mouse cohort was large (47 mice aged 5 to 17 months) and sufficiently powered to detect statistically significant differences across the entire cohort, there were not sufficient numbers within different age groups to make meaningful comparisons of potential effects by age. This is especially true for the older mice, where we lacked any wildtype mice for comparison. Since glaucoma is known to be acutely dependent upon age in humans, it will be important to more thoroughly investigate aged mice with sufficient numbers. Lastly, mouse eyes and retinas differ considerably from humans. While the mouse serves as a useful tool for glaucoma research, it is possible that systems critical in humans do not exist in mice. With all of that said, our work suggests that increased Piezo1 activity in mice does not lead to glaucoma-like phenotypes.Our experiments in mice showed no significant impact of increased Piezo1 e756del variant is a frequent polymorphism present in individuals of African descent, but that this specific variant may be unlikely to have a significant role in influencing glaucoma phenotypes, based on our analyses of relatively large cohorts of both humans and mice that were sufficiently powered to detect these phenotypic differences. However, given that PIEZO1 channels have been found in the trabecular meshwork of human eyes17, and that glaucoma is clearly a pressure-sensitive disease, further investigations of Piezo1 variants in glaucoma are still warranted. This is especially true because the Piezo1 locus is not easily studied in genome-wide association studies (GWAS), which mainly use single nucleotide polymorphisms to determine associations and encounter challenges when evaluating more complex loci. For example, the locus containing e756del has multiple short tandem repeats, and therefore imputation of this mutation into current GWAS datasets is not straightforward11. Analyzing Piezo1 variants in tandem with other known loci identified from prior GWAS analyses in glaucoma may yield further insights. Another potential future direction would be to further evaluate the effects of Piezo1 on capillary density measured with OCTA, an imaging modality that can provide additional information about early detection of glaucomatous changes43. At the time of this analysis, few individuals in our clinical cohort had undergone OCTA testing, so we were not able to generate sufficient power for comparison. However, this may be a promising route of investigation in the future with additional testing. In summary, much work remains to continue developing our understanding of PIEZO1 and mechanotransduction in the pathophysiology and management of glaucoma and other conditions.Taken together, our work confirms that the Supplementary Information


Effect) and 8 without (No effect). Samples were collected at baseline, after 6 and 12 months. Samples from the transplants (Donor) served as a comparator. In total 66 recipient samples and 17 donor samples were subjected to deep metagenomic sequencing, and taxonomic and functional analyses were performed. Alpha diversity measures showed a significantly increased diversity and evenness in the IBS groups compared to the donors. Taxonomic profiles showed higher relative abundance of phylum Firmicutes, and lower relative abundance of phylum Bacteroidetes, compared to donors at baseline. This profile was shifted toward the donor profile following FMT. Imputed growth rates showed that the resulting growth pattern was a conglomerate of donor and recipient activity. Thirty-four functional subclasses showed distinct differences between baseline samples and donors, most of which were shifted toward a donor-like profile after FMT. All of these changes were less pronounced in the No effect group. We conclude that FMT induces long-term changes in gut microbiota, and these changes mirror the clinical effect of the treatment. The study was registered in ClinicalTrials.gov (NCT02154867).Irritable bowel syndrome (IBS) is a common disorder of the lower gastrointestinal tract. The pathophysiology is far from settled, but a gut microbial dysbiosis is hypothesized to be a contributing factor. We earlier published a randomized double-blind placebo-controlled clinical trial on fecal microbiota transplantation (FMT) for IBS – the REFIT trial. The present data set describes the engraftment and includes participants from the study who received active FMT; 14 participants with effect of FMT ( IBS presents as one of the three phenotypes: IBS with diarrhea (IBS-D), constipation (IBS-C), or mixed (IBS-M). IBS is a very common complaint with an estimated global prevalence of 11.2%, but higher in Northern Europe, and more frequent among young women than in men.2 The current hypothesis on IBS pathophysiology focuses on the interaction between gut microbiota and host factors such as immune system activation, altered neuroendocrine signaling, and gut mucosal barrier function.3 This hypothesis is supported by the notion that IBS-like traits can be observed in rats following fecal transplant from IBS patients.4A key challenge in IBS research is that the pathophysiology is largely unknown, and remains controversial. Interaction between host and gut microbiota is considered a new clinical frontier. Experimental evidence and theoretical considerations imply that disturbances in this interaction may have multiple effects as diverse as obesity, metabolic syndrome, liver cirrhosis, inflammatory bowel disease, and colorectal cancer.3 antagonists, antispasmodics, and antibiotics) are available treatments of IBS. However, the drugs with best documentation show low to moderate effect, and the dietary interventions have not been evaluated extensively.5 In Europe, the agreed intervention of choice is in general dietary interventions, like low FODMAP.6 Documentation of its efficacy is, however, relatively poor, 7 and the theoretical framework is not completely understood. Several mechanisms of action have been suggested including both microbial shifts in profile and metabolic output, and changes in the intestinal regulatory systems like serotonin-producing cells (reviewed by Staudacher and Whelan8).Both diet regimens ; low fructose; gluten-free), food supplements , probiotics, and pharmacologic treatments .11 Consequently, altering the gut microbiome by fecal microbiota transplantation (FMT) has been suggested as a possible treatment option in IBS.12–14It has been hypothesized that a 15 It included 90 IBS patients in a randomized double-blind placebo-controlled clinical trial of donor FMT vs. placebo in a 2:1 randomization. Patients with IBS-D or IBS-M defined by the ROME III criteria, scored as moderate to severe according to the IBS severity scoring system (IBS-SSS), were enrolled locally by general practitioners in northern Norway. The donor feces was freshly processed, and was used the same day, or frozen for later use. The participants’ own preprocessed and frozen feces served as placebo. The transplant was administered by a colonoscope to the cecum. The main outcome measure was self-rated clinical effect assessed by the IBS-SSS (reduction of >75 points). The trial showed significant clinical effects 3 months after administration of FMT, with very few and minor self-limited adverse effects until 1 year post-FMT.Our previously published REFIT trial provided the first proof of concept for FMT in IBS patients.16 The mode of delivery may be a key issue, as classic FMT shows promise, while capsule delivery does not.17 In order to understand this observed variability, it is necessary to characterize the intervention of classic FMT in detail.However, the effect of FMT in IBS is contested, as not all studies agree on its efficacy.Here, we provide a data set generated by deep metagenomic sequencing of fecal samples collected before and after FMT during the REFIT trial.Effect), and 8 had none or only a transient effect of the intervention (termed No effect). Seventeen samples from the transplants (termed Donors) used for treatment of the 22 participants, from two individual donors, acted as comparator group in the present setup. The baseline characteristics for the selected study participants are presented in Stool was collected at three time points from the 83 participants who completed the REFIT study. The present data set was constructed to investigate the effects of engraftment of the donor transplant; hence, we picked only participants from the active treatment group who received donor transplants. Only 22 participants with complete sample sets were available due to low compliance in delivery of fecal samples after the treatment. Of these, 14 had good effect . The No effect group had significantly lower richness at baseline (P = .0049 for Observed) compared to the Effect group, with values comparable to the Donors (P = 1.0000 for Observed). At 12 months, the richness in the No Effect group increasingly resembled that of the Effect group (P = 1.0000 for Observed). However, looking at diversity measures that include evenness (Shannon and Simpson), both IBS participant groups showed higher diversity at baseline compared to the Donors , and these measures did not change significantly with time.Measures of diversity and evenness are presented in Donor, Effect, and No Effect groups prior to FMT and after 12 months  was performed on beta diversity in the fecal samples from the 2 months , left pae points , right pDonor and IBS patient samples were widely different  (In order to identify taxa that changed significantly in the (DESeq2) , S1). AtEubacterium sp., 0 of 2 Clostridium sp., 11 of 12 Ruminococcus sp., 2 of 2 Klebsiella sp., and 2 of 2 Lactobacillus sp. had a decreased abundance after FMT. However, considering baseline counts of these species, the resulting SCFA production capacity (based on mean baseline counts by fold change) increased by a factor 6.7 after FMT. Akkermansia muciniphila had a significantly reduced abundance at 12 months.In total, 170 species showed a significantly different abundance in response to FMT  <0.01 and log2fold change <-2 or >2) (Table S1); of these, 128 species increased in abundance and 42 decreased in abundance after FMT. Twenty-four of the 36 species belonging to the Firmicutes phylum showed a reduced abundance following FMT . In phylum Bacteroidetes, 64 of 67 species increased in abundance after FMT . Of species known to be involved in the production of short-chain fatty acids (SCFA), 3 of 4 Effect group moved toward the Donor profile over time after FMT treatment. At 12 months, however, the profiles were still clearly distinct. Notably, the most profound change was observed between baseline and 6 months after the FMT treatment, The orthogonal partial least squares projection to latent structures (OPLS) analysis showed that the taxonomic profile in the Donor samples, and from the Effect group prior to treatment and after 12 months, were estimated using the growth rate index (GRiD).18 In total, GRiD reported 959 species for the 47 samples. After filtering of low-frequent species (occurring <15 times in <30 samples), and including only species with an FDR < 0.00005, we identified 27 species that displayed a significantly different growth rate in the three groups, Donor samples and in samples taken after FMT. The largest group of species found were belonging to the phylum Bacteroidetes (15/27), followed by different species of the phylum Firmicutes (8/27), among these Coprococcus catus, a known butyrate producer.Growth rates for bacterial species identified in the Effect group after 12 months, compared to the corresponding baseline samples  showed a significant differential abundance in samples from the  samples . ThirteeDonors and recipients (baseline sample vs. Donor sample used for each participant), as well as individual changes (12 months vs. baseline) in each of the 34 functional classes, for each participant. The baseline differences between Donors and recipients were quite similar in Effect and No effect groups . At 12 months, however, convergence toward the Donor profile was more profound in the Effect group than in the No effect group . Notably, Carbohydrates was the only functional class with a positive change in the Effect group, which could not be found in the No effect group. However, this difference was not significant. A further sub-analysis focused on the Carbohydrates cluster revealed subtle differences in systems involved in D-Galacturonate and D-Glucuronate utilization, D-ribose utilization, L-rhamnose utilization, Lactose and Galactose uptake and utilization, and Lactose utilization . Next, a multivariate regression model of the functional level 3 classes was built. Overall, the OPLS model  showed on model ). Eleven orphans ).Figure Donors. For subjects who experienced an effect of the treatment, a trend of convergence toward the Donor microbiome profile after FMT was shown; this trend was however less clear for subjects with no treatment effect. This trend was seen both on the taxonomic and on the functional level by the dimension reduction analysis and was even more clearly demonstrated by the multivariate regression (OPLS) models. Selected features on the phyla level, such as the fraction of Bacteroidetes and Firmicutes, showed the same trend )Donor samples, the baseline profile of IBS patients found in the present data set shows a relative dominance of Firmicutes over Bacteroidetes, while Donor samples show the opposite pattern. This finding supports an earlier report using similar methodology, 11 and some studies using 16S ribosomal ribonucleic acid (16S rRNA) sequencing.20 However, our Firmicutes:Bacteroidetes result is challenged by other studies where 16S rRNA sequencing demonstrates the exact opposite balance in IBS,21–23 or no distinct IBS profile.24 Factors such as race, regional diet, and methodology may have had an impact on the taxonomic profiles reported.25 A recent review on 16S rRNA sequencing data addresses the Firmicutes:Bacteroidetes schism, as well as the question of diversity, concluding that most data sets support the increased Firmicutes:Bacteroidetes ratio in IBS, while a lower alpha diversity is a more prevalent finding in IBS.26 Further support for the hypothesis that microbiota composition may be a key feature in IBS pathophysiology can be drawn from our longitudinal data showing increasing relative abundance of Bacteroidetes, and decreasing relative abundance of Firmicutes during the time course of the study. Thus, the microbiome profile change mirrors the clinical effect observed after FMT.Compared to Ruminococcus sp. and Bifidobacterium sp.27 increased after FMT in the Effect group. This fits well with the notion of lower SCFA in IBS patients, as reviewed by Camilleri.28Moreover, the present data set showed that, based on taxonomic data, the short-chain fatty acid (SCFA) producing species such as Akkermansia muciniphila was significantly reduced at 12 months after FMT, a signal also reported in a recent case series.29 This species is known to have various effects on host T-cell immunology in mice, 30 and has been linked to a decrease in the integrity of the intestinal mucus layer.9 Furthermore, it has been shown to be among the five taxa associated with the production of 10% of fecal metabolites.31The abundance of Effect group at baseline, the heat map plot . For the No effect group, a similar pattern of combined growth rate profiles could be seen, though involving much fewer bacterial species, and possibly leaving the overall impact on the recipient microbiome too weak to induce a clinical effect.In the map plot ) shows ranalyses  and 6. T8 A recent study found reduced levels of inflammatory cytokines, an altered gut microbiota profile, and reduced levels of short-chain fatty acids to be associated with symptom relief from a low-FODMAP diet.32 Here, we can demonstrate that relative to Donor stool, the fecal samples from IBS patients have a lower representation of carbohydrate-related subsystems. In the Effect group, increases in D-Galacturonate and D-Glucuronate utilization, D-ribose utilization, L-rhamnose utilization, and Lactose and Galactose uptake and utilization were observed after 12 months. The L-rhamnose pathway was recently reported to be among the top five microbial pathways associated with 53% of fecal metabolites found in 479 unrelated individuals.31 Thus, the functional output of our FMT treatment closes the gap to the Donor profile in the Effect group. This change is not seen in the No effect group. All other subsystem clusters show a tendency of convergence toward the Donor profile in both the Effect and No effect groups.The involvement of certain functional subsystems raises interesting questions about the basic pathophysiology of IBS. When we consider the role of a FODMAP-reduced diet in IBS treatment, SCFA and carbohydrate signals are of special interest. FODMAPs are a group of short-chain carbohydrates hypothesized to induce symptoms, possibly by altered colonic fermentation of these compounds, leading to a change in the functional output, which then induces gut symptoms.et al. .23 On the functional level, the Acetyl-CoA fermentation pathway leading to production of the short-chain fatty acid butyrate33 was reduced at baseline.Strains that are known SCFA producers were relatively poorly represented in the baseline samples, a finding also reported by Pozuelo 34 Also, the functional subsystem involved in synthesis of folate was significantly more abundant, which is thought to enhance the growth of commensals such as Lactobacillus sp.35 Subsystems involved in inorganic sulfur assimilation, 36 transport of Zinc, Manganese,37 and uptake of heme also changed significantly after FMT. The acquisition of metal ions is an essential survival factor for all microorganisms, both in the general environment and in the human host.38The observed increase in menaquinone pathways after FMT has interesting implications for growth patterns, and for the bacterial environment, as menaquinones represent a major class of growth factors for several bacterial species in the human gut.et al.39 However, the temperature rise was relatively short and affected all samples in a similar manner. Thus, the observed systematic changes in the recipient groups probably reflect a genuine shift. Further, the observed shift of microbial profile might also reflect changes in diet, which is known to affect the bacterial composition almost on a daily basis. However, diet registrations in the clinical study did not reveal any systematic changes in FODMAP intake during the course of the study, although these were only registered at two time-points. Our microbiome characterization only addresses the luminal microbiota, whereas mucosa-associated microbes may be quite important (16). We included non-constipated IBS patients with a broad age and BMI span in order to retain a high clinical translation value for the results. However, in the present data set, this may introduce added complexity to the results due to hormonal effects and weight-related differences in the gut microbiome. Finally, the clinical study used only two donors, resulting in a very tight clustering of the Donor samples. This is unlikely to reflect the actual variability found in “normal” microbiomes.The strengths of this study are: Firstly, our study design allowed microbiome analysis in two dimensions: Comparison between donor and IBS, and over time after the FMT intervention enabling both the generation of an “IBS profile,” and a proof of concept that microbiome changes mirror symptom improvement. Secondly, deep metagenomic sequencing enables an analysis of both taxonomy and functional features of the microbiome, yielding a better understanding of these components in IBS pathophysiology. There are also some weaknesses to be addressed: The sample size is quite small, which reduces the statistical power of the analyses. Also, the exploratory nature of the study precludes any firm conclusions. Importantly the transient freezer failure  may have induced changes in the microbiome profiles, as reported by Tedjo Donors). Subjects who experienced symptom relief after FMT showed a more explicit convergence toward the Donor sample profiles when compared to subjects who had no effect of the intervention. This supports the hypothesis that intestinal dysbiosis is a key component in IBS pathophysiology, as well as the notion that FMT could be a possible treatment for IBS.In conclusion, we have found distinct differences in microbiome composition between IBS patients and controls . We randomly assigned participants to active or placebo FMT (2:1). The transplant was either freshly processed and used the same day, or previously stored in a freezer. Each participant’s own feces served as placebo. A dose of 8 mg loperamide was administered orally 2 h before endoscopy, to help participants retain the transplant. The transplant  was administered to the cecum by colonoscope. The primary endpoint was a symptom relief of more than 75 points, as assessed by IBS-SSS, 3 months after FMT. Stool samples were collected from Donors at baseline, and from participants at baseline and after 6 and 12 months.The REFIT study was described in detail in the primary publication.All samples were frozen without additives in 1.7 mL Eppendorf tubes at −40°C. At one point during storage, the freezer had a technical failure, during which the temperature of the samples may have briefly exceeded 0°C (duration ≤12 h).Sequencing was performed at the Genomics Support Center Tromsø (GSCT) at UiT the Arctic University of Norway. DNA isolation was performed on a QIAcube instrument  using a QIAamp Fast DNA Stool Mini Kit (QIAgen). DNA concentration was measured on a Qubit 3 instrument . Library preparation was done with Nextera XT DNA library kit  using an input of 1 ng DNA. The samples were then sequenced on an Illumina NextSeq550 instrument with a NextSeq 500/550 High output v2 kit (300 cycles) (Illumina), generating 2*150 base-pair reads. The sequencing generated a mean of 27 271 415 (range 2 933 179 − 74 426 851) reads per sample.40 and the data were filtered using the default settings for AfterQC, 41 with additional trimming of the 15 first nucleotides of each read. Optical sequence duplicates were removed with the optical distance of 40 using Clumpify.42 Fastq Screen40 was used, together with the human genome (GRCh38), to filter host contamination. The unsynchronized PE sequence files were repaired using Repair.42The quality of the sequence data was investigated using FastQC, 43 against the pre-indexed NCBI BLAST nr, including fungi and microbial eukaryotes as reference database, and with the Greedy mode (higher sensitivity but longer run-time) and default parameter settings. The taxonomic abundance data from each sample – as generated by the Kaiju classification of the sequence reads – were merged into an abundance matrix and imported into Phyloseq44 along with the corresponding metadata.Taxonomic assignments of the pre-processed sequence reads were performed using Kaiju version 1.6.245 aligning reads against the pre-indexed DB_98 database with DIAMOND 0.9.14.46 The functional classification results obtained for each SEED  subsystem level were imported into Phyloseq, and the prune_taxa function was applied to retain only subsystems with absolute abundance >0.01%. The data were transformed into fractional abundances for multivariate analysis and visualization in STAMP.47 Differential abundance analysis of the SEED subsystems between groups was calculated using DESeq2.48 Only functional subsystems with differential abundance at a cut off log2 fold change (FC) >0.5, and adjusted p-value (FDR) ≤0.05 were reported.Functional assignments of the pre-processed sequence reads were performed using SUPER-FOCUS version 0.32,48 Differentially abundant species were identified at a log2FC cut off of <-2 to >2, and an FDR cutoff of <10−4.The species diversity  in all samples was explored for absolute abundance values, using observed taxa, Chao1, Shannon, and Simpson indexes, and plots were generated using the plot_richness function. Taxa not seen more than five times in at least 20% of the samples – in the total dataset – were removed using the filter_taxa function. Differential abundance analysis between samples from participants who responded to treatment after 12 months, and the corresponding baseline samples, was performed on the taxa remaining after abundance filtering the data with DESeq2.−5 were used. The inter-individual differences (beta diversity) were calculated using the Bray–Curtis dissimilarity index on relative abundance values and explored using nMDS ordination plots generated by the Phyloseq package in R. The taxa remaining after abundance filtering were agglomerated at each taxonomic rank, using the tax_glom function to generate taxonomic bar plots, and subjected to multivariate analysis. Finally, relative abundance plots were generated, using the filtered data, for phyla at a cut off >0.01 relative abundance per sample group.The filtered abundance data were transformed to fractional abundances, and only taxa with a mean fractional abundance >10R2Xcum, R2Ycum, and Q2cum, where R2Xcum is the cumulative modeled variation in X. R2Ycum is the amount of variation in X correlated to Y (response matrix), and Q2cum is the cumulative predicted ability of the model. The validity and degree of overfitting of the OPLS models were assessed by conducting analysis of variance testing of cross-validated predictive residuals (CV-ANOVA). An obtained P-value lower than 0.05 was considered as an indication of a significant model. Variables were ranked according to their scaled regression coefficients from the OPLS models.The data were log transformed and Pareto scaled. Multivariate analysis was carried out using SIMCA software . Supervised orthogonal partial least squares projection to latent structures (OPLS) was applied, to facilitate the interpretation of changes in the composition of bacterial species and functional group profiles along the time points . The performance of the OPLS models was described by 18 was run. The samples were mapped against the stool-specific database49 using a minimum genome coverage cutoff = 0.2, enabling reassignment of ambiguous reads using Pathoscope2.50 In total, this analysis reported 959 species for the 47 samples. Species that occurred <15 times in <30 samples were eliminated from the further analysis, that was carried out on the remaining 340 species. Statistical testing on the ratios produced by GRiD for each species, per sample, was performed using ANOVA, and the results were visualized in STAMP.47 Only species at FDR cut off <0.00005 were included (n = 29) in the heat map.To calculate the growth rate of species identified in the samples, GRiDClick here for additional data file.