This study aimed at evaluating the effect of swimming before and during pregnancy on rats born with intrauterine growth restriction (IUGR) and their offspring. For this, nondiabetic and streptozotocin-induced severely diabetic (SD) pregnant rats were mated and generated offspring with appropriate  and small (IUGR) for pregnancy age, respectively. Following that, C and IUGR groups were further distributed into nonexercised control (C), exercised control (Cex), nonexercised IUGR (IUGR), and exercised IUGR (IUGRex). IUGR rats presented lower mating rate than control rats. Regardless of physical exercise IUGR rats presented decreased body weight from birth to lactation. At 90 days of life, IUGR rats presented glucose intolerance. Maternal organ weights were increased and relative adiposity of IUGRex rats was lower than Cex. IUGR and IUGRex offspring presented reduced body weight than C and Cex, respectively. IUGRex dams presented an increased rate of appropriate for pregnancy age newborns. IUGEex male and female offspring relative brain weight was increased compared with Cex. Therefore, swimming before and during pregnancy prevented glucose intolerance, reduced general adiposity, and increased maternal and offspring organ weight in rats, showing the benefit of physical exercise for IUGR rats. Maternal body composition has important effects on the offspring. Extremes of maternal body composition in pregnancy are associated with adverse long-term offspring outcomes . Several Diabetes Mellitus [Experimental models using laboratory animals are relevant to expand and improve the understanding of the pathophysiological mechanisms involved in an inappropriate intrauterine environment. Therefore, it is necessary to develop adequate experimental models . SeveralMellitus –20.Maternal hyperglycemia during pregnancy causes complications for both mother and offspring. Previous studies showed that adult female rats with chemically induced diabetes by streptozotocin (STZ) presented high glycemia (>300 mg/dL: severe diabetes) and their offspring were born small for pregnancy age (SPA) due to intrauterine growth restriction (IUGR) , 20, whiSeveral procedures are employed to control maternal glycemia and prevent embryofetal development impairment. Traditionally, the most used therapeutic resource to control blood glucose is the association between diet and insulin. However, the efficacy of alternative aprroaches, such as exercise, is being tested , 24. In Considering the negative results obtained by Volpato et al.  in the o ad libitum. The rats were maintained in Laboratory of Experimental Research on Gynecology and Obstetrics under controlled conditions .Female and male Wistar rats  weighing approximately 200 grams (g) were housed in a certified animal care. Food and water were providedSevere diabetes was induced at adult life of female rats (approximately at 90 days of age) by beta cytotoxic drug . STZ was dissolved in a citrate buffer  and intravenously (i.v.) administered at a dose of 40 mg/kg body weight –20. ContAfter vaginal delivery, all newborns (NB) were examined for sex determination by the anogenital distance, which is about twice larger in the male than in the female . Followi gavage at a final dose of 2 g/kg body weight. Following that, the blood samples were obtained from a cut tip tail for glycemic determinations using a specific glucosemeter at 30, 60, and 120 minutes (min) [At mornings of days 90 and 120 of life, the maternal body weights were recorded and oral glucose tolerance test (OGTT) was performed. For OGTT, after fasting for 6 hours, glycemia was verified (timepoint 0); then a glucose solution (200 g/L) was administered byes (min) .C (control): nonexercised APA female rats;Cex (exercised control): APA female rats exercised prior to and during pregnancy;IUGR (intratuerine growth restriction): nonexercised SPA female rats;IUGRex (intratuerine growth restriction): SPA female rats exercised prior to and during pregnancy.For distribution of control and IUGR rats, submitted or not to swimming, the experimental groups were denominated:At day 90 of life, one month before the mating period, C and IUGR rats were randomly selected to begin swimming program modified of other two exercise protocols: as the swimming Volpato et al. (2006)  and simiAt day 120 of life, all groups  of rats were submitted for mating using similar proceedings to mother rats. During pregnancy, the pregnant rats were maintained in the individual cages. The exercised groups (Cex and IUGRex) continued swimming program during this period until day 20 of pregnancy.At mornings of days zero (early pregnancy), 7 (embryonic period), 14 , and 20 of pregnancy , the maternal body weights and postprandial glycemia were determined for evaluation of swimming effect. All blood samples were obtained by venous puncture of the tail. Blood glucose concentrations were measured by conventional glucometer and these values were expressed in mg/dL.At the day 17 of pregenacy, OGTT was performed in all these rats to evaluate the glucose tolerance following the methodology described in On day 1 after vaginal delivery, the mother rats and their offspring were weighed. Then, the sexing and body weight classification of these newborns were performed as described in At days 5 and 9 of lactation, the dams and their offspring were again weighed and blood samples were obtained by venous puncture of the tail for blood glucose concentrations were measured by conventional glucometer (mg/dL).At day 10 after delivery, the dams and their offspring were weighed and anesthetized with sodium pentobarbital . The maternal and newborn heart, lung, pancreas, liver, adipose tissues  were collected. These organs were dissected and weighed to obtain the relative weight (absolute weight/body weight × 100). Regarding adipose tissues, the calculation of total fat  and relative weight . ad libitum. After that, these dams were weighed at the beginning (T1) and end (T2) of the 4 h period. The indirect calculation of milk production (g) was the weight (T1) and weight (T2) [At 7:00 AM at days 5 and 9 of lactation, pups were removed from the mothers for 4 h and their dams ateght (T2) .t-test for normal distribution and Mann-Whitney for abnormal distribution of data were used to compare only two groups. The proportion data were analyzed by Fisher's exact test. The oral glucose tolerance test was analyzed by Gamma distribution followed by repeated measures. SAS software (version 9.3) was applied for all statistical analyses. p < 0.05 was considered as statistical significance limit.The data were presented as mean ± standard deviation. To avoid overinfluence of data from a single mother in study, the females for each group came from different litters. Respecting that the homogeneity among experimental units is one of the basics of experimental design and considering that SPA and APA are biologically different organisms, the comparison between sedentary SPA versus sedentary APA and exercited SPA versus exercited was performed. Student's unpaired The Ethics Committee on Animal Experiments of the Botucatu Medical School,UNESP, approved all experimental procedures performed in this study (Protocol number: 938/2012).At 90 days old, it was oberved that the blood glucose levels were increased in the timepoints 30 and 60 minutes (min) in the IUGR group  compared to those of control group  . With 12p < 0.05) compared to their respective control groups (C and Cex) (and Cex) . During and Cex) . During and Cex) .It was verified that there was no change regarding the litter size and the number of newborns among experimental groups .The experimental groups showed a milk mean production on day 5 of lactation in the control group (3.33 ± 1.12 g); Cex (4.93 ± 3.12 g); IUGR (4.40 ± 0.80 g) and IUGRex (3.91 ± 12.02 g) and on day 9 in the control group (3.83 ± 3.18 g); Cex (2.50 ± 7.20 g); IUGR (3.90 ± 3.92 g) and IUGRex (5.48 ± 2.55 g). There was no statistically significant difference in milk production among experimental groups.In relation to parental generation, 64 female rats were injected with streptozotocin and of these 100% presented blood glucose concentration above 300 mg/dL. In the nondiabetic group, 7 rats received citrate buffer and 100% of them presented glycemia below 120 mg/dL. At adult life, all nondiabetic female rats and 27 diabetic female rats mated. Of these, all nondiabetic rats (100%) and 13 severe diabetic rats reached at term pregnancy. The severe diabetic dams presented lower alive fetuses compared to nondiabetic rats .p < 0.05). The relative weight of pancreas and lung was increased in IUGRex group compared to the group Cex. There was no statistically significant difference in the relation to relative weights of liver and mammary gland among the different experimental groups (p > 0.05)  .The relative weight of the adipose tissues  was reduced in IUGRex rats compared to Cex rats. Regarding peritoneal and periovariane adipose tissues, it was verified that there was a reduction in relative weight in IUGRex rats compared to the IUGR group. The relative weight of sternal adipose tissue was also reduced in IUGR group compared to the C group .In relation to total fat, the IUGR group showed a decrease compared to control group (C) and the IUGRex group also showed reduced adiposity compared to Cex and IUGR groups. The relative fat was reduced in IUGRex rats in relation to Cex and IUGR rats .The females and males newborns showed reduced body weights  in IUGR and IUGRex groups in relation to C and Cex groups, respectively .The glycemic mean of female newborns at day 5  and day 9  presented no difference compared to days 5 and 9. The glycemic mean of male newborns at day 5  and day 9  presented also no difference compared to days 5 and 9, regardless of the groups where mothers were inserted.p > 0.05) with the blood glucose levels of their newborns (data not shown).The maternal blood glucose levels were not correlated . The male offspring showed an increase in heart weight in IUGRex group compared to those of Cex rats .In the present study, the rats born with intrauterine growth restriction (IUGR) presented glucose intolerance at adulthood. These findings corroborate previous study of our laboratory, where diabetic rats presented two or more timepoints superior to 140 mg/dL during OGTT at day 90 of life . Diabetes Mellitus onset, and regulates lipid metabolism [After 30 and 50 days of swimming application, the rats of IUGRex group showed reduced blood glucose levels in OGTT at day 120 of life and 17 of pregnancy, respectively, showing that exercise was beneficial for these rats. Physical exercise and insulin physiologically stimulate glucose transport in skeletal muscle , 38. Exetabolism , 29. Thetabolism . Anothertabolism . In our In the present experiment, regardless of practicing swimming, the rats of IUGR group were born and remained with lower weights throughout the experiment , showing how these rats showed no growth. The catch-up growth can be defined as a realignment of individual genetic growth potential after intrauterine growth retardation (IUGR) . Accordiβ-cell development plays a major role in the subjects who fail to undergo catch-up growth. Corvino et al. [The absence of catch-up growth may play important role in protecting them from adverse metabolic outcomes in the long term and to prevent the deterioration of in vivo insulin action that occurs with age and, as a result, glucose levels are more easily maintained , corroboo et al.  also verIn our study, the rats born with IUGR who practiced swimming presented weight gain during pregnancy and reduced total and relative adiposity, showing beneficial effect of exercise for these rats. This could prevent obesity onset, which develops in adulthood of offspring born of IUGR , 44. HowRegarding the relative weights of maternal organs, our results showed that the relative weights of heart, pancreas, and lung of exercised IUGR rats were similar to the control group. In a model of uteroplacental insufficiency in rats, it was observed that these dams generated restricted females newborns and it was verified that organ relative weights (heart and pancreas) were also not different between control and restricted pregnant groups at day 19 of pregnancy .In this study, it was found no changes in litter size. Similarly, it was demonstrated that the total  F2 litter size was not different between control and restricted rats in others investigations , 13.In relation to F2 male and female newborns from mothers restricted (IUGR), regardless of the swimming practice, there was a higher percentage of newborns classified as adequate for pregnancy age (data not shown), showing that the exercise was not harmful for offspring growth of rats. In contrast with the results of Damasceno et al., 2013 , and VolThe glycemia of female and male newborn showed no changes in the perinatal period. It has been shown in our study that the male and female newborns of IUGRex mothers group showed an increase in the relative weight of the heart, brain, and lung and increased relative weight of brain and lung  in the IUGR mothers group was also observed. Brain sparing is a feature of intrauterine growth retardation (IUGR), which implies that there is a redistribution of metabolic supply so that body growth slows to a greater extent than brain growth . Our finIn summary, there is ample evidence that an abnormal intrauterine environment can induce alterations in fetal metabolism with persisting consequences in late life and successive generations. Our data show that the newborns from diabetic rats were born with IUGR and developed glucose intolerance at adulthood. However, when these rats were subjected to a swimming program before and during pregnancy, the intolerance glucose was prevented. Besides, there was a reduced adiposity general preventing the possibilities of developing an obesity status, an increased organ weight in maternal organism and in offspring, and increased rate of newborn classified as adequate for pregnancy age, showing the beneficial effect of physical exercise in IUGR rats throughout two successive generations.Studies to translate findings of animal models to human practice are found in the literature but the transgenerational studies using adult intrauterine growth restricted rats are rare, especially considering pregnant and submitting to physical exercise. Then this investigation was performed with adult intrauterine growth restricted rats submitted to swimming program before and during pregnancy to evaluate the exercise effect on maternal organisms and their offspring. Our findings showed that the swimming program prevented glucose intolerance of adult intrauterine growth restricted rats and obesity and favored an increase in organs and body weight of their offspring, suggesting beneficial effect of swimming program in two rat successive generations.


Avena sativa L. as a surrogate weed). Isotopic tracer 15N was used to measure the fertilizer N uptake efficiencies of the wheat genotypes and weed. A field experiment, a split-plot design with four replications, was conducted during two consecutive growing seasons in a typical Mediterranean environment. In the absence of interspecific competition, few differences in either total N uptake (range: 98–112 kg N ha−1) or the 15N fertilizer recovery fraction (range: 30.0–36.7%) were observed among the wheat genotypes. The presence of competition, compared to competitor-free conditions, resulted in reductions in grain yield (49%), total N uptake (29%), and an 15N fertilizer recovery fraction (32%) that were on average markedly higher in modern varieties than in old ones. Both biomass and grain reductions were strongly related to the biomass of the competitor (correlation coefficients > 0.95), which ranged from 135 to 573 g m−2. Variations in both grain and biomass yield due to interspecific competition were significantly correlated with percentage of soil cover and leaf area at tillering, plant height at heading, and total N uptake, thus highlighting that the ability to take up N from the soil played a certain role in determining the different competitive abilities against weed of the genotypes.Choosing genotypes with a high capacity for taking up nitrogen (N) from the soil and the ability to efficiently compete with weeds for this nutrient is essential to increasing the sustainability of cropping systems that are less dependent on auxiliary inputs. This research aimed to verify whether differences exist in N uptake and N fertilizer recovery capacity among wheat genotypes and, if so, whether these differences are related to a different competitive ability against weeds of wheat genotypes. To this end, 12 genotypes, varying widely in morphological traits and year of release, were grown in the presence or absence of interspecific competition (using  Triticum durum Desf.) carried out in Italy over the past 70 years has involved the constant release of cultivars selected to perform well under intensive crop management and characterized by an increased yield potential  or organic systems .To the best of our knowledge, no information is available on (i) whether wheat genotypes vary in their ability to compete with weeds for N or (ii) what effect different N uptake capacities can have on a genotype's ability to compete with weeds. Thus, the present study had as objectives to verify whether differences in N uptake and N fertilizer recovery exist among wheat genotypes that differ in their year of release and, if so, to verify whether these differences are related to a different competitive ability against weeds. To this end, 12 genotypes, chosen to include large variability in plant growth habit, grain yield potential, and year of release, were grown in the presence or absence of interspecific competition. Isotopic tracer −1 clay, 227 g kg−1 silt, and 248 g kg−1 sand; pH 8.2; 16.8 g kg−1 total C and 1.78 g kg−1 total N).A field experiment was conducted during two consecutive growing seasons (2008–09 and 2009–10) at the experimental farm Pietranera, located about 30 km north of Agrigento, Italy . In both growing seasons, the soil was a Chromic Haploxerert with a clay texture , followed by spring . There is a dry period from May to September. The mean air temperatures are 15.9°C in autumn, 9.7°C in winter, and 16.5°C in spring. Weather data were collected from a weather station located within 200 m of the experimental site.T. durum Desf.; 1 of bread wheat, T. aestivum L., Maiorcone) that varied widely in their morpho-phenological traits (Table Avena sativa L.) was chosen as a surrogate weed to obtain a homogenous weed density across the experimental plots. We used A. sativa as a surrogate weed rather than a real wild weed  to ensure reliability and uniformity of emergence and synchronous development . Before the experiment began, the soil was plowed in August and harrowed after the first autumn rainfalls. Phosphate fertilizer was applied before sowing at 69 kg P2O5 ha−1 as triple superphosphate. In both years, plots were sown at the end of December, using 350 germinable wheat seeds per square meter. In the relevant subplots , oat was seeded in the same row as the wheat to maximize interspecific competition. Oat was planted at 100 germinable seeds per square meter. Ammonium sulfate fertilizer was applied at the time of seed emergence. Plots were labeled with 15N fertilizer (80 kg N ha−1 as (NH4)2SO4 with an isotopic enrichment of 1.33 atom%) added to a 1.20-m2  area in the middle of each subplot, following the application procedure described by Høgh-Jensen and Schjoerring (15N-labeled area) received equivalent amounts of unlabeled fertilizer. All natural weeds in both weedy and weed-free plots were removed by hand.The experiments were set up in a split-plot design with four replications. Main plots were planted with 12 wheat genotypes . Plants and tillers of each species were counted and separated into leaves, stems, and spikes; the fresh weight of each sample was determined and the leaf area of the leaves was immediately measured using a leaf area meter (LI-COR LI-3100C Area Meter). All samples were dried in a forced air oven at 60°C for 36 h and weighed. The plant height, percentage of soil cover , and lodging  for each wheat genotype grown in both the presence and absence of interspecific competition were also recorded. Moreover, leaf habit  in a fast running mill (Retsch ZM 100), and analyzed for total N and 15N enrichment . Plant height, lodging, grain yield and yield components , and N grain content were recorded for both wheat and oat.At seed maturity, the area of each subplot labeled with 15N enrichment of biomass were used to calculate the labeled-fertilizer N recovery (15NREC) on an area basis (kg N ha−1) and a percentage basis, according to Hauck and Bremner , 15Nfp was the atom% 15N in the fertilized plants, 15Nnfp was the atom% 15N in the unfertilized plants, 15Nfert was the atom% 15N in the fertilizer, and f was the fertilizer rate (kg N ha−1).where For all measured variables normality was tested using Shapiro–Wilk test of normality. All variables corresponding to proportions were arcsine transformed before analysis to ensure a better fit with the Gaussian law distribution. Data from each year were analyzed separately, and homogeneity of variances was assessed using Bartlett's test before combined analyses were performed.Two separate analyses were performed for each measured variable. In the first one, data were analyzed according to a split-plot design with genotype (G) as the main-plot treatment, interspecific competition (IC) as a subplot factor, and year and replicates as random factors. Considering also that G can be aggregated into two groups [Old (released before 1915) vs. Modern; Table In the presence of interaction between G and IC, G means at the same or different IC levels can be compared, and the appropriate LSD isFinally a Pearson correlation analysis was performed to highlight the relationships among morpho-agronomic traits  and competitive ability against weeds (measured either as percent decreases in grain and biomass yields in weedy compared to weed-free plots or as weed biomass in weedy plots). All analyses were carried out in the R environment , with higher values observed in the old genotypes compared to the modern varieties Table . HoweverP < 0.05). For plant biomass, decreases due to the presence of a competitor were on average 27% for the old genotypes and 47% for the modern varieties. Differences observed among wheat genotypes for biomass production were related to the biomass production of the competitor, which ranged from 127 g m−2  to 475 g m−2  to 573 g m−2 (Simeto). On the whole, the surrogate weed accumulated more biomass when grown with the modern genotypes than the old ones. Among the modern varieties, the surrogate weed produced less biomass when grown with Valbelice (378 g m−2) and Capeiti (397 g m−2), the tallest of the modern cultivars, than when grown with the other wheat genotypes.Wheat genotypes differed widely in plant height Table , which r−1 on average, respectively; Table −1 (Claudio) to 112 kg N ha−1 (Cappelli), and recovery of 15N fertilizer ranged from 30.0% (Scorsonera) to 36.7% (Biancuccia). Interspecific competition caused a general decrease in grain protein yield, N uptake, and N recovery but with differing intensities among genotypes. In particular, the presence of a competitor resulted on average in a reduction in grain protein yield of 27% in old genotypes and 62% in modern varieties; a reduction in N uptake of 14% in old genotypes and 45% in modern varieties; and a reduction in the recovery of 15N fertilizer of 18 and 47% for the old and modern genotypes, respectively. Decreases in both N uptake and recovery of 15N fertilizer were associated with proportional increases in both parameters in the competitor; in fact, they were greater when the surrogate weed grew with the modern varieties than when it grew with the old genotypes.Significant differences among wheat genotypes were observed in grain protein content, with the old genotypes having the highest values (on average 13.7 vs. 11.7% for the modern varieties). When a competitor was absent, grain protein yield was significantly higher for the modern varieties than for the old ones  and weed biomass , reducing the availability of resources  to the competitor, rather than their different abilities to tolerate competition (measured by the ability to maintain yield in the presence of weeds relative to the weed-free condition) and reductions in the contested resources. On the whole, the differences in grain yield observed between the old and modern genotypes, which were very high in weed-free conditions, were canceled out when genotypes were grown in the presence of weed competition. Moreover, while in weed-free conditions the modern varieties showed a grain protein yield markedly higher than the old ones, in weedy conditions the opposite was true. The latter result confirms that the old genotypes were more capable than the modern varieties to compete with weeds for N. These facts could be of interest when one is choosing which variety of durum wheat to grow, and it is particularly important for low-input or organic systems in which weed control is often a serious problem. Moreover, the use of genotypes with high weed suppression ability, reducing weed seed dissemination, can offer a clear advantage to subsequent crops, thus improving the overall efficiency of the cropping system.The presence of a competitor resulted on average in a reduction in both N uptake and the recovery of Moreover, the results of the present study showed a positive correlation between wheat plant stature at maturity and competitive ability against weeds, as already reported by many authors  when they were contested. This greater ability was positively related to the percentage of soil cover and leaf area at tillering, plant height at heading, and N accumulation capacity, all traits that were expressed more in the old genotypes than the modern ones, inasmuch the most competitive modern genotypes, Capeiti and Valbelice, both had some characteristics in common with the old genotypes. These results could have practical implications for cultivar choice, particularly in organic farming, in which weed control is often a serious problem, and given that the use of a variety high in competitiveness with weeds could have positive effects on subsequent crops due to reductions in the soil weed seed-bank.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative  Genomic imprinting disturbance can give rise to complex congenital disorders affecting growth, metabolism and behaviour. Here the authors report mutations in NLRP5, which suggests a connection between imprinting, maternal reproductive fitness and zygotic development. Cis-acting mutations, that is, affecting only one imprint and the gene(s) controlled by it, are associated with a range of clinically defined human-imprinting disorders, while trans-acting mutations may affect establishment or maintenance of multiple imprinting marks across the genome, and thus may have a wider impact on developmentNLRP2 and ZFP57 have been identified in rare cases of MLID3On fertilization, the genome undergoes genome-wide epigenetic reprogramming to supersede the developmental programmes of the sperm and oocyte with that of the developing zygote. A small number of gametic epigenetic marks are impervious to this reprogramming, and survive in the organism as genomic imprints, regulating genes under their control according to their parent of originNLRP7 or KHDC3L56NLRP7 and KHDC3L are maternal-effect genes, expressed from the maternal genome and required for zygotic development before activation of the zygotic genomeNLRP5 or its murine counterpart Mater, was the first-described maternal-effect gene in miceImprinted loci throughout the genome are paternalized in molar pregnancies; like imprinting disorders these can occur in sporadic or heritable forms, and the best-established cause of recurrent molar pregnancy is mutation of NLRP5 variants also have periods of infertility, and reproductive outcomes including miscarriage and reported molar pregnancy. NLRP5 is a novel candidate gene causing MLID, with deleterious maternal-effect variants in 15% of our cohort.We performed whole-exome sequencing of a large cohort of MLID patients, to seek genetic causes of the epigenetic errors in MLID. This led to the identification of NLRP5 variants in five mothers of patients with MLID. As well as offspring with MLID, though without consistent clinical presentation or consistent epimutations, some mothers with NLRP5 was the only gene in which rare and novel variants were found in more than two individuals. Using Sanger sequencing, NLRP5 was sequenced in 14 further MLID patients and 19 mothers. In total, 39 MLID patients and 33 mothers were sequenced for NLRP5 in this study , 10 with Silver–Russell syndrome  and 7 with transient neonatal diabetes mellitus (MIM #601410). In addition, parental samples were exome sequenced for four BWS–MLID, five SRS–MLID and four transient neonatal diabetes mellitus–MLID patients, and the mother of two siblings with MLID (SRS and BWS presentations). NLRP5 variants were detected in 5 of 33 unrelated MLID imprinting disorder pedigrees, where maternal samples were available  was identified within the NACHT  domain. Strikingly, the duplication was found neither in the proband with SRS–MLID, nor in her twin sister, but in their mother. Sanger sequencing identified an additional novel variant in family 5, NM_153447.4:c.1699A>G , which was present heterozygously in the proband  and homozygously in her mother. Copy number variation analysis by digital PCR confirmed there to be two copies of NLRP5 exon 7 in the mother , and hypermethylation of the neighbouring somatic GNASAS differentially methylated region (DMR), methylated on the paternal allele in inverse relation to the hypomethylation of the ICR. The distribution and severity of methylation disturbance varied between patients; and, as expected given the divergent clinical presentations of probands in families 1 and 2, there were also significant differences in the imprinted loci affected despite the shared genetic exposure to NLRP5 variants.NLRP5 variants with similar damaging potential were identified in The Database of Short Genetic Variation (dbSNP version 138) and are presented in The amino-acid conservation, pathogenicity and location within the polypeptide for each variant are illustrated . The amiNLRP5 mutations. The mother of family 1 had biallelic mutations affecting the NACHT and LRR domains, both predicted to be deleterious. She had no healthy offspring, two children with different imprinting disorders (SRS–MLID at 24 years and BWS–MLID at 30 years), and multiple pregnancy losses with three unrelated partners, including one termination for a presumed molar pregnancy. The mother of family 2 had biallelic variants in the LRR domain, one a truncation and the other predicted to be deleterious. She had two healthy offspring, followed by a period of reproductive wastage, a child with BWS–MLID (at the age of 35 years) and a child with non-specific developmental and marked behavioural problems diagnosed as autism with extreme separation anxiety, born when she was 39 years of age. The mother of family 3 had two NLRP5 variants in cis, one near and one within the DAPIN domain. The mother had one healthy child before the proband at the age of 34, presenting with BWS–MLID. The offspring of family 4 have previously been describedNLRP5 variants were clinically healthy, and none had methylation disturbance at imprinted loci , compared with a median age of 30.1 years among other mothers of MLID patients . No offspring of NLRP5-variant mothers were conceived by assisted reproductive technology (ART), compared with 2 of 20 other MLID (where data were available). One of seven offspring of NLRP5-variant mothers was a monozygous twin, compared with 6 of 26 other MLID (The median age of mothers affected by her MLID .Nlrp5 (Mater) was the first-described maternal-effect gene, with maternal ablation causing developmental arrest at the two-cell stage in miceNLRP5 depletion results in arrested zygote development before the 16-cell stage15NLRP7 and NLRP2) and their binding partners are also associated with a spectrum of reproductive wastage and stochastic disturbance of genomic imprinting471718NLRP7 being enriched within the NACHT and LRR domains18NLRP5 mutation in our cohort is underestimated, because further variants  may remain to be identified, which may contribute to the affectedness of some of the families described here. Our findings do not exclude the possibility that variants in other genes may contribute to the MLID seen in the families described here. It is also possible that some MLID cases may be caused by compound heterozygosity of variants in NLRP5 and its functional partners. Further exome analysis of patients and mothers is ongoing to address these questions. In addition, functional analysis is required to determine how NLRP5 coding variants alter the interactions between NLRP5 and its binding partners, and whether these reduce the developmental competence of the oocyte, or compromise its intrinsic biology or stability, or deplete the oocyte pool in affected females. Moreover, given the existence of other rare but potentially deleterious missense and nonsense variants within its coding sequence  and the median maternal age for all births in the general population 29.5 years . It is well recognised that the progressive decline in female reproductive fitness may reflect non-genetic factors such as the chronological age of the mother, or the reproductive age of the oocyte. Dankert et al.in vitro aged oocytes show a decline in the abundance of transcripts including NLRP5, suggesting that delay in fertilization may jeopardize oocyte fitness.The phenotypes of affected offspring were variable, with growth and neurodevelopmental problems. While the majority of probands had clinically recognizable imprinting disorders, two of seven cases had clinical features not consistent with a diagnosis of any imprinting disorder. Therefore, it is likely that in general, such patients remain undiagnosed. Although patient numbers in this study are too low to describe trends, the severity of clinical outcome seemed to reflect both the severity of NLRP5 involvement were conceived by ART, though one sibling (family 4) was conceived by ART, which may be indicative of reproductive difficulties. By comparison, 2 of 20 other children in the MLID cohort were conceived by ART, against a population rate of ∼2% . The relationship between imprinting disorders, ART and age of parents is complex. ART is often sought by parents with fertility problems, and it may be that the risk of imprinting disorders is associated less with ART per se than with the reduced fertility of the parents or the increase in age following a delay in conception. The small case numbers in this study and the potential for ascertainment bias preclude definitive conclusions, but our observations suggest that collection of detailed data on ART, twinning, fertility history and parental ages in families affected by MLID may reveal some novel associations.The affected offspring of mothers with NLRP5 variants included a D-MZ twin pair. Some imprinting disorders are associated with an elevated rate of twinning, almost invariably monozygotic and discordant, and enriched for MLID, but the causal relationship between imprinting disorders and twinning has remained unclearNLRP5 mutations in five mothers of offspring with MLID suggests links between maternal-effect genes, maternal reproductive fitness, epigenetic and developmental reprogramming of zygotes, and reproductive outcomes. We suggest that NLRP5 mutations may have been overlooked in the past as a cause of imprinting disorders or reproductive problems because their effects encompass both, and might not be captured by studies of either alone; an integrated investigation of the relationship between NLRP5, imprinting disorders and reproductive wastage is now warranted. Furthermore, maternal effect genes in general may be underestimated as a cause of developmental problems in children. Recent technological advances in the epigenetic analysis of gametes and zygotes23The finding of www.southampton.ac.uk/geneticimprinting/informationpatients/imprintingfindingoutwhy.page, accessed on September 2013), with the exception of the patient in family 4 and patients 17–23 who were consented into the research study ‘Disorders caused by imprinting defects' funded by the Bundesministerium für Bildung und Forschung (BMBF grant 01GM1513), and approved by the Ethical committee of the University Hospital Aachen, Germany.All patients were consented into the research study ‘Imprinting disorders—finding out why'  through the UK Comprehensive Local Research network (www.southampton.ac.uk/geneticimprinting/informationpatients/imprintingfindingoutwhy.page).Because the consent framework of the IDFOW study does not encompass the deposition of whole-exome sequence data in an open-access repository, whole-exome sequence files are instead deposited in a restricted-access repository under doi:10.5258/SOTON/378548. Data access are managed by the Wessex Imprinting Governance Team, to which application can be made by contacting the Corresponding Author, or the Study Administrator  to produce binary sequence alignment format (BAM) files and Picard (v1.95) was used to remove duplicate reads. Local indel realignment and base quality recalibration were performed using the Genome Analysis Toolkit (GATK v3.0–0) before the realigned and recalibrated BAM files were used to determine single-nucleotide variants including SNPs and indel (insertion–deletion) alleles. GATK was used to predict and genotype variants for each sample, raw variant calls were outputted in variant call format file, and variant filtration was performed for both SNPs and indels to remove low quality and potentially false-positive variants. Variant data were annotated using Annovar (v 2013Aug23) and KggSeq (v 0.6).NLRP5 was used to confirm exome variants, establish their inheritance, fill gaps in exome coverage and screen a further 14 patients  and 19 mothers of individuals with MLID.Sanger sequencing of ExoSAP to degrade any remaining primers, before sequencing with M13 forward and reverse primers using BigDye 1.1chemistry (Applied Biosytems). Sequencing reactions were analysed on an ABI Prism 3130XL sequencer (Applied Biosystems).M13 universal tagged primers were designed to 14 of the 15 exons of NLRP5 see . Exons wCo-amplification of exons 5 and 6 was observed with both primer sets due to their highly similar sequence and required the use of internal sequencing primers with exon-specific terminal-3′ bases see  to generhttp://www.ensembl.org/Homo_sapiens/Tools/VEP), Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) and PROVEAN v1.1.3 (http://provean.jcvi.org/index.php), applied with standard procedures and settings and presented in NLRP5 variants in dbSNP138. Nonsense variants and variants predicted to be pathogenic by one or more programmes are presented in The pathogenicity  scores of the variants identified was predicted using the online tools Ensembl Variant Effect Predictor  analysis was performed on bisulfite-converted DNA . MSP was used to determine DNA methylation at 17 DMRs of imprinted genesP value  <0.05 across a minimum of three consecutive CpGs within 2,000 nucleotides; the additional requirement of M values between −1 and +1 in normal controls was applied to enrich differential methylation at loci consistent with genomic imprinting. Regions associated with known imprinted genes (http://igc.otago.ac.nz/home.html) or loci previously identified as disregulated in individuals with MLIDGenome-wide DNA methylation analysis was generated using the Illumina Infinium HumanMethylation450 BeadChip  and a single-sample analysis pipelineFor digital PCR, genomic DNAs were fragmented by MseI digestion (New England Biolabs) for five normal controls and one sample where a novel homozygous variant was identified in a region of homozygosity by Sanger sequencing. Droplet digital PCR amplification of two regions of exon 7 generated a 91- and 90-bp amplicon, respectively, in 20-μl reactions containing 64-ng digested DNA, using 900 nM primers  × copy number of KRAS reference (=2).Droplets were generated using the QX200 Droplet Generator (Bio-Rad Laboratories Inc.) and 70 μl Droplet Generation Oil (Bio-Rad Laboratories Inc.). Droplet PCR cycling conditions were 95 °C for 10 min, then 40 cycles of 94 °C for 30 s, 55 °C for 60 s and final enzyme deactivation at 98 °C for 10 min. Fluorescent droplets containing amplified products were read using the QX200 Droplet Reader (Bio-Rad Laboratories Inc.). Copy number variation of In family 1, patient 1 , was born at 32 weeks gestation with a birth weight of 1,040 g (<0.4th centile). She had a cleft palate, a right unilateral cleft lip and bilateral fifth finger clinodactyly. There were feeding difficulties for the first 6 months of life requiring nasogastric tube feeds. By two years of age minimal body asymmetry was documented ; her height and weight were on the 0.4th centile with a head circumference on the 9th centile. A diagnosis of SRS was made and confirmed using routine NHS testing for hypomethylation at H19. In childhood, she showed mild developmental delay and behavioural difficulties including poor sleep and self harm. By 9 years of age her height, weight and head circumference were all on the 25th centile without treatment, and the asymmetry was less marked. There were ongoing concerns regarding her behaviour and she required significant additional help in a mainstream primary school, including a statement of educational need and, speech and language therapy. Her mother reported that throughout her childhood, patient 1 showed episodic excess sweating, but there have been no documented episodes of hypoglycaemia. Investigations including calcium, phosphate and parathyroid hormone were normal.Patient 2 in family 1 (BWS), the maternal half-brother of patient 1, was born at 36 weeks gestation with a birth weight of 3,425 g (between the 91st and 98th centiles), and a head circumference of 33.7 cms (50th centile). During the pregnancy, antenatal ultrasound scans identified a cystic appearance of the placenta and by 30 weeks, macroglossia was diagnosed. Post-delivery maternal β-HCG levels were normal. At birth macroglossia was confirmed and he was also reported to have bilateral ear creases and a natal tooth. During the neonatal period there was one documented episode of hypoglycaemia. By the age of three months, Patient 2 had developed an umbilical hernia. He was diagnosed with BWS and this was confirmed on routine NHS testing to show a loss of methylation at ICR2. There was no hemihypertrophy. At around 1 year of age a renal ultrasound scan revealed discrepant size of the kidneys, but no evidence of a tumour.There was no further family history of SRS, BWS or any other imprinting disorder; however, the mother of these two children had suffered significant pregnancy related morbidity. She had six pregnancy losses, with three unrelated partners, and one medical termination of pregnancy for a presumed molar pregnancy, associated with a developing fetus . Aside from the pregnancy losses, there was no other medical history of note: she herself was born with a normal birth weight to non-consanguineous parents. Her own mother had suffered three miscarriages.th centile) and by 13 years his height was 161.7 cm (75th centile) and weight 55 kg (90th centile). He had a naevus flammeus on his forehead. He was noted to have asymmetry, with the right side of the face, tongue and right leg being larger than the left (2-cm difference in foot length), and showed a mild positional scoliosis.In family 2, patient 1, (BWS) was the third of four children, born when the mother was 35 years of age. He was born at 32 weeks of gestation with a birth weight of 1,700 g, following a diagnosis of preeclampsia in his mother. The placenta was described as showing mesenchymal dysplasia. He had an uneventful neonatal period, requiring tube feeding initially but not ventilation, and was discharged at 4 weeks of age. He was admitted following an episode of apnoea at 3 months, and at that time was diagnosed with macroglossia, though surgical resection was not performed. He was also noted to have an umbilical hernia, undescended testes and hemihypertrophy. A diagnosis of BWS was made and confirmed on routine NHS testing to show hypomethylation at ICR2. He had an episode of pneumonia at 5 months and was diagnosed with asthma. He had delayed development, walking at 18 months and was diagnosed with speech delay and subsequently dyslexia and dyscalculia. He required an educational statement and is currently educated at a special school. On examination at 8 years 5 months, his height was 126.5 cm (25th centile), weight 28.9 kg (50th–75th centile) and occipital frontal circumference (OFC) 54 cm . At 11 months he weighed 14.2 kg and a diagnosis of BWS was considered because of excessive weight gain and a forehead naevus flammeus; but routine testing was negative. He walked at 17 months and started to say single words at 2 years, but did not progress to speaking in sentences until 4 years, and marked expressive speech delay was diagnosed. He required special education, with an educational statement of need. At the age of 8 years and 6 months, he had markedly unusual behaviour, with extreme separation anxiety such that he was able to attend school only on three mornings a week. He was diagnosed with autism, with episodes of severe anger. He was constantly hungry, but did report satiety after eating, and there was no history of stealing food. He suffered from gastrointestinal reflux and severe constipation. His general health was good. On examination aged 8 years and 6 months his OFC was 58 cm (98–99.6th centile), his height at 134.8 cm was on the 75th centile, and his weight was 54.4 kg well above the 99.6th centile. He had a round face with long, narrow palpebral fissures, a short nose with anteverted nares, normal ears and tongue, no clinical asymmetry. He had hyperextensible fingers but no evidence of shortening of the metacarpals. He was prepubertal. The rest of the examination was normal.There were two older siblings with normal development and growth  born 3 years and 2 years before patient 1. The older male sibling had mild anxiety in early childhood but is doing well at school with well above-average educational attainment. Mother suffered one miscarriage before patient 1 and 3 miscarriages before patient 2.The proband in family 3 was the son of young, healthy and unrelated parents. He was born at 39 weeks gestation with a birth weight of 3,500 g (50th centile), birth length of 53 cm (90–97th centile) and a head circumference of 36 cm (50th–90th centile). At birth he was confirmed to have macroglossia, cheek and tongue right-side hemihyperplasia, a naevus flammeus of the forehead and occipital region. A diagnosis of BWS was made but no episodes of hypoglycaemia were reported during the neonatal period. At around 2 years, diastasis recti was observed, but surgery was not needed. At the age of two and a half years the child had age-appropriate psychomotor development and abdominal scanning was normal. He has one healthy elder brother, and no miscarriages were documented.The proband in family 4 has previously been published as patient 3 ref. . The femThe family history was unremarkable. The German parents were not consanguineous; the maternal age at birth was 30 years and paternal age was 33 years. The twins were the product of a normal conception, but their elder  sibling was the product of assisted reproductive therapy and has normal growth and psychomotor development.The proband in family 5 has previously been reportedNo details of health or reproductive issues are reported among other family members, except for atrial septal defect and ventral septal defect in one sibling.Accession codes: Exome sequence data for the patients in families 1–4 have been deposited in http://eprints.soton.ac.uk/id/eprint/378548 under the accession codes 378548.How to cite this article: Docherty, L. E. et al. Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans. Nat. Commun. 6:8086 doi: 10.1038/ncomms9086 (2015).Supplementary Figure 1 and Supplementary Tables 1-6Targeted DNA methylation testingImprinted loci identified by Illumina Infinium Human450 methylation array


We present here a methodology that enables accurate determination of delivered to cell dose metrics. This methodology includes (1) standardization of engineered nanomaterial (ENM) suspension preparation; (2) measurement of ENM characteristics controlling delivery to cells in culture; and (3) calculation of delivered dose as a function of exposure time using the ISDD model. The approach is validated against experimentally measured doses, and simplified analytical expressions for the delivered dose (Relevant In Vitro Dose (RID)f function) are derived for 20 ENMs. These functions can be used by nanotoxicologists to accurately calculate the total mass (RIDM), surface area (RIDSA), or particle number (RIDN) delivered to cells as a function of exposure time.There is a great need for screening tools capable of rapidly assessing nanomaterial toxicity. One impediment to the development of reliable in vitro dosimetry methodology for a number of ENMs (measured delivered to cell dose within 9% of estimated).The proposed methodology was used to derive the effective density, agglomerate diameter and RID functions for 17 industrially-relevant metal and metal oxide ENMs, two carbonaceous nanoparticles, and non-agglomerating gold nanospheres, for two well plate configurations (96 and 384 well plates). For agglomerating ENMs, the measured effective density was on average 60% below the material density. We report great variability in delivered dose metrics, with some materials depositing within 24 hours while others require over 100 hours for delivery to cells. A neutron-activated tracer particle system was employed to validate the proposed in vitro systems for nanotoxicology. The mixed experimental/computational approach to cellular dosimetry proposed and validated here can be used by nanotoxicologists to accurately calculate the delivered to cell dose metrics for various ENMs and in vitro conditions as a function of exposure time. The RID functions and characterization data for widely used ENMs presented here can together be used by experimentalists to design and interpret toxicity studies.Our findings confirm and extend experimental and computational evidence that agglomerate characteristics affect the dose delivered to cells. Therefore measurement of these characteristics is critical for effective use of  Nanoe number ,20-22. Min vitro cytotoxicity study, ENM powders are usually suspended in liquid media for application to cells. Once suspended in liquid, ENMs often form large fractal agglomerates [In a typical omerates -26 thereomerates ,27. Nanoomerates ,28,29. Domerates , Notablyomerates ,29-33.2 suspended in culture media , Cohen et al. estimated delivery of the entire administered dose to cells in culture can take up to hundreds of hours, indicative of the great importance of ENM interactions in physiological fluid and their subsequent effect on particle delivery to cells [in vitro dosimetry in nanotoxicology studies, a challenge recently overcome by our group [Until recently, this phenomena had been demonstrated and quantified experimentally for a very limited number of nanoparticles ,33-35, ato cells ,24. Morein vitro systems. The accuracy of the VCM has been validated by comparison to established, high accuracy, but more costly and time consuming methods [in vitro nanotoxicologists in an fast and cost effective manner is now possible. Coupled with existing dosimetry algorithms such as the In vitro Sedimentation, Diffusion and Dosimetry model (ISDD) [in vitro toxicity screens currently employed in nanotoxicology studies, which are based on administered to cell dose [The recently developed Harvard Volumetric Centrifugation Method (VCM) is a simple, user- friendly method for experimentally determining the effective density of agglomerates under the conditions of study for  methods ,27. Withl (ISDD) ,27,29 paell dose ,37. It min vitro particle dosimetry that takes into account the particokinetics in an in vitro system and enables accurate determination and reporting of delivered to cell dose metrics. Simple mathematical equations, referred to as Relevant In Vitro Dose (RID)f functions that represent the rate of delivery of each particle under the given experimental conditions as a function of exposure time, are derived based on the proposed methodology for twenty widely used ENMs and are ready for use by toxicologists. The term “relevant” here refers to the dose actually delivered to cells, rather than the typically reported administered dose of particles in suspension. The reported RIDf functions can be used by in vitro nanotoxicologists to accurately calculate the particle mass (RIDM), particle surface area (RIDSA), or particle number (RIDN) delivered to cells as a function of exposure time.We present here an integrated methodology for 2, CeO2, Fe2O3, Fe3O4, and TiO2) using the Harvard VENGES generation system [Seventeen metal oxide ENMs covering representative oxides across the periodic table, two low aspect ratio carbonaceous nanoparticles, and non-agglomerating gold nanospheres typically used for biomedical applications were selected for study. Several of the materials were synthesized in house by flame spray pyrolysis  in RPMI tissue culture media supplemented with 10% fetal bovine serum (RPMI/10%FBS) is reported in Table The measured agglomerate effective density for each ENM suspended in RPMI media was determined by the previously established Harvard Volumetric Centrifugation Method (VCM), and are also summarized in Table EV) values significantly lower than that of their material density (ρENM). This is a clear indication of the formation of porous agglomerates containing large amounts of trapped intra-agglomerate media. Non-agglomerating gold nanospheres exhibited a ρEV value only slightly less than the density of elemental gold (17.73 vs 19.3 g/cm3), consistent with minimal agglomeration, as expected. In general ρEV for all ENMs investigated correlated with raw material density, which may suggest that agglomerates of these ENMs are composed of comparable relative proportions of raw ENM and trapped media. For example, the lowest effective densities were measured for particles with the lowest material densities , and the highest effective densities were measured for particles with the material densities greater than 5 g/cm3 .As expected, due to the fractal nature of flame-generated metal oxide ENMs, these materials exhibited effective density , the total mass dose, M (μg), can be calculated as:For an ENM suspension of known mass concentration, V is the volume of exposure media (ml) applied directly to the cells in culture.where N (#), can be calculated from the total mass, M, hydrodynamic radius, rH , and agglomerate effective density, ρE, (g/cm3), assuming spherical agglomerates, as:The total particle number dose, SA (cm2), can then be calculated assuming spherical agglomerates as:Total surface area dose, in vitro toxicity of SiO2 nanoparticles and the areal density of nanoparticle mass delivered to cells over the exposure duration [in vitro is therefore more accurately represented in relation to the delivered dose for any selected dose metric, rather than the typically reported administered mass concentration of ENMs in suspension.It is worth noting that cellular response to a biologically active material reflects the quantity of the substance actually coming into contact with the cells. For example, Wittmaack recently reported significant correlation between duration . Sharma duration . CellulaUsing the recently characterized material-media specific parameters for agglomerate hydrodynamic diameter and effective density (see Table The estimated f(t) function was then fitted as a Gompertz sigmoidal function as previously described by the authors  as follo−1) is the material-media specific deposition fraction constant, and t (hr) is exposure duration.Where α (hrsIn Vitro Dose (RID)f functions for delivered mass, number and surface area as a function of time can be derived by combining Equation 4 with Equations 1, 2 and 3 as follows:The Relevant M, μg):For particle mass delivered to cells .For particle number delivered to cells ,For total particle surface area delivered to cells , and agglomerate effective density (ρe) are reported in Table RID functions are provided here for all twenty ENMs and conditions used in this study. The necessary parameter values, consisting of the deposition fraction constant (α), agglomerate size  [dH, and ρEV, should be measured over the time of exposure. These time- resolved values should then be utilized by the transport simulation model to accurately estimate delivered dose. Considering mass loss due to dissolution will likely result in a net decrease in effective density, any dosimetry calculations for soluble materials based on effective density measured immediately after sample preparation would provide an overestimate of particle deposition over time. In addition, for completion of dosimetry calculations, both the particulate and soluble components must be correctly identified and considered separately as previously described [It is necessary to note that for materials that undergo significant dissolution over the course of the study, changes in agglomerate diameter and effective density that result from mass loss due to dissolution must be resolved over time and addressed in the fate and transport algorithm in order to accurately estimate delivered dose . A recen3) . To ensuescribed . The ISD2, and SiO2 coated CeO2) were applied to transwell insert membranes with 3 μm pores  used in this study as the fate and transport algorithm in deriving the RID functions has been validated for a limited number of materials including non-agglomerating fluorescently labeled polystyrene beads of various agglomerate diameter ,34, and −1), and the time required to deliver 90% of the administered dose, t90 (h) for each ENM-media-concentration system are summarized in Table The simple mathematical RID functions obtained here for the extensive panel of ENMs can be used by nanotoxicologists to accurately estimate the delivered to cell dose metrics  as a function of time. The material and well geometry specific deposition fraction constant, α h−, and the2O3 exhibited the highest alpha parameter and shortest t90, consistent with its relatively large agglomerate diameter and effective density . In contrast, VENGES SiO2 exhibited a low alpha parameter and long t90, consistent with its relatively small agglomerate diameter and effective density . This trend is consistent with previous reports in the literature of measured target dose in vitro[In general, ENMs having greater values for both effective density and agglomerate diameter were expected to deposit more rapidly than those with smaller values for both properties. For example, Cr90), in hours (h), for all materials investigated, in two well plate geometries (96 and 384 well plate). These results confirm and significantly extend evidence that that the dose rates and target cell doses can vary significantly depending on the material, media, and well plate properties, and emphasize the importance of characterizing ENM-suspensions for both agglomerate diameter and effective density. For the materials investigated in this study, the time required to deliver particles to cells in vitro can differ by a factor of 20, and can range from <10 hours up to >100 hours , and materials exhibiting slow transport that achieve significantly lower delivered doses It is necessary to note that the reported dosimetry data for all ENMs was estimated based on agglomerate size and density measured immediately following sample preparation, assuming no agglomerate changes over time due to dissolution. The dissolution of a large panel of metal and metal oxide ENMs (many of which are included in this study) suspended in cell culture media following 24 hour incubation has been previously reported , and onlOne potential limitation to the proposed dosimetry approach is the lack of validation for high aspect ratio nanomaterials such as carbon nanotubes. Considering the many assumptions of spherical agglomerates in the estimation of particle fate and transport via diffusion and sedimentation, the mobility of high aspect ratio materials may not be so easily estimated. Carbon nanotubes can form large irregularly shaped agglomerates in liquid suspension, the mobility of which may in fact be accurately estimated from the agglomerate diameter and effective density. Our group is currently working to characterize the mobility of such materials and determine whether the proposed approach may be applicable.in vitro studies [It must also be noted that agglomerate diameter may be greatly influenced by the mass concentration of particles in solution. The RID functions derived in this study were generally valid for a mass concentration range of 0-50 μg/ml (data not shown), which is within the usual levels used for  studies ,39. Concin vitro nanotoxicology.The protein corona is another important factor influencing nanoparticle agglomeration, particle delivery to cells, and cellular uptake and trafficking -46. Our in vitro screening assays requires identification of equivalent doses between in vitro and in vivo systems. One proposed approach was recently reported that utilized the multiple-path particle dosimetry model (MPPD) to estimate the deposited and retained dose of ENMs in the alveolar regions of exposed animals. These doses were then compared with cellular responses measured at equivalent doses of ENMs delivered to cells in vitro [in vitro and affect hazard ranking for some ENMs and endpoints. More importantly, there is consensus in the nanotoxicology field that there is a need to bridge the gap between in vitro and in vivo models, which requires reporting of biological response data in vitro and in vivo on the same dose scale- the amount of material deposited to cells/tissue, rather than the administered mass dose metric currently used in nanotoxicology studies. The proposed dosimetric approach can be a valuable and easy to use tool for nanotoxicologists in their quest of understanding the toxicological implications of ENMs.Examples from the recent literature demonstrate the significant impact of dosimetry on interpreting the results of various in vitro assays. These principles were first described in detail several years ago ,29,31, ain vitro ,48, Teegin vitro. Our group recently demonstrated for a small panel of neutron activated ENMs that particle uptake and trafficking is dependent on particle delivery to cells, and provided electron micrographs and DLS data characterizing the agglomeration state of internalized and exocytosed particles [in vitro, and it will be useful to compare the dose response curves based on administered, delivered, and internalized doses.Direct measurement of the internalized cellular dose is another dose metric worth investigating to further elucidate the mechanisms of nanomaterial toxicity articles . Future in vitro dosimetry methodology and the derived RID functions provide a tool for nanotoxicologists to accurately calculate the particle mass (RIDM), particle surface area (RIDSA), or particle number (RIDN) delivered to cells in culture as a function of exposure time. This methodology is based on direct measurement of agglomerate parameters, and is an improvement upon previous work relying on unvalidated estimates for agglomerate effective density [in vitro conditions can assist nanotoxicologists in addressing dosimetry issues in their in vitro screening studies. Adoption of the proposed dosimetry methodology will be a major step towards the development of inexpensive, accurate, and reproducible in vitro screening assays, and will overcome potential inaccuracies that may arise from reporting dose as simply the nominal media concentrations. Consideration of the variability of delivered dose may hold large implications for the interpretation of previously reported high-throughput toxicity screens of large panels of ENMs [Our integrated  density . Numeric of ENMs ,37, and 2, Fe2O3, and CeO2 ENM powders were generated in-house by flame spray pyrolysis using the Harvard Versatile Engineered Nanomaterial Generation System (VENGES) recently developed by the authors [2 and TiO2: EVONIK, Essen, Germany; Al2O3, Cr2O3, Fe2O3, Mn2O3, and Zr2O3: US Research Nanoaterials Inc., Houston, TX; CoO: Skyspring Nanomaterials Inc., Houston, TX; Gd2O3 and SiO2: Nanostructured and Amorphous Materials Inc., Houston, TX; CeO2: Meliorum Technologies Inc., Rochester, NY). Carbon nanohorns and Printex-90 carbon black particles were donated by Dr. Dhimiter Bellow at University of Massachussetts Lowell, and characterized previously in the literature [ENMs investigated are listed in Table  authors ,38. Additerature ,50.4 in a round bottom flask was heated to a rolling boil with vigorous stirring. 50 ml of 38.8 mM sodium citrate solution was added rapidly. After a color change from pale yellow to purple, indicating formation of gold nanoparticles, boiling was continued for another 15 minutes, after which the heating source was removed and the suspension stirred for an additional 15 minutes. The suspension was filtered through 0.45 μm syringe filters and stored at 4°C.Spherical monodisperse gold nanospheres, which were donated by Dr. Srinivas Sridhar at Northeastern University, were prepared in suspension as previously described . BrieflySSA, defined as the particle surface area per mass (m2/g), was determined by the nitrogen adsorption/Brunauer-Emmett-Teller (BET) method using a Micrometrics Tristar 3000  for each ENM. The equivalent primary particle diameter, dBET, was calculated, assuming spherical particles, asFor powdered ENMs specific surface area, ρp is the particle density, which was obtained for each particle from the densities of component materials, at 20°C, reported in the CRC handbook of Chemistry and Physics [where  Physics . ENM powCR) was determined following a previously established protocol [CR, corresponding to the lowest agglomeration state. From each curve, an ENM-specific DSECR is estimated by determining the DSE value at which the dispersed ENMs were within 10% of their observed minimum hydrodynamic diameter as measured by DLS. To determine the stability of the sonicated suspension over time, hydrodynamic diameter measurements were repeated for several hours following sonication.The material specific delivered sonication energy required to achieve stable monodisperse agglomerates in suspension  tubes  and centrifuged at 2,000 × eviously . Media deviously .in vitro sedimentation, diffusion and dosimetry (ISDD) model developed by Hinderliter et al.[fD(t) as previously described [dH, measured by DLS in the test media, at 50 μg/ml, the media column height , temperature (310 K), media density, (1.00 g/cm3), media dynamic viscosity (0.00074 Pa s) [fD (t) was fit to a Gompertz sigmoidal equation,The er et al., was useescribed ,24. In a74 Pa s) , and admt is time (h), and α is an ENM- and media-specific deposition fraction constant (h−1). Solving Equation (9) for the time t at which the fraction fD(t) of administered particles is delivered yieldswhere t90, for each ENM dispersion using the specific deposition function constants, α, and an fD(t) value of 0.90.Equation (10) was used to calculate the time required for delivery of 90% of the administered dose, 141Ce ENMs  were stored in irradiation tubes. 141Ce is a gamma emitter with a half-life of 32 days, and the successful production of radioactive ENMs following irradiation was confirmed by gamma energy spectrometry using a Packard gamma counter . ENMs were quantified as either percentage of total administered mass dose, based on total measured radioactivity . Concentration calibrations were performed for each ENM by measuring radioactivity for a measured and known total ENM mass in suspension, and measuring radioactivity of dilutions by half down to 5 ng.ENM powders were irradiated with neutrons for up to 24 hours at the Nuclear Reactor Laboratory , and the radioactive 100 μl of ENM suspensions at a concentration of 12.5 μg/ml were applied to transwells without cells, for 2, 4, and 24 hours ; ρENM: Material density (g/cm3); f(t): Fraction of administered particles deposited onto cells as a function of time; α: Material-media specific deposition fraction constant (hrs−1); t90: Time required to deliver 90% of the administered particle dose (h).ENM: Engineered nanomaterials; VCM: Volumetric Centrifugation Method; RIDM: Relevant in vitro dose function for particle mass delivered to cells; RIDN: Relevant in vitro dose function for particle number delivered to cells; RIDSA: Relevant in vitro dose function for particle surface area delivered to cells; ISDD: In vitro sedimentation, diffusion and dosimetry model; TEM: Transmission electron microscopy; VENGES: Versatile Engineered Nanomaterial Generation System; BET: Brunauer-Emmett-Teller; DSECR: Critical delivered sonication energy; DLS: Dynamic light scattering; PCV: Packed cell volume; SF: Stacking factor; ρEV: Agglomerate effective density (g/cmNone of the authors have any competing financial interests in the work described in this manuscript.JMC co-developed the dosimetry methodology, carried out ENM suspension characterization, dosimetric modeling, and dosimetry validation experiments, and drafted the manuscript. JGT provided nanomaterials for characterization, provided the ISDD model, and drafted the manuscript. PD supervised the project, co-developed the dosimetry methodology, prepared VENGES nanomaterials, and drafted the manuscript. All authors read and approved the final manuscript.Experimental setup for dosimetry methodology validation experiments.Click here for file


The heterogeneity and poor prognosis associated with gliomas, makes biomarker identification imperative. Here, we report autoantibody signatures across various grades of glioma serum samples and sub-categories of glioblastoma multiforme using Human Proteome chips containing ~17000 full-length human proteins. The deduced sets of classifier proteins helped to distinguish Grade II, III and IV samples from the healthy subjects with 88, 89 and 94% sensitivity and 87, 100 and 73% specificity, respectively. Proteins namely, SNX1, EYA1, PQBP1 and IGHG1 showed dysregulation across various grades. Sub-classes of GBM, based on its proximity to the sub-ventricular zone, have been reported to have different prognostic outcomes. To this end, we identified dysregulation of NEDD9, a protein involved in cell migration, with probable prognostic potential. Another subcategory of patients where the IDH1 gene is mutated, are known to have better prognosis as compared to patients carrying the wild type gene. On a comparison of these two cohorts, we found STUB1 and YWHAH proteins dysregulated in Grade II glioma patients. In addition to common pathways associated with tumourigenesis, we found enrichment of immunoregulatory and cytoskeletal remodelling pathways, emphasizing the need to explore biochemical alterations arising due to autoimmune responses in glioma. Gliomas are the most aggressive CNS tumours with poor prognosis2Gliomas have been sub-typed based on various molecular markers like IDH1, 1p/19q co-deletion, amplification of EGFR amplification, loss of PTEN, MGMT etc. to predict the prognosis of the patients, with due consideration of parameters like patient’s age and complete histopathological profileTraditionally, gliomas have been diagnosed either by imaging techniques, histopathology or bothIn this study, we performed screening of sera from healthy controls and various grades of glioma patients using human proteome arrays containing more than 17000 proteins . To the 2 = 0.99) (2 = 0.904) was observed between the signal-intensities from different microarrays processed on different days using the same serum sample  . Similarm sample .After the background correction and normalization, the data seemed to be median centric , therebyThe expression values of all the proteins from the healthy controls were compared to that of different grades of gliomas . In thisWhen the lists of differentially expressed proteins were compared with each other, we identified sets of proteins which were differentially expressed in more than one grade . For exaThe comparison of SVZp versus SVZn  revealedAnother analysis with IDH1 mutation as focus, revealed 22 proteins significantly dysregulated in a comparison of Grade II WT versus IDH1p . Among tGrade II Gliomas were found to be enriched in invasive pathways like cytoskeleton based cell adhesion and migration, cytoskeleton remodelling, chemotaxis, etc. . MetabolTLR signalling and histone phosphorylation were among the top enriched Gene Ontology (GO) processes in case of comparison of high grade gliomas with healthy subjects while aorta morphogenesis was one of the top hits in this category, in case of low grade gliomas . On enri®Pathway Analysis (IPA) was used to analyse the most significant interactions of differentially expressed proteins with special focus to the involvement of classifier proteins. Interaction networks like “cell death and survival, inflammatory response, cell to cell signalling and interaction” and “developmental disorder, hereditary disorder, neurological disease” network were implicated in Grade II  . SVM is Another panel of 10 proteins was elucidated which differentiated healthy samples from Grade III samples with 89% sensitivity and 100% specificity at an AUC value of 1 and classification accuracy of 93%. This panel included 9 up-regulated proteins such as C14orf80, GCK, HSD17B14, LYPLAL1, MAGEA4, MLX, RTN4, SNX1 and TEX264 and 1 down-regulated protein, ARHGAP17 .Similarly, another set of 10 proteins, was found to differentiate the healthy samples from Grade IV samples with 94% sensitivity, 73% specificity, AUC value of 0.975 and classification accuracy of 88%. The panel included proteins like SNX1, IGHG1 and C11orf74, which were up-regulated and down-regulated proteins like C17orf57, CIB1, RCSD1, CDH26, PQBP1, EYA1 and ZHX3 .Through the intra-grade analysis of Grade IV i.e. SVZp versus SVZn, we identified a set of 10 classifier proteins which helped distinguish SVZp samples from SVZn with 77% sensitivity and 95% specificity and with an AUC value of 0.975. These classifier proteins included 6 proteins, PGM2, DR1, HIBADH, GPBP1, EEF1A1 and LOC339685, which were overexpressed in SVZp and 4 proteins, TMOD4, FAM120B, NEDD9 and GMEB1, which were overexpressed in SVZn .The multidimensional scaling (MDS) plots of healthy samples versus different grades of gliomas revealed a separation of samples albeit with some mis-classification error . SimilarA robust classifier panel could not be deduced to distinguish IDH1p and WT subjects in any grade while maintaining stringent statistical thresholds; however few of the significant proteins exhibited interesting trends in the IDH1p and WT cohorts .Glioma is one of the most common and aggressive types of primary brain tumourset al.et al.A holistic observation of this analysis revealed four proteins, IGHG1, EYA1, SNX1 and PQBP1, which were found to be significantly dysregulated in all the grades compared to the healthy individuals. Of these four proteins, SNX1 contributed to the classification of healthy controls and all the three grades of gliomas. On the other hand IGHG1, EYA1 and PQBP1 were found to help in the classification of Grade II and Grade IV from healthy individuals. The expression of IgG heavy region has been reported in various cancerous cell lines. However, the reasons for its expression are still not very clear1819IDH1 gene is widely associated with good prognosis of glioma patients. One of the primary reasons for a prolonged survival is that the lower levels of NADPH found in the IDH1p patients. The lower levels of NADPH make the tumour more responsive to irradiation and chemotherapyIDH1 mutation is more frequent in younger patients, compared to that of olderDifferentiating sub-categories of a given grade of cancer is extremely important in order to understand underlying mechanisms in the manifestation of the disease. In this study, we tried to identify differentially expressed proteins between the sub-categories of Grade IV i.e. SVZp and SVZn. NEDD9 has been known to cause invasiveness in lung cancer and melanoma28Biochemical pathways form the framework, which leads the molecular and biological processes in a system to function optimally36394345The findings from our data point towards the potential lists of classifiers, which are differentially expressed in various grades of gliomas compared to that of healthy control samples. To the best of our knowledge, this is a first study of its kind where more than 17,000 proteins were screened for their autoantigenicity using glioma serum samples on a protein microarray platform. The pathway analysis from potential TAAs opens a new avenue, which needs to be pursued in depth as to how and why these proteins are targeted by the immune system. This also gives us an understanding of the likely functionalities to be compromised depending on the targeted biochemical pathways. The findings from our study, thus, lay the foundation for early diagnosis of gliomas and prognostics.The tumour serum samples collected and experiments conducted in this study were approved as part of an institutional review board approved study  in accordance with approved guidelines. Patients with radiologically suspected gliomas were enrolled after giving written informed consent.n = 17), III (n = 18) and IV (n = 34) patients and healthy individuals (n = 15) we used Human Proteome arrays (HuProt™ arrays) (Johns Hopkins University). The median age of the patients with Grade II, III and IV were 32, 32.5 and 57 years with a standard deviation of 15, 12 and 11 years, respectively. Further patient details are enlisted in the Blood was collected prior to the first surgery and before patients receiving any kind of treatment. The collected serum samples were aliquoted into smaller volumes and stored at −80 °C until further use. Prior to the assay, the serum samples were thawed on ice. For the evaluation of serum samples from Glioma grades II  and negative controls  were spotted in duplicates on the microarraysThe HuProt™ arrays comprised of ~17000 unique full-length proteins printed in duplicates. The expression of these 17000 GST-tagged proteins was done in Prior to the screening of the serum samples for the detection of autoantibody signatures , the quaIn order to obtain a high signal-to-noise ratio one needs to optimize the assay. We used 3% non-fat milk powder in TBST , 3% BSA in TBST, SuperBlock (Pierce) and 3% BSA in SuperBlock. Similarly, various dilutions of serum and secondary antibody were tried.Finally, the protocol which gave us the best intrachip and interchip reproducibility and low background and high signal-to-noise ratio was used to further evaluate the serum samples. For deducing the intrachip reproducibility, we used the signal intensities from the duplicate spots of the same protein, while the interchip reproducibility was calculated using the signal intensities from the identical spots on two different microarray slides processed using same serum sample on different days . The prohttps://www.r-project.org/).The processed microarrays were scanned with GenePix 4000B Microarray Scanner (Molecular Devices). The image processing and the data acquisition of microarrays were done using GenePix Pro 7 (Molecular Devices). In order to identify differentially expressed proteins, the raw data needs to be pre-processed so as to adjust variation between arrays, thus adjusting for any effects not arising due to biological differencesPre-processing the data involves two steps: background correction and normalization. Background correction was performed using the ‘nec’ method present in ‘limma’. ‘nec’ performs ‘normexp-by-control’ background correction utilizing the negative controls only. An offset of 100 is added to the final adjusted values in order to reduce the variability of the log ratios. This data is then normalized across arrays using ‘normalizebetweenarrays’ method with the ‘quantile normalization’ function in limma. Quantile normalization normalizes the value in two or more data sets by making the distribution of the probe intensities identical, statistically by extending the concept of Q-Q plots to n-dimensions, where n is the number of proteins, in our case n = 17000.The data having been pre-processed is then analysed for selecting differentially expressed genes. ‘limma’ makes use of moderated t-statistic to test the null hypothesis that the genes are not differentially expressed. These tests are adjusted for multiple hypothesis testing using the ‘Benjamin-Hochberg’(BH) correction method. Though limma was primarily developed for DNA microarrays, it has been used for analysing protein microarraysThe proteins with a Log-fold change more than 0.5 or less than −0.5 and with an adjusted p value less than 0.05 were selected to be potentially differentially expressed. The chosen cut off values essentially focuses on genes with higher fold changes which are also significant statistically. Thus, in grade II versus control comparison the threshold was maintained at p value less than 0.05. For grade III versus healthy and Grade IV versus healthy comparisons the threshold was maintained at adjusted p value less than 0.05. The shortlisted proteins were then put to pathway analysis to determine if they were a part of a pathway that might have strong implications for tumourigenesis. By applying recursive feature elimination using SVMs with k-fold cross-validation we elucidated a panel of 10 classifier proteins, which enable differentiation of healthy samples from glioma samples. For visualization of the samples being discriminated using these panels, we used MDS. The 10 dimensional classifier space was mapped to a 3 dimensional space using a non-isometric MDS algorithm.®Pathway Analysis .After identifying the significant proteins from various comparisons, we proceeded further for the identification of enriched pathways and gene ontology terms using GeneGoMetacore™ software (Thomson Reuters). The significance level of 0.05 was used for this analysis and FDR corrected values at p < 0.05 were used as a threshold for these analysis. We, further, wanted to investigate the protein-protein interactions among the significant proteins identified from various grades of gliomas for which we used QIAGEN’s IngenuityHow to cite this article: Syed, P. et al. Autoantibody Profiling of Glioma Serum Samples to Identify Biomarkers Using Human Proteome Arrays. Sci. Rep.5, 13895; doi: 10.1038/srep13895 (2015).