In vitro sex differences in dendritic morphology are driven in part by estrogen-dependent mechanisms, as evidenced by decreased dendritic complexity in male hippocampal neurons cultured in phenol red-free media or in the presence of an estrogen receptor antagonist. Evidence that sex influences dendritic morphogenesis in two models of neurodevelopment in a region-specific manner has significant mechanistic implications regarding sex biases in NDDs.Altered dendritic morphology is common in neurodevelopmental disorders (NDDs), many of which show sex biases in prevalence, onset and/or severity. However, whether dendritic morphology varies as a function of sex in juvenile mice or primary neuronal cell cultures is largely unknown even though both are widely used models for studying NDDs. To address this gap, we quantified dendritic morphology in CA1 pyramidal hippocampal and adjacent somatosensory pyramidal cortical neurons from male and female postnatal day (P)28 C57BL/6J mice. As determined by Sholl analysis of Golgi-stained brain sections, dendritic arbors of male hippocampal neurons are more complex than females. Conversely, dendritic morphology of female cortical neurons is more complex than males. In primary neuron-glia co-cultures from P0 mouse hippocampi, male neurons have more complex dendritic arbors than female neurons. Sex differences are less pronounced in cortical cultures.  For example, ASD is almost five times more common among boys (1 in 42) than girls (1 in 189)5; ADHD symptoms differ in boys and girls with boys tending to show externalizing problems while girls exhibit internalizing problems8; and men tend to develop schizophrenia earlier and in a more severe form than women9. However, despite the importance of sex in NDDs, the mechanisms underlying sex differences are not completely understood. Addressing this gap is necessary for improving preventative, diagnostic and therapeutic strategies for NDDs.While neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD), attention deficit and hyperactivity disorder (ADHD) and schizophrenia affect both sexes13, and permanent sex differences in the brain are established during development by gonadal hormones17. Quantitative sex differences observed shortly after birth include overall size and organization of brain regions, the number and architecture of cells, and neurochemistry26. In rodent models, sex differences are especially evident in regions of the brain important for reproduction and sexual behavior, but are also observed in regions involved in cognition, learning and memory, such as the hippocampus and cortex27, which correlates with sex differences in cognitive function in adult rats28. Sex differences in the hippocampus and cortex are of particular interest since these regions are implicated in the etiology of multiple NDDs31. Sex differences in the brain are maintained at later developmental stages and into adulthood, as exemplified by human neuroimaging studies demonstrating that the hippocampus and cortex continue to develop in a sex-dependent manner during adolescence during which time the volume of the hippocampus continues to increase in males but decreases in females35.Inherent sex differences in neurodevelopment may contribute to the sex bias observed in NDDs. The vertebrate brain is organized in a sex-dependent manner38, which is often perturbed in NDDs40. Further, genes important for regulating dendritic shape and size during development are implicated in NDD etiology40. Sex differences in dendritic morphology have been observed in the neurotypical adult brain. For example, in adult rats, the dendrites of cortical neurons are longer, more complex and have greater spine density in males compared to females42. In the CA3 hippocampus, dendritic morphology is more complex in female versus male neurons in the proximal portion of the arbor, while the distal dendritic arbor is more complex in male versus female neurons43. However, detailed analysis of dendrite morphology between sexes is lacking for mouse models of juvenile development and for in vitro models of neurodevelopment, both of which are experimental models commonly used for investigating pathogenic mechanisms of and therapeutic strategies for NDDs44.More recent data demonstrate sex differences not only in brain structure, but also in neuronal cytoarchitecture, specifically the morphology of dendrites. This is critically important because dendritic morphology is a major determinant of neuronal connectivity45. Here, we extend those studies to further characterize sex differences in the dendritic morphology of CA1 pyramidal hippocampal neurons and determine whether sex differences are generalized to pyramidal neurons in the somatosensory cortex of P28 mice. To determine whether sex differences are also observed in vitro, we quantified dendritic arborization in primary cell cultures derived from the hippocampus and neocortex of male versus female mouse pups at P0, which are widely used models for characterizing factors that influence dendritic arborization47.Our preliminary studies of Golgi-stained neurons in the hippocampus of P28 C57BL/6J mice suggested that the basilar dendritic arbor of CA1 pyramidal hippocampal neurons is significantly more complex in male compared to female hippocampal neurons49. The dendritic morphology of Golgi-stained neurons in brain sections from P28 C57BL/6J mice was quantified using Sholl analysis, a method in which concentric rings are placed at fixed intervals from the soma and dendritic intersections counted at each ring. Sholl plots obtained from Golgi-stained neurons were analyzed using a mixed-effects model to account for the structure of the data, in which multiple neurons were analyzed per animal, as well as the autoregressive covariance structure of the Sholl profile45. Dendritic arborization was analyzed as the area under the curve (AUC) in Sholl plots.For these studies, we used Golgi staining to visualize individual pyramidal neurons in the CA1 hippocampus and adjacent somatosensory cortex of P28 male and female juvenile mice, an age previously examined for effects of early life stresses, including environmental exposures, on neurodevelopment2 vs. female 270 ± 16 μm2, p = 0.3).Representative photomicrographs of Golgi-stained CA1 pyramidal hippocampal neurons and the corresponding Sholl plots are shown in Fig. versus male neurons . Because experimental evidence indicates a positive correlation between body size and the extent of dendritic arborization in sympathetic neurons50, and since body mass has been correlated with neuron cell size in the brain51, we quantified body mass and found no significant differences between the sexes at the stage examined  6, high cell density cultures were transfected with a plasmid encoding microtubule-associated-protein-2B (MAP2B) fused to enhanced green fluorescent protein (EGFP) under conditions of low transfection efficiency in order to visualize the complete dendritic arbor of individual neurons52. Cultures were then fixed on DIV 9 and the dendritic arbors of EGFP-positive cells were quantified by Sholl analysis and morphometric analyses of dendrite number and length.Dissociated cultures of primary neurons are a widely used experimental model for investigating mechanisms underlying normal and aberrant dendritic morphogenesisThe dendritic morphology of primary hippocampal neurons varies between the sexes Fig. . Specifiin vitro by quantifying the number and length of primary dendrites and non-primary dendrites, with the latter defined as those that branch off the primary dendrites . The total number, sum length and mean length of primary dendrites did not differ significantly between male and female hippocampal neurons  release into the media, and by live dead analysis of cells stained with calcein AM and propidium iodide, which label live vs. dead cells, respectively. There were no significant differences in cell viability between male and female hippocampal , estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2) and aromatase (Cyp19a1) in our model systems. All four transcripts were detected in the hippocampus and cortex of P28 mice and in primary cell cultures derived from these brain regions  and distal  portions of the arbor43. This is important to consider since it has previously been shown that in utero and lactational exposure to environmental neurotoxicants, specifically, polychlorinated biphenyls (PCBs), decreases the dendritic complexity of basilar CA1 pyramidal hippocampal neurons in juvenile male rats (P22), but only in distal portions of the arbor59. Interestingly, when examined in adult male rats (P60), in utero and lactational exposure to PCBs causes a significant increase in complexity, but again only in the distal portion of the arbor59. This study only examined male rats, which raises the intriguing question of whether underlying sex-dependent differences in proximal-distal dendritic complexity contribute to sexually dimorphic effects of neurotoxicants such as PCBs. These results also underscore the importance of not relying on a single aspect of dendritic morphology when studying sex-dependent effects of environmental neurotoxicants since effects may vary depending on where in the dendritic arbor data are collected.In the developing mouse hippocampus, robust sex differences in dendritogenesis were observed in vivo and in vitro models. In contrast to hippocampal neurons, we observed more subtle sex differences in cortical neurons. In the P28 brain, sex differences consisted primarily of female cortical neurons exhibiting greater dendritic complexity than male cortical neurons. In cultured cortical neurons, sex differences were limited, with female neurons exhibiting longer but fewer primary dendrites than male neurons. The difference in age and heterogeneity of neurons present in the somatosensory cortex at the time of analysis likely explain the differences in the extent and type of sex differences observed in cortical neurons in vivo and in vitro. The somatosensory cortex consists of a heterogeneous population of neurons organized in a multi-layered structure with neurons at different stages of maturation within the layers60. For in vivo analyses, we focused on P28 pyramidal neurons within Layer 4–5. For in vitro studies, the entire P0 cortex is dissociated to establish primary cultures thus making it technically impossible to only focus on a single layer. Moreover, not all layers are represented in the primary cultures since neurons of the upper layers are still migrating in at P060. Thus it is not surprising that trends in sex differences for pyramidal neurons of the somatosensory cortex in each model were more variable and the correlation between the types of sex differences observed in vivo vs. in vitro were not as pronounced as in the case of the hippocampus. In vivo, differences in the maturation of somatosensory cortical neurons may explain the observed sex difference of females having greater dendritic complexity than males, which was the opposite trend observed in P28 CA1 pyramidal hippocampal neurons. The prefrontal cortex is one of the later brain regions to mature, with dendritic maturation and pruning occurring in this brain region primarily during adolescence61. In rats, dendritic complexity in the cortex increases in puberty from P20 to P3561; however, rates of growth differ between the sexes. Ramification of prefrontal cortex neurons occurs earlier in females than males and also differs in location, with females showing growth across the dendritic arbor while males exhibit the most growth in the middle portion of the dendritic arbor61. This may explain why we saw greater complexity in female vs. male cortical neurons at P28 while others have reported the opposite in studies of adult rat cortex, in which male neurons exhibit increased dendritic length, complexity and spine density compared to female neurons62.A notable finding in this study was that sex differences in dendritic morphology varied regionally. In the hippocampus, male neurons exhibited significantly more complex dendritic arbors than female neurons in both 63. These data suggest that the mechanisms driving sex differences in dendritic arborization, at least in hippocampal pyramidal neurons involve estrogen, an observation consistent with a significant body of literature indicating that the growth and maturation of neurons is hormone-dependent, with estrogen playing a major role13, as well as numerous studies demonstrating that estrogens increase dendritic spine number and density in adult male rat neurons of the dentate gyrus, and that this effect can be completely blocked by addition of ER antagonist, ICI64. These effects are especially evident in the hippocampus where adult male rat estradiol levels are higher in the hippocampus than in the systemic circulation65.Mechanistic studies of sex differences in primary neuron-glia co-cultures indicate that these differences are not hardwired, but rather they are malleable, as evidenced by the impact of changing culture conditions on male dendritic arbors. Specifically, sex differences in dendritic arborization in cultured hippocampal neurons are mitigated by removing the estrogenic compound phenol red from the culture medium, or by pharmacological antagonism of estrogen receptors. These findings are consistent with previous studies showing that estrogen augments neurite outgrowth from explants of the hypothalamus or preoptic areain vitro experiments have a practical implication in that they show that culture conditions commonly used in neurodevelopmental studies can significantly influence dendritic morphology. Therefore, interpretation of data generated using single sex or mixed sex cultures should include a consideration of the influence of media components or other endocrine signaling components in the culture system. It should be noted that phenol red-free media is not meant to be interpreted here as a ‘hormone free’ culture condition. Primary cultures of rat hippocampal neurons are capable of producing hormones, including estrogen, in vitro66, and common growth supplements added to growth media often contain corticosterone and progesterone. It would be valuable to determine levels of steroid hormones present in standard growth media, phenol free growth media, the cells themselves or carried over from brain tissue during dissection.The findings from our 67. We did not observe differences between the sexes with respect to the number of primary dendrites in pyramidal neurons from the CA1 hippocampus or adjacent pyramidal somatosensory cortex in P28 brain sections or in pyramidal neurons in hippocampal cell cultures. This suggests that the sex differences in dendritic arborization observed in these models likely occur during the later stage of dendrite maturation, which is consistent with literature suggesting that primary dendritogenesis is largely determined by intrinsic factors whereas dendritic maturation is strongly influenced by extrinsic factors. What is not clear from these studies is whether the sex differences we observed reflect differences between the sexes in dendritic branching and elongation or in dendritic pruning. During the dynamic process of establishing neural connectivity, dendrites extend and eliminate branches at a high rate70. Pruning occurs in both sexes, however, evidence suggests that the rates may vary. For example, dendritic pruning occurs in the medial prefrontal cortex of female, but not male, rats from P35 to P9071. Dendritic branch survival also depends on the strength of the synaptic connections established. Branches are preferentially formed in regions of active synapse formation and the rates of addition and pruning are affected by neural electrical activity72. Whether sex differences in pruning or neural electrical activity contribute to the brain region specific sex differences in primary and non-primary dendrite arbor morphology observed remain to be determined.Dendritic development can be broadly separated into two phases: (1) primary dendritogenesis which includes initiation of dendritic growth and extension of primary dendritic shafts; and (2) dendritic maturation which encompasses dendrite branching and elongation, spine formation and pruning28. The dentate gyrus granule cell layer of the adult rat hippocampus is also larger in male compared to female animals, and is correlated with performance in behavioral tasks of learning and memory, such as the radial arm maze and Morris Water Maze, where it is recognized that male rats perform better than female rats74. Human studies also indicate that hippocampal volumes increase in both sexes during adolescence but eventually diverge, increasing in males and slightly decreasing in females35. Future studies to address the growth trajectories of these endpoints in mice would greatly add to our knowledge of sex differences in dendrite morphology of the developing brain and help in guiding studies that employ these models to investigate NDDs.Our findings capture a snapshot of the sex differences observed in mouse CA1 hippocampal and adjacent somatosensory cortical neurons during early postnatal development. Whether these differences persist into later stages of development was not examined here, however, evidence from rodent and human data suggest that at least some of these differences persist and have functional consequences. Adult rat studies illustrate that CA1 pyramidal cell field volume and soma size are greater in male vs. female neurons40, our data suggest that inherent sex differences in dendritic morphology, which is a critical determinant of neuronal connectivity31 may in part contribute to the sex bias observed in NDDs. In line with our findings, human studies examining functional connectivity in the brain indicate that not only do typically developing males and females have different patterns of connectivity  but also that male and female ASD patients differ in their patterns of connectivity75. Thus, a pattern of hyperconnectivity is observed in ASD female vs. typical female, whereas a pattern of hypoconnectivity is observed in ASD male vs typical male, suggesting that ASD females shift towards a male pattern while ASD males shift towards a female pattern75. Disruption in neural masculinization/feminization patterns in ASD has also been reported for neuroanatomy76, task based brain activity77 and cognitive profiles78. In summary, our findings establish that there are significant sex differences in the dendritic morphology of typically developing CA1 pyramidal hippocampal and adjacent pyramidal somatosensory cortical neurons in juvenile mice and in primary culture. These results highlight the importance of examining sexually dimorphic effects of genetic mutations and environmental stressors on dendritic morphology in models of neurodevelopment, particularly in the context of NDDs where sex-specific patterns of connectivity likely impact NDD risk and/or severity.The question in the field remains as to what drives the sex bias in the onset, prevalence and severity for many NDDs. Given the central role of neuronal connectivity in many of these NDDsad libitum. Female mice were paired overnight with males to obtain timed-pregnant dams.All procedures involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of California Davis Animal Care and Use Committee (IACUC protocol numbers 18853 and 18682). C57BL/6 J wild type mice were purchased from Jackson Labs  and housed in clear plastic cages containing corn cob bedding. Mice were maintained on a 12 h reverse light and dark cycle at 22 ± 2 °C. Feed  and water were available 59. Additionally, criteria for the CA1 hippocampus were that the cell body was located in the middle third of the thickness of the section and in the somatosensory cortex cell body was located in Layer 4–5. Pyramidal neurons were selected based on their cytoarchitecture, including a clearly identifiable axon and apical dendrite. Basilar dendritic arbors of selected neurons were traced using NeuroLucida  and arbor complexity was quantified by branch structure analysis and Sholl analysis using NeuroLucida Explorer 59.Golgi staining was performed using the FD Rapid GolgiStain kit (FD NeuroTechnologies Inc.) according to the manufacturer’s instructions with the following modifications. After immersion in Solution C, brains were transferred to 10% sucrose in phosphate-buffered saline (PBS) for 3–4 h at 4 °C, and then stored in 30% sucrose in PBS for 3–4 days at 4 °C until sectioned. Prior to sectioning, brains were transferred to 70% ethanol in distilled water for 4 h at 4 °C. Brains were cut into 100-µm thick coronal sections using a vibratome . Sections were mounted on gelatin-subbed slides and stained according to the manufacturer’s instructions. Brightfield image stacks of CA1 hippocampal pyramidal neurons and pyramidal somatosensory cortical neurons adjacent to the CA1 hippocampus were acquired with an IX-81 inverted microscope  using MetaMorph Image Analysis Software  by an individual blinded to sex. Criteria for selection of neurons for Golgi analyses have been described previously52. Sex determination was confirmed by examination of the sexually dimorphic lower urinary tract to confirm the presence of testes or ovaries81. Cortical and hippocampal neurons were plated on German glass coverslips  precoated with poly-L-lysine (Sigma-Aldrich) at a density of 83,000 cells/cm2 and maintained at 37 °C in NeuralQ Basal Medium  or NeuralQ Basal Medium minus phenol red  supplemented with 2% GS21  and 1% GlutaMAX  under 5% CO2. On DIV 4, half the volume of culture medium was replenished and cytosine β-D-arabinofuranoside hydrochloride (Ara-C)  added at a final concentration of 2.5 μM to curb glial cell proliferation. On DIV 6, cells were transfected with plasmid encoding microtubule-associated-protein-2B (MAP2B) fused to either enhanced green fluorescent protein (EGFP) or red fusion (MAP2B-FusRed) construct 82 using Lipofectamine-2000  according to the manufacturer’s protocol. DIV 7 cultures were treated for 48 h with vehicle  to mimic common dosing paradigms used in assessing dendritic growth in response to environmental contaminants, drugs and other molecular targets53. In a subset of studies, DIV 7 cultures were treated for 48 h with either vehicle or the estrogen receptor antagonist ICI 182, 780 at 1 μM . At DIV 9, cultures were fixed with 4% paraformaldehyde and mounted to glass slides using ProLong Gold antifade reagent with DAPI (ThermoScientific). Slides were imaged using ImageExpress Micro XL high content imaging system  to obtain automated non-biased acquisition of EGFP or FusRed positive neurons from each group83. The dendritic complexity of individual neurons was quantified using ImageJ software84 with Sholl analysis plug-in v3.4.2 85 and with NeuronJ plug-in86 to quantify dendritic length, magnification used for image analysis was 0.65 microns per pixel.Primary neuron-glia co-cultures dissociated from the neocortex and hippocampus of P0 mouse pups were prepared as previously describedCell viability was tested in DIV 9 cultures. Lactate dehydrogenase (LDH) release was measured using the CytoTox-ONE™ Homogenous Membrane Integrity Assay  per the manufacturer’s instructions. Cell viability was also assessed in separate cultures co-stained with calcein-AM  and propidium iodide  to identify live vs. dead cells, respectively. Lysis control wells were incubated with 0.2% Triton X for 2 min. After a 30 min at 37 °C incubation in calcein AM and propidium iodide, cultures were washed in phosphate-buffered saline (PBS) and imaged using the ImageXpress Micro XL high content imaging system (Molecular Devices). The number of calcein-AM and propidium iodide-stained cells was quantified using MetaXpress 5.0 software (Molecular Devices), and the % live cells determined as the number of calcein-AM stained cells divided by the sum of the number of calcein-AM and propidium iodide-stained cells per field.Ar) F 5′-GATGGTATTTGCCATGGGTTG-3′ and R 5′-GGCTGTACATCCGAGACTTGTG-3′; estrogen receptor alpha (Esr1) F 5′-GCTGTGCACCCTGGAAATAAC-3′ and R 5′-CTCGACGACCAATGACCTCTC-3′; estrogen receptor beta (Esr2) F 5′-CATCCAGAAGATGTGGTGCTG-3′ and R 5′-GCTGCCTGTGGAAGACAAAG-3′; aromatase (Cyp19a1) F 5′- TGAGACGATTCCAGGTGAAGAC-3′ and R 5′- TGTCCTCATTTGGGTGCAAG-3′; peptidylprolyl isomerase a (Ppia) F 5′-TCTCTCCGTAGATGGACCTG-3′ and R 5′-ATCACGGCCGATGACGAGCC-3′; hypoxanthine guanine phosphoribosyl transferase (Hprt) F 5′-GGAGAGCGTTGGGCTTACCT-3′ and R 5′-CGGCAAAAAGCGGTCTGAGG-3′; and phosphoglycerate kinase 1 (Pgk1) F 5′- CCCATGCCTGACAAGTACTCC-3′ and R 5′-ACAGGCATTCTCGACTTCTGG-3′. Relative mRNA abundance was determined by the ΔΔCt method as described previously87 and normalized to the geometric mean of references genes Ppia, Hprt and Pgk1 abundance.RNA was purified from tissues using the Qiagen RNeasy kit  and from DIV 9 cultures using TRIzol reagent (ThermoFisher Scientific) according to the manufacturer’s instructions. RNA was reverse transcribed using the SuperScript VILO cDNA synthesis kit (ThermoFisher Scientific) according to manufacturer’s instructions. Real time PCR was performed in 12.25 μl reactions containing 6 μl POWER SYBR Green PCR Master Mix (ThermoFisher Scientific), 0.2 μM PCR primers (0.25 μl of 10 μM stock), 5 μl of cDNA and 1 μl of water and amplified using an Applied Biosystems 7900HT Fast thermocycler at the Real-Time PCR Research and Diagnostics Core Facility (UC Davis). PCR conditions were 50 °C for 2 min, 95 °C for 10 min, then 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec and 72 °C for 30 sec. QPCR was conducted on n = 6–8 tissues/wells per sex per brain region from at least three mice from independent litters or three dissections from independent litters using gene specific primers for androgen receptor ) covariance structure. All analyses were conducted using SAS® software version 9.4 of the SAS System for Windows® . For multi-level data, if an outcome variable did not appear to be normal based on histograms, summary statistics and residual plots, or if unequal variances were observed, the appropriate transformation was used to achieve approximate normality and Satterthwaite degrees of freedom were used in the mixed model to account for unequal variance. For all other endpoints, data were assessed for normality and homogeneity of variance using the Shapiro-Wilks test and F test respectively using GraphPad Prism v 6.07 . Differences between groups were assessed using Student’s T-test or Student’s T-Test with Welch’s correction for parametric data and by the Mann Whitney U test for nonparametric data. Cell viability assays were analyzed using a one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test for parametric data or Kruskal-Wallis test with Dunn’s multiple comparison test for nonparametric data. Area under the curve for the number of dendritic intersections, distance from soma of peak intersection (peak X), and maximum dendritic intersections (peak Y) values were calculated for Sholl profiles using built in area under the curve analysis in GraphPad Prism Software. Data are reported as mean ± SEM for Sholl plots and box and whisker plots indicate the mean as a (+) and whiskers are 10–90th percentile, p values ≤0.05 were considered significant.Golgi stained neurons from P28 brain sections were analyzed as described previouslyThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


Patterns of gene expression of human pregnancy are poorly understood. In a trial of vitamin D supplementation in pregnant women, peripheral blood transcriptomes were measured longitudinally on 30 women and used to characterize gene co-expression networks.Studies suggest that increased maternal Vitamin D levels may reduce the risk of asthma in early life, yet the underlying mechanisms have not been examined. In this study, we used a network-based approach to examine changes in gene expression profiles during the course of normal pregnancy and evaluated their association with maternal Vitamin D levels.The VDAART study is a randomized clinical trial of vitamin D supplementation in pregnancy for reduction of pediatric asthma risk. The trial enrolled 881 women at 10–18 weeks of gestation. Longitudinal gene expression measures were obtained on thirty pregnant women, using RNA isolated from peripheral blood samples obtained in the first and third trimesters. Differentially expressed genes were identified using significance of analysis of microarrays (SAM), and clustered using a weighted gene co-expression network analysis (WGCNA). Gene-set enrichment was performed to identify major biological pathways.Comparison of transcriptional profiles between first and third trimesters of pregnancy identified 5839 significantly differentially expressed genes (FDR<0.05). Weighted gene co-expression network analysis clustered these transcripts into 14 co-expression modules of which two showed significant correlation with maternal vitamin D levels. Pathway analysis of these two modules revealed genes enriched in immune defense pathways and extracellular matrix reorganization as well as genes enriched in notch signaling and transcription factor networks.Our data show that gene expression profiles of healthy pregnant women change during the course of pregnancy and suggest that maternal Vitamin D levels influence transcriptional profiles. These alterations of the maternal transcriptome may contribute to fetal immune imprinting and reduce allergic sensitization in early life.NCT00920621clinicaltrials.gov  Pregnancy is associated with many biological changes. While the structural and physiological changes are well characterized, the associated underlying molecular changes are less understood. The Barker hypothesis first posited that maternal dietary influences in pregnancy affect fetal development and can increase the risk of chronic disease later in life . Within Fetal growth is thought to be a multifactorial process that is modulated by maternal, fetal, placental and environmental factors . Studiesst trimester to the 3rd trimester. Our key concern is to illuminate the relationship between maternal levels of Vitamin D (VitD) and variation in gene transcription patterns. Our study is based on a nested cohort of 30 participants in the Vitamin D Antenatal Asthma Reduction Trial (VDAART), a multicenter randomized, controlled trial of vitamin D supplementation in pregnancy. VDAART has been registered in clinicaltrials.gov by ID# NCT00920621. The design, method and results for the primary outcome of the trial are published [We conducted a nested cohort study involving 30 healthy first trimester (10–18 weeks of gestation) women aged 18–39 years that were recruited as part of a larger multicenter, randomized, double-blind, controlled clinical trial from 3 different centers, Boston, San Diego and St. Louis . VDAART ublished , 22. Theublished .rd trimester at 32–38 weeks of gestation.Peripheral blood samples were collected at enrollment (at 10–18 weeks of gestation) and during the 3All specimens were analyzed at the Channing Laboratory DCC as previously described . The metTotal RNA was isolated from whole blood using the Paxgene Blood RNA Kit (Qiagen®) according to manufacturers protocol. The Ambion Globin Clear kit (Ambion®) was used to remove alpha and beta globin mRNA from the sample. This is done for whole blood samples in order to increase the sensitivity of gene expression assays, improving the detection rate of expressed genes. The RNA was quantified using Nanodrop 8000 and checked for high integrity before preparation of cDNA (first strand synthesis). The quality was assessed using the Agilent 2100 Bioanalyzer, which provides a good estimate of RNA concentration and purity of the sample as assessed by the RNA Integrity Number (RIN). An RIN ≥ 8 was deemed acceptable.Gene expression was assessed using the Illumina GX–Direct Hybridization Assay and Biotinylated–cRNA prepared according to manufactures protocol . Briefly, 200ng total RNA was reverse transcribed to synthesize first- and second-strand cDNA followed by a single in-vitro transcription that incorporates biotin-labeled nucleotides. The quality and quantity of the purified cRNA was assessed and samples hybridized onto Illumina HumanHT-12 v4 Expression Bead Chip. The chips were washed, blocked and stained with strepdavidin-Cy3 and scanned using Illumina’s Bead Array Reader. Quality control analysis and reports were created using Illumina’s GenomeStudio 2011.1 Software; Gene Expression Module version 3.2.7; iCheck version 0.5.0.2 among the 12 GC arrays ranging from 0.961 to 0.995, and pairwise R2 among duplicate and triplicate arrays ranging from 0.856 to 0.996. Probes analyzed were confined to those annotated to autosomal chromosomes. Background adjustment, log2 transformation and quantile normalization were performed using the functions lumiB, lumiT, and lumiN in R BioConductor's library lumi. Pairs of first/third trimester samples were hybridized to arrays on the same chip, blocked together to mitigate potential chip effects. The final expression data was further filtered to yield a final expression set of 36,116 probes with 60 samples, representing 30 subjects with samples collected at 10–18 weeks (M1) and 30–38 weeks (M2), respectively.The overall VDAART pilot gene expression data consist of 144 arrays from 12 HumanHT-12_V4_0_R2_15002873_B chips. Each chip has one genetic control (GC) array and 11 sample arrays. Each array has measures of gene expression levels for 47,304 sample gene probes and 882 quality control probes. We excluded 13 technically failed arrays, and one frankly outlying sample. Statistics on control arrays indicated good reproducibility of the protocol, with pairwise Rhttp://www.r-project.org). In conjunction with this platform, several Bioconductor tools for detailed gene expression analysis (http://www.bioconductor.org) were also used. Differential expression analysis was carried out using the Bioconductor package “siggenes” and a two class, paired case significance of analysis of microarrays (SAM) performed [Gene expression measurements were analyzed using the publicly available open source statistical computing and graphics software tool “R” . WGCNA rhttp://genemania.org). To this end, we first matched transcripts to their corresponding official human gene symbols and uploaded these to the GeneMANIA website. The options Genetic, Physical and Predicted interactions were applied and the attributes Consolidated Pathways, InterPro and Transcriptional-Factor-Targets-2013 selected. The query was run with the setting: “equal by network" and with number of related genes set to “0”. This ensured that only module genes measured in our study were used in the pathway analysis. The analysis consisted of those genes for which gene symbols were recognized by the webtool, while any duplicate genes and those that did not match any annotated Gene Symbol were excluded. A biological network analysis was performed on the modules of interest using the “Consolidated Pathway” function as described by GeneMANIA [http://bioinfo.vanderbilt.edu/webgestalt). P-values for differential network or gene-set expression were obtained via the hypergeometric method, adjusted for multiple comparisons using the Benjamini & Hochberg FDR measure.Modules with significant correlation to VitD levels were chosen for further characterization to identify relevant biological pathways using the GeneMANIA platform  and a transcription factor network enrichment analysis performed. The “Transcription Regulation Workflow” creates sub-networks that are centered on transcription factors as described by MetaCore ™. Each sub-networks contains nodes from module of interest that are labeled (seed nodes). Transcription factors sub-networks are ranked by a p-value and interpreted in terms of Gene Ontology (GO).We used the web accessible MetaCore™ platform from Thompson Reuters to perform additional module network analysis to generate a list of key transcription factors networks and their targets within modules of interest. Transcripts within modules of interest were converted to Entrez IDs and uploaded to the MetaCore™ website at D levels of 47.2 nmol/L and third trimester levels of 79nmol/L (Table 1). We compared this nested cohort to the remaining VDAART study cohort of 846 women and found no significant differences in population characteristics (Table 2).We examined changes in gene expression profiles over the course of pregnancy in a cohort of 30 pregnant women all of whom contributed whole blood samples in both the first and the third trimesters of pregnancy. The cohort was 80% African American and 20% Caucasian, with mean age of 25.2 years and mean pre-pregnancy body mass index (ppBMI) of 32.61kg/mFig 1). Of these, 2473 transcripts were significantly increased expression in third relative to first trimester, while 3366 transcripts had decreased expression in the third, relative to the first, trimester. Up and down-regulated transcripts were ranked based on the lowest p-value. Top differentially expressed genes (S1 Table).To identify differentially expressed transcripts, we used the significance analysis of microarrays (SAM) algorithm of Tusher et al.  to compuTo enable a comprehensive analysis of the expressed gene list that takes into account all significantly differentially expressed transcripts, we used a well described (unsupervised) methodology of gene correlation network analysis to cluster transcripts into groups of highly interconnected modules based on topological overlap mapping, Weighted Gene Correlation Network Analysis (WGCNA) .Fig 2). Each module varies in size, consisting of different numbers of transcripts per module and is represented by a single eigengene value ; this “eigengene vector” is a theoretical average gene expression value based on actual expression levels of all transcripts in the module. To identify modules with transcripts of greatest clinical relevance, the eigengene values of each module was further correlated to various maternal traits. This led to the identification of three modules , each consisting of 241, 287 and 61 transcripts, respectively, that were significantly correlated to maternal pre-pregnancy body mass index (ppBMI), race and VitD levels at both early (enrollment) and late time points (3rd trimester). Specifically, our data show that the green module is positively correlated with both baseline VitD and late VitD levels with correlation coefficients of 0.35 (p = 0.05) and 0.45 (p = 0.01), respectively. Similarly, the yellow module was significantly correlated with late VitD levels, with a correlation coefficient of 0.38 (p = 0.04) while the salmon module is positively correlated to maternal ppBMI, and negatively correlated to both baseline VitD level and mother’s race, with correlation coefficients of 0.41 (p = 0.02), -0.41 (p = 0.02) and -0.44 (p = 0.07) respectively . After adjusting for the effects of VitD using multiple regression analysis, race and ppbmi were no longer significantly correlated with the salmon module.The weighted gene correlation network analysis clustered 5839 transcripts into 14 co-expression modules, with the gray module representing those transcripts with the lowest connectivity scores  of the 241 transcripts associated with the green module and 61 transcripts of the salmon module, we used the open source, web based GeneMANIA analysis tool to perform gene-set enrichment and identify major biological pathways using enrichment maps as accessed through the Consolidated Pathways option. Of the 61 genes uploaded to GeneMania, 46 (75%) were recognized as genes by the webtool, Overall, the consolidated pathway analysis revealed an enrichment of 11 of the 46 genes in a variety of biological pathways largely involved in immune defense, extracellular matrix reorganization and degradation, as well as activation of metalloproteinase genes . In addition to the pathway enrichment analysis, we performed a functional enrichment analysis using Gene Ontology (GO). The salmon module was associated with a variety of significant GO terms, such as extracellular matrix reorganization, secretory granule as well as heparin and glycosaminoglycan binding (S2 Table).Following WGCNA analysis, we wished to explore in more detail the nature of the green and the salmon module as these showed significant correlation with VitD levels at the 1Fig 4). Of the 241 genes, 184 (76%) had recognizable gene symbols (on GeneMANIA). Of the 184 annotated genes, 18 were found to be associated with known pathways such as glutathione biosynthesis, Notch Signaling pathway and E2F transcription factor network, among others. Functional GO enrichment of the Green module genes yielded significant GO functional terms before, but not after adjusting for multiple testing (S3 Table).Similarly, pathway analysis of the Green module resulted in the enrichment of several biological pathways (S4 Table).To gain additional insight into possible transcription factor networks identified by pathway enrichment, we used the MetaCore™ software to examine whether key transcription factor networks and their gene targets are overrepresented in the green module. To this end, we uploaded Entrez IDs of the 241 green module transcripts onto the MetaCore platform that converted to 202 annotated, unique, gene symbols. Subsequent transcription factor network analysis of these 202 genes identified 21 different transcription factors that were either significantly enriched in the green module or acted on target genes within the green module (−20), contained 73 total nodes of which all 73 nodes (100%) were derived from the original data set (green module). In contrast, the c-Myc transcription factor sub-network, which was also found to be significantly enriched (p<10−20), was found to have a total of 49 nodes of which 48 (98%) were derived from the green module data set (identified as “seed nodes”). Indeed, our data show that of the 21 transcription factor sub-networks, only the CREB1 network was fully contained within the module, with both the transcription factor (CREB1) and a high number of target genes (n = 72) with known interactions being present .The top five sub-networks were represented by, cAMP responsive element binding protein 1(CREB1), Myc Proto-Oncogene Protein (c-Myc), tumor protein p53, zinc finger protein (ZNF) 143 and glucocorticoid receptor-alpha . The total nodes for each sub-network were calculated and the number of seed nodes (number of genes from the green module) identified. Our data show, that the CREB1 transcription factor sub-network represented the sub-network with the lowest p-value [st and the 3rd trimester. Of these, 57% (3366 probes) were down regulated in later gestation while 42% (2473 probes) were up regulated . The relatively large number of differentially expressed transcripts likely reflects the dramatic physiological changes associated with pregnancy that ultimately enable the maternal support of a developing human fetus [In this study, we have conducted a longitudinal whole genome expression analysis over the course of pregnancy ranging from the 1 (VDAART). Initialan fetus .Fig 2). Correlating gene modules with clinical traits enables identification of those traits that likely impart significant influence on gene expression profiles during pregnancy. In this regard, our data show that 2 of the 14 modules, green and salmon , were significantly associated with serum vitamin D levels both, during the 1st and 3rd trimester of pregnancy. The salmon model was also found to be negatively correlated with race and positively correlated with pre-pregnancy BMI . While these observations suggest a mixed influence over gene expression profiles that are driven by all 3 covariates, subsequent multiple regression analysis revealed that two of the variables (pre-pregnancy BMI and race) behaved in a collinear fashion, as their effects become non-significant after adjustment for serum VitD level. As a result, our analysis suggests that transcriptional profiles of the salmon and green module are significantly associated with maternal vitamin D levels during pregnancy.To derive meaningful biological information from large gene lists, we employed an unsupervised network approach based on co-expression analysis using WGCNA . This apTo gain insight into the biological significance of the two modules , we performed a pathway enrichment analysis. The analysis revealed that the salmon module is enriched in genes largely involved in antimicrobial processes and immune defense mechanisms. In addition, we observed a significant enrichment for genes associated with cellular rearrangement as well as collagen and extra cellular matrix deposition/reorganization. Interestingly, this module was enriched for genes responsive to vitamin D regulation, particularly neutrophil associated genes such as defensins, antimicrobial peptide cathelicidin (CAMP) and Neutrophil Elastase (ELANE) , 30. In p = 3.9X10-193). CREB1 is a major transcription factor that regulates over 73 genes (of various functions) within the green module  and is associated with a variety of GO Biological processes. Interestingly, CREB1 has been shown to interact with C/EBP family of transcription factors [Similar pathway analysis of the green module revealed biological pathways such as cell cycle regulation, transcription factor networks, glutathione regulation and several metabolic pathways. To examine the nature of the transcriptional networks more closely, we conducted additional (supervised) pathway enrichment analysis and hypothesized that pregnancy related transcriptional changes are likely associated with the activation of various transcription factors. The resulting network analysis identified 21 significant transcriptional networks with cyclic AMP Responsive Element Binding Protein 1 (CREB1) ranked as the top, most significant transcription factor network Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


A-to-I RNA editing is an important process for generating molecular diversity in the brain through modification of transcripts encoding several proteins important for neuronal signaling. We investigated the relationships between the extent of editing at multiple substrate transcripts  in brain tissue obtained from adult humans and rhesus macaques. Several patterns emerged from these studies revealing conservation of editing across primate species. Additionally, variability in the human population allows us to make novel inferences about the co-regulation of editing at different editing sites and even across different brain regions.The online version of this article (doi:10.1186/s13041-017-0291-1) contains supplementary material, which is available to authorized users. Imp. Imp2]. eceptors  4]to chto ch2C receptors , GLUR4  10] [10]2C r [10]2C receptors  indicaterhesus macaque brains supporting the notions that the precise extent of editing at each of these substrates is conserved, tightly regulated, and functionally important. These results provide evidence that editing at certain substrates may be co-regulated within certain anatomical contexts and regulated by divergent mechanisms in others. When comparing ADAR mRNA expression levels with editing profiles we observe no direct relationship between mRNA expression and the extent of editing at any site analyzed in this study, supporting the hypothesis that A-to-I editing is regulated downstream of ADAR expression. Interestingly, we did observe a direct relationship between the mRNA expression levels of ADAR1 and ADAR2 mRNA transcripts over a large dynamic range in both species. Furthermore, observations in the human population indicate that certain individuals harbor multi-substrate deficiencies in A-to-I editing that generalize across each of the analyzed substrates while others display apparent deficiencies in editing at only a few substrates in certain neuroanatomical contexts.For this study, we simultaneously quantify the extent of editing at several substrates in cortical and striatal tissue samples by targeted multiplex transcript analysis . Interesp > .05) did not reveal any evidence that the parameters discussed here are significantly influenced by diagnosis, sex, age or PMI . After observing that 5HT2C editing was not altered in the PWS we decided to leverage the remaining available tissue to investigate other aspects of the regulation of RNA editing presented in this study. After we analyzed editing in each tissue sample, we first verified that PWS diagnosis, sex, age, and postmortem interval (PMI) did not have an effect on patterns of editing or RNA expression. Correlation analysis  were analyzed by multiplexed targeted transcript analysis which takes advantage of massively parallel sequencing on the MiSeq sequencing platform . RNA editing was detected at each site in all brain samples from humans and monkeys. The mean extent of editing observed for each respective editing site in these two species were generally similar, however considerably more variability was observed in the human population compared to the monkey at each substrate . Additional interspecies differences were observed for the extent of editing at various substrates in specific brain regions, these are summarized in Fig. While the mean extent of editing at most sites was found to be similar in monkey and human, the extent of editing at GLUR4-Flip and GLUR4-Flop transcripts is higher in both brain regions in the monkey (Cortex: Flip +25 p < .01). CADPS editing was significantly lower in the cortex than in striatum in both species (p < .01)  Fig. . The conWhen profiling the extent of editing in the human cohort we noticed higher levels of variability than we typically observe in rodent cohorts . This wap < .0001, r2 = .8, Fig. Our observations support the previously reported model that editing at each of the five sites on 5HT2C transcripts are co-regulated . The extp > .001 r2 = .62). Thus, the extent of editing at GLUR4-Flip is a strong predictor of the extent of editing at GLUR4-Flop. This direct relationship could be related to the fact that these transcripts are generated by alternative splicing of the same pre-mRNA . Re. Re12]. The FASTX-Toolkit was used to split the reads according each of the 24 different barcodes. For each read, we then used a script for pairwise alignments against all of the reference sequences allowing for only one mismatch (at the editing site) and assigned each match to its corresponding candidate gene. We then counted the nucleotide composition at the editing position and considered any adenosine at the corresponding site “not edited” and any guanosine “edited”, other nucleotides were detected at a very low rate presumably due to sequencing or polymerase errors and were not counted.F5 was detected using a Fam labeled Taqman probe and primer set (Mm00484202_m1) and human ADAR expression was detected using probe and primer sets ADAR1 Hs00241666_m1) and ADAR2 (Hs00953724_m1), and rhesus ADAR expression was detected with probes for ADAR1 (Hs00241666_m1) and ADAR2 (Rh00955199_m1). For qRT-PCR, first strand cDNA was synthesized using random primers using the High-Capacity Reverse Transcription Kit and expression was analyzed according to manufacturer’s instructions for Taqman real-time PCR  on a CFX96 platform . Parallel amplification efficiencies were verified by PCR miner algorithm [1666_m1 aT-test for Prader-Willi diagnosis and sex; or linear regression for the continuous variables ADAR expression, age, and PMI. No significant effect was identified for any of these variables (p > .05). Standard statistical analysis (t-test) was used for pair wise comparisons. When analyzing correlations we used Pearson’s product moment to determine p-values.Analysis of covariance was performed in human samples by plotting the extent of editing at each respective site by Prader-Willi diagnosis, age, sex, and PMI. Covariance was determined by Additional file 1: Figure S1.A Comparing extent of editing at each site between males and females in both cortex and striatum does not reveal any significant effects on the extent of editing . B Comparing the extent of editing at each site between patients diagnosed with PWS and and normal controls in both cortex and striatum does not reveal any significant effects on the extent of editing . C Linear regression analysis comparing the age of each individual at the time of death does not reveal any significant effects on the extent of editing (p > .05). D Linear regression analysis comparing the post mortem interval to the extent of editing at each site does not reveal any significant effects on the extent of editing (p > .05). (DOCX 1894 kb)Plots for covariant analysis of various factors that were performed to determine the potential effects of sex, PWS diagnosis, age, and postmortem interval on the extent of editing at each respective site in the human cohort. Additional file 2: Figure S2.A Comparing the ADAR1 mRNA expression levels as determined by real-time PCR analysis to the extent of editing at each site does not reveal any significant effect on the extent of editing (p > .05). B Comparing the ADAR2 mRNA expression levels as determined by real-time PCR analysis to the extent of editing at each site does not reveal any significant effect on the extent of editing (p > .05). (DOCX 1356 kb)Plots for linear regression analysis for the effect of ADAR mRNA expression on the extent of editing at each site in humans. Additional file 3: Figure S3.A Comparing the ADAR1 mRNA expression levels as determined by real-time PCR analysis to the extent of editing at each site does not reveal any significant effect on the extent of editing (p > .05). B Comparing the ADAR2 mRNA expression levels as determined by real-time PCR analysis to the extent of editing at each site does not reveal any significant effect on the extent of editing (p > .05). (DOCX 960 kb)Plots for linear regression analysis for the effect of ADAR mRNA expression on the extent of editing at each site in monkeys. Additional file 4: Figure S4.p > .05). (DOCX 200 kb)Plots comparing the observed spontaneous activity of monkeys used in these studies to the extent of editing at each respective site. The two activity groups did not have significantly different extent of editing at any of the analyzed sites (


Chronic kidney disease (CKD) is a syndrome characterized by progressive and irreversible deterioration of renal function linked to slow destruction of renal parenchyma, eventually terminating in death when sufficient number of nephrons are damaged. Oxidative stress is commonly observed in CKD patients resulting from an imbalance between overproduction of reactive oxygen species (ROS) and impairment of defence mechanisms. Grape seed extract (GSE) is a polyphenolic mixture exhibiting antioxidant and anti-inflammatory properties. We conducted an interventional pilot study of supplementation with GSE capsules  or placebo  on CKD patients. Blood and urine samples were collected at baseline and after a six-month-long supplementation period to determine some renal function biomarkers, as well as antioxidant, anti-inflammatory and haematological parameters. GSE improved glomerular filtration rate (GFR) and proteinuria, increased the anti-oxidant status as assessed by high plasma catalase and superoxide dismutase and also lowered lipoperoxidation and carbonylation. GSE ameliorated inflammation by decreasing CRP, triglyceridemia and counteracted anemia and thrombocytopenia. Supplementation with 2 g GSE/day for six months improved some kidney function parameters of CKD patients and this beneficial effect of GSE seems to be mediated at least partly by its antioxidant and anti-inflammatory properties. Chronic kidney disease (CKD) is a slow and progressive decline of kidney function that is often linked to long standing diseases such as diabetes and hypertension . It is Grape seed extract (GSE) is a complex polyphenolics mixture containing flavonoïds, non flavonoïds, proanthocyanidins exhibiting multi-organ protection in various experimental settings . For inThe present work aims to test the ability of GSE to improve kidney dysfunction of CKD patients from various renal stages i.e stage 2, 3, 4. Data mainly show that although used at a low dosage, GSE improves several kidney function parameters as well as the anti-oxidative status of CKD patients after a 6-month-supplementation period.CKD patients with renal failure  were recruited as volunteers at the haemodialysis unit of the Regional Hospital of Menzel Bourguiba for a randomized double blind and placebo control pilot study. All patients suffered from diabetic nephropathy and hypertension. They were treated with insulin (two doses per day), received captopril 50 mg (2 tablets/day) for hypertension, furosemide 40 mg (asylix 2 tablets/day) for nephropathy, folic acid (foldine 2 tablets/ day) and iron (2 tablets/day) for anemia. Key exclusion criteria included smokers and haemodialysis patients receiving antioxidant supplements in the previous six months. All patients were asked to keep their habits and medication as usual and were dispatched into two groups: experimental and placebo. Patients in the placebo group (n = 10) were distributed as follows:2)3 patients were from stage 2 (GFR ≤ 90 ml/min/1.73m2)4 patients were from stage 3 (GFR ≤ 60 ml/min/1.73m2)3 patients were from stage 4 (GFR ≤ 30 ml/min/1.73mPatients in the GSE group (n = 23) were distributed as follows:6 patients were from stage 2 11 patients were from stage 36 patients were from stage 4The GSE group was daily supplemented with six capsules of GSE, each capsule containing 350 mg of grape seed powder. Two bottles of 90 capsules each were provided to each patient for one month during the entire six months long study which lasted from May to October 2011. The placebo group received starch containing capsules in the same conditions. Blood samples were withdrawn after an overnight fasting at the beginning of the study and after 6 months of supplementation with either GSE or placebo. Urines were also collected at baseline and at the end of the experimentation for proteinuria determination. The study protocol was approved by the ethics committee on clinical research of the Regional Hospital of Menzel Bourguiba and informed consent was obtained for all participants. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000 and 2008.Vitis vinifera from northern Tunisia. Seeds were carefully separated from skin, washed, dried and grounded with an electric grounder until a fine powder was obtained. After mixing with 3 % colloïdal silica as excipient, the powder was conditioned into capsules weighting approximately 350 mg. Total polyphenolics content as well as polyphenol composition were determined previously  of al., 2014).2O2  method. Plasma was processed by centrifugation at 3000 rpm for 10 min and used for the determination of renal function biomarkers as urea and uric acid using commercial kits from Biomaghreb, Tunisia. Urine urea and uric acid were also determined and proteinuria evaluated using the biuret method . Oxidatal., 1979), free ial., 1979) as wellal., 1979) and glual., 1979).Inflammation was evaluated using plasma CRP and LDH. Triglyceridemia and cholesterolemia were measured using commercial kits from Biomaghreb . P value less than 0.05 was considered signiﬁcant.We reported in Figure 1GSE also affected several plasma biomarkers of the antioxidant status. GSE slightly decreased plasma MDA by -14.96 % Figure 2A. We reported in Table 1Table 2We reported in Table 3The present work is a preliminary and pilot study dealing with the effect of GSE treatment on CKD patients of various renal failure stages. Data mainly show that even when used at a low dosage, GSE exerts a clear beneficial effect on some renal function biomarkers as well as on the antioxidant status of CKD patients. To our knowledge, our work is the first one to describe such relevant and hopeful clinical trials data for million CKD patients worldwide. First of all GSE improves some kidney function parameters as it enhances GFR and clearly lowers proteinuria. This result is of utmost importance as reduction in proteinuria to the lowest achievable level is an important predictor of long term renal protection , as it The beneficial effect of GSE on renal function seems to be mediated, at least partially, by its antioxidant and anti-inflammatory properties. After a six-month-period of treatment, GSE counteracts both plasma lipoperoxidation and carbonylation as well as CRP and LDH, and clearly increases plasma CAT and SOD . MoreovRemarkably that in our present case, GSE improvement in kidney function of CKD patients has been obtained at a low GSE dosage of 35 mg/kg if we consider the weight of the starting grape seed powder, and highly lower if we consider the polyphenol content. Our laboratory has recently demonstrated the efficiency of high dosage GSE till 4 g/kg) in multi-organ protection against various insults as obesity , stro g/kg in Nevertheless the current interventional pilot study suffers from several limitations. First the sample size is too small to be of robust statistical significance. Although GSE was used at low dosage, a significant improvement in the decline of some kidney function biomarkers after a six-month-treatment period was obtained. Duration of the study should be extended to increase blood and urine sampling and a ten fold higher GSE dosage (350 mg/kg) should be tested to reach improvement of statistical significance. Owing to its tremendous safety, GSE is increasingly used at high dosage (more than 500 mg/kg) according to our own data  or to cIn conclusion, due to the multi-organ safety and beneficial health effects of grape polyphenols, clinical trials using high GSE dosage should be urgently planned to confirm these preliminary and hopeful data.The authors declare no conflict of interest.


Mitochondrial diseases are defined by a respiratory chain dysfunction and in most of the cases manifest as multisystem disorders with predominant expression in muscles and nerves and may be caused by mutations in mitochondrial (mtDNA) or nuclear (nDNA) genomes. Most of the proteins involved in respiratory chain function are nuclear encoded, although 13 subunits of respiratory chain complexes (together with 2 rRNAs and 22 tRNAs necessary for their translation) encoded by mtDNA are essential for cell function. nDNA encodes not only respiratory chain subunits but also all the proteins responsible for mtDNA maintenance, especially those involved in replication, as well as other proteins necessary for the transcription and copy number control of this multicopy genome. Mutations in these genes can cause secondary instability of the mitochondrial genome in the form of depletion (decreased number of mtDNA molecules in the cell), vast multiple deletions or accumulation of point mutations which in turn leads to mitochondrial diseases inherited in a Mendelian fashion. The list of genes involved in mitochondrial DNA maintenance is long, and still incomplete. The number of mitochondria in the cell varies and depends on the cell type and energy requirement, where cells with greater energy needs have more mitochondria than cells with smaller needs . Additionally, according to the newest discoveries, human mtDNA also encodes three short peptides, humanin, gau proteins and MOTS-c, with important biological functions, e.g., humanin plays a significant role in protecting neurons from apoptosis in Alzheimer’s disease  required for the proper function of all mitochondrial biochemical pathways, including the rest of the subunits of respiratory complexes, are encoded by nuclear genes. This means that mitochondrial DNA expression, maintenance, copy number regulation, and repair processes rely on the nuclear genome  and adult patients (1:4300 affected or at risk)  are inherited in a Mendelian fashion  are collectively called mitochondrial maintenance diseases or mtDNA depletion syndromes. The main feature of those disorders is rearrangement of the mitochondrial genome seen as multiple deletions of mitochondrial DNA molecules (the presence of multiple classes of mtDNA molecules of different lengths) and/or decrease of mtDNA copy number in cells, known as mtDNA depletion . The number of mtDNA molecules in one nucleoid is still being discussed — it seems to be one to a few. Obviously, replication plays the main role in the maintenance of mitochondrial DNA  of replication was suggested but it was partially incorrect due to artifacts which occurred during the preparation process. An updated version of the SDM is called the RNA intermediate throughout the lagging-strand (RITOLS) model. Both models imply the presence of two origins of replication , one on the heavy (H) strand and one on the light strand (L), called OH and OL respectively . This model implies that synthesis is initiated bidirectionally from multiple origins of replication at ori zone (ori z). The leading H strand is synthesized continuously and the lagging L strand is formed without delay as Okazaki fragments  (Holt and Reyes POLG gene) and its processivity factor (encoded by the POLG2 gene) together with Twinkle helicase (encoded by the TWNK gene), DNA replication helicase/nuclease 2 (encoded by the DNA2 gene), single-stranded DNA binding protein 1 (encoded by the SSBP1 gene), primase and polymerase (DNA-Directed) (encoded by the PRIMPOL gene), and mitochondrial genome maintenance exonuclease 1 (encoded by the MGME1 gene) play the key role in mitochondrial DNA maintenance and replication processes . Polγ is the only DNA polymerase involved in mtDNA replication present in the human mitochondrion  and a homodimer of accessory subunits POLG2 encoded by the POLG2 gene  , a linker domain (419–755 amino acids), and 5′-3’polymerase activity responsible for replication (756–1239 amino acids) , a fatal infant disease with epilepsy and drug induced liver failure, mitochondrial DNA depletion syndrome 4B  with gastrointestinal involvement, mitochondrial recessive ataxia syndrome , and relatively benign progressive external ophthalmoplegia autosomal dominant 1 (MIM 157640) and autosomal recessive 1 (MIM 258450)  develops as a result of mutations in the POLG2 gene.Mutations affecting polymerase gamma result in a wide range of genetic syndromes with many mtDNA mutations, deletions, multiple deletions, and depletion of mitochondrial DNA . TWINKLE is a mitochondrial 5′-3′ helicase necessary for replication of human mitochondrial DNA  encoded by the TWNK gene studied in cell cultures and deletor mice resulted in blocking of the replication process, accumulation of intermediates and finally in multiple mtDNA deletions . SSBP1 interacts with polymerase gamma and helicase Twinkle and strengthens their functions (Hudson and Chinnery Stabilization of the replication fork through preventing binding strands of a replicated fragment of DNA from forming secondary structures and degradation is the main role of single stranded DNA binding protein 1 (SSBP1) encoded by the Saccharomyces cerevisiae RIMI null mutants (RIMI encodes ssDNA binding protein)  gene from Drosophila melanogaster   encodes nuclear and mitochondrial primase and DNA directed polymerase. PRIMPOL plays a key role in mtDNA replication initiation. Moreover, it enables the replication machinery to replicate past DNA lesions , e.g., in apurinic/apyrimidinic sites (AP sites). This protein is present both in the nucleus and mitochondria. Silencing of the PRIMPOL gene in human fibroblasts leads to multiple mtDNA deletions and depletion. A PRIMPOL mouse knockout is viable but mtDNA replication deficiency is observed on the cellular level. This confirms that absence of PRIMPOL has multiple adverse effects on mtDNA synthesis . It has nuclease, helicase, and ATPase activity and interacts with polymerase gamma by stimulating its catalytic activity. DNA2 removes RNA primers and stabilizes mtDNA structure during the replication process; thus, playing an important role in the maintenance of mitochondrial DNA. Moreover, DNA2 participates in repair of small DNA lesions induced by oxidation, alkylation or spontaneous hydrolysis and is critical for long-patch base-excision repair (LP-BER)  and dominant progressive external ophthalmoplegia with mitochondrial DNA deletions (MIM 615156).Mutations in MGME1 gene  encodes mitochondrial genome maintenance exonuclease 1 (MGME1) protein probably involved in mtDNA repair  during replication and enables the processing of mtDNA ends due to the ability to cleave dsDNA in both 5′-3′ and 3′-5′ directions. Thus this exonuclease can cut 5′ and 3′ flaps. Moreover, MGME1 may enhance exonuclease activity of polymerase gamma .RNASEH1 gene  is an endonuclease involved in DNA replication and repair processes both in the nucleus and mitochondria, but in mitochondria it is the only ribonuclease of that type while in the nucleus there are two (besides ribonuclease H2). Ribonuclease H1 specifically digests double–stranded DNA-RNA hybrids and is necessary to produce primers for mtDNA replication.Ribonuclease H1, encoded by the Rnaseh1 leads to embryonic lethality with mtDNA depletion   and HSP2  is responsible for transcription of the mitochondrial genome and also provides primers for mtDNA replication, therefore all changes in the enzyme structure (or transcription complex) may impact mtDNA stability. POLRMT together with TFAM, TEFM, and TFB2M forms a transcription complex  is the mitochondrial transcription factor A coding gene also known as TCF6L3 or mtTFA. TFAM is a key activator of mitochondrial transcription, plays an important role in mitochondrial DNA replication and copy number regulation, and is crucial for mitochondrial biogenesis. TFAM expression and turnover depends on the interaction between POLMRT, TFAM, and mitochondrial DNA . A mutation in the TFAM gene has been recently described as a cause of neonatal liver failure with mtDNA depletion  is responsible for transcript elongation. TEFM forms a complex with mitochondrial RNA polymerase, interacts with its catalytic domain, enhances processivity  is a part of the mtDNA transcription complex  to deoxyribonucleoside diphosphates (dNDP) – basic elements for DNA synthesis  phosphorylates deoxythymidine, deoxycytidine, and deoxyuridine, while deoxyribonucleoside kinase (dGK) phosphorylates deoxyguanosine and deoxyadenosine  encodes a mitochondrial matrix enzyme – thymidine kinase 2 (TK2). TK2 is an enzyme essential for mtDNA maintenance, catalyzes the rate-determining step of the pyrimidine salvage pathway  and mitochondrial DNA depletion syndrome 2   with a hot spot in exon 5  were present in DNA isolated from muscle biopsies from patients with myopathy. They also observed a dramatic decrease of mtDNA copy number in cells. Structural analysis of the enzyme showed that missense mutations were linked with binding affinities of dTMP and dCTP  provides phosphorylated purines necessary for mtDNA synthesis (Jullig and Eriksson Deoxyguanosine kinase (dGK), another matrix enzyme, encoded by the In silico analysis suggested that the most frequent mutations in the gene affect the structure of dGK. Biochemical analysis of the activity of dGK isolated from skeletal muscles from myopathic patients showed that mutations may impair the enzyme function  encodes ribonucleotide reductase regulatory TP53 inducible subunit M2B (p53R2), a part of ribonucleotide reductase. This cytoplasmic enzyme is responsible for conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates and is an element of the de novo nucleotide biosynthesis pathway. Ribonucleotide reductase is composed of large R1 and small R2 subunits. There are two types of R2 subunits in the cell. One is present during the S phase of the cell cycle only, the second one, encoded by RRM2B, provides the basal level of deoxyribonucleoside diphosphates. mtDNA replication, generally independent of cell cycle, takes place not only during the S phase, p53R2 protein is crucial for mtDNA synthesis.RRM2B lead to mtDNA depletion  (MIM 612075), mitochondrial DNA depletion syndrome 8B (MNGIE type) (MIM 612075), and progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant 5 (MIM 613077).Diseases associated with mutations in the TYMP gene . This cytosolic enzyme of the salvage pathway catalyzes the cleavage of thymidine into thymine and 2-deoxy-α-D-ribose-1-phosphate and plays an important role in catabolic processes  is another protein important for nucleotide biosynthesis encoded by the TYMP lead to accumulation of nucleosides and an imbalance in the mitochondrial nucleotide pool and results in MNGIE type autosomal recessive mitochondrial DNA depletion syndrome 1 (MIM 603041)  encoding a heart muscle specific isoform of a solute carrier family 25  member 4 also known as ANT1 was the first gene in which mutations responsible for mtDNA instability were described  (MIM 609283) seem to show a dominant-negative effect on the molecular level, while the phenotype caused by recessive ones ) (MIM 615418) is more similar to the one obtained for knockout mice.Mutations in OPA , MFN1 (mitofusin 1), and MFN2 (mitofusin 2) encoding proteins involved in fusion, FIS1 , DNM1L , and MFF  important for proper mitochondrial division  gene encodes mitochondrial dynamin like GTPase. OPA1 plays an important role in mtDNA maintenance, mutations in the OPA1 gene lead to mtDNA multiple deletions , optic atrophy plus syndrome (MIM 125250) and autosomal recessive Behr syndrome (MIM 210000). In one consanguineous family, mitochondrial DNA depletion syndrome 14  (MIM 616896) due to a homozygous OPA1 mutation has been described.More than 200 mutations in the MFN1 gene  encodes a transmembrane GTPase localized in the mitochondrial outer membrane. MNF1 (mitofusin 1) forms homomultimers and heteromultimers with MFN2 (mitofusin 2) and together they are responsible for outer mitochondrial membrane fusion. There are no human diseases attributed to MFN1 mutations.The MFN1, the MFN2 gene  encodes a transmembrane GTPase localized in the mitochondrial outer membrane and shares high homology with MFN1. Two transcriptional forms are known. Mitofusin 2 was described as a protein enabling close contact of mitochondria with the endoplasmic reticulum. Recently the opposite was found: depletion of MFN2 results in a closer contact with ER  but also autosomal dominant hereditary motor and sensory neuropathy VIA (MIM 601152) and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A2B (MIM 617087). mtDNA depletion and multiple deletions were observed in muscles and fibroblasts from patients with AD Charcot-Marie-Tooth disease caused by MFN2 mutations  encodes another member of the GTPase family: dynamin-1-like protein which regulates mitochondrial function and plays a crucial role in the division, fusion, and fission of mitochondria. DNM1L forms an oligomeric ring at the division spot that narrows and splits the mitochondrial tubule  development, its absence causes abnormality in embryonic development. Nonsense mutations disturb central nerve system development. Yoon et al.  and knock-out mice. Mutations in the DNM1L gene cause encephalopathy which is lethal due to defective mitochondrial and peroxisomal fission (type 1) .The n et al.  have shoMFF gene encodes the mitochondrial fission factor , which is an outer membrane protein required for localization of DNM1L and division of mitochondria. MFF protein together with FIS1 are responsible for recruitment of DNM1L to the division site .Mutations in FIS1 gene  acts independently of MFF. No diseases caused by FIS1 mutations have been described.FIS1 – tetratricopeptide repeat domain-containing protein 11 encoded by the Besides the above-mentioned genes encoding proteins involved in the processes with more or less well described influence on mtDNA stability, there are multiple other genes not involved in any of these processes in which mutations lead to mtDNA deletions or depletion. Here we mention only a few of them with the highest impact on human health.MPV17  mutations were described as a cause of autosomal recessive mitochondrial depletion syndrome ten years ago  encodes paraplegin which is a component of the mitochondrial AAA protease. Spastic paraplegia 7 takes part in many cellular functions like ribosome assembly regulation, membrane trafficking, protein folding, intracellular motility, organelle biogenesis, and proteolysis. Mutations in SPG7 historically have been attributed to spastic paraplegia 7, autosomal recessive (MIM 607259) but recently were found to be an important factor in mitochondrial diseases (Sánchez-Ferrero et al. SPG7 gene is greater than in TWNK, OPA1, and POLG genes (Pfeffer et al. The As we mentioned at the beginning of this review, mitochondrial diseases are very difficult to diagnose due to complex genotype–phenotype relationships, also called a blended phenotype. This means that mutations in one gene can lead to different clinical phenotypes and mutations in different genes can lead to the same signs and symptoms (Wortmann et al. Mitochondrial diseases affect each individual differently. Although mitochondrial disease primarily affects children, adult onset is becoming more common Fig. .Fig. 2ThMitochondrial diseases are a heterogeneous group of diseases. Age of onset is very different, from infants to the fifth decade of life. Symptoms involve multiple tissues and most of them may be progressive. Genetic background of this group of diseases in most cases is still unknown. The list of genes involved in mitochondrial DNA maintenance is long and still incomplete.TK2 mutations.Although therapeutic options are still limited, effective diagnosis on the clinical and molecular level opens the way to proper treatment. In some cases, targeted therapy is possible such as nucleotide supplementation in the case of patients with


Neurofibromatosis type 1 (NF1) is associated with systemic vascular disease, and it can also affect intracranial vasculature in a small percentage of patients. Very rarely, NF1 may co-present with hypoplasia of the internal carotid artery (ICA). Prior reports have documented NF1 with bilateral optic gliomas and a unilateral hypoplastic internal carotid artery; however, we report a case with the aforementioned findings in addition to a right-sided lentiform mass. This case report further suggests a common congenital pathway related to neurofibromin loss of function resulting in both nerve sheath tumors and cerebrovascular anomalies. Neurofibromin loss of function can result in systemic malignancies and vascular abnormalities . NeurofiCase series have demonstrated NF1 with non-specific cerebrovascular dysplasia in as few as two percent of patients -9. MoreoOur patient is an 18-year-old female with known NF1 and bilateral optic gliomas who presented to the neurology department for evaluation of a hypoplastic left ICA demonstrated on a prior CT angiogram at an outside hospital. Given the rarity of a hypoplastic ICA in NF1 patients, workup was initiated with particular attention given to alternate etiologies such as Moyamoya syndrome. The patient had a long-standing history of headaches, chronic left-greater-than-right vision loss, intermittent vertigo, numbness, tingling, and pain. A physical exam confirmed her visual symptoms and demonstrated 4+/5 right upper extremity weakness, hyporeflexia of the left triceps reflex, and diffuse café au lait spots. The patient underwent magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) imaging of the brain Figures .The patient then underwent a diagnostic six-vessel cerebral angiogram (DCA) that demonstrated a complete left internal carotid occlusion distal to the branch of the ophthalmic artery. In addition, the entire left middle cerebral artery (MCA) and anterior cerebral artery (ACA) vascular territories were supplied by the right internal carotid via a robust anterior communicating artery Figures -5.The findings on the DCA excluded the diagnosis of Moyamoya syndrome and further characterized the complete occlusive disease of her left carotid artery with total perfusion of her anterior circulation via the right ICA through a very robust anterior communicating artery. Moreover, a computerized tomography (CT) of her head revealed hypoplasia of the internal carotid canal suggesting a long-standing congenital lesion. The additional lentiform mass was radiographically determined as not exerting mass effect and warranted scheduled surveillance imaging without further intervention. The patient has since been conservatively followed by the neurology service.This case demonstrates the presence of an intracranial mass in addition to a hypoplastic ICA and optic gliomas in an NF1 patient. This constellation of findings is extremely rare and further demonstrates a clinical example in which loss of function in neurofibromin results in both intracranial and systemic nerve sheath tumors and congenital vascular anomalies. This clinical presentation highlights the importance of further understanding the genetic pathways leading to neurologic tumors and vascular abnormalities. Moreover, this case suggests that further screening for intracranial or congenital malformations in the setting of neurofibromatosis is warranted to optimize management of vascular risk factors to minimize vascular neurologic injury.Although neurofibromatosis is exceedingly rare, this disease can co-present with vascular manifestations, and this case report suggests the workup of congenital and acquired intracranial vascular disease for this patient population.