Increased expression of chemokine and chemokine receptor genes that modulate B and T cell recruitment to the mucosa were in part dependent on NOD2, TLR, and signaling adaptor protein MyD88. Together the results suggest GRA induces ILF through cooperative signals provided by bacterial ligands under normal conditions to induce B cell recruitment to ILF to the gut, but that the relative contribution of these signals differ between ileum and colon.18β-glycyrrhetinic acid (GRA) is a pharmacologically active component of licorice root with documented immunomodulatory properties. We reported that GRA administered orally to mice induces B cell recruitment to isolated lymphoid follicles (ILF) in the small intestine and shortens the duration of rotavirus antigen shedding. ILF are dynamic lymphoid tissues in the gut acquired post-natally upon colonization with commensal bacteria and mature through B cell recruitment to the follicles, resulting in up-regulation of IgA synthesis in response to changes in the composition of microbiota. In this study, we investigated potential mechanisms by which GRA induces ILF maturation in the ileum and the colon using mice depleted of enteric bacteria and a select group of mice genetically deficient in pattern recognition receptors. The data show GRA was unable to induce ILF maturation in ileums of mice devoid of commensal bacteria, MyD88 Glycyrrhizin is rapidly hydrolyzed in the gut by bacterial glucuronidases to GRA + B cells framed by CD11c+ dendritic cells (DC), and few CD3+ T cells. Upon induction with appropriate signals, B cells are recruited to ILF to form germinal centers that displace the lamina propria, and by mechanisms not entirely understood, develop a follicle-associated epithelium containing M cells similar to PP We reported that GRA administered orally to mice induces B cell recruitment to isolated lymphoid follicles (ILF) in the gut and does so in the absence of external antigenic stimulus −/− mice, mature ILF are hypertrophic in the colons Signals that stimulate induction and maturation of ILF have been revealed through studies in knockout mice as well as germ-free, and differentially colonized mice We reported that GRA administered orally induces ILF maturation in the ileum and in addition, shortens the duration of rotavirus antigen shedding in the mouse model 18β-glycyrrhetinic acid was purchased from Sigma-Aldrich. Stock solutions were prepared to a concentration of 100 mg/mL in DMSO and diluted to working concentration in calcium and magnesium-free phosphate buffered saline (PBS). Endotoxin levels were measured by Limulus Amoebocyte Lysate Assay , and the final amount of endotoxin present in doses delivered to mice was <0.025 EU.−/−, and MyD88−/− mice were obtained from Jackson Laboratories. Animals were co-housed according to treatment group under SPF conditions, with ad libitum access to food and water. All experiments were performed when animals were eight weeks of age and all groups contained five mice/group. GRA treatments and rotavirus infections were performed as previously described 5 shedding dose 50 (SD50) of murine rotavirus strain EW was administered by oral gavage on day two. EW does not cause diarrhea or other illness in mice >15 days of age, and infection is measured by antigen shedding in feces. Mice were sacrificed on day eleven by overdose CO2 inhalation.All animal experiments were performed according to the NIH Guidelines for Care and Use of Animals with protocol approval from the Montana State University (Protocol number 2011-44) and Washington State University (Protocol number 00453-004) Institutional Animal Care and Use Committees. Male C57Bl/6, C3H/HeJ, NOD2Tissue sections from ileum and colon were harvested as described previously Bacteria were ablated from the intestines of C57Bl/6 mice as previously described cis reporter and phRL renilla luciferase reporter plasmids (Promega). Twenty-four hours post-transfection, cells were treated with GRA and reporter gene expression was measured with the Dual-Glo Luciferase Assay (Promega). Data are presented as percent response relative to the NOD2 ligand muramyl dipeptide control set to 100%. Data were analyzed by unpaired t test. Error bars represent standard error of the mean of three separate experiments; p<0.05.HEK293 cells were cultured in DMEM containing 10% FBS. Cells were co-transfected with NOD2 expression plasmid pUNO-mNOD2 (InVivoGen), pNFκB-luc-2 Profiler™. Custom arrays included Cxcr5, Ccl19, Ccl21b, Cxcl13, Lta, Ltb, Ccr6, Ccr7, Ccr9, Il10, Il6, Ccl20, and Ccl25. One µg of RNA was reverse transcribed with RT2 First Strand kit (SABiosciences) and PCR reactions were performed on an Eppendorf Realplex 4s under reaction conditions of 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for one minute. Data are from a minimum of three mice per group and are expressed as fold-change over vehicle-treated animals. Fold-changes >2 were considered significant.Gene expression in intestinal tissue induced by GRA was measured by RT-qPCR and custom gene arrays as previously described + cell recruitment to lamina propria ILF , DC (CD11c), and T cells (CD3) in the ileum revealed that GRA induced B220pria ILF . Howeverpria ILF . Colonicpria ILF , and in bacteria . Small I+ cell clusters in the ileums of rotavirus infected C57Bl/6 mice nine days post-infection, and sizes of B220+ aggregates were enhanced by GRA treatment + cell aggregates in both ileum and colon , nor did it activate NOD1-mediated reporter gene expression in transient transfections (data not shown). However, GRA did activate NOD2-mediated gene expression  intestinal inflammatory homeostasis −/− mice suggest TLR signaling is not required for up-regulation of gene expression, but is required for B cell recruitment to ILF because ILF were not observed. Activation of NOD2 also is implicated because GRA was unable to induce chemokine and receptor gene expression in ileums of NOD2−/− mice, nor were B cells recruited to ILF even though these mice are normally colonized. Cellular responses to NOD2 activation are propagated through activation of NFκB in vitro and in vivoCollective interpretation of data regarding GRA-induced gene expression and B cell recruitment to ILF leads us to propose that a mechanistic role for both TLR signaling and NOD2 signaling differs between small intestine and colon. GRA-induced chemokine and chemokine receptor gene expression in ileums of antibiotic treated mice and MyD88−/− mice, whereas B cell recruitment was induced in the former, but not the latter. The lack of induced gene expression and ILF maturation in colons of MyD88−/− mice points to a role for TLR signaling, or alternatively, signaling through the IL1R. In contrast, although gene expression was up-regulated in colons of NOD2−/− mice, robust B cell recruitment to ILF was not observed. The difference in gene expression in ileum and colon compared to the other mouse models is not clear, but in the NOD2−/− mice, induction may be driven more by signaling through TLR in the presence of expanded bacterial colonization in these mice, although such differences recently have been called to question In contrast to the ileum, GRA did not induce chemokine and chemokine receptor gene expression in colons of antibiotic treated or MyD88in vitroin vivo following exposure by the oral route have not been reported.ILF function as inductive sites for IgA synthesis in part in response to changes in gut microbiota in vitro and in vivo brings forth the intriguing possibility that GRA induces secretion of antimicrobial peptides that result in alterations in the composition of the microbiota. NOD2 is highly expressed in Paneth cells localized in the small intestinal crypts and play a prominent role in controlling both commensal and pathogenic bacteria through up-regulated expression of antimicrobial peptides Evidence for NOD2 activation both Glycyrrhizin and GRA are inhibitors of 11β-hydroxsteroid dehydrogenase 1 (11βHSD1) which catalyzes conversion of cortisone to cortisol, resulting in suppression of inflammation + T cells are significantly increased in PP in GRA treated, rotavirus infected C57Bl/6 mice and endpoint anti-rotavirus serum antibody titers were slightly increased. In the previous study, an apparent increase in B220+ cell aggregates in rotavirus infected mice that were not treated with GRA was noted, and the sizes of these aggregates were enhanced in GRA treated, infected mice. Rotavirus infection in the current study did not result in B cell recruitment either the presence or absence of GRA in any of the mouse models, suggesting increases in B cell recruitment to ILF as a result of rotavirus infection requires all of the components tested here, including pattern recognition receptors and signaling adaptor MyD88. Studies to determine mechanisms by which GRA reduces the duration of rotavirus antigen shedding, including T cell proliferation and generation of rotavirus-specific IgA are ongoing.Orally delivered GRA shortens the duration of rotavirus antigen shedding in the adult mouse model


Despite the clinical promise of CAR therapy in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address this question and a priori define the therapeutic potential of T cell based therapies. An integration of the existing clinical data suggests that the efficacy of ACT is likely balanced by two requirements of the T cell infusion product: immediate cytotoxicity to enable eradication of the tumor and the ability to persist and establish memory to ensure long term benefit.T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR) have shown considerable promise in clinical trials even in tumors refractory to all other treatment methods. In particular, the use of CAR T cells rendered specific for CD19 demonstrated significant anti-tumor effects in patients with CD19in vitro. Although there were no discernible differences using a population level assay, we demonstrate using TIMING that significantly more CAR T cells bearing the CD8a hinge participate in serial killing. This superior efficacy was confirmed in a mouse model in which CART cells containing the CD8a extracellular domain were superior in controlling the disease.Using our novel single cell multiparametric assay, Timelapse Imaging Microscopy In Nanowell Grids (TIMING), we compared the efficacy of two second generation (CD28 endodomain) CD19 specific CAR constructs, bearing CD8a and IgG4 hinge respectively, by tracking their interaction with NALM6 tumor cells In order to identify biomarkers which distinguish between killer  CAR T cells and non-killers, we performed single cell multiplexed gene expression profiling (96 genes) subsequent to a TIMING assay. Across three separate donors, killer CAR T cells consistently expressed higher transcript levels of the cytotoxic molecule Granzyme B, the costimulatory receptor CD137 (41BB) and the regulatory receptor TIM3 (HAVCR2).By utilizing multicolor flow cytometry, we confirmed that CD137 was overexpressed in cytotoxic CAR T cells and that its stimulation was associated with higher cytotoxicity and lower expression level of the immunoregulatory receptors CTLA4 and PD1. By comparison, TIM3 was not enriched in killer CAR T cells but rather was a likely marker for effector status since blocking TIM3 boosted cytotoxic responses.In aggregate, these results demonstrate the utility of our TIMING single cell methodology in uncovering not only the dynamic profile of T cell behavior but in also uncovering the phenotypic biomarkers of CAR T cells with superior functional efficacy.


WDR45) gene on chromosome Xp11 resulting in reduced autophagic flux. This study describes the clinical and neuropathological features of a female 51 year old BPAN case. The clinical history includes learning disability and progressive gait abnormalities since childhood followed by progressive dystonic features in young adulthood. Brain imaging revealed generalised brain atrophy and bilateral mineralisation of the globus pallidus and substantia nigra.Beta-propeller protein associated neurodegeneration (BPAN) is associated with mutations in the WD repeat domain 45  immunoblotting of brain homogenates indicated autophagic activity and may support the role of WDR45 in autophagy.The widespread Alzheimer-type tau pathology in this disease indicates that this should be considered as a tauopathy and adds further support to the proposal that impaired autophagy may have a role in tauopathies.The online version of this article (doi:10.1186/s40478-015-0221-3) contains supplementary material, which is available to authorized users. PANK2); PLA2G6-associated neurodegeneration (PLAN) due to mutations in phospholipase A2, group VI (PLA2G6), fatty-acid hydroxylase-associated neurodegeneration (FAHN) associated with mutations in fatty acid 2-hydroxylase (FA2H), mitochondrial membrane protein-associated neurodegeneration (MPAN) due to mutations in chromosome 19 open reading frame 12 (C19orf12), Kufor-Rakeb-Syndrome (KRS) due to ATPase type 13A2 (ATP13A2), aceruloplasminemia due to mutations in ceruloplasmin (CP), hereditary neuroferritinopathy due to mutations in ferritin-L subunit gene FTL1, Coasy protein-associated neurodegeneration associated with mutations in bifunctional coenzyme A synthase (COASY) and Woodhouse-Sakati-Syndrome (WSS) due to mutations in DCAF17 [PKAN, PLA2G6, COASY and FA2H have roles in fatty acid metabolism, C19orf12 encodes a protein with a role in mitochondrial function and fatty acid metabolism. CP and FTL1 have functions in iron homeostasis, DCAF17 regulates transcription and ATP13A2 is involved in apoptosis and magnesium metabolism [The dysregulation of brain iron metabolism leading to accumulation of iron in the basal ganglia and substantia nigra is a characteristic feature of the group of inherited neurological disorders classified under the term neurodegeneration with brain iron accumulation (NBIA). The main clinical features of NBIA include progressive dystonia, dysarthia, spasticity, parkinsonism and optic atrophy. The age of onset and rate of progression of the disease varies. Several genetic forms have been identified: pantothenate kinase-associated neurodegeneration (PKAN) with mutations in pantothenate kinase 2 (de novo mutations in the gene encoding WD repeat containing protein 45 (WDR45) have been associated with an X-linked dominant form of NBIA causing static encephalopathy of childhood with neurodegeneration in adulthood (SENDA), now reclassified as BPAN (β-propeller protein-associated neurodegeneration), or NBIA5 [WDR45 encodes a beta-propeller scaffold protein which has a role in autophagy and autophagic flux has been found to be reduced in lymphoblasts from BPAN patients [Recently or NBIA5 –5. Clinior NBIA5 , 5. Duripatients .WDR45 demonstrated the hallmark feature of NBIA, with iron accumulation in the substantia nigra and in the globus pallidus together with axonal spheroids, gliosis and severe neuronal loss. In the cerebellum significant depletion of Purkinje cells with axonal torpedoes was observed. Tau positive neurofibrillary tangles (NFTs) were described in several brain regions [The neuropathological description of a previously reported case with mutation in WDR45 [We report the detailed neuropathological description of a case with the previously reported loss-of-function mutation, p.Leu232Alafs*53 in The brain was donated to the Queen Square Brain Bank for neurological disorders (QSBB) where all cases are collected for research according to ethically approved protocols and are stored under a licence from the Human Tissue Authority. The left half of the brain was fixed in 10 % neutral buffered formalin before being dissected in the coronal plane, examined and blocks selected for paraffin wax embedding and histology.Paraffin-embedded sections (8 μm) were cut and stained using routine histological methods. Sections were stained using haematoxylin and eosin (H&E), Gallyas silver and Luxol fast blue/cresyl violet. IHC staining was performed using an avidin-biotin complex protocol using the antibodies and pre-treatments detailed in Additional file Neuronal loss was assessed on H&E stained sections and scored semi-quantitatively . Tau pathology was assessed using AT8 antibody and the tau burden was scored semi-quantitatively for tau positive neuropil threads, pre-tangles and NFTs .Sarkosyl-insoluble tangle PHF-tau was isolated from brain samples as previously described . InsolubSamples of frontal cortex from the WDR45 case and three age and sex matched controls selected from the archive of the QSBB were homogenised in 50 mM Tris–HCl pH7.5, 150 mM NaCl, 5 mM EDTA, 1 % tritonX-100, 2 % SDS containing complete protease inhibitor and phosphatase inhibitor (Roche). Tissue lysates were obtained after centrifugation of homogenised samples at 10000 g for 15 min. Protein concentration was determined using Bio-RAD protein assay kit. Protein lysates (40 μg) were analysed by SDS-PAGE on 4–12 % Bis-Tris gel  and transferred onto nitrocellulose membranes (Amersham). Membranes were blocked in 5 % BSA in phosphate buffered saline-tween for 1 h at room temperature, before being probed overnight at 4 °C with LC3 and beta-actin primary antibodies. The following steps of the protocol were as described above. The immunoblots were repeated twice with similar results.The female patient had learning difficulties as a child with progressive gait abnormalities. In school, other children would tease her for her “funny gait” as well as for being unable to keep up in class. By the age of 13, she moved to a special school for handicapped children. At age 19, she left school and continued to live in a specialised community for mentally and physically challenged people. Due to increasing behavioural difficulties at age 36 she moved to a social service hostel from where she attended a day centre several times a week.Her movement disorder symptoms started deteriorating significantly at the age of 29 with slowing of gait and reduction of arm-swing whilst walking. Even though there were initial doubts in the diagnosis of Parkinson’s disease, she was started on Pergolide and later on Levodopa, both with equivocal and unpredictable effects with the subsequent development of dyskinesias and severe fluctuation of mobility. She deteriorated rapidly and developed dystonic features with an inward turning of the left foot, a bilateral akinetic-rigid syndrome with orofacial stereotypies during her thirties. Due to increasing behavioural problems, social withdrawal, reduced attention span and motivation a combination of cognitive and behavioural therapy was started in 1998. She was treated with Sertraline, and later with Amitriptyline, Velofaxine, Slisulpiride, and Carbamazepine, all with little effect. She had disturbed sleep with early morning awakening and behavioural problems with long off-phases. In the later stages of the disease she was wheelchair-bound and had urinary and faecal incontinence.Other medical problems included slowly evolving difficulties with her eyesight with intermittent double-vision. She had a right corneal graft at the age of 32 – unfortunately no detailed further information on the underlying pathology of this was available. There was no family history except her mother who received a diagnosis of bipolar disorder and schizophrenia and died of a sudden brain haemorrhage when the proband was in her twenties.Brain computerised tomography showed abnormal bilateral generalised mineralisation of the globus pallidus and substantia nigra, likely to represent iron deposition on subsequent MRI. Other investigations were normal except for WDR45 gene sequencing which showed a hemizygous mutation .The fresh brain weighed 1,117 g. External examination of the left half brain showed frontal atrophy with preservation of parietal, temporal and occipital lobes. Coronal slices revealed mild dilatation of the frontal horn of the lateral ventricle and patchy thinning of the cortical ribbon in the frontal lobe. In the occipital cortex an area of cavitation with the appearance of an old infarct involved the primary visual cortex. The white matter in the anterior frontal and temporal lobes showed mild atrophy. The globus pallidus was reduced in size with blurring of the distinction between internal and external parts and showed dark discolouration anteriorly Fig. , arrow. Histological examination Fig.  showed mNeuronal populations in the hippocampus, amygdala, striatum, thalamus and subthalamic nucleus were well preserved. Examination of the globus pallidus showed extensive mineralisation of vessels including arteries, veins and capillaries. Perl’s staining for iron was positive in areas of mineralisation and also in scattered cells with macrophage morphology. IHC staining for glial fibrillary acidic protein (GFAP) showed marked gliosis of the globus pallidus and there was also an increase in CD68-immunoreactive microglia. No axonal swellings were identified.Examination of the midbrain showed very severe loss of pigmented neurons in the substantia nigra accompanied by gliosis, macrophage infiltration and marked deposition of Perl’s positive pigment. Numerous axonal swellings were present and were highlighted with IHC for amyloid precursor protein (APP), neurofilaments  and ubiquitin. In the pons, the locus coeruleus showed mild neuronal loss with the pontine nuclei being well preserved. In the medulla the Xth and XIIth cranial nerve nuclei as well as the inferior olivary nucleus were well preserved. Axonal swellings were observed in the gracile and cuneate nuclei. The cerebellum showed mild Purkinje cell depletion with good preservation of the dentate nucleus and hemispheric myelin. There was no Aβ deposition in the hippocampus or neocortex. No α-synuclein pathology was found in brainstem or limbic regions and there was no TDP-43 pathology in limbic structures.Tau pathology was extensive in the form of NFTs, pre-tangles and neuropil threads affecting cortical regions, white matter, deep grey nuclei, hippocampus, amygdala, brainstem structures and the cerebellum. Very rare coiled bodies were present in the sub-cortical white matter but there was no accumulation of tau in astrocytes. The type of tau pathology and semi-quantitative analysis of the tau burden in different brain regions is summarised in Table Immunohistochemial staining using isoform specific antibodies demonstrated the expression of both 3- and 4-repeat tau isoforms. Immunoblotting of tau showed a pattern similar to that of AD with the classical triplet band of 3R- and 4R- tau isoforms compared to the 3R- tau isoforms predominantly observed in PiD (Fig. WDR45 mutation was available quantitation of immunoblots and statistical analysis was not performed and clear alteration of autophagic activity could not be demonstrated.In the mutation case and 3 controls LC3I and LC3II could be demonstrated by immunoblotting confirming autophagic activity Fig. . As onlyWDR45. This patient has previously been reported in genetic studies as case HH8 [We report the neuropathological details of a 51 year old female with a clinical phenotype compatible with BPAN and positive for the hemizygous loss-of-function mutation, p.Leu232Alafs*53 in The major pathological findings were severe neuronal loss with gliosis affecting the substantia nigra, iron deposition in the substantia nigra and globus pallidus, axonal swellings in the substantia nigra, gracile nucleus and cuneate nucleus together with very extensive phospho-tau deposition similar to that previously been reported . We inveC. elegans [WDR45 mutation. As autophagy is known to contribute to the degradation of tau it seems likely that impaired autophagy in BPAN may underlie the severe tau pathology seen in this disease and supports the notion that autophagy may be important in the pathogenesis of other forms of tauopathy [In neurodegenerative disorders, disruption of autophagy inhibits elimination of abnormal and toxic protein accumulation and can promote cellular stress and death. WDR45 belongs to a large family of WD40 repeat protein and is believed to be an important regulator of autophagy as documented in yeast  and C. e elegans . LC3-I iauopathy .We have described a case of BPAN and documented the neuropathological changes confirming a characteristic pattern of neuronal loss, most severe in the substantia nigra with iron deposition in both the globus pallidus and substantia nigra. Axonal swellings were present in the substantia nigra, gracile nucleus and cuneate nucleus. Impaired autophagic flux could not be demonstrated in a single case however, this could be the basis of future investigations. The striking widespread tau deposition was shown for the first time to be composed of fibrillar tau of Alzheimer-type and suggests that this disease may be considered as a tauopathy.


Herein, towards the goal of developing a safe and more efficacious high field T1 MRI CA for these applications, we report the sub-acute toxicity and contrast enhancing capabilities of a novel nanoparticle MRI CA comprising of manganese (Mn2+) intercalated graphene nanoparticles functionalized with dextran  in rodents. Sub-acute toxicology performed on rats intravenously injected with Mangradex at 1, 50 or 100 mg/kg dosages 3 times per week for three weeks indicated that dosages ≤50 mg/kg could serve as potential diagnostic doses. Whole body 7 Tesla MRI performed on mice injected with Mangradex at a potential diagnostic dose  showed persistent (up to at least 2 hours) contrast enhancement in the vascular branches . The results lay the foundations for further development of Mangradex as a vascular and cellular/ molecular MRI probe.Current clinical Gd T1, and transverse relaxation time T2. Various imaging pulse sequences have been developed to highlight the endogenous differences in T1 or T2 relaxation times of bodily fluids and tissues to enhance contrastMagnetic resonance imaging (MRI) is one of the most powerful clinical imaging techniques in diagnostic medicine that non-invasively renders exquisite anatomical details of soft and hard tissues for improved diagnosis of many pathologies. In clinical MRI, the nuclear magnetic resonance (NMR) signals from water protons in living tissues are used to image organs and disease sites in three dimension (3D) ranging from brain lesions and tumors throughout the body to myocardial infarctions13+)-based small molecule complexes dominate the MRI CA market 3+-based MRI CAs are considered as “first pass” agents as they are detectable by MRI within the first few minutes in the circulatory system after injection to rapidly extravasate and are subsequently eliminated via the kidneysT1T1 variations in image acquisition of the k-space sampling, causing image artifacts73+-macromolecule complexes have been designed as advanced intravascular or blood pool agents with longer blood half-life and slower extravasation into the interstitial space compared to first-pass agents to first-pass agents. One agent (Ablavar® (Gadofosveset Trisodium)9r1 relaxivities (an important measure of imaging efficacy defined as change in the relaxation rate (R1 = 1/T1 s−1) of the water protons per unit concentration of CA (mM)) of current clinical MRI CAs, with minimum detectable concentration of tens of μM to provide satisfactory contrast enhancement, are suboptimal for advanced MRI-based molecular and cellular imaging applications12143+-based small molecule “first pass” agent or large molecule blood pool MRI CAs show significant drop in relaxivity3+)-based clinical CAs have been reported to induce nephrogenic systemic fibrosis (NSF) in patients with moderate to severe renal insufficiency and vascular or metabolic disorders1617T1 MRI CA that allows significant contrast enhancement at substantially lower dosages than (Gd3+)-based clinical CAs for advanced applications, such as blood pool or cellular or molecular imaging, would constitute a significant advancement.Gadolinium Gd+-based sT1 and T2 MRI CAs, respectively19Other than gadolinium, the elements manganese and iron have also been employed in the design of T1 MRI CAs, including complexes of gadolinium with carbon nanostructures such as fullerenes (gadofullerenes), carbon nanotubes and graphene, have been proposed as T1 CAs1520242526272829in vitro relaxivity of these complexes at various magnetic fields. A few studies have also reported proof-of-principle in vivo efficacy at high magnetic fields (>1.5 Tesla) after systemic injection into normal rodents or direct tumor injection in rodent models of cancer. In the case of gadolinium carbon nanostructure complexes, these studies have only been reported with gadofullerenesNanoparticle-based 2+) ions as novel T1 MRI CAs3233in vitro physicochemical properties of Mangradex32in vivo small animal intravenous (IV) dose range-finding, expanded acute toxicity and pharmacokineticsin vitro and in vivo small animal studies to characterize its hematological and vasoactive effectsin vitro relaxometry and phantom MRI results demonstrate that Mangradex exhibits relaxivity values ~20 fold greater (92 mM−1S−1) than current clinical contrast agents (~4.5 mM−1S−1)Recently, we reported the synthesis and characterization of graphene nanoparticles (a.k.a. graphene nanoplatelets) intercalated with manganese  was performed on sham and experimental animals that survived the entire 3 week duration of the study. A complete blood count (CBC) and established serum biochemistry assays (comprehensive lipid and metabolic panel) were performed and analyzed for the 1, 50 and 100 mg/kg doses of Mangradex, dextran, mannitol and sham. The results of the experimental groups were compared with sham, mannitol and dextran group, and also with the normal range of blood parameters of Wistar rats 2+ ions/kg). With the level of anatomical details achievable by our MR imaging protocol (200-μm isotropic resolution) in combination with our homemade MRI probe enabling full mouse body coverage, we were able to examine most of the main organs and the major vascular branches. The results from the serial MRI performed in contrast agent-free subjects followed by serial acquisitions at 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 minutes post-injection in all mice clearly demonstrated the blood-pool nature of our compound Mangradex. As illustrated by the example shown in A to D corresponds to either coronal (A&D) or axial (B&C) sections at various levels of the mouse body as follow: A) covers the upper body including the head, neck, heart and lungs; B) is an axial slice re-orientation obtained from the same lung and heart area described in A) while in C) the section covers the lung and liver region; D) is a coronal view from the lower abdominal region that includes the kidneys and the spleen. As evidenced by the example shown, there is an obvious signal enhancement starting from the first image set acquired 12-min. post-injection. The greatest tissue contrast emanated from the vascular space in the various regions of the body as well as from the cardiac chambers seen in row A) with a long axis view of the heart & B) through its short axis section. This tissue contrast was most notable within the large vascular branching: head & neck in row A), liver in row C) as well as highlighting the abdominal aorta and inferior vena cava in row D). This enhancement was maintained throughout the two-hour period in which all the subjects were examined every 12-min. and where only a subset of the imaging time points corresponding to 12-, 36- and 120-min. post-injection are shown in D).3+ ion concentration = 455 nanomoles/kg). A), these signal enhancements gradually decayed (data not shown) and vanished by the last imaging time point corresponding to 120-min. Expectedly, the kidneys demonstrated immediate and subtle bright enhancement consistent with the renal excretion known to Ablavar following injection. The subtle increase in signal from kidneys can be partially distinguished in Fig. 3, row B). Importantly, the injection of either Mangradex or Ablavar resulted in gradual and consistent enhancement of the bladder as shown in row A&B.The qualitative comparison of image contrast efficacy and pharmacokinetics was assessed using Maximum-Intensity-Projection of the serial 3D image datasets and by identifying regions of MRI signal enhancement induced by the presence of either Mangradex or the clinical blood pool agent Ablavar® at the prescribed clinical dose  from regions easy to delineate anatomically thanks to the well-defined tissue contrast obtained and illustrated by the example in in vitro physio-chemical characterization studies show that this nanoparticle formulation is hydrophilic, thermally stable under physiological conditions, and when mixed with the excipient mannitol, iso-osmolar and iso-viscous to blood, and forms stable colloidal dispersions in deionized water and biological fluids (buffers and blood) up to 100 mg/ml concentrationsThe overall objectives of this study were (1) to identify the no-observed adverse effect level (NOAEL) within the previously identified maximum tolerated dose (MTD) range that could be used to help identify the potential diagnostic dose, and (2) to evaluate its efficacy as an MRI CA in healthy mice at a potential diagnostic dose. Our previous FDA and International Conference on Harmonization (ICH) guidelines on preclinical safety studies of MRI CAs have also emphasized that repeat dose chronic toxicity studies be conducted to identify potential therapeutic dosages for first-in-human trials363738The necropsy findings were consistent with acute toxicity results, where rats with mortality at higher dosages (>125 mg/kg) showed presence of Mangradex in pulmonary capillaries with symptoms similar to pulmonary congestionMangradex related unfavorable reaction in the circulatory system can affect the immune system and/or hematological factors. Accordingly, standard serum chemistry test and complete blood panel analyses were performed. The results of blood chemistry parameters for Mangradex dosages and controls were compared with sham and published data by Charles River Laboratoriesin vivo MRI studies, the choice of 25 mg/kg dose was based on the outcomes of all the in vitro and in vivo (including the repeat dose) safety studies32333332in vivo toxicity studies was 50 mg/kg ≤MTD < 125 mg/kgin vivo MRI was less than half of the MTD. The clinical Gd3+-based blood pool agent Ablavar was used as control. The longer blood circulation of Ablavar compared to monomeric imaging agents such as Gd-DTPA or Gd-DOTA is attributed to its strong and high degree of reversible binding to serum albumin, while the blood circulation of Mangradex could be because of its hydrophilic properties, which is consistent with our previous in vitro studies showing that the partition coefficient of Mangradex is –0.18, suggesting that majority of the contrast agents remain in liquid phase than in lipid (tissue in vivo)For the in vivo rodent studies indicated immediate and very strong vascular signal contrast enhancement that was prolonged for more than 2-hours evidenced by the serial imaging depicted by the example shown in row B. Other covalently or non-covalently functionalized graphene formulations have shown analogous pharmacokinetics in mice following intravenous administration where sustained recirculation and excretion via the biliary and renal pathways were observed442 (R2 = 1/T2) to R1 (R1 = 1/T1) relaxivities of Mangradex. This R2/Rl alteration is commonly observed in iron oxide-based agents2+/kg dose of 455 nanomoles/kg; 66X and 220X lower than clinical dose of Ablavar®  after the first pass would be ~300 ppb or 45 femtomoles /voxel. Our previous relaxometric studies indicate that the dynamics of water within nanostructures with multiple layers of graphene and the interaction between water and Mn2+ ions in these confined spaces, affect the T1 relaxation mechanism, and could be responsible for the observed increase in contrast enhancementq) and water molecule residence lifetime (τM) coordinated with the Mn2+ ions, and the rotational correlation time (τR) of Mangradex. The combination of significant contrast enhancement at low detectable concentrations achieved at a high field strength taken together with the longer residence times in the blood introduces Mangradex not only as a great addition to the existing blood pool agent, but also suggests its suitability to be incorporated in the design of preclinical cellular/molecular imaging probes to monitor the progression of vascular pathologies.Numerous clinical studies have demonstrated the efficacy of Ablavar® in strongly enhancing blood vessels for MRA examinations–2.5% mixture of O2/air, via inhalation). Control groups were given IV injection of mannitol , and dextran  with the same dosing regimen. Blood pressure and heart rate were measured using a non-invasive occlusion type tail cuff system  once per week after administration of Mangradex. The parameters evaluated included survival, clinical observations, body weight and diet observation. All the animals were sacrificed using 100% CO2 inhalation after 3 weeks and blood was obtained by cardiac puncture technique. All the major organs  from each animal were harvested and prepared for histology analysis.All the experimental protocols were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Stony Brook University, NY. The 21 - day sub-acute toxicity study at three doses, was conducted in a total of 44 Wistar rats  weighing 200–250 g. Three groups of 8 rats  had received Mangradex injections intravenously via the tail vein three times per week at the doses of 1 mg/kg, 50 mg/kg and 100 mg/kg of body weight. During injections the animals were anesthetized using isoflurane  for histologic evaluation. Digital photomicroscopy was performed using a bright field microscope at 400X magnification. Histologic assessment of the tissue sections were performed by an experienced diagnostic and research anatomic pathologist (KRS).2 inhalation and ~8 ml blood was collected from each animal by cardiac puncture for blood analysis. Serum chemistry test and complete blood panel analyses were performed in a Clinical Laboratory Improvement Amendments (CLIA) certified facility at Stony Brook University Hospital.From the total of 8 animals per dose of Mangradex, controls and sham, 6 animals  were used for serum chemistry analysis. Animals were euthanized by 100% COAll data were analyzed for statistical significance by One-way analysis of variance (ANOVA) using Graphpad Prism . The criterion for statistical significance was p ≤ 0.05.2+ ion. The injection volume of Mangradex and clinically approved Ablavar  was similar (50 μl). Ablavar diluted in saline dose per kg of mouse weight was 455 nanomoles Gd3+ ion.For contrast enhanced-MRI studies, 10 mg/ml solution of Mangradex was prepared by dissolving 10 mg of lyophilized Mangradex powder in distilled deionized water. D-Mannitol  was added to make Mangradex solution iso-osmol to blood. The formulation was UV sterilized for 30 min. The dose for Mangradex per kg of mouse weight was 25 mg/kg or 455 nanomoles MnFor imaging, C57BL/6 wild type female mice  with weight ranging from 25 g to 30 g were used. All mice were maintained in agreement with procedures approved by the Institutional Animal Care and Use Committee at New York University School of Medicine. Mice were anesthetized with isoflurane gas delivered via a vaporizer/anesthesia setup : 5% isoflurane in air for induction followed by 0.5–1.5% isoflurane in air via nosecone for maintenance. Following anesthetic induction, each animal was catheterized via femoral artery to infuse the contrast agent using a polyethylene tubing PE-10 line . Each subject was then placed supine and restrained on a custom made body holder bed with the cannula tubing extended long enough to enable remote infusion using a PHD-2000 computer-controlled syringe pump , while the animal was centered within the magnet bore. Body temperature was maintained between 35–37 °C with a warm air thermostat system while all basic physiologic signals (temperature and breathing rate) of the freely-breathing animals were monitored continuously . Respiratory motion was monitored using a pneumatic pillow fixed to the abdomen and the body temperature was measured via a temperature probe. The whole mouse bed was then subsequently inserted within a home-built radiofrequency (RF) coil. This circularly polarized MRI probe was developed in-house to resonate at a proton frequency of 300-MHz in both transmit and receive modes with dimensions  insuring a homogenous RF coverage of the adult whole mouse body.in vivo micro-MRI (μ-MRI), a modified three-dimensional (3D) spoiled gradient echo (GE) sequence was used to acquire an additional self-gated signal on the readout dephasing gradient within each TRAV) = 3 and imaging time ~12 min. Flip angle (FA) = 34° was chosen to provide the greatest T1-enhancement contrastMRI experiment were conducted using a 7-Tesla (7T) micro-MRI system consisting of a 7T 200-mm horizontal bore magnet  interfaced to a Bruker Biospec Avance-2 console . The system was equipped with an actively shielded gradient coil . For T1-weighted 3D-GE MRI scans of 12 min. duration each, were performed serially during every imaging session in mice to compare qualitatively the bio-distribution and pharmacokinetics of Mangradex  with FDA-approved clinical intravascular agent Ablavar® (Gadofosveset trisodium). The protocol consisted of acquiring an initial image set serving as the contrast-free baseline followed by the infusion of the blood-pool contrast agent and serial scanning of nine image datasets at 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 minutes post-injection in all subjects.In order to compare qualitatively the image contrast efficacy and enhancement kinetics of Mangradex with the clinical blood pool agent Ablavar®, 3D image datasets from both mouse groups were analyzed by generating Maximum-Intensity-Projection (MIP) throughout time course of the imaging session. In this approach, the MIP algorithm enables pixels with the highest value crossed via a chosen angle throughout the 3D image set to be projected for display on a 2D plane. This method permitted the visual rendering of the tissues where the contrast agent was distributed due to the signal enhancement it induced by its presence.Quantitative comparison on tissue contrast was also assessed between both agents by calculating signal-to-noise ratios (SNRs) and contrast-to-noise ratios (CNRs) in various regions of interest (ROI) including the major large vessels throughout the whole body, the bladder and muscle. The SNR in each of these regions was calculated as SI/Stdev where, SI is the mean signal intensity within the ROI and Stdev is the standard deviation of the signal intensity within the background noise (air). The standard deviation of the noise was obtained by carefully choosing the largest possible ROI placed outside the subject in the image background while avoiding ghosting/aliasing or eye movement artifact regions.(CA)-SI(muscle))/Stdev(Noise) where, SI(CA) is the mean signal intensity in the region where the contrast agent (CA) is localized in either the major vessels or in the bladder, SI(muscle) is the mean signal intensity in the muscle region. Pre-contrast SNRs were subtracted from post-contrast SNRs to minimize differences in baseline signals from animal to animal. Similarly, pre-contrast CNRs were subtracted from post-contrast CNRs. SNRs and CNRs calculations were performed for each animal and then average values were reported for each group (n = 4).The CNR was defined relative to the muscle region as  parameters. The formulations at low paramagnetic ion concentrations allow at high magnetic fields significant and sustained contrast enhancement of major vascular branches. Taken together, these results establish the basis for further employing Mangradex in studying vascular disease models and subsequently into higher species to facilitate its eventual clinical translation.How to cite this article: Kanakia, S. et al. Towards An Advanced Graphene-Based Magnetic Resonance Imaging Contrast Agent: Sub-acute Toxicity and Efficacy Studies in Small Animals. Sci. Rep. 5, 17182; doi: 10.1038/srep17182 (2015).


A basic premise in behavioural ecology is the cost-benefit arithmetic, which determines both behavioural decisions and evolutionary processes. Aggressive interactions can be costly on an energetic level, demanding increased energy or causing injuries, and on a psychological level, in the form of increased anxiety and damaged relationships between opponents. Here we used urinary glucocorticoid (uGC) levels to assess the costs of aggression in wild chimpanzees of Budongo Forest, Uganda. We collected 169 urine samples from nine adult male chimpanzees following 14 aggressive interactions (test condition) and 10 resting events (control condition). Subjects showed significantly higher uGC levels after single aggressive interactions compared to control conditions, likely for aggressors as well as victims. Higher ranking males had greater increases of uGC levels after aggression than lower ranking males. In contrast, uGC levels showed no significant change in relation to aggression length or intensity, indicating that psychological factors might have played a larger role than mere energetic expenditure. We concluded that aggressive behaviour is costly for both aggressors and victims and that costs seem poorly explained by energetic demands of the interaction. Our findings are relevant for studies of post-conflict interactions, since we provide evidence that both aggressors and victims experience a stress response to conflict. A central principle in behavioural ecology is that the function and evolution of behaviour is analysed in terms of economic logic as costs and benefits, which determines an individual’s fitness , 2. CostOncorhynchus mykiss [Urosaurus ornatus [Parus major [Canis lupus [Cavia porcellus [Helogale parvula [Macaca assamensis [M. sylvanus [Pan troglodytes [Homo sapiens [The relationship between high GC levels and aggressive behaviour has been established in a wide range of taxa . In sapiens , 25). OnIn many non-human primates there is an inverse linear relationship between dominance rank and aggression received , resultiGiving or receiving of aggression affects the subsequent behaviour of opponents. Prior aggression sometimes attracts further aggression  and formPan troglodytes) caused increased urinary glucocorticoid (uGC) levels in victims and aggressors. We compared uGC levels related to aggression with uGC levels of samples related to pre-aggression periods. We also investigated the effect of duration and intensity of aggression on uGC levels, since fighting duration is correlated with energy use, and contact aggression can more easily lead to injuries than non-contact aggression. Finally we investigated the effects of dominance rank in relation to aggression. Given evidence of reproductive skew in male chimpanzees with dominant males siring more offspring [Here, we investigated whether single events of aggression in male chimpanzees  of the Sonso community living in the Budongo Forest , Uganda, between February 2008 and July 2010. The Sonso community has been observed continuously since 1990 [We observed nine adult male chimpanzees  from approximately 7 a.m. to 5 p.m. through the forest, recording the party behaviour using all occurrence sampling  for aggrUrinary GC levels were measured at the Lab for field-endocrinology at the Max Planck Institute for Evolutionary Anthropology using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), applying a method that measures 23 endogenous steroids in small quantities of primate urine . Samples3H-labeled cortisol, the peak recovery of 3H-labeled cortisol metabolites in chimpanzee urine was between ∼2 and 4.8 hours after administration [relative uGC level where we divided the uGC level of samples related to the target behaviour (peak-period samples) through the uGC levels of samples collected in the pre-target behaviour times window (pre-period samples). The pre-period urine samples were excreted between 0 min (t0) and 135 min (t1) after the start of the target behaviour, representing the peak uGC secretion of behaviour preceding the aggression or the resting. The peak-period urine samples were excreted between 135 min after the start (t1) and 270 min after the end of the target behaviour , representing the peak uGC secretion of the aggression or resting event. We calculated the relative uGC level for each event, representing the percentage of how high the uGC level was during the peak-period in comparison to the uGC level during the pre-period. Therefore, we divided the mean hormone level of peak-period samples through the hormone level of the pre-period samples: relative uGC level = (mean uGC peak period / mean uGC pre period) x 100 for each subject for each series of urine samples . However, since urine is stored in the bladder and excreted after varying latencies, some urine samples were expected to contain urine from the two adjacent time periods. To address this problem, we assigned these samples to the time period in which the greatest proportion of urine was likely to have been excreted . Specifically, if the sample was collected within 30 min after the pre-period (t1) or the peak-period (t2), this was considered to be a potential ‘overlap zone’ . ‘Overlap-zone’ samples could be assigned to periods on either side of the period changing point depending on the latency between the sample excreted in the ‘overlap zone’ and the previous urination. If Δt between the sample in the ‘overlap zone’ and t1 (or t2 respectively) was smaller than between the last sample before t1 (or t2) and t1 (or t2), we classified the sample in the ‘overlap zone’ as a sample from the period before t1 (or t2 respectively). In all other cases the sample was classified as belonging to the time period in which it was excreted .The adult males observed during our study had an average urination interval ± SD = 78 min ± 32. We defined GC clearance in urine of chimpanzees following the results of . Using 3stration . Althougstration , absolutIn order to examine the influence of various predictor variables on changes in urinary GC levels, we ran General Linear Mixed Models (GLMM: ) using RWe ran several GLMMs. In the model including all the data, relative uGC level was the response variable and subject identity was the random factor. We ran additional models which including only the samples relating to aggressive events. This was in order to examine behavioural variation related to aggression (which obviously did not occur for resting events). Due to the small sample size, we were unable to fit all predictor variables into one model. Therefore we looked at the effects of each of the predictor variables on the relative uGC level separately. To establish the significance of the full model we used a likelihood ratio test, comparing the full model with the respective null model comprising only the random effect and the intercept. When models were insignificant, we considered the predictor variables to have no clear effect on the response variable.The ethics committee of the School of Psychology, University of St. Andrews, UK, approved this non-invasive behavioural and hormonal study with the Sonso chimpanzees located around Budongo Conservation Field Station  in the Budongo Forest Reserve, Uganda. In accordance with ethical guidelines we kept 7m distance to the chimpanzees, never interacted with chimpanzee subjects and collected urine with plastic bags, when subjects were sitting > 10m high in the trees, or from leafs after subjects had voluntarily moved. Research was conducted under the permits by the Uganda Wildlife Authority (TDO/33/02) and Uganda National Council for Science and Technology (NS 181). Chimpanzees are an endangered species (IUCN red list) and under the protection of Uganda Wildlife Authority.χ2 = 5.35, df = 1, p = 0.021). Males’ relative uGC levels were significantly higher after aggression (mean relative uGC ± SD = 112% ± 28) than after resting for >30 min . Combining the target behaviour with the subject’s role during the aggression  showed that uGC levels tended to be higher  in both aggressors (mean relative uGC ± SD = 107% ± 12) and victims  than after resting. Estimates show that whilst differences in uGC levels were large for both aggressors and victims against resting, differences were small between aggressor and victim conditions  with higher ranking males showing greater elevation of relative uGC levels after aggression . In contrast we did not find effects due to aggression intensity (contact or non-contact) or aggression duration (min.), although it should be noted that negative effects could be a result of small sample sizes .Further, we investigated in three separate GLMMs whether the subject’s rank (rank 1–9), aggression duration (min.) or aggression intensity (contact or non-contact aggression) affected the relative uGC levels. Only subject’s dominance rank showed a significant effect (Male chimpanzee aggressors and victims had larger increases in uGC levels after single aggressive interactions than after resting. In addition, higher ranking male opponents had greater increases in uGC levels than lower ranking males. Sample sizes were small but our results are comparable to previous studies showing raised plasma cortisol in both winners and losers in humans , rodentsThe proximate reasons for the increased uGC levels in our study remain unclear. In fish, plasma cortisol increased after fights in both winners and losers, but winners showed only increased plasma cortisol levels 5 min after the end of the fights, while losers still had increased plasma cortisol levels 24h after the fight . If injuHowever, we suggest this is unlikely to be the only possible explanation, for the following reasons. First, a study in humans has shown that competitive martial arts matches release significantly more plasma cortisol than comparable non-competitive exercise , suggestWith the above in mind, it is very unlikely that the energetic stress hypothesis  alone caSocial stress is often caused by a lack of control and predictability, such as the exclusion from food or other resources, or the threat of recurring aggression , 11. ThiOur results further our understanding of conflict management in animals. Several studies on primates have shown an increase of self-directed behaviours in aggressors and victims –49, (but


Accumulating evidence from animal and human studies suggests that vitamin D is involved in many functions of the human reproductive system in both genders, but no comprehensive analysis of the potential relationship between vitamin D status and Assisted Reproduction Technologies (ART) outcomes is currently available. On this basis, the purpose of this systematic review and meta-analysis was to perform an in-depth evaluation of clinical studies assessing whether vitamin D status of patients undergoing ART could be related to cycle outcome variables. This issue is of interest considering that vitamin D deficiency is easily amenable to correction and oral vitamin D supplementation is cheap and without significant side effects. Surprisingly, no studies are currently available assessing vitamin D status among male partners of couples undergoing ART, while seven studies on vitamin D status of women undergoing controlled ovarian hyperstimulation (COH) for ART were found and included in the review. Results show that vitamin D deficiency is highly prevalent among women undergoing COH, ranging from 21% to 31% across studies conducted in Western countries and reaching 75-99% in Iranian studies. Data on vitamin D deficiency (25-hydroxyvitamin D serum levels <20 ng/ml) in relation to ART outcomes could be extracted from three studies and included in the meta-analysis, yielding a common risk ratio (RR) of 0.89 (95% CI 0.53-1.49) and showing a lower but not statistically significant likelihood of clinical pregnancy for vitamin-D-deficient women compared with vitamin-D-sufficient patients. In conclusion, there is insufficient evidence to support the routine assessment of vitamin D status to predict the clinical pregnancy rate in couples undergoing ART. The partly conflicting results of the available studies, potentially explaining the lack of statistical significance for a negative influence of vitamin D deficiency on clinical pregnancy rate, are likely secondary to confounders and insufficient sample size, and further larger cohort and randomised controlled studies are required. Accumulating evidence from animal and human studies suggests that vitamin D is involved in many functions of the human reproductive system in both genders. While crucial roles for vitamin D in human infertility, pregnancy and neonatal growth have been extensively reviewed elsewhere -5 no comA role for vitamin D in ovarian steroidogenesis is well established , and rec1. Vitamin D deficiency has been suggested to contribute to the development of insulin resistance and impaired glucose clearance in Polycystic Ovary Syndrome (PCOS). Indeed, even if the issue of whether PCOS itself may be associated with altered levels of vitamin D is still controversial, lower 25-hydroxyvitamin D [25(OH)D] levels characterise PCOS women suffering from metabolic disturbances including obesity, metabolic syndrome and insulin resistance ,8. Consiin vitro could not be categorized neither as vitamin D deficient nor as vitamin D sufficient and was therefore not considered. Studies categorising patients only based on follicular fluid (FF) rather than serum levels of 25(OH)D were excluded from the meta-analysis in order to contain the heterogeneity.For each study, the following information was extracted: first author's last name; year of publication; country of origin; number of subjects; design of the study; statistical analysis; outcomes assessed; confounding factors considered; results. The only cycle outcome parameter assessed in all studies was CPR. Outcome data on CPR were therefore retrieved from individual reports and pooled estimation and meta-analysis were performed as follows. The number of patients who achieved a clinical pregnancy among groups of women with and without serum 25(OH)D deficiency (<20 ng/ml) was obtained from each study and used in the meta-analysis. A clinical pregnancy was defined as the observation of an intrauterine gestational sac following IVF/ICSI procedures and fresh embryo transfer. In one study , patientThe heterogeneity Cochrane Q and the I2 statistics were calculated, showing a moderate heterogeneity (I2 = 53%); for this reason the DerSimonian-Laird random-effects model, which incorporates both within- and between-study variations was used in the meta-analysis ,36. Due in vitro studies n = 1, reviews without original data report, n = 6; Figure Figure ≥30 ng/ml) . Results are shown in Figure When pooling data for meta-analysis, data on the effect of vitamin D serum deficiency <20 ng/ml) on CPR after IVF/ICSI procedures were extracted ,40-42. C0 ng/ml oNo data are currently available regarding vitamin D and semen performance in regards to clinical outcomes including fertilization rate, embryo quality, pregnancy rate and LBR in the context of IVF treatments. Further studies conducted on spermatozoa from infertile men are utterly needed to allow the identification of a potential role of vitamin D in influencing treatment outcomes for couples undergoing IVF for male factor infertility. Of note, based on the available data in infertile men in general, there is an ongoing randomized controlled trial (RCT) by Blomberg Jensen’s group  testing the benefits of vitamin D supplementation on semen parameters.The issue of whether vitamin D levels are reliable predictors of ART outcomes is still controversial. Evidence is still poor, because no RCTs are currently available and results of existing small cohort studies are very heterogeneous. Consequently, the meta-analysis performed herein fails to identify a significant association between vitamin D status and CPR after ART. Marked differences in exposure categorisation, analytic approaches, outcomes considered and general methodological design characterize the identified studies. More specifically:1. Main outcomes varied greatly among the various studies including both biological and clinical outcomes such as embryo parameters and pregnancy rates.2. A matter of concern may be the threshold used to define insufficiency. The commonly accepted cut-off value for vitamin D deficiency [serum 25(OH)D < 20 ng/ml] has been established based on bone health in the general population, and may not be appropriate to the peri-conceptional period, with some authors proposing that circulating levels of 25(OH)D should be as high as 40–60 ng/ml during gestation . Therefo3. Some clinical conditions that are commonly encountered among IVF candidates  have been related to significantly altered serum 25(OH)D levels. These conditions are also associated with relevant differences in prognosis with regards to IVF outcomes. While the potential confounders represented by age and BMI can be mostly excluded because all studies properly controlled for both covariates in their statistical analyses , most of the available studies do not give sufficient information to exclude potential biases caused by the different prevalence of the other factors .4. Not all the currently available studies have controlled for potential confounders affecting implantation and pregnancy rates such as the number and quality of embryos transferred.5. Variations exist in the assays used for 25(OH)D assessment among studies as demonstrated by measurement variability and large discrepancies in concentrations observed in different laboratories .6. Finally, relevant factors such as duration of vitamin D deficiency or source of vitamin D   have nevDue to the presence of several sources of controversy in the interpretation of the relationship between vitamin D deficiency and poor IVF outcome, we have employed the nine criteria proposed by Austin Bradford Hill, which still stand as foundation milestones for addressing causation conditions . Five  and low (500 IU) dose vitamin D supplementation on 1000 women initiating IVF treatment in Sweden in the registered prospective double-blind randomised trial entitled “Vitamin D during IVF” [ClinicalTrials.gov: NCT01019785], but results are still not available. On the other hand, there is the need for independent trials in different populations as the benefits of supplementation may markedly differ among populations.ART: Assisted reproductive technology; BMI: Body mass index; COH: Controlled ovarian hyperstimulation; CP: Clinical pregnancy; CPR: Clinical pregnancy rate; FF: Follicular fluid; ICSI: Intra-cytoplasmic sperm injection; IVF: In vitro fertilization; LBR: Live birth rate; OR: Odds ratio; RR: Risk ratio; VDR: Vitamin D receptor.The authors declare that they have no competing interests.The review was conceived by PV and MC. Data were collected, analyzed and interpreted by VSV, PV, EP and LP, who also drafted the article. ES, AP and MC made substantial contributions to further interpretation and discussion of data and to article revision. All authors read and approved the final manuscript.


Eupneic breathing in mammals is periodically interrupted by spontaneous augmented breaths (sighs) that are characterized by a biphasic larger-amplitude inspiratory burst followed by post-sigh apnea. Previous in vitro studies in newborn rodents have demonstrated that the respiratory oscillator of the pre-Bötzinger complex (preBötC) can generate the distinct inspiratory-related motor patterns for both eupnea- and sigh-like activity ,2. Howev2+ oscillations and the eupnea subnetwork generates fast oscillations mainly driven by activation/inactivation of the persistent Na+ current .Furthermore, we used this model to make predictions that were subsequently tested on the isolated preBötC in brainstem slice preparations. Through a combination of our in vitro and in silico approaches we found that 1), sigh events are less sensitive to network excitability than eupneic activity, 2)The combination of voltage-gated calcium current and persistent sodium current control the sigh period of, and 3), specific parameters of Ih activation set the low sensitivity to excitability in the sigh neuronal subset. Altogether, our results strongly support the hypothesis that distinct subpopulations within the preBötC network are responsible for sigh and eupnea rhythmogenesis.The model reproduces basic features of simultaneous sigh and eupnea generation: two types of bursts differing in terms of shape, amplitude and frequency of occurrence and mimics the effect of glycinergic synapses blockade. We designed a two-compartment computational model for sigh and eupnea subpopulations of neurons with several different parameters reflecting distinct burst generating mechanisms. The sigh subpopulation generates a low frequency rhythm based on slow intracellular Ca


P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low  While coronary artery disease (CAD) is due in part to environmental and metabolic factors, about half of the risk is genetically predetermined. Genome-wide association studies in human populations have identified approximately 150 sites in the genome that appear to be associated with CAD. The mechanisms by which mutations in these regions are responsible for predisposition to CAD remain largely unknown. To begin to explore how disease-specific gene sequences and disease gene function promotes pathology, we have mapped the loci and genes that are downstream of the transcription factor TCF21, which is strongly associated with CAD. By identifying genes that are regulated by TCF21 we have been able to link together multiple other CAD associated genes and begin to identify the critical molecular processes that mediate atherosclerosis in the blood vessel wall and contribute to the genesis of ischemic cardiovascular events. Recent large-scale GWAS have identified 46 genome-wide significant CAD loci and a further 104 independent variants associated at a 5% false discovery rate (FDR), yet the biological and disease-relevant mechanisms for these associations remain largely unknown [Tcf21 is expressed in mesodermal cells in the proepicardial organ that give rise to coronary artery smooth muscle cells (SMC) and loss of Tcf21 results in increased expression of smooth muscle markers by cells on the heart surface consistent with premature SMC differentiation [Tcf21 in the fate decisions of a precursor cell for SMC and cardiac fibroblast lineages [Tcf21 is important for expansion of the SMC compartment of the coronary circulation, with persistent Tcf21 expression being required for cardiac fibroblast development [Tcf21 is a member of the basic helix-loop-helix (bHLH) TF family and is critical for the development of a number of cell types during embryogenesis of the heart, lung, kidney, and spleen . Tcf21 intiation . Knockoulineages . These delopment .To better understand the cellular functions of TCF21 in the SMC lineage, and to gain insights into how such functions might contribute to CAD risk, we performed chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq), examined the downstream target loci and genes that harbor TCF21 binding sites, and employed bioinformatic and experimental approaches to investigate how the target genes work together to mediate the risk of CAD. Pathway analysis of downstream target genes revealed that TCF21 regulates cell-cell and cell-matrix interactions as well as growth factor signaling pathways. Also, we found TCF21 target regions to be over-represented among CAD associated loci, and that genes in these regions assemble into pathways that mediate fundamental processes such as cell cycle, chromatin remodeling, and growth factor signaling. Taken together, these studies elucidate disease-associated genes and pathways that lie downstream of TCF21, and show how SMC related processes may be responsible for a substantial portion of the genetic risk for CAD.Our primary interest in these studies was the transcriptional network of TCF21-regulated genes contributing to the development of human CAD. Because of the known role of TCF21 in the embryonic development of coronary vascular SMC, we undertook these ChIP-Seq experiments in primary cultured human coronary artery SMC (HCASMC). Furthermore, we selected culture conditions that maintain these cells in the synthetic, undifferentiated state that most closely reflects the disease phenotype . Two polChIP-Seq using Ab1 identified 10,523 peaks while Ab2 identified 4,900 peaks that largely overlapped with those identified by Ab1. These two sets of peaks were within 50 kb of 12,226 and 7,150 genes, respectively . To bettIL6R, SH2B3, and SMG6. Due to this overlap, as well as the similarities in peak binding patterns described above, we intersected the two datasets to refine the number of peaks to those identified by both antibodies  performed in this laboratory  and withTCF21 gene expression. We retrieved 2705 co-expression modules, each containing highly co-regulated genes, derived from 108 coexpression networks constructed from many different tissues in human and mouse populations [TCF21 and that identified peaks functionally regulate target gene expression.To investigate whether genes with binding peaks are directly regulated by TCF21, we took advantage of existing co-expression networks to investigate overlap between ChIP-Seq identified target genes and those genes that track with ulations . We thenulations . Taken tTo investigate the molecular and cellular processes downstream of TCF21 and possible mechanisms of disease association, studies were conducted to look for over-representation of TCF21 target genes among well annotated regulatory pathways. Here, we analyzed the common peak set (Ab_shared) with the Genomic Regions Enrichment of Annotations Tool (GREAT) [P = 1e-1010) (P = 1e-627), and identified as identical to the motif recognized by bHLH factor Olig2, likely representing a second motif that is recognized by TCF21. Interestingly, an additional enriched TF binding motif was also identified in approximately 30% of peaks, corresponding to the bZIP motif TGA(G/C)TCA (P = 1e-336) that is known to bind the AP-1 family of TFs. Other motifs of interest included those that mediate binding of TEAD and CEBP transcription factor families. Graphing the distribution of these motifs in comparison to the summits of TCF21 peaks suggested that AP-1 and possibly ATF1 factors bind in a bimodal pattern flanking TCF21, suggesting a possible steric binding relationship between TCF21 and these bZip factors  . This se factors . These mTCF21, and disruption of this pathway by disease-associated allelic variation in the related binding site may account in part for the CAD susceptibility observed at this locus [P<4.12e-215, fold enrichment = 2.84), and employing the same methods with HAoSMC DHS regions as background, TCF21 overlap with JUN and JUND peaks remained significant .We have previously shown that JUN and other AP-1-related transcription factors transactivate is locus . To explWT1 gene and the developmental growth factor PDGFRB gene   and evaluated them in the context of their functional annotation. Functional SNPs for TCF21 target genes as defined by the RegulomeDB Category I  showed the highest fold enrichment for SNPs with CAD GWAS P-values < 0.01 . Category II SNPs  also showed highly significant enrichment for low P-values   which have been identified through studies in a variety of tissues investigating the genetic basis of gene expression ,16,29–31.60e-40) . All anai) coronary artery disease phenotypes, ii) risk factors that are known to be associated with CAD, iii) non-atherosclerotic vascular diseases that are not specifically associated with CAD, iv) primarily inflammatory disease phenotypes that are known to involve molecular pathways that are also linked to CAD, v) disease phenotypes related to tissues where TCF21 is known to not be expressed and which were predicted negative controls. Also, we focused analyses on traits with the largest number of associated variants, with the goal of strengthening the statistical analysis, and primarily employed traits/phenotypes with at least 20 associated variants. Phenotypes or traits that passed P<0.05 from both methods were deemed significant, yielding a combined cutoff of P<0.0025. Considering ~20 disease sets were tested, this combined statistical cutoff is equivalent to a Bonferroni-corrected P<0.05.We noted previously that a number of the CAD loci that have reached genome wide significance contain TCF21 peaks , and reaP-values. The methodology for this permutation-based analysis is provided in the Methods section.In the first analysis, we investigated over-representation of TCF21 binding region genes among CAD locus genes. As a preliminary analysis, to test for possible confounding, we tested whether TCF21 binding is by chance more likely to be near genes mapped for various GWAS phenotypes by running an enrichment analysis between genes linked to TCF21 binding sites and all GWAS genes from the GWAS Catalog . As showP = 0.014) and these results did not change substantially with exclusion of lipid trait genes  (P<1.0e-03) and again this did not change substantially with removal of lipid trait variation . We also found that the candidate sets of GWAS genes associated with the CAD related trait platelet number and a disease phenotype related to a dysfunctional immune system, inflammatory bowel disease (IBD), also showed a high degree of enrichment for TCF21 target genes among the GWAS gene sets. At Bonferroni corrected P<0.05 (raw P<0.0022) in this test, height, CAD extended, CAD extended no lipid, IBD, and platelet phenotypes reached statistical significance. Importantly, we found little evidence of enrichment of TCF21 target genes among GWAS candidate genes for risk factors blood pressure, lipids, and glucometabolic related traits.Employing genes in the GWAS catalog associated with CAD phenotypes, enrichment was found for TCF21 target genes among CAD genes compared to a background of all GWAS genes  . When CA2 greater than or equal to the true data. Results from this analysis showed that SNPs in TCF21 peaks have significantly greater LD than expected by chance with SNPs for CAD related phenotypes: CAD (permutation P = 0.0209) and for CAD Extended (permutation P = 0.0086) that analyzed GWAS SNPs plus those from CARDIoGRAM+C4D  terms annotated for the “biological processes” category. For the TCF21 target CAD genes , the top terms that reached significance were disease ontology terms related to migration, including “regulation of cell migration,” “regulation of locomotion,” as well as those related to cell division, “regulation of proliferation,” and metabolism, “regulation of phosphorous metabolic and signaling processes,” regulation of protein amino acid phosphorylation” . Only 3 Further, we investigated the interaction of TCF21 peak CAD genes with other genes with the STRING algorithm that integrates protein-protein interaction, text mining and genomic data to link candidate genes into a related network . For thiWe focused ChIP-Seq experiments on smooth muscle cells based on previous findings demonstrating the role of TCF21 in coronary vascular development during embryogenesis and its relevance for the CAD phenotype. During development, TCF21 is expressed in SMC progenitors but not in endothelial or other cell types present in vascular disease lesions . VasculaTCF21 expression [We provide evidence for cooperation of TCF21 with other transcription factors through the finding of other TF binding motifs in the ChIP-Seq peak sequences. We found evidence for enrichment of JUN (AP-1) and several other bZip factor binding motifs, including CEBP and ATF1, in the TCF21 peaks. There was also enrichment for binding sites recognizing the TEAD family of transcription factors that have been linked to signaling through the Hippo pathway that regulates developmental and cancer related cellular processes . We founpression . These fpression , provideTCF21 risk for CAD [These ChIP-Seq studies have identified a number of other CAD associated loci and genes that are bound by TCF21, identifying a disease related transcriptional network. To investigate the hypothesis that TCF21 targets a greater than expected number of CAD-related genes we performed enrichment analysis at both the gene and SNP level with GWAS data. These studies identified a greater than expected number of TCF21 targets among CAD GWAS genes, and showed that TCF21 peak region SNPs are in greater linkage disequilibrium with CAD GWAS SNPS than expected. Since the ChIPseq data was generated in HCASMC, a cell type found in the coronary artery, the results of CAD loci enrichment is consistent with the hypothesis that TCF21 regulates a greater than expected number of CAD genes in the vessel wall and that its effect on disease risk may be due in part to its regulation of the network of TCF21 target CAD associated genes. Consistent with TCF21 CAD target genes being active primarily in the vascular wall, we saw no difference in enrichment between the CAD analyses with and without lipid genes or SNPs being included in the analysis, and no enrichment was found for vascular risk factor traits “LDL cholesterol” and “Total cholesterol.” The enrichment for “Platelets” is not surprising given the known association of platelet number with vascular disease events, and this association could explain some portion of the  for CAD –43. HumaSurprisingly, both the gene- and SNP-level enrichment GWAS analyses also found TCF21 target genes to be over-represented among those associated with height and immune related IBD. Although SMC would seem unlikely to be involved in the height and IBD phenotypes, we speculate that enrichment of TCF21 target genes within these other phenotypes is real and reflects overlapping biology between the physiology/pathophysiology of these phenotypes and that of CAD. For instance, short stature has been repeatedly linked to an elevated risk for CAD, suggesting the presence of a substantial overlap in the biological pathways that determine height as well as the risk of CAD –46. In sOur analyses provide multiple lines of circumstantial evidence supporting a role for the identified TCF21 related transcriptional network in the etiology of CAD. First, the TCF21 target genes are highly enriched for molecular processes with established relationships to CAD, such as growth factor binding, cell adhesion, vascular development, inflammation, and PDGF signaling. Second, the TCF21 target genes are enriched for established CAD genes identified through GWAS. Third, the functional SNPs of TCF21 target genes are enriched for, and tend to be in significant LD with, SNPs associated with CAD in GWAS. Fourth, the finding that the TCF21 transcriptional network is enriched in other traits is consistent with a biological program that contributes to a number of disease-relevant tissue-specific cellular processes. However, further work is required to firmly establish our hypothesis that the TCF21 transcriptional gene network contributes to CAD risk. If TCF21 binding to other CAD loci is important for the disease risk mechanism, one may be able to identify epistatic effects between polymorphisms in or near TCF21 and other CAD SNPs in the TCF21 target regions. At an experimental level it will be important to link TCF21 binding sites in target loci to function of the causal variant and the causal gene, and to investigate how TCF21 binding may regulate the causal mechanisms at other CAD loci. Finally, it will be important to functionally assess the predicted TCF21 gene network by perturbation in vitro and in vivo with experimental cell culture and animal models. Taken together, these approaches may provide significant new insights into how a disease associated transcription factor may work through other disease-associated loci to modulate disease risk.Human primary Coronary Artery Smooth Muscle Cells  were purchased from Lonza  and cultured in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics  for 3 passages. Technical replicate samples were cultured from the same stock vial but harvested 24h apart. The crosslinked nuclei of all replicates were thereafter processed in parallel.Ten million HCASMC cells per condition were crosslinked for 10 min with 1% formaldehyde. Cells were lysed for 10 min on ice . Nuclei were then lysed for 10 min on ice . Crosslinked chromatin was sheared for 3x5 min by sonication using a Bioruptor  and pre-cleared with 10 μ g anti-rabbit IgG pre-immune serum  using protein G sepharose beads . TCF21 ChIP was performed overnight in IP dilution buffer , using 14 μ g anti-TCF21  or Abcam, Cambridge, UK, #ab49475, Lot No. 153899 (Ab2)) or anti-rabbit IgG respectively. JUN and JUND ChIP was performed with Santa Cruz Biotechnology sc-1694 and sc-74 respectively. Beads were washed twice with buffer 1 , once with buffer 2  and twice with TE buffer. Bound chromatin was eluted from the beads twice with 100 mM NaHCO3 containing 1% SDS. After reverse-crosslinking, RNaseA and proteinase K digestion, chromatin was cleaned up using the Qiagen PCR purification kit .Follow-up confirmatory ChIP was performed with immunoprecipitated and reverse-crosslinked chromatin from HCASMC that was prepared as described above. Quantitative real-time PCR  was performed with primers specific for ChIP-Seq peak sequences using SYBR Green (Applied Biosystems) assays and fold enrichment was calculated by measuring the delta Ct—delta Ct IgG. Melting curve analysis was also performed for each ChIP primer pair.Original data has been deposited at GEO, accession number: GSE61369.Q-value) for subsequent analysis. We utilized the Genomic Regions Enrichment of Annotations Tool (GREAT) [Sequence data were aligned to the reference genome (hg19) using BWA . In the  (GREAT)  to analy (GREAT) . Genes oTo more broadly assess the co-expression patterns among the TCF21 target genes, we retrieved 2,705 co-expression modules derived from 108 coexpression networks constructed from many different tissues in human and mouse populations . The enrATAC-Seq was performed with slight modifications to published protocol . BrieflyTo discover DNA binding motifs enriched among TCF21 peaks, HOMER findMotifsGenome.pl script was used to search for the known TRANSFAC motifs and to create de novo motifs from 4852 TCF21 ChIP-Seq binding regions (Ab_Shared regions) . All sofP<0.05 as true HAoSMC DHS signal (n = 121731). The overlaps among TCF21 binding regions, ATAC-Seq, and HAoSMC DHS peaks were evaluated through one-sided Fisher exact test. In the overlap between TCF21 ChIP-Seq and ATAC-Seq peaks, we only focused on the ATAC-Seq signals with high-confidence fold-enrichment (mfold>200).We retrieved the HAoSMC DHS data from the ENCODE project and selected peaks with We tested co-activity between TCF21 and the AP-1 factors JUN and JUND by evaluating the level of overlap (using BEDTools) between ChIP-Seq binding regions corresponding to each TF. Peaks from each dataset were first reduced to those that overlap with open chromatin as identified with our HCASMC ATAC-Seq or ENCODE HAoSMC DHS data. In each analysis, a one-sided Fisher’s exact test was used to determine whether the observed peak overlap between TCF21 and JUN or between TCF21 and JUND was statistically greater when compared against a background of all HCASMC ATAC-Seq or HAoSMC DHS peaks.P-values of association (P<0.01) with CAD when compared to the proportion of low P-values observed for all functional SNPs examined in CARDIoGRAM, using a Fisher's exact test. The distribution of the P-values of these two sets of SNPs was also tested using a one-sided Kolmogorov-Smirnov (KS) test. For this analysis, we identified 189,002 functional SNPs (eSNPs) through eQTL experiments involving liver, brain, blood, human aortic endothelial cells (HAEC), and adipose tissues [http://www.regulomedb.org/). Functional SNPs from RegulomeDB were further classified into various categories based on multiple measures of functional implication according to the database, with category 1 SNPs having the highest number of independent functional measures. TCF21 target genes were first mapped to their associated eSNPs or RegulomeDB SNPs and then compared with the corresponding background functional SNPs of all genes for enrichment of low P-value associations with CAD. For both analyses, LD structure was addressed by pruning the eQTL and ENCODE SNPs. This was done by removing those with r2 > 0.3 to derive independent SNPs.We utilized the publicly available summary level association results (~2.5 million HapMap SNPs) of the CARDIoGRAM consortium involving 22,233 CAD cases and 64,762 controls to further analyze the relevance of the TCF21 target genes to CAD . We expl tissues –33,55,56To investigate possible over-representation of TCF21 binding regions among CHD GWAS loci, we conducted analyses at both the gene and variant level, looking for enrichment of TCF21 target genes among CHD GWAS loci, and for enrichment of average linkage disequilibrium between TCF21 peak SNPs and GWAS loci SNPs.We first assessed whether there was a spurious association of TCF21 target genes with CAD due to confounding, by looking for an over-representation of these genes among those associated with GWAS loci genes in general. For this analysis, we downloaded the NHGRI GWAS catalog and extracted candidate GWAS genes for all diseases or phenotypes curated in the catalog. Enrichment of candidate GWAS genes among the TCF21 target genes located in the TCF21 peaks was estimated using one-sided Fisher’s exact test. We then looked for an over-representation of CAD candidate genes discovered thorough recent GWAS studies among genes targeted by TCF21. The list of CAD candidate genes we considered (“CAD”) was also collated from the NHGRI GWAS catalog and includes all genes assigned to a SNP in the catalog for phenotypes relevant to CAD including "coronary heart disease", "myocardial infarction", and "coronary artery calcification". For a second category (“CAD extended”) this list was supplemented with genes identified through the CARDIoGRAMplusC4D study, which derive from analysis of Metabochip data and for this reason have not been included in the GWAS catalog . Lipid mFor both GWAS enrichment methods we also included additional phenotypes as described in the Result section to test the specificity of CAD signal enrichment in TCF21 targets. These phenotypes included cholesterol measures related to CAD , diseases known to be associated with dysfunction of the arterial vessel wall or myocardial infarction , migraine, high blood pressure, platelet number, and Kawasaki's disease), height, which has been associated with CAD, glucometabolic related phenotypes insulin resistance (insulin resistance measures), β-cell function (B function glucose), diseases associated with a dysfunctional immune system (IBD), systemic lupus erythematosus, celiac disease), neuropsychiatric disorders (schizophrenia and Parkinson’s disease), breast cancer and macular degeneration which are well represented in the GWAS catalog but unlikely to be associated with CAD.To correct for any potential bias in the large numbers of GWAS genes for certain traits such as height and CAD, we implemented a permutation strategy by generating 1000 random GWAS gene sets of matching size for each trait to derive permutation-based enrichment P-values. For example, there are 414 GWAS genes for height, and we generated 1000 gene sets each containing 414 randomly selected GWAS genes from the pool of 8277 GWAS genes in the GWAS catalog. In addition to the enrichment P-value obtained with Fisher’s exact test, (Ptrait) for the observed GWAS gene set of each trait we also calculated 1000 enrichment P-values from the 1000 random gene sets . Ptrait was then compared against the 1000 P-values from the random gene sets to derive a permutation-based enrichment P-value as defined by /1000. This was done for each of the CAD phenotypes and each additional phenotypes/traits to correct for the different numbers of GWAS genes between phenotypes.2 value to TCF21—GWAS SNP pairs [2 values for a given SNP pair did not agree, we used the average of the two. Using custom software developed in the R language, we calculated the average of all r2 values between TCF21 peak—trait SNPs pairs, which was denoted as the “true average” for the association between TCF21 peak SNPs and that particular trait. We then computed 10,000 null averages, each time permuting the trait labels in the GWAS catalog and determining the r2 average between TCF21 peak SNPs and an equal number of SNPs now incorrectly associated with the trait of interest. In this analysis, if a TCF21 peak SNP was in LD with multiple GWAS SNPs, only the maximum r2 value of these associations was taken into calculating the r2 average for a given trait. This filter ensured that each TCF21 SNP mapped onto at most one GWAS trait SNP, although a GWAS trait SNP was allowed to map onto multiple TCF21 SNPs.For the TCF21-GWAS LD analysis, we developed a custom pipeline to assess the linkage between variants in TCF21 peaks and variants associated with disease by GWAS. The same GWAS traits/phenotypes were investigated in this analysis as for the gene enrichment analysis. SNPs were identified from the GWAS catalog, where we excluded any GWAS studies that did not contain a cohort of European descent. TCF21 peak SNPs were defined as those 1000 Genomes phase 1 variants that are polymorphic in Europeans and overlap with a TCF21 peak . We usedNP pairs . In the For pathway analysis, the DAVID algorithm  was emplS1 FigDistribution of TCF21 peaks identified by Ab1 (A) and Ab2 (B) in relation to transcription start site (TSS). Distribution of TCF21 peaks identified by Ab1 (C) and Ab2 (D) in relation to structural gene features.(TIF)Click here for additional data file.S2 FigChIP was performed with separately isolated chromatin from HCASMC derived from a different donor, employing PCR primers flanking a number of TCF21 peaks.(TIF)Click here for additional data file.S1 Table(PDF)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.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.