Foxf2: A Novel Locus for Anterior Segment Dysgenesis Adjacent to the Foxc1 Gene

Anterior segment dysgenesis (ASD) is characterised by an abnormal migration of neural crest cells or an aberrant differentiation of the mesenchymal cells during the formation of the eye's anterior segment. These abnormalities result in multiple tissue defects affecting the iris, cornea and drainage structures of the iridocorneal angle including the ciliary body, trabecular meshwork and Schlemm's canal. In some cases, abnormal ASD development leads to glaucoma, which is usually associated with increased intraocular pressure. Haploinsufficiency through mutation or chromosomal deletion of the human FOXC1 transcription factor gene or duplications of the 6p25 region is associated with a spectrum of ocular abnormalities including ASD. However, mapping data and phenotype analysis of human deletions suggests that an additional locus for this condition may be present in the same chromosomal region as FOXC1. DHPLC screening of ENU mutagenised mouse archival tissue revealed five novel mouse Foxf2 mutations. Re-derivation of one of these (the Foxf2W174R mouse lineage) resulted in heterozygote mice that exhibited thinning of the iris stroma, hyperplasia of the trabecular meshwork, small or absent Schlemm's canal and a reduction in the iridocorneal angle. Homozygous E18.5 mice showed absence of ciliary body projections, demonstrating a critical role for Foxf2 in the developing eye. These data provide evidence that the Foxf2 gene, separated from Foxc1 by less than 70 kb of genomic sequence (250 kb in human DNA), may explain human abnormalities in some cases of ASD where FOXC1 has been excluded genetically

Gene-Based Tests of Association

Genome-wide association studies (GWAS) are now used routinely to identify SNPs associated with complex human phenotypes. In several cases, multiple variants within a gene contribute independently to disease risk. Here we introduce a novel Gene-Wide Significance (GWiS) test that uses greedy Bayesian model selection to identify the independent effects within a gene, which are combined to generate a stronger statistical signal. Permutation tests provide p-values that correct for the number of independent tests genome-wide and within each genetic locus. When applied to a dataset comprising 2.5 million SNPs in up to 8,000 individuals measured for various electrocardiography (ECG) parameters, this method identifies more validated associations than conventional GWAS approaches. The method also provides, for the first time, systematic assessments of the number of independent effects within a gene and the fraction of disease-associated genes housing multiple independent effects, observed at 35%–50% of loci in our study. This method can be generalized to other study designs, retains power for low-frequency alleles, and provides gene-based p-values that are directly compatible for pathway-based meta-analysis

Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist

<p>Abstract</p> <p>Background</p> <p>The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia.</p> <p>Methods</p> <p>First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion.</p> <p>Results</p> <p>Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ΔT (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ΔT > -3°C for more than 12 hours.</p> <p>Conclusions</p> <p>Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.</p

Modulating Pharmacokinetics, Tumor Uptake and Biodistribution by Engineered Nanoparticles

Inorganic nanoparticles provide promising tools for biomedical applications including detection, diagnosis and therapy. While surface properties such as charge are expected to play an important role in their in vivo behavior, very little is known how the surface chemistry of nanoparticles influences their pharmacokinetics, tumor uptake, and biodistribution.Using a family of structurally homologous nanoparticles we have investigated how pharmacological properties including tumor uptake and biodistribution are influenced by surface charge using neutral (TEGOH), zwitterionic (Tzwit), negative (TCOOH) and positive (TTMA) nanoparticles. Nanoparticles were injected into mice (normal and athymic) either in the tail vein or into the peritoneum.Neutral and zwitterionic nanoparticles demonstrated longer circulation time via both i.p. and i.v. administration, whereas negatively and positively charged nanoparticles possessed relatively short half-lives. These pharmacological characteristics were reflected on the tumor uptake and biodistribution of the respective nanoparticles, with enhanced tumor uptake by neutral and zwitterionic nanoparticles via passive targeting

Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines

Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1–7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12–15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45−) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis

Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3−/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3−/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3−/− mice. Lipid metabolism disorders in Sik3−/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice

Environmental chemical stressors as epigenome modifiers:a new horizon in assessment of toxicological effects

In eukaryotic cells, chromatin transformation from euchromatin into heterochromatin as a means of controlling gene expression and replication has been known as the ?accessibility hypothesis?. The interplay of epigenetic changes including histone modifications, DNA methylation, RNA interference (RNAi) and other functional epigenetic components are intricate. It is believed that these changes are well-programmed, inherited and can be modified by environmental contaminant stressors. Environmentally-driven epigenetic alterations during development, e.g. embryonic, foetal or neonatal stage, may influence disease susceptibility in adulthood. Therefore, understanding how epigenome modifications develop in response to environmental chemicals and, how epigenetic-xenobiotic interactions influence human health will shed new insights into gene-environment interactions in the epidemiology of several diseases including cancer. In this review, we consider studies of chemical modifiers including nutritional and xenobiotic effects on epigenetic components in vitro or in vivo. By examining the most-studied epigenome modifications and how their respective roles are interlinked, we highlight the central role of xenbiotic-modified epigenetic mechanisms. A major requirement will be to study and understand effects following environmentally-relevant exposures. We suggest that the study of epigenetic toxicology will open up new opportunities to devise strategies for the prevention or treatment of at-risk populations

Stochastic Drift in Mitochondrial DNA Point Mutations: A Novel Perspective Ex Silico

The mitochondrial free radical theory of aging (mFRTA) implicates Reactive Oxygen Species (ROS)-induced mutations of mitochondrial DNA (mtDNA) as a major cause of aging. However, fifty years after its inception, several of its premises are intensely debated. Much of this uncertainty is due to the large range of values in the reported experimental data, for example on oxidative damage and mutational burden in mtDNA. This is in part due to limitations with available measurement technologies. Here we show that sample preparations in some assays necessitating high dilution of DNA (single molecule level) may introduce significant statistical variability. Adding to this complexity is the intrinsically stochastic nature of cellular processes, which manifests in cells from the same tissue harboring varying mutation load. In conjunction, these random elements make the determination of the underlying mutation dynamics extremely challenging. Our in silico stochastic study reveals the effect of coupling the experimental variability and the intrinsic stochasticity of aging process in some of the reported experimental data. We also show that the stochastic nature of a de novo point mutation generated during embryonic development is a major contributor of different mutation burdens in the individuals of mouse population. Analysis of simulation results leads to several new insights on the relevance of mutation stochasticity in the context of dividing tissues and the plausibility of ROS ”vicious cycle” hypothesis

A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes